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A mathematical model for simulating the phase-based transmissibility of a novel coronavirus https://doi.org/10.1186/s40249-020-00640-3 SHA: 018269476cd191365d6b8bed046078aea07c8c01 Authors: Yin, Tian-Mu Chen; Jia, Rui; Qiu-Peng, Wang; Ze-Yu, Zhao; Jing-An, Cui; Ling Date: 2020 DOI: 10.1186/s40249-020-00640-3 License: cc-by Abstract: Background As reported by the World Health Organization, a novel coronavirus (2019-nCoV) was identified as the causative virus of Wuhan pneumonia of unknown etiology by Chinese authorities on 7 January, 2020. The virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020. This study aimed to develop a mathematical model for calculating the transmissibility of the virus. Methods In this study, we developed a Bats-Hosts-Reservoir-People transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model. The next generation matrix approach was adopted to calculate the basic reproduction number (R 0) from the RP model to assess the transmissibility of the SARS-CoV-2. Results The value of R 0 was estimated of 2.30 from reservoir to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. Conclusions Our model showed that the transmissibility of SARS-CoV-2 was higher than the Middle East respiratory syndrome in the Middle East countries, similar to severe acute respiratory syndrome, but lower than MERS in the Republic of Korea. Text: On 31 December 2019, the World Health Organization (WHO) China Country Office was informed of cases of pneumonia of unknown etiology (unknown cause) detected in Wuhan City, Hubei Province of China, and WHO reported that a novel coronavirus (2019-nCoV), which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses on 11 February, 2020, was identified as the causative virus by Chinese authorities on 7 January [1] . It is reported that the virus might be bat origin [2] , and the transmission of the virus might related to a seafood market (Huanan Seafood Wholesale Market) exposure [3, 4] . The genetic features and some clinical findings of the infection have been reported recently [4] [5] [6] . Potentials for international spread via commercial air travel had been assessed [7] . Public health concerns are being paid globally on how many people are infected and suspected. Therefore, it is urgent to develop a mathematical model to estimate the transmissibility and dynamic of the transmission of the virus. There were several researches focusing on mathematical modelling [3, 8] . These researches focused on calculating the basic reproduction number (R 0 ) by using the serial intervals and intrinsic growth rate [3, 9, 10] , or using ordinary differential equations and Markov Chain Monte Carlo methods [8] . However, the bat origin and the transmission route form the seafood market to people were not considered in the published models. In this study, we developed a Bats-Hosts-Reservoir-People (BHRP) transmission network model for simulating the potential transmission from the infection source (probably be bats) to the human infection. Since the Bats-Hosts-Reservoir network was hard to explore clearly and public concerns were focusing on the transmission from Huanan Seafood Wholesale Market (reservoir) to people, we simplified the model as Reservoir-People (RP) transmission network model, and R 0 was calculated based on the RP model to assess the transmissibility of the SARS-CoV-2. The reported cases of SARS-CoV-2, which have been named as COVID-19, were collected for the modelling study from a published literature [3] . As reported by Li et al. [3] , the onset date of the first case was on 7 December, 2020, and the seafood market was closed on 1 January, 2020 [11] . The epidemic curve from 7 December, 2019 to 1 January, 2020 was collected for our study, and the simulation time step was 1 day. fourth-order Runge-Kutta method, with tolerance set at 0.001, was used to perform curve fitting. While the curve fitting is in progress, Berkeley Madonna displays the root mean square deviation between the data and best run so far. The coefficient of determination (R 2 ) was employed to assess the goodness-of-fit. SPSS 13.0 (IBM Corp., Armonk, NY, USA) was employed to calculate the R 2 . The Bats-Hosts-Reservoir-People (BHRP) transmission network model The BHRP transmission network model was posted to bioRxiv on 19 January, 2020 [12] . We assumed that the virus transmitted among the bats, and then transmitted to unknown hosts (probably some wild animals). The hosts were hunted and sent to the seafood market which was defined as the reservoir of the virus. People exposed to the market got the risks of the infection (Fig. 1) . The BHRP transmission network model was based on the following assumptions or facts: a) The bats were divided into four compartments: susceptible bats (S B ), exposed bats (E B ), infected bats (I B ), and removed bats (R B ). The birth rate and death rate of bats were defined as n B and m B . In this model, we set Ʌ B = n B × N B as the number of the newborn bats where N B refer to the total number of bats. The incubation period of bat infection was defined as 1/ω B and the infectious period of bat infection was defined as 1/γ B . The S B will be infected through sufficient contact with I B , and the transmission rate was defined as β B . b) The hosts were also divided into four compartments: susceptible hosts (S H ), exposed hosts (E H ), infected hosts (I H ), and removed hosts (R H ). The birth rate and death rate of hosts were defined as n H and m H . In this model, we set Ʌ H = n H × N H where N H refer to the total number of hosts. The incubation period of host infection was defined as 1/ω H and the infectious period of host infection was defined as 1/γ H . The S H will be infected through sufficient contact with I B and I H , and the transmission rates were defined as β BH and β H , respectively. c) The SARS-CoV-2 in reservoir (the seafood market) was denoted as W. We assumed that the retail purchases rate of the hosts in the market was a, and that the prevalence of SARS-CoV-2 in the purchases was I H /N H , therefore, the rate of the SARS-CoV-2 in W imported form the hosts was aWI H /N H where N H was the total number of hosts. We also assumed that symptomatic infected people and asymptomatic infected people could export the virus into W with the rate of μ P and μ' P , although this assumption might occur in a low probability. The virus in W will subsequently leave the W compartment at a rate of εW, where 1/ε is the lifetime of the virus. d) The people were divided into five compartments: susceptible people (S P ), exposed people (E P ), symptomatic infected people (I P ), asymptomatic infected people (A P ), and removed people (R P ) including recovered and death people. The birth rate and death rate of people were defined as n P and m P . In this model, we set Ʌ P = n P × N P where N P refer to the total number of people. The incubation period and latent period of human infection was defined as 1/ω P and 1/ω' P . The infectious period of I P and A P was defined as 1/γ P and 1/γ' P . The proportion of asymptomatic infection was defined as δ P . The S P will be infected through sufficient contact with W and I P , and the transmission rates were defined as β W and β P , respectively. We also assumed that the transmissibility of A P was κ times that of I P , where 0 ≤ κ ≤ 1. The parameters of the BHRP model were shown in Table 1 . We assumed that the SARS-CoV-2 might be imported to the seafood market in a short time. Therefore, we added the further assumptions as follows: a) The transmission network of Bats-Host was ignored. b) Based on our previous studies on simulating importation [13, 14] , we set the initial value of W as following impulse function: In the function, n, t 0 and t i refer to imported volume of the SARS-CoV-2 to the market, start time of the simulation, and the interval of the importation. Therefore, the BHRP model was simplified as RP model and is shown as follows: During the outbreak period, the natural birth rate and death rate in the population was in a relative low level. However, people would commonly travel into and out from Wuhan City mainly due to the Chinese New Year holiday. Therefore, n P and m P refer to the rate of people traveling into Wuhan City and traveling out from Wuhan City, respectively. In the model, people and viruses have different dimensions. Based on our previous research [15] , we therefore used the following sets to perform the normalization: In the normalization, parameter c refers to the relative shedding coefficient of A P compared to I P . The normalized RP model is changed as follows: The transmissibility of the SARS-CoV-2 based on the RP model In this study, we used the R 0 to assess the transmissibility of the SARS-CoV-2. Commonly, R 0 was defined as the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population [13, 16, 17] . If R 0 > 1, the outbreak will occur. If R 0 < 1, the outbreak will toward an end. In this study, R 0 was deduced from the RP model by the next generation matrix approach [18] . The multiple of the transmissibility of A P to that of I P . The parameters were estimated based on the following facts and assumptions: a) The mean incubation period was 5.2 days (95% confidence interval [CI]: 4.1-7.0) [3] . We set the same value (5.2 days) of the incubation period and the latent period in this study. Thus, ω P = ω' P = 0.1923. b) There is a mean 5-day delay from symptom onset to detection/hospitalization of a case (the cases detected in Thailand and Japan were hospitalized from 3 to 7 days after onset, respectively) [19] [20] [21] . The duration from illness onset to first medical visit for the 45 patients with illness onset before January 1 was estimated to have a mean of 5.8 days (95% CI: 4.3-7.5) [3] . In our model, we set the infectious period of the cases as 5.8 days. Therefore, γ P = 0.1724. c) Since there was no data on the proportion of asymptomatic infection of the virus, we simulated the baseline value of proportion of 0.5 (δ P = 0.5). d) Since there was no evidence about the transmissibility of asymptomatic infection, we assumed that the transmissibility of asymptomatic infection was 0.5 times that of symptomatic infection (κ = 0.5), which was the similar value as influenza [22] . We assumed that the relative shedding rate of A P compared to I P was 0.5. Thus, c = 0.5. e) Since 14 January, 2020, Wuhan City has strengthened the body temperature detection of passengers leaving Wuhan at airports, railway stations, long-distance bus stations and passenger terminals. As of January 17, a total of nearly 0.3 million people had been tested for body temperature [23] . In Wuhan, there are about 2.87 million mobile population [24] . We assumed that there was 0.1 million people moving out to Wuhan City per day since January 10, 2020, and we believe that this number would increase (mainly due to the winter vacation and the Chinese New Year holiday) until 24 January, 2020. This means that the 2.87 million would move out from Wuhan City in about 14 days. Therefore, we set the moving volume of 0.2 million per day in our model. Since the population of Wuhan was about 11 million at the end of 2018 [25] , the rate of people traveling out from Wuhan City would be 0.018 (0.2/11) per day. However, we assumed that the normal population mobility before January 1 was 0.1 times as that after January 10. Therefore, we set the rate of people moving into and moving out from Wuhan City as 0.0018 per day (n P = m P = 0.0018). f) The parameters b P and b W were estimated by fitting the model with the collected data. g) At the beginning of the simulation, we assumed that the prevalence of the virus in the market was 1/100000. h) Since the SARS-CoV-2 is an RNA virus, we assumed that it could be died in the environment in a short time, but it could be stay for a longer time (10 days) in the unknown hosts in the market. We set ε = 0.1. In this study, we assumed that the incubation period (1/ ω P ) was the same as latent period (1/ω' P ) of human infection, thus ω P = ω' P . Based on the equations of RP model, we can get the disease free equilibrium point as: In the matrix: By the next generation matrix approach, we can get the next generation matrix and R 0 for the RP model: The R 0 of the normalized RP model is shown as follows: Our modelling results showed that the normalized RP model fitted well to the reported SARS-CoV-2 cases data (R 2 = 0.512, P < 0.001) (Fig. 2) . The value of R 0 was estimated of 2.30 from reservoir to person, and from person to person and 3.58 from person to person which means that the expected number of secondary infections that result from introducing a single infected individual into an otherwise susceptible population was 3.58. In this study, we developed RP transmission model, which considering the routes from reservoir to person and from person to person of SARS-CoV-2 respectively. We used the models to fit the reported data in Wuhan City, China from published literature [3] . The simulation results showed that the R 0 of SARS-CoV-2 was 3.58 from person to person. There was a research showed that the R 0 of SARS-CoV-2 was 2.68 (95% CI: 2.47-2.86) [8] . Another research showed that the R 0 of SARS-CoV-2 was 2.2 (95% CI: 1.4-3.9) [3] . The different values might be due to the different methods. The methods which Li et al. employed were based on the epidemic growth rate of the epidemic curve and the serial interval [3] . Our previous study showed that several methods could be used to calculate the R 0 based on the epidemic growth rate of the epidemic curve and the serial interval, and different methods might result in different values of R 0 [26] . Our results also showed that the R 0 of SARS-CoV-2 was 2.30 from reservoir to person which was lower than that of person to person. This means that the transmission route was mainly from person to person rather than from reservoir to person in the early stage of the transmission in Wuhan City. However, this result was based on the limited data from a published literature, and it might not show the real situation at the early stage of the transmission. Researches showed that the R 0 of severe acute respiratory syndrome (SARS) was about 2.7-3.4 or 2-4 in Hong Kong, China [27, 28] . Another research found that the R 0 of SARS was about 2.1 in Hong Kong, China, 2.7 in Singapore, and 3.8 in Beijing, China [29] . Therefore, we believe that the commonly acceptable average value of the R 0 of SARS might be 2.9 [30] . The transmissibility of the Middle East respiratory syndrome (MERS) is much lower than SARS. The reported value of the R 0 of MERS was about 0.8-1.3 [31] , with the inter-human transmissibility of the disease was about 0.6 or 0.9 in Middle East countries [32] . However, MERS had a high transmissibility in the outbreak in the Republic of Korea with the R 0 of 2.5-7.2 [33, 34] . Therefore, the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS transmitted in the Republic of Korea. To contain the transmission of the virus, it is important to decrease R 0 . According to the equation of R 0 deduced from the simplified RP model, R 0 is related to many parameters. The mainly parameters which could be changed were b P , b W , and γ. Interventions such as wearing masks and increasing social distance could decrease the b P , the intervention that close the seafood market could decrease the b W , and shorten the duration form symptoms onset to be diagnosed could decrease 1/γ. All these interventions could decrease the effective reproduction number and finally be helpful to control the transmission. Since there are too many parameters in our model, several limitations exist in this study. Firstly, we did not use the detailed data of the SARS-CoV-2 to perform the estimation instead of using the data from literatures [3] . We simulated the natural history of the infection that the proportion of asymptomatic infection was 50%, and the transmissibility of asymptomatic infection was half of that of symptomatic infection, which were different to those of MERS and SARS. It is known that the proportion of asymptomatic infection of MERS and SARS was lower than 10%. Secondly, the parameters of population mobility were not from an accurate dataset. Thirdly, since there was no data of the initial prevalence of the virus in the seafood market, we assumed the initial value of 1/100 000. This assumption might lead to the simulation been under-or over-estimated. In addition, since we did not consider the changing rate of the individual's activity (such as wearing masks, increasing social distance, and not to travel to Wuhan City), the estimation of importation of the virus might not be correct. All these limitations will lead to the uncertainty of our results. Therefore, the accuracy and the validity of the estimation would be better if the models fit the first-hand data on the population mobility and the data on the natural history, the epidemiological characteristics, and the transmission mechanism of the virus. By calculating the published data, our model showed that the transmissibility of SARS-CoV-2 might be higher than MERS in the Middle East countries, similar to SARS, but lower than MERS in the Republic of Korea. Since the objective of this study was to provide a mathematical model for calculating the transmissibility of SARS-CoV-2, the R 0 was estimated based on limited data which published in a literature. More data were needed to estimate the transmissibility accurately.

As of January 17, how many people were tested for body temperature?

1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A (H1N1) viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: [email protected] The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

Are viruses in the first and third waves of the 1918 swine flu pandemic same or derived from the virus from the second wave of the swine flu?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

What does the disruption of the ciliary movement during viral infection may cause?

Epidemiology of HBoV1 infection and relationship with meteorological conditions in hospitalized pediatric patients with acute respiratory illness: a 7-year study in a subtropical region https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048719/ SHA: f2f78c95ab378a31bd35dc1de84e0ec75eb7ce1b Authors: Liu, Wen-Kuan; Liu, Qian; Chen, De-Hui; Tan, Wei-Ping; Cai, Yong; Qiu, Shu-Yan; Xu, Duo; Li, Chi; Li, Xiao; Lin, Zheng-Shi; Zhou, Rong Date: 2018-07-16 DOI: 10.1186/s12879-018-3225-3 License: cc-by Abstract: BACKGROUND: Human bocavirus 1 (HBoV1) is an important cause of acute respiratory illness (ARI), yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients (≤14 years old), with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 (49.2%) were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 (2.2%) were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients (45.2%, 112/248). A significant difference in the prevalence of HBoV1 was found in patients in different age groups (p < 0.001), and the peak prevalence was found in patients aged 7–12 months (4.7%, 56/1203). Two HBoV1 prevalence peaks were found in summer (between June and September) and winter (between November and December). The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections. Text: Human bocavirus 1 (HBoV1), which belongs to family Parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 [1, 2] . HBoV1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. The prevalence of HBoV1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness (ARI), according to different studies [3] [4] [5] [6] [7] . Serological and nucleic acid test results are generally consistent [8] [9] [10] [11] , showing HBoV1 infection is very common. HBoV1 can cause both upper respiratory illness (URI) and lower respiratory illness (LRI) [12] [13] [14] [15] [16] [17] [18] . Infection with HBoV1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms [15, 19] . In some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms [18, 20] . Clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications [18] [19] [20] [21] [22] . In some cases, patients develop severe respiratory injury symptoms, which can be fatal [21, 23] . HBoV1 can be detected in fecal samples [24] , blood samples [25, 26] , urine [27, 28] , cerebrospinal fluid [29] [30] [31] , river water [32] and sewage [33, 34] , indicating that HBoV1 may be associate with a variety of diseases. Current in vitro studies modeling tissue-like airway epithelial cells cultures show HBoV1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy [35] [36] [37] , similar to lung injury tissue changes in vivo. There is currently no vaccine or specific treatment for this virus; prevention and treatment of HBoV1-related diseases still require further research. The prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses [38] . Studying the epidemiology of HBoV1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. In this study, we investigated the epidemiology of HBoV1 infection in children (≤14 years old) hospitalized with ARI in a subtropical region in China over a 7-year period. In addition, we collected climate data to determine if there was a relationship between HBoV1 prevalence and meteorological conditions. This study will add to existing epidemiological data on HBoV1 and its relationship with climate conditions in subtropical regions and will play a positive role in HBoV1 control and prevention. The study sites were three tertiary hospitals in Guangzhou, southern China (Longitude: E112°57′ to E114 03′; Latitude N22°26′ to N23°56′). Inclusion criteria were pediatric patients (≤14 years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. Chest radiography was conducted according to the clinical situation of the patient. Throat swab samples were collected from the enrolled patients between July 2009 and June 2016 for routine screening for respiratory viruses, Mycoplasma pneumoniae (MP), and Chlamydophila pneumoniae (CP). The samples were refrigerated at 2-8°C in viral transport medium, transported on ice and analyzed immediately or stored at − 80°C before analysis, as described previously [15, 39] . Meteorological data for Guangzhou, were collected from July 2009 to June 2016, from the China Meteorological Administration, including the monthly mean temperature (°C), mean relative humidity (%), rainfall (mm), mean wind speed (m/s), mean air pressure (hPa), mean vapor pressure (hPa), sunshine duration (h). Real-time PCR for HBoV1 and common respiratory pathogen detection DNA and RNA were extracted from the respiratory samples using the QIAamp DNA Mini Kit and QIAamp Viral RNA Mini Kit (Qiagen, Shanghai, China), respectively, in accordance with the manufacturer's protocols. Taqman real-time PCR for HBoV1 was designed based on the conserved region of the NP1 gene, as described previously [15] . Common respiratory pathogens, including respiratory syncytial virus (RSV), influenza A virus (InfA), influenza B virus (InfB), four types of parainfluenza (PIV1-4), adenovirus (ADV), enterovirus (EV), human metapneumovirus (HMPV), four strains of human coronavirus (HCoV-229E, OC43, NL63 and HKU1), human rhinovirus (HRV), MP and CP were detected simultaneously as previously reported [40] . Data were analyzed using Chi-squared test and Fisher's exact test in SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Correlation with climate data was analyzed using multiple linear regression analysis. All tests were two-tailed and a p value < 0.05 was considered as statistically significant. Eleven thousand three hundred ninety-nine pediatric patients (≤14 years old) hospitalized with ARI were enrolled in the study between July 2009 and June 2016. The male-to-female ratio was 1.82:1 (7361:4038) and the median age was 1.75 years (interquartile range 0.75-3.83). Overall, 86.5% (9857/11399) of patients were under the age of 5 years. All the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 (49.2%) were positive for one or more of those pathogens (Table 1) , and had a median age of 1.50 years (interquartile range 0.67-3.00). The male-to-female ratioes were 1.94: 1 (3698:1908) in pathogen-positive patients and 1.72: 1 (3663:2130) in pathogen-negative patients (p = 0.002). Two hundred forty-eight of 11,399 patients (2.2%) tested positive for HBoV1 infection. Of the HBoV1-positive patients, 112 (45.2%) were co-infected with other pathogens, most frequently with RSV (11.7%, 29/248) ( Table 1 ). The median age was 1 year (interquartile range 0.75-1.83). The male-to-female ratio was 2.54:1 (178:70) in HBoV1-positive patients and 1.81:1 (7183:3968) in HBoV1-negative patients (p = 0.019). To clarify the age distribution of HBoV1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. There was a significant difference in the prevalence of HBoV1 in patients in different age groups (p < 0.001) and the peak prevalence was found in patients aged 7-12 months (4.7%, 56/1203) (Fig. 1) . In this study, we monitored the prevalence of HBoV1 in patients (≤14 years old) hospitalized with ARI from July We collected meteorological data for Guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of HBoV1. Guangzhou, which is located in southern China (longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′), has a maritime subtropical monsoon climate. Between July 2009 and June 2016, the mean temperature was 21.8 ± 5.8°C (mean ± standard deviation), humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hPa and vapor pressure was 21.3 h ± 7.4 hPa. Between 2009 and 2016, the mean temperature from May to September was greater than 25°C (Fig. 3) . For multiple linear regression analysis of HBoV1 prevalence and meteorological conditions correlation, independent variables of mean air pressure (adjusted R 2 = 0.793, p < 0.001) and mean vapor pressure (adjusted R 2 = 0.929, p < 0.001), which linearly associated with mean temperature, and rainfall (adjusted R 2 = 0.278, p < 0.001), which strongly correlated with mean relative humidity, were excluded. The independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. The effect of temperature had a delay therefore mean temperature in the preceding month (mean temperature 1 month before) was also included as an independent variable in the analysis ( Table 2) . Both regression models were established (p < 0.001) and the adjusted R 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. HBoV1 prevalence was positively correlated with temperature (coefficient = 0.259 in the current temperature model (p = 0.002), coefficient = 0.328 in mean temperature in the preceding month model (p < 0.001)). Conversely, HBoV1 prevalence was negatively correlated with relative humidity (coefficient = − 0.126 in the current temperature model (p = 0.024), coefficient = − 0.083 in the temperature delay model (p = 0.039)) ( Table 2 ). ARI is one of the most common human diseases, predominantly caused by different respiratory viruses [41, 42] . One of these viruses, HBoV1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas [3] [4] [5] [6] [7] 43] . In general, the prevalence of viruses varies because of factors such as Multiple linear regression analysis was performed using HBoV1 monthly prevalence as the dependent variable, monthly mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables Data captured in bold are highly significant geographical location, climatic conditions, population and social activity [38] . Epidemiology of HBoV1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring [15, 16, 39, 44] . To describe the epidemiology of HBoV1 in Guangzhou, we collected throat swabs from 11,399 children (≤14 years old), hospitalized with ARI and monitored HBoV1 and other common respiratory pathogens over a 7-year period (Table 1 ). In the current study, 86.5% (9857/11399) of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ARI, consistent with previous reports [45, 46] . Overall, 49.2% (5606/11399) of patients tested positive for one or more respiratory pathogens, 2.2% (248/11399) of patients were tested with HBoV1 infection (Table 1) . A higher prevalence of HBoV1 was detected in male patients compared with female patients (p = 0.019), consistent with previous reports [15, 16, 39, 44] . Co-infection with HBoV1 and other pathogens is common [14, 15] . In our study, 45.2% (112/248) of HBoV1-positive patients also tested positive for other pathogens (Table 1 ). This may be partly caused by coinciding epidemics of HBoV1 and other pathogens. In our study, the HBoV1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference (Fig. 2) . Current research shows that HBoV1 infection can lead to the collapse of the first line of defense of airway epithelium [35] [36] [37] , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. Whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. The characteristics of the HBoV1 infection are likely to be a good model for studying the effects of co-infections. In this study, there was a significant difference in prevalence of HBoV1 in patients of different ages (p < 0.001). The majority of HBoV1 infections occurred in patients under 2 years old and the peak frequency of HBoV1 infection occurred in patients aged 7-12 months (Fig. 1) , consistent with previous serological and epidemiological reports on the virus [8-11, 15, 16, 39, 44] . This might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. The distribution of HBoV1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. Many factors affect the epidemiology of pathogens, such as geographical location and local climate. Guangzhou, a central city and main transport hub in southern China, is located in a subtropical region. Guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period (Fig. 3) . In this study, two HBoV1 peaks were observed (Fig. 2) . The large prevalence peaks of HBoV1 infection occurred between June and September of each year, which are the summer months in Guangzhou, with mean temperatures of higher than 25°C (Fig. 3) . Small peaks of HBoV1 infection occurred in winter, between November and December of each year. This seasonal distribution is similar to the prevalence in subtropical regions reported previously [47] , but different from the HBoV1 epidemics in temperate regions, which mostly occur in winter and spring [15, 16, 39, 44] , as well as from tropical regions, such as India, where no obvious epidemic season has been found [48] . To analyze the correlation between HBoV1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with HBoV1 monthly prevalence as the dependent variable and mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables (Table 2) . Both regression models were established (p < 0.001) and the adjusted R 2 value (0.373) of the temperature dorp 1 month model was greater than the adjusted R 2 value (0.231) of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. Both of the models showed that the prevalence of HBoV1 was significantly correlated with temperature and relative humidity ( Table 2 ). In detail, HBoV1 prevalence was positively correlated with temperature, that is consistent with previous reports [47, 49] . Conversely, HBoV1 prevalence was negatively correlated with relative humidity, this was different from a previous report in Suzhou [47] , which may be related to Guangzhou high humidity (mean monthly relative humidity was 77.2 ± 7.3%) (Fig. 3) . It is common for pathogen prevalence to fluctuate over time because of a variety factors. In this study, HBoV1 prevalence was relatively low in 2013 to 2014. It might be partly related to the relatively higher mean relative humidity during this period (Fig. 3) . Climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between HBoV1 infection and wind speed or sunshine duration were found in this study (p > 0.05) ( Table 2) . Some limitations of this study should be noted. First, because our study mainly focused on HBoV1 circulation in hospitalized patients with ARI, HBoV1 in outpatients and the asymptomatic population were not included. Second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. Third, the study was only conducted in three hospitals and may not be representative of the overall population. Our study has provided a better understanding of the epidemiology of HBoV1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of HBoV1 infections.

What percentage of patients were positive for at least one respiratory pathogen?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What was the result of the test of efficacy of PIV5 in murine challenge?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What general types of vaccines are available?

Pre-existing immunity against vaccine vectors – friend or foe? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542731/ SHA: f5bdf18567bb3760e1ce05008135f0270badbd5c Authors: Saxena, Manvendra; Van, Thi Thu Hao; Baird, Fiona J.; Coloe, Peter J.; Smooker, Peter M. Date: 2013-01-27 DOI: 10.1099/mic.0.049601-0 License: cc-by Abstract: Over the last century, the successful attenuation of multiple bacterial and viral pathogens has led to an effective, robust and safe form of vaccination. Recently, these vaccines have been evaluated as delivery vectors for heterologous antigens, as a means of simultaneous vaccination against two pathogens. The general consensus from published studies is that these vaccine vectors have the potential to be both safe and efficacious. However, some of the commonly employed vectors, for example Salmonella and adenovirus, often have pre-existing immune responses in the host and this has the potential to modify the subsequent immune response to a vectored antigen. This review examines the literature on this topic, and concludes that for bacterial vectors there can in fact, in some cases, be an enhancement in immunogenicity, typically humoral, while for viral vectors pre-existing immunity is a hindrance for subsequent induction of cell-mediated responses. Text: In the fields of medicine and veterinary medicine, there are numerous live, attenuated bacterial and viral vaccines in use today worldwide. The safety and efficacy of such vaccines is well established and allows further development as vector systems to deliver antigen originating from other pathogens. Various attenuated bacteria, including Escherichia coli, Vibrio cholerae, lactic acid bacteria (LAB), specifically Lactococcus lactis, Mycobacterium, Listeria, Shigella and Salmonella, have been tested for the targeted delivery of heterologous antigens of bacterial, viral and parasitic origin into a variety of animal hosts (Bahey-El-Din et al., 2010; Innocentin et al., 2009; Johnson et al., 2011; Tobias et al., 2008 Tobias et al., , 2010 Tobias & Svennerholm, 2012) . Bacteria such as E. coli and lactic acid bacteria have recently gained favour, as E. coli is a commensal and lactic acid bacteria are present in most fermented food items and are therefore naturally present in the host. They are also a much safer option than traditional attenuated vaccines in children and immunecompromised people. As this review discusses the effects of pre-existing immune responses to attenuated vaccines, further discussion of LAB and E. coli as potential vectors will not be undertaken; however, the reader is directed to several interesting reviews (Bermú dez-Humarán et al., 2011; Wells & Mercenier, 2008) . Intracellular bacteria from the genera Mycobacterium (Guleria et al., 1996) , Listeria (Gentschev et al., 2001) , Shigella (Levine et al., 1997) and Salmonella (Dougan et al., 1987) are considered to be suitable candidates for the delivery of vaccine antigens due to their capability to induce robust T cell immune responses (Alderton et al., 1991; Lo et al., 1999; Mastroeni et al., 2001; Mittrücker & Kaufmann, 2000; Nauciel, 1990) . Salmonella is one genus that has been well examined as a vector, building on the extensive research available on the micro-organism's physiology and pathogenesis (Basso et al., 2000; Killeen & DiRita, 2000; Sirard et al., 1999; Ward et al., 1999) . There exist several commercial vaccines that are used as anti-Salmonella vaccines in humans and animals (e.g. Ty21a for typhoid fever in humans, several Salmonella serovars against salmonellosis in chickens and other animals). The general strategy for vectoring heterologous antigen is depicted in Fig. 1 . The first clinical trial of a recombinant, which was conducted over 20 years ago using an attenuated Salmonella as a delivery vector, led to the widespread testing of this bacterium as a mucosal delivery system for antigens from non-Salmonella pathogens (Dougan et al., 1987) . These studies have demonstrated the utility of live bacteria to deliver expressed antigens and DNA vaccines to the host immune system (Atkins et al., 2006; Husseiny & Hensel, 2008; Jiang et al., 2004; Kirby et al., 2004) . Since then several other intracellular bacterial vectors have been successfully tested for their capability to deliver a variety of antigens from various pathogens, as well as vaccination against cancer. One genus which has been widely tested as vector is Listeria. Listeria species are Gram-positive intracellular food-borne pathogens. The advantages of Listeria are that it can invade a variety of cells, including antigen presenting cells (APCs). After invading the host cell, Listeria resides inside the phagosome; however, it can escape the phagosome with the help of listeriolysin O (LLO; Hly) and reside in the cytoplasm of the cells, thereby efficiently presenting antigen to both CD8 and CD4 T cells (Cossart & Mengaud, 1989; Kaufmann, 1993; Pamer et al., 1997) . Several studies have demonstrated the effectiveness and ease of using Listeria monocytogenes to deliver heterologous vaccine antigens and DNA vaccines Jensen et al., 1997; Johnson et al., 2011; Peters et al., 2003; Shen et al., 1995; Yin et al., 2011) . Similarly, various viral vectors have been successfully tested for their capability to deliver heterologous vaccine antigens, and this generally results in the induction of strong CTL immune responses. In the veterinary field, there are numerous viral vector vaccines that are currently licensed for use in livestock and domesticated animals. These recombinant vaccines are based on both DNA viruses (such as fowlpox virus-based vaccines which target avian influenza virus and fowlpox virus, or vaccinia virusbased vectors against the rabies virus in wildlife) and RNA viruses [such as Newcastle disease virus-based vaccines to be used in poultry or yellow fever virus (YFV)-based vaccines to be used in horses against West Nile virus] (Draper & Heeney, 2010) . Based on the safety record in the veterinary field, many viruses have been studied for human use as a vector in vaccine development (Beukema et al., 2006; Esteban, 2009; Schirrmacher & Fournier, 2009; Stoyanov et al., 2010; Weli & Tryland, 2011) . Amongst them, YFV (YF-17D strain) was the first to be licensed for use in humans, where the cDNAs encoding the envelope proteins of YFV were replaced with the corresponding genes of an attenuated Japanese encephalitis virus strain, SA14-14-2 (Appaiahgari & Vrati, 2010; Rollier et al., 2011) . Poxviruses are also studied extensively as candidate vectors for human use, among which attenuated derivatives of vaccinia virus [such as modified vaccinia virus Ankara (MVA) and New York attenuated vaccinia virus NYVAC strains] are the most promising vectors (Esteban, 2009; Gó mez et al., 2008; Rimmelzwaan & Sutter, 2009 ). They are ideal candidate vectors due to their large DNA-packing capacity and their thermal and genetic stability (Minke et al., 2004) . The NYVAC vector has been shown to induce CD4 + T cell-dominant responses, and MVA induces both CD4 + and CD8 + T cell responses (Mooij et al., 2008) . The adenovirus (Ad) vector is another of the most widely evaluated vectors to date to express heterologous antigens, due to ease of production, safety profile, genetic stability, the ease of DNA genome manipulation, and the ability to stimulate both innate and adaptive immune responses and induce both T and B cell responses (Alexander et al., 2012; Fitzgerald et al., 2003; Gabitzsch & Jones, 2011; Lasaro & Ertl, 2009; Vemula & Mittal, 2010; Weyer et al., 2009) . They have been extensively examined as a delivery vector in several preclinical and clinical studies for infectious diseases such as anthrax, hepatitis B, human immunodeficiency virus (HIV)-1, influenza, measles, severe acute respiratory syndrome (SARS), malaria and tuberculosis M. Saxena and others (Chengalvala et al., 1994; Gao et al., 2006; Hashimoto et al., 2005; Hsu et al., 1992; Limbach & Richie, 2009; Radosevic et al., 2007; Shiver et al., 2002) . However, before vectored vaccines can be used in the human population they need to satisfy several important criteria. Safety is a major concern, as even a low level of toxicity is unacceptable (of course the minor discomfort that accompanies many vaccinations is normal). Secondly, a vaccine should be inexpensive, so that it can be administered to a large population at minimal cost, and this is particularly important in resource-poor countries (Killeen & DiRita, 2000) . Similar constraints apply to veterinary vaccines, with cost often an even more important consideration. Finally, long-lasting cellular and (where appropriate) humoral immune responses to the vectored antigen must be induced following administration of these vaccines, preferably with a single dose (Atkins et al., 2006) . As some of the vectors in use will have been seen by the host immune system prior to vaccination, whether the presence of pre-existing immune responses is detrimental for the further development of a vector-based vaccine scheme, or can augment responses to the vectored antigen, needs to be considered in detail. This is the subject of this review. In discussing the possible effects on pre-existing immunity, the natural immunity to the vector needs to be considered. Therefore, considering a vector such as Salmonella, if a host has previously been infected there will exist robust B and T memory responses, and as such, when a vaccination is delivered, an anamnestic response to the Salmonella antigens will be induced (while the response to the vectored antigen will be a primary response). This will theoretically reduce the exposure of the heterologous antigen to the immune system, as the vector is rapidly cleared. Surprisingly, as will be seen in some of the examples given below, this can have results that differ depending on the magnitude of the response to the vectored antigen. Similarly, for virally vectored antigens, the existence of pre-existing immunity to the vector (particularly neutralizing antibody) will restrict delivery of the virus into cells, thereby effectively reducing the dose of the vectored antigen. Again, this might be expected to result in a reduction in the antigenicity of the vectored antigen. In the case of bacterial vectors, the effect of pre-existing immune responses has only been tested using Salmonella serovars and Listeria spp. Concern that prior immunological experience of the host with either the homologous Salmonella vector strain or a related strain might compromise its ability to deliver heterologous vaccine antigen was first raised in 1987 (Dougan et al., 1987) . Bao and Clements subsequently reported experimental evidence of the consequences of prior exposure of animals to the vector strain (Bao & Clements, 1991) . This work showed that both serum and mucosal antibody responses against the foreign antigen were in fact upregulated in animals with prior exposure to the vector strain. Whittle & Verma (1997) reported similar findings. Mice immunized via the intra-peritoneal route with a Salmonella dublin aroA mutant expressing heterologous antigen after being exposed to the same vector showed a higher immune response to the vectored antigen in comparison to mice without any immunological memory against the vector. Subsequently, several studies have been conducted to examine the effect of pre-existing immunity in the host against Salmonella. These results are summarized in Table 1 . The various reports are contradictory in their findings and seem to paint a rather confusing picture. Some studies concluded that pre-existing immunity against the Salmonella vector leads to stronger immune responses against the delivered antigen (Bao & Clements, 1991; Jespersgaard et al., 2001; Kohler et al., 2000a, b; Metzger et al., 2004; Saxena et al., 2009; Sevil Domènech et al., 2008; Whittle & Verma, 1997) , with others considering pre-existing immunity to be a limiting factor in the long-term use of Salmonella as an efficient vector for antigen delivery (Attridge et al., 1997; Gahan et al., 2008; Roberts et al., 1999; Sevil Domènech et al., 2007; Vindurampulle & Attridge, 2003a, b) . A slight majority of the studies listed in Table 1 (10 versus eight) indicate the upregulation of immune responses after animals have been exposed to either homologous or related strains before the delivery of heterologous antigen using a Salmonella vector. A study by Metzger and co-workers on human volunteers using Salmonella Typhi as a vector suggested that there was no change in the T cell immune response against the heterologous antigen in human volunteers who were exposed to empty vector in comparison with volunteers who were immunologically naive of the vector strain (Metzger et al., 2004) . In these subjects, humoral responses were moderately elevated in preexposed individuals. Similarly, Saxena et al. (2009) indicated higher humoral and T cell responses in mice pre-exposed to homologous or heterologous Salmonella strains. The interleukin 4 (IL4) response was significantly higher when the animal host was exposed to the homologous strain, whereas pre-exposure to a related species did not have such an impact on IL4 responses. Conversely interferon (IFN)-c responses were higher, irrespective of the strain to which mice were pre-exposed. This study also indicated that the presence of homologous or heterologous opsonizing antibodies leads to a higher uptake of Salmonella by macrophages in vitro, which may explain the higher immune responses in exposed mice. As may be expected, uptake was higher when homologous sera were used as the opsonin rather than heterologous sera. This is depicted in Fig. 2 . Conversely, there are reports that indicate that pre-existing immunity against the bacterial vector downregulates immune responses against the delivered heterologous antigen using similar or related vectors. Attridge and coworkers reported that the presence of immunity against the bacterial vector prior to the delivery of vectored antigenic Microbiology 159 protein can downregulate immune responses in mice against the delivered antigen (Attridge et al., 1997) . Similar results were reported by Roberts et al. (1999) and Vindurampulle & Attridge (2003a, b) . However, the latter authors found that the hypo-responsiveness could be largely eliminated by exposing animals to the foreign antigen prior to vectorpriming (Vindurampulle & Attridge, 2003b) . Unfortunately, this would appear to be impractical for an immunization regimen! A study presented by Gahan et al. (2008) immunized mice with S. Typhimurium expressing C fragment of tetanus toxin antigen from an expression plasmid or as a DNA vaccine. Vaccinated mice developed humoral responses to LPS and tetC (for the plasmid-bearing vaccines). Animals from all groups (including a previously unvaccinated group) were immunized on day 182 with Salmonella expressing tetC. At this time, the anti-LPS and tetC titres were beginning to wane. Fourteen days after the second immunization, the colonization of various mouse organs was assessed. The ability to colonize was found to be significantly reduced in groups that had been previously vaccinated with Salmonella. In view of this finding, it was perhaps not surprising that at day 210 the LPS titres were not significantly different between groups receiving one or two vaccinations. More interestingly, mice that had been primed with Salmonella alone, and then boosted with Salmonella expressing tetC, induced much lower anti-tetC responses than mice that had not been primed. This argues strongly that prior immunological immunity to the vector can seriously dampen subsequent antigen-specific humoral responses. Whether the same is true for cellular responses was not evaluated. Other studies have evaluated cellular responses. A study by Sevil Domènech and colleagues reported that pre-existing anti-vector immunity seriously compromises CD8 + responses in mice when exposed to a similar strain used as vector (Sevil Domènech et al., 2007) . In contrast, another study by the same authors reported that animals exposed to related vectors induce much higher CD8 + responses when compared with animals which do not have any pre-existing Salmonella immunity (Sevil Domènech et al., 2008) . The difference between these two studies was that in the first, the prime and boost were with identical serovars, while in the second study, different serovars were used. This may point to a way of avoiding downregulation of CD8 responses by pre-existing immunity. This is important, as one of the advantages of using Salmonella (an intracellular pathogen) is that strong cellular immune responses can be induced. It must be noted that in the case of Salmonella vaccines, effects other than strictly immunological responses (particularly adaptive responses) should be considered. In the context of innate immunity, it was shown that administration of non-virulent Salmonella to gnobiotic pigs eliminated disease following challenge with a virulent strain (Foster et al., 2003) . Interestingly, protection was not by competitive exclusion, as the virulent strain was in high numbers in the gut but did not distribute systemically. The protection was proposed to be mediated by the infiltration of a large number of polymorphonuclear leukocytes into the gut, and although perhaps impractical as a general prophylactic (as the time between vaccination and infection is short), this may be an option for short-term or perhaps therapeutic vaccination (as reviewed by Foster et al., 2012) . Chickens (Gallus gallus) are a natural animal reservoir for Salmonella, which makes them an important source of Salmonella-associated gastroenteritis in humans. The ability to use oral Salmonella vaccines to immunize against heterologous pathogens would be of enormous benefit to Uptake of STM-1 by J774 macrophages, relative to the highest uptake percentage. X, Opsonized with naive sera; m, opsonized with serum from mice exposed to Salmonella enteriditis; &, opsonized with serum from mice exposed to STM-1. Pre-existing immunity against vaccine vectors the poultry industry in both broiler and layer flocks. Both vertical and horizontal transmission is associated with Salmonella in chickens (Liljebjelke et al., 2005) . Vertical transmission via in ovo transmission is particularly important, because if there is prior exposure to the vaccine strain, subsequent vaccination using an oral Salmonella vector could be severely compromised. A considerable number of studies on cross-protective immunity and competitive exclusion have been undertaken in chickens. Protective cross-reactive immunity against Salmonella strains has been demonstrated against both homologous and heterologous challenges (Beal et al., 2006) , although cross-serogroup protection was not strong. Furthermore, a recent study reported that pretreatment of newly hatched chickens with different Salmonella strains could produce a complete invasioninhibition effect on any subsequent exposure to both homologous and heterologous strains (Methner et al., 2010) . Pre-exposure with a highly invasive form of Salmonella Enteritidis caused a large influx of heterophils to the caecal mucosa in 1-day-old chicks, and subsequent heterologous caecal colonization was inhibited for a period of 48 h (Methner et al., 2010) . The implications of this kind of colonization-inhibition study on the immunological status of the affected chickens are yet to be fully elucidated. It should be noted that the studies listed in Tables 1 and 2 are controlled laboratory studies, with the possibility of a competitive exclusion component to immunity not discussed. Similarly studies of L. monocytogenes and the effects of preexisting immune responses indicate conflicting results. A study by Bouwer et al. (1999) indicates that pre-existing immune responses against the Listeria vector do not diminish immune responses against the delivered heterologous antigen, and a similar study by Starks et al. (2004) also concluded that prior exposure of mice to the empty Listeria vector did not influence anti-cancer immune responses when a similar mutant was used as a carrier of a melanoma cancer antigen. Similar findings were reported by Whitney et al. (2011) in rhesus macaques in which L. monocytyogens was used as a carrier of gag-HIV antigen. Conversely, studies by Stevens et al. (2005) in which L. monocytogens was used to deliver feline immunodeficiency virus (FIV) gag protein and as a carrier of DNA vaccines to vaccinate cats against FIV envelope protein indicated lower immune responses against the delivered antigen in cats exposed to empty Listeria vector in comparison with naive animals (Stevens et al., 2005) . Similar findings have been reported by Tvinnereim et al. (2002) and Leong et al. (2009) . However, taken together, these studies conclude that prior exposure of host animals to empty vector does not abrogate immune responses to the vectored antigen, but only reduces them somewhat. Only the study by Vijh et al. (1999) indicated that exposure to the empty vector may completely abrogate immune responses against the delivered antigens (Vijh et al., 1999) . However, these studies also indicate that downregulation of antigenspecific immune responses is highly dependent on dose and time. Leong et al. (2009) also demonstrated that the negative impact of vector-specific immune responses can also be countered by repeated immunization with the same vaccine and dose; this in effect leads to higher priming of naive T cells against the delivered antigen. Of course, such repeated vaccination may not be practicable in real-world situations. Despite the many advantages which viral vectoring can offer, pre-existing immunity is a major obstacle of many viralvectored vaccines, such as Ad serotype 5 or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40-45 % and 70 % (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009) . Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009; Pine et al., 2011) . In a large-scale clinical trial (STEP) of an Ad serotype 5 (AdHu5)-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had pre-existing neutralizing antibodies to AdHu5 (Buchbinder et al., 2008) . For an HSV-1-based vector vaccine, it has been demonstrated that pre-existing anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccine-encoded antigen (Hocknell et al., 2002; Lauterbach et al., 2005) . However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSVencoded antigen were not affected by prior HSV immunity (Brockman & Knipe, 2002) . Similarly, pre-existing immunity to poliovirus has little effect on vaccine efficacy in a poliovirus-vectored vaccine (Mandl et al., 2001) . Different effects of pre-existing immunity on the efficacy of recombinant viral vaccine vectors are summarized in Table 2 . There are several approaches to avoiding pre-existing vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro & Ertl, 2009) , heterologous prime-boost approaches (Liu et al., 2008) , homologous reimmunization (Steffensen et al., 2012) and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch & Jones, 2011; Roberts et al., 2006) . The inhibitory effect of pre-existing immunity can also be avoided by masking the Ad vector inside dendritic cells (DCs) (Steffensen et al., 2012) . In addition, mucosal vaccination or administration of higher vaccine doses can overcome pre-existing immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003) . As we search for new vaccine approaches for the array of pathogens for which none is yet available, revisiting proven vaccines and developing these further has gained M. Saxena and others momentum. Hence, attenuated bacteria and viruses which have a long history of efficacy and safety are being brought into use. While very attractive, a common theme in these experimental approaches has been the limitations that preexisting immunity to the vector may pose. However, as this examination of the relevant literature shows, there is a rather confusing picture, with some studies in fact indicating that pre-existing immunity may be a friend, rather than foe. Few studies using viral vectors have reported on the influence of pre-existing immunity on humoral responses. Generally speaking, for bacterial-delivered antigens, the humoral responses were influenced by pre-existing immunity, with slightly more studies finding augmentation rather than diminution. Why is there variation? This may be due to several factors, including the type of Salmonella used and its invasiveness. Dunstan and colleagues tested the ability of six isogenic Salmonella serovar Typhimurium strains harbouring different mutations for their ability to induce immune responses against the C fragment of tetanus toxin and concluded that the strain which had the least ability to colonize Peyer's patches induced the lowest immune responses (Dunstan et al., 1998) . Similarly, the boosting time and nature of the antigen used might be important. Attridge and colleagues indicated the importance of boosting time. In one experiment, boosting mice at 10 weeks led to complete inhibition of antibody responses against the delivered heterologous antigen; however, when the mice were boosted at 4 weeks, the downregulation of antibody responses was not so prominent (Attridge et al., 1997) . A similar study conducted by Kohlers and colleagues shows that boosting at 7 weeks after pre-exposing animals to empty vector leads to lower antigen-specific IgG and secretory IgA responses; however, boosting at 14 weeks leads to higher IgG and secretory IgA responses (Kohler et al., 2000b) . This is in conflict with the above result, although it should be mentioned that they used different Salmonella species. Vindurampulle and Attridge also examined the impact of the Salmonella strain and the nature of the antigens used. In their study, they used S. Dublin and Salmonella Stanley aroA mutants to deliver E. coli K88 and LT-B antigens, and concluded that the effect of pre-existing immunity depends on both the strain used and the type of antigen delivered (Vindurampulle & Attridge, 2003b) . All these studies on the effect of pre-existing immunity discuss the impact on humoral responses. Sevil Domenech and colleagues reported that pre-exposing animals to the homologous Salmonella vector leads to a significant reduction in CD8 + responses; however, exposure of animals to a heterologous strain leads to significantly higher CD8 + responses (Sevil Domènech et al., 2007 , 2008 . Saxena and colleagues also reported that antigenspecific T cell responses were either similar or significantly higher, with no downregulation in T cell responses observed after pre-exposing mice to either homologous or heterologous strains (Saxena et al., 2009) . For viral vectors, the impact of cell-mediated immunity was more pronounced, and as depicted in Table 2 , almost always resulted in a reduction in the subsequent immune response. Presumably this is because viruses will induce neutralizing antibody on the first dose, and in subsequent doses this antibody will limit the number of transduced cells, therefore limiting the responses. This is particularly a problem with a common viral vector such as Ad, where a large proportion of the population will have immunological memory against common serotypes (Lasaro & Ertl, 2009) . As these authors conclude, it will be possible to utilize such vectors only by developing vaccines from alternative serotypes. It may be that a vector such as Pre-existing immunity against vaccine vectors attenuated influenza virus, with the ability to easily develop reassortants, will be useful in this context. In addition, immunological memory in the form of opsonizing antibody certainly plays an important role in the early uptake of Salmonella by macrophages and DC. This may be beneficial, as the live bacterial vector used for delivery purposes harbours mutations in genes encoding proteins responsible for their survival in the animal host. This not only encumbers their ability to cause disease, making them safe live vectors, but also limits the number of replications. The presence of opsonizing antibodies should mean a higher level of bacterial uptake, leading to higher presentation to the immune system and therefore a better immune response. We have previously shown that this is indeed the case (Saxena et al., 2009 ) (depicted in Fig. 2 ). It would be of great benefit to address these issues not only in mice but also in other organisms such as chickens, which are the most likely host to be targeted for the use of live Salmonella vectors, specifically where the vaccines are developed for use in livestock and poultry. To summarize, bacterial vectors such as Salmonella and viral vectors such as Ad show great promise as delivery vehicles for heterologous antigens; however, prior exposure to the vector must be considered. By judicious selection of the strain/serotype it will be possible to avoid the negative effects and it may indeed be possible to positively influence the response, particularly for humoral immunity.

What are important criteria for selecting vaccine delivery vectors?

Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.

When is this especially true?

Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.

What leads to oxidative stress in the body?

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459876/ SHA: f0b1fa4036434b57c8307d43c39a4193f7e8053a Authors: Morokutti-Kurz, Martina; König-Schuster, Marielle; Koller, Christiane; Graf, Christine; Graf, Philipp; Kirchoff, Norman; Reutterer, Benjamin; Seifert, Jan-Marcus; Unger, Hermann; Grassauer, Andreas; Prieschl-Grassauer, Eva; Nakowitsch, Sabine Date: 2015-06-08 DOI: 10.1371/journal.pone.0128794 License: cc-by Abstract: BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted We investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)

Is there a dose-dependent response to carageenan and Zanamavir intranasal therapy?

Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.

What is the advantage of adenovirus?

Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.

What have hantaviruses been identified in potentiating?

Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.

What are Furin, along with other PCSK family members implicated in?

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.

How were the assays confirmed?

1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A (H1N1) viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: [email protected] The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

What was the death rate in the first wave of the 1918 swine flu pandemic?

Host resilience to emerging coronaviruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/ SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4 Authors: Jamieson, Amanda M Date: 2016-07-01 DOI: 10.2217/fvl-2016-0060 License: cc-by Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome. Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] . In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: [email protected] REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care. Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses. Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] . The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] . The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] . Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] . One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] . Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge. Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] . REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately. A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV. The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. • Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome. • Antivirals have limited effects on the course of the infection with these coronaviruses. • There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus. • Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health. • Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience. • The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients. Papers of special note have been highlighted as:

What is the role of topoisomerase I in improving host resilience in viral lung infections?

1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A (H1N1) viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: [email protected] The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

What is an unique feature of the 1918 swine flu?