Question: What are the observed mutations in the SARS-CoV-2 genome and how often do the mutations occur?

Title: Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions Content: Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive repository. Up until March 27, 2020, we downloaded 53 illumina datasets, mostly from China, USA (Washington DC) and Australia (Victoria). Of 30 high quality datasets, 27 datasets (90%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA sequencing samples. Sequencing samples from USA in GenBank present this signature with 50% allele frequencies among samples. Australian mutation signatures were more diverse than USA samples, but still, clonal events were found in those samples. Mutations in the helicase and orf1a coding regions from SARS-CoV-2 were predominant, among others, suggesting that these proteins are prone to evolve by natural selection. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.

Title: Insights on early mutational events in SARS-CoV-2 virus reveal founder effects across geographical regions Content: Here we aim to describe early mutational events across samples from publicly available SARS-CoV-2 sequences from the sequence read archive and GenBank repositories. Up until 27 March 2020, we downloaded 50 illumina datasets, mostly from China, USA (WA State) and Australia (VIC). A total of 30 datasets (60%) contain at least a single founder mutation and most of the variants are missense (over 63%). Five-point mutations with clonal (founder) effect were found in USA next-generation sequencing samples. Sequencing samples from North America in GenBank (22 April 2020) present this signature with up to 39% allele frequencies among samples (n = 1,359). Australian variant signatures were more diverse than USA samples, but still, clonal events were found in these samples. Mutations in the helicase, encoded by the ORF1ab gene in SARS-CoV-2 were predominant, among others, suggesting that these regions are actively evolving. Finally, we firmly urge that primer sets for diagnosis be carefully designed, since rapidly occurring variants would affect the performance of the reverse transcribed quantitative PCR (RT-qPCR) based viral testing.

Title: Comment on “Genetic variants and source of introduction of SARS‐CoV‐2 in South America” Content: I read with great interest a recent study by Poterico and Mestanza1 who described mutations in 30 SARS-CoV-2 genomes from South American countries (Argentina, Brazil, Chile, Colombia, Ecuador, and Peru) This article is protected by copyright. All rights reserved.

Title: Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant Content: BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.

Title: Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant Content: BACKGROUND: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. METHODS: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from December 2019 to mid-March 2020. SARS-CoV-2 reference genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann–Whitney and Fisher-Exact tests were used to assess statistical significance. RESULTS: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly observed in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on February 9th, 2020 while a different mutation in RdRp changing its amino acid composition emerged on February 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2–5], otherwise they have a median of 1 mutation [range: 0–3] (p value < 0.001). CONCLUSIONS: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.

Title: SARS-CoV-2 variants: Relevance for symptom granularity, epidemiology, immunity (herd, vaccines), virus origin and containment? Content: The origin of the SARS-CoV-2 virus remains enigmatic. It is likely to be a continuum resulting from inevitable mutations and recombination events. These genetic changes keep developing in the present epidemic. Mutations tending to deplete the genome in its cytosine content will progressively lead to attenuation as a consequence of Muller's ratchet, but this is counteracted by recombination when different mutants co-infect the same host, in particular, in clusters of infection. Monitoring as a function of time the genome sequences in closely related cases is critical to anticipate the future of SARS-CoV-2 and hence of COVID-19.

Title: Overwhelming Mutations or SNPs of SARS-CoV-2: A Point of Caution Content: • Mutation studies hits cause for the superior infectious rate of SARS-CoV-2. • SARS-CoV-2 is evolving rapidly with number of mutations and SNPs. • Mutations were found in the ACE2 binding region of SARS-CoV-2 spike glycoprotein. • ORF1ab, ORF8 and spike glycoprotein regions of SARS-CoV-2 are highly mutated. • Overwhelming mutations or SNPs of SARS-CoV-2 alerts drug treatment options.

Title: Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission Content: The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has a capability of gaining rapid mutations as the virus spreads. Whole genome sequencing data offers a wide range of opportunities to study the mutation dynamics. The advantage of increasing amount of whole genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity and to investigate the implications of those mutations in protein stability and viral transmission. Four proteins, surface glycoprotein, nucleocapsid, ORF1ab and ORF8 showed frequent mutations, while envelop, membrane, ORF6 and ORF7a proteins showed conservation in terms of amino acid substitutions. Some of the mutations across different proteins showed co-occurrence, suggesting their functional cooperation in stability, transmission and adaptability. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Analysis of protein structure stability of surface glycoprotein mutants indicated viability of specific variants and are more prone to be temporally and spatially distributed across the globe. Similar empirical analysis of other proteins indicated existence of important functional implications of several variants. Analysis of co-occurred mutants indicated their structural and/or functional interaction among different SARS-COV-2 proteins. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing and containment of the disease.

Title: Genome-Wide Identification and Characterization of Point Mutations in the SARS-CoV-2 Genome Content: OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019 and has been rapidly spreading worldwide. Although the causal relationship among mutations and the features of SARS-CoV-2 such as rapid transmission, pathogenicity, and tropism, remains unclear, our results of genomic mutations in SARS-CoV-2 may help to interpret the interaction between genomic characterization in SARS-CoV-2 and infectivity with the host. METHODS: A total of 4,254 genomic sequences of SARS-CoV-2 were collected from the Global Initiative on Sharing all Influenza Data (GISAID). Multiple sequence alignment for phylogenetic analysis and comparative genomic approach for mutation analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA), and an in-house program based on Perl language, respectively. RESULTS: Phylogenetic analysis of SARS-CoV-2 strains indicated that there were 3 major clades including S, V, and G, and 2 subclades (G.1 and G.2). There were 767 types of synonymous and 1,352 types of non-synonymous mutation. ORF1a, ORF1b, S, and N genes were detected at high frequency, whereas ORF7b and E genes exhibited low frequency. In the receptor-binding domain (RBD) of the S gene, 11 non-synonymous mutations were observed in the region adjacent to the angiotensin converting enzyme 2 (ACE2) binding site. CONCLUSION: It has been reported that the rapid infectivity and transmission of SARS-CoV-2 associated with host receptor affinity are derived from several mutations in its genes. Without these genetic mutations to enhance evolutionary adaptation, species recognition, host receptor affinity, and pathogenicity, it would not survive. It is expected that our results could provide an important clue in understanding the genomic characteristics of SARS-CoV-2.

Title: Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp Content: Since its emergence in Wuhan, China in late 2019, the origin and evolution of SARS-CoV-2 have been among the most debated issues related to COVID-19. Throughout its spread around the world, the viral genome continued acquiring new mutations and some of them became widespread. Among them, 14408 C>T and 23403 A>G mutations in RdRp and S, respectively, became dominant in Europe and the US, which led to debates regarding their effects on the mutability and transmissibility of the virus. In this study, we aimed to investigate possible differences between time-dependent variation of mutation densities (MDe) of viral strains that carry these two mutations and those that do not. Our analyses at the genome and gene level led to two important findings: First, time-dependent changes in the average MDe of circulating SARS-CoV-2 genomes showed different characteristics before and after the beginning of April, when daily new case numbers started levelling off. Second, this pattern was much delayed or even non-existent for the “mutant” (MT) strain that harbored both 14408 C>T and 23403 A>G mutations. Although these differences were not limited to a few hotspots, it is intriguing that the MDe increase is most evident in two critical genes, S and Orf1ab, which are also the genes that harbor the defining mutations of the MT genotype. The nature of these unexpected relationships warrant further research.

Title: Genomic, geographic and temporal distributions of SARS-CoV-2 mutations Content: The COVID-19 pandemic is the most significant public health issue in recent history. Its causal agent, SARS-CoV-2, has evolved rapidly since its first emergence in December 2019. Mutations in the viral genome have critical impacts on the adaptation of viral strains to the local environment, and may alter the characteristics of viral transmission, disease manifestation, and the efficacy of treatment and vaccination. Using the complete sequences of 1,932 SARS-CoV-2 genomes, we examined the genomic, geographic and temporal distributions of aged, new, and frequent mutations of SARS-CoV-2, and identified six phylogenetic clusters of the strains, which also exhibit a geographic preference in different continents. Mutations in the form of single nucleotide variations (SNVs) provide a direct interpretation for the six phylogenetic clusters. Linkage disequilibrium, haplotype structure, evolutionary process, global distribution of mutations unveiled a sketch of the mutational history. Additionally, we found a positive correlation between the average mutation count and case fatality, and this correlation had strengthened with time, suggesting an important role of SNVs on disease outcomes. This study suggests that SNVs may become an important consideration in virus detection, clinical treatment, drug design, and vaccine development to avoid target shifting, and that continued isolation and sequencing is a crucial component in the fight against this pandemic. Significance Statement Mutation is the driving force of evolution for viruses like SARS-CoV-2, the causal agent of COVID-19. In this study, we discovered that the genome of SARS-CoV-2 is changing rapidly from the originally isolated form. These mutations have been spreading around the world and caused more than 2.5 million of infected cases and 170 thousands of deaths. We found that fourteen frequent mutations identified in this study can characterize the six main clusters of SARS-CoV-2 strains. In addition, we found the mutation burden is positively correlated with the fatality of COVID-19 patients. Understanding mutations in the SARS-CoV-2 genome will provide useful insight for the design of treatment and vaccination.

Title: Large scale genomic analysis of 3067 SARS-CoV-2 genomes reveals a clonal geo-distribution and a rich genetic variations of hotspots mutations Content: In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variant sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 38 recurrent non-synonymous mutations, including ten hotspot mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed certain genotypes specific to the geographic location. We also found co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets We have also created an inclusive unified database (http://genoma.ma/covid-19/) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.

Title: Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission Content: The recent pandemic of SARS-CoV-2 infection has affected more than 3.0 million people worldwide with more than 200 thousand reported deaths. The SARS-CoV-2 genome has the capability of gaining rapid mutations as the virus spreads. Whole-genome sequencing data offers a wide range of opportunities to study mutation dynamics. The advantage of an increasing amount of whole-genome sequence data of SARS-CoV-2 intrigued us to explore the mutation profile across the genome, to check the genome diversity, and to investigate the implications of those mutations in protein stability and viral transmission. We have identified frequently mutated residues by aligning ~660 SARS-CoV-2 genomes and validated in 10,000 datasets available in GISAID Nextstrain. We further evaluated the potential of these frequently mutated residues in protein structure stability of spike glycoprotein and their possible functional consequences in other proteins. Among the 11 genes, surface glycoprotein, nucleocapsid, ORF1ab, and ORF8 showed frequent mutations, while envelop, membrane, ORF6, ORF7a and ORF7b showed conservation in terms of amino acid substitutions. Combined analysis with the frequently mutated residues identified 20 viral variants, among which 12 specific combinations comprised more than 97% of the isolates considered for the analysis. Some of the mutations across different proteins showed co-occurrences, suggesting their structural and/or functional interaction among different SARS-COV-2 proteins, and their involvement in adaptability and viral transmission. Analysis of protein structure stability of surface glycoprotein mutants indicated the viability of specific variants and are more prone to be temporally and spatially distributed across the globe. A similar empirical analysis of other proteins indicated the existence of important functional implications of several variants. Identification of frequently mutated variants among COVID-19 patients might be useful for better clinical management, contact tracing, and containment of the disease.

Title: SARS-CoV2 envelope protein: Non-synonymous mutations and its consequences Content: In the NCBI database, as on June 6, 2020, total number of available complete genome sequences of SARS-CoV2 across the world is 3617. The envelope protein of SARS-CoV2 possesses several non-synonymous mutations over the transmembrane domain and (C)-terminus in 15 (0.414%) genomes among 3617 available SARS-CoV2 genomes. More precisely, it is to be mentioned that 10(0.386%) out of 2588 genomes from the USA, 3(0.806%) from Asia, 1 (0.348%) from Europe and 1 (0.274%) from Oceania contain the missense mutations over the envelope proteins of SARS-CoV2 genomes. The C-terminus motif DLLV has been changed to DFLV and YLLV in the proteins QJR88103 (Australia: Victoria) and QKI36831 (China: Guangzhou) respectively, which might affect the binding of this motif with the host protein PALS1.

Title: Global genetic diversity patterns and transmissions of SARS-CoV-2 Content: Background: Since it was firstly discovered in China, the SARS-CoV-2 epidemic has caused a substantial health emergency and economic stress in the world. However, the global genetic diversity and transmissions are still unclear. Methods: 3050 SARS-CoV-2 genome sequences were retrieved from GIASID database. After aligned by MAFFT, the mutation patterns were identified by phylogenetic tree analysis. Results: We detected 17 high frequency (>6%) mutations in the 3050 sequences. Based on these mutations, we classed the SARS-CoV-2 into four main groups and 10 subgroups. We found that group A was mainly presented in Asia, group B was primarily detected in North America, group C was prevailingly appeared in Asia and Oceania and group D was principally emerged in Europe and Africa. Additionally, the distribution of these groups was different in age, but was similar in gender. Group A, group B1 and group C2 were declined over time, inversely, group B2, group C3 and group D were rising. At last, we found two apparent expansion stages (late Jan-2020 and late Feb-2020 to early Mar-2020, respectively). Notably, most of groups are quickly expanding, especially group D. Conclusions: We classed the SARS-CoV-2 into four main groups and 10 subgroups based on different mutation patterns at first time. The distribution of the 10 subgroups was different in geography, time and age, but not in gender. Most of groups are rapidly expanding, especially group D. Therefore, we should attach importance to these genetic diversity patterns of SARS-CoV-2 and take more targeted measures to constrain its spread.

Title: Implications of SARS-CoV-2 Mutations for Genomic RNA Structure and Host microRNA Targeting Content: The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.

Title: Implications of SARS-CoV-2 Mutations for Genomic RNA Structure and Host microRNA Targeting. Content: The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.

Title: Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 Content: SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 5 2020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

Title: Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could affect viral autophagy Content: BACKGROUND: SARS-CoV-2 is a new coronavirus that has spread globally, infecting more than 150000 people, and being declared pandemic by the WHO. We provide here bio-informatic, evolutionary analysis of 351 available sequences of its genome with the aim of mapping genome structural variations and the patterns of selection. METHODS: A Maximum likelihood tree has been built and selective pressure has been investigated in order to find any mutation developed during the SARS-CoV-2 epidemic that could potentially affect clinical evolution of the infection. FINDING: We have found in more recent isolates the presence of two mutations affecting the Non-Structural Protein 6 (NSP6) and the Open Reding Frame10 (ORF 10) adjacent regions. Amino acidic change stability analysis suggests both mutations could confer lower stability of the protein structures. INTERPRETATION: One of the two mutations, likely developed within the genome during virus spread, could affect virus intracellular survival. Genome follow-up of SARS-CoV-2 spread is urgently needed in order to identify mutations that could significantly modify virus pathogenicity.

Title: International expansion of a novel SARS-CoV-2 mutant Content: Letter to the editor. There is no abstract. The summary was showed: SARS-CoV-2 has inevitably mutated during its pandemic spread to cause unpredictable effects on COVID-19 and complicate epidemic control efforts. Here we report that a novel SARS-CoV-2 mutation (ORF3a) appears to be spreading worldwide, which deserves close attention.

Title: Investigating the genomic landscape of novel coronavirus (2019-nCoV) to identify non-synonymous mutations for use in diagnosis and drug design Content: This study presents a comprehensive phylogenetic analysis of SARS-CoV2 isolates to understand discrete mutations that are occurring between patient samples. The analysis unravel various amino acid mutations in the viral proteins which may provide an explanation for varying treatment efficacies of different inhibitory drugs and a future direction towards a combinatorial treatment therapies based on the kind of mutation in the viral genome.

Title: SARS-CoV2 envelope protein: Non-synonymous mutations and its consequences Content: Abstract In the NCBI database, as on June 6, 2020, total number of available complete genome sequences of SARS-CoV2 across the world is 3617. The envelope protein of SARS-CoV2 possesses several non-synonymous mutations over the transmembrane domain and (C)-terminus in 15 (0.414%) genomes among 3617 available SARS-CoV2 genomes. More precisely, it is to be mentioned that 10(0.386%) out of 2588 genomes from the USA, 3(0.806%) from Asia, 1 (0.348%) from Europe and 1 (0.274%) from Oceania contain the missense mutations over the envelope proteins of SARS-CoV2 genomes. The C-terminus motif DLLV has been changed to DFLV and YLLV in the proteins QJR88103 (Australia: Victoria) and QKI36831 (China: Guangzhou) respectively, which might affect the binding of this motif with the host protein PALS1.

Title: Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity Content: Monitoring the mutation dynamics of SARS-CoV-2 is critical for the development of effective approaches to contain the pathogen. By analyzing 106 SARS-CoV-2 and 39 SARS genome sequences, we provided direct genetic evidence that SARS-CoV-2 has a much lower mutation rate than SARS. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. The discrepant phylogenies for the spike protein and its receptor binding domain proved a previously reported structural rearrangement prior to the emergence of SARS-CoV-2. Despite that we found the spike glycoprotein of SARS-CoV-2 is particularly more conserved, we identified a mutation that leads to weaker receptor binding capability, which concerns a SARS-CoV-2 sample collected on 27th January 2020 from India. This represents the first report of a significant SARS-CoV-2 mutant, and raises the alarm that the ongoing vaccine development may become futile in future epidemic if more mutations were identified. Highlights Based on the currently available genome sequence data, we proved that SARS-COV-2 genome has a much lower mutation rate and genetic diversity than SARS during the 2002-2003 outbreak. The spike (S) protein encoding gene of SARS-COV-2 is found relatively more conserved than other protein-encoding genes, which is a good indication for the ongoing antiviral drug and vaccine development. Minimum Evolution phylogeny analysis revealed the putative original status of SARS-CoV-2 and the early-stage spread history. We confirmed a previously reported rearrangement in the S protein arrangement of SARS-COV-2, and propose that this rearrangement should have occurred between human SARS-CoV and a bat SARS-CoV, at a time point much earlier before SARS-COV-2 transmission to human. We provided first evidence that a mutated SARS-COV-2 with reduced human ACE2 receptor binding affinity have emerged in India based on a sample collected on 27th January 2020.

Title: Implications of SARS-CoV-2 mutations for genomic RNA structure and host microRNA targeting Content: The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2 permissive host cells, we identified twelve separate target sequences in the SARS-CoV-2 genome; eight of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host could be constrained by host miRNA defence. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation.

Title: An overview of the genetic variations of the SARS-CoV-2 genomes isolated in Southeast Asian countries Content: Monitoring the mutation dynamics of human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical in understanding its infectivity, virulence and pathogenicity for development of a vaccine. In an "age of mobility", the pandemic highlights the importance and vulnerability of regionalisation and labour market interdependence in Southeast Asia. We intend to characterise the genetic variability of viral populations within the region to provide preliminary information for regional surveillance in the future. By analysing 142 complete genomes from South East Asian (SEA) countries, we identified three central variants distinguished by nucleotide and amino acid changes.

Title: An overview of the genetic variations of the SARS-CoV-2 genomes isolated in Southeast Asian countries. Content: Monitoring the mutation dynamics of human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical in understanding its infectivity, virulence and pathogenicity for development of a vaccine. In an "age of mobility", the pandemic highlights the importance and vulnerability of regionalisation and labour market interdependence in Southeast Asia. We intend to characterise the genetic variability of viral populations within the region to provide preliminary information for regional surveillance in the future. By analysing 142 complete genomes from South East Asian (SEA) countries, we identified three central variants distinguished by nucleotide and amino acid changes.

Title: The genomic variation landscape of globally-circulating clades of SARS-CoV-2 defines a genetic barcoding scheme Content: We describe fifteen major mutation events from 2,058 high-quality SARS-CoV-2 genomes deposited up to March 31st, 2020. These events define five major clades (G, I, S, D and V) of globally-circulating viral populations, representing 85.7% of all sequenced cases, which we can identify using a 10 nucleotide genetic classifier or barcode. We applied this barcode to 4,000 additional genomes deposited between March 31st and April 15th and classified successfully 95.6% of the clades demonstrating the utility of this approach. An analysis of amino acid variation in SARS-CoV-2 ORFs provided evidence of substitution events in the viral proteins involved in both host-entry and genome replication. The systematic monitoring of dynamic changes in the SARS-CoV-2 genomes of circulating virus populations over time can guide therapeutic and prophylactic strategies to manage and contain the virus and, also, with available efficacious antivirals and vaccines, aid in the monitoring of circulating genetic diversity as we proceed towards elimination of the agent. The barcode will add the necessary genetic resolution to facilitate tracking and monitoring of infection clusters to distinguish imported and indigenous cases and thereby aid public health measures seeking to interrupt transmission chains without the requirement for real-time complete genomes sequencing.

Title: Genomic characterization of a novel SARS-CoV-2 Content: A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with human to human transmission and extreme human sickness has been as of late announced from the city of Wuhan in China. Our objectives were to mutation analysis between recently reported genomes at various times and locations and to characterize the genomic structure of SARS-CoV-2 using bioinformatics programs. Information on the variation of viruses is of considerable medical and biological impacts on the prevention, diagnosis, and therapy of infectious diseases. To understand the genomic structure and variations of the SARS-CoV-2. The study analyzed 95 SARS-CoV-2 complete genome sequences available in GenBank, National MicrobiologyData Center (NMDC) and NGDC Genome Warehouse from December-2019 until 05 of April-2020. The genomic signature analysis demonstrates that a strong association between the time of sample collection, location of sample and accumulation of genetic diversity. We found 116 mutations, the three most common mutations were 8782C>T in ORF1ab gene, 28144T>C in ORF8 gene and 29095C>T in the N gene. The mutations might affect the severity and spread of the SARS-CoV-2. The finding heavily supports an intense requirement for additional prompt, inclusive investigations that combine genomic detail, epidemiological information and graph records of the clinical features of patients with COVID-19.

Title: We shouldn’t worry when a virus mutates during disease outbreaks Content: Mutation. The word naturally conjures fears of unexpected and freakish changes. Ill-informed discussions of mutations thrive during virus outbreaks, including the ongoing spread of SARS-CoV-2. In reality, mutations are a natural part of the virus life cycle and rarely impact outbreaks dramatically.

Title: An updated analysis of variations in SARS-CoV-2 genome Content: A novel pathogen, named SARS-CoV-2, has caused an unprecedented worldwide pandemic in the first half of 2020. As the SARS-CoV-2 genome sequences have become available, one of the important focus of scientists has become tracking variations in the viral genome. In this study, 30366 SARS-CoV-2 isolate genomes were aligned using the software developed by our group (ODOTool) and 11 variations in SARS-CoV-2 genome over 10% of whole isolates were discussed. Results indicated that, frequency rates of these 11 variations change between 3.56%–88.44 % and these rates differ greatly depending on the continents they have been reported. Despite some variations being in low frequency rate in some continents, C14408T and A23403G variations on Nsp12 and S protein, respectively, observed to be the most prominent variations all over the world, in general, and both cause missense mutations. It is also notable that most of isolates carry C14408T and A23403 variations simultaneously and also nearly all isolates carrying the G25563T variation on ORF3a, also carry C14408T and A23403 variations, although their location distributions are not similar. All these data should be considered towards development of vaccine and antiviral treatment strategies as well as tracing diversity of virus in all over the world.

Title: Genomic characterization of a novel SARS-CoV-2 Content: Abstract A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) associated with human to human transmission and extreme human sickness has been as of late announced from the city of Wuhan in China. Our objectives were to mutation analysis between recently reported genomes at various times and locations and to characterize the genomic structure of SARS-CoV-2 using bioinformatics programs. Information on the variation of viruses is of considerable medical and biological impacts on the prevention, diagnosis, and therapy of infectious diseases. To understand the genomic structure and variations of the SARS-CoV-2. The study analyzed 95 SARS-CoV-2 complete genome sequences available in GenBank, National MicrobiologyData Center (NMDC) and NGDC Genome Warehouse from December-2019 until 05 of April-2020. The genomic signature analysis demonstrates that a strong association between the time of sample collection, location of sample and accumulation of genetic diversity. We found 116 mutations, the three most common mutations were 8782C>T in ORF1ab gene, 28144T>C in ORF8 gene and 29095C>T in the N gene. The mutations might affect the severity and spread of the SARS-CoV-2. The finding heavily supports an intense requirement for additional prompt, inclusive investigations that combine genomic detail, epidemiological information and graph records of the clinical features of patients with COVID-19.

Title: The origin and underlying driving forces of the SARS-CoV-2 outbreak Content: BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019-12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

Title: Genome-wide variations of SARS-CoV-2 infer evolution relationship and transmission route Content: In the epidemic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the issues of mutation, origin, typing and the effect of mutation on molecular detection remain to be unrevealed. In order to identify the evolutionary relationship of SARS-CoV-2 and evaluate the detection efficiency of primers that are currently used in different countries, we retrieved genomic sequences of 373 SARS-CoV-2 strains from multiple databases and performed genome-wide variation analysis. According to the nucleotide C28144T variation, the SARS-CoV-2 can be divided into group A (117 strains) and group B (256 strains). The spike protein gene (S gene) coding region 1841 (total 23403) A1841G, formed a B1 subgroup (40 strains) in group B, of which 30 strains were from European and American countries in March (especially Washington, USA). These mutations are likely to be influenced by the environment or the immunization selection pressure of different populations. Although the mutation is not in the receptor binding region (RBD) and alkaline cleavage region, it may also affect the ability of transmission and pathogenicity; however, the significance is not yet clear. As the ratio of A / B strains in the epidemic months showed an increasing trend (0.35: 1 in January, 0.62: 1 in February and 0.76: 1 in March), it seems that the transmissibility of group A strains becomes stronger with time. Based on the variation of 11 nucleotide sites during the epidemic process, it is speculated that the Washington strain is more like an ancestor type, and the Wuhan strain is the offspring of the group A virus strain. By comparing the detection capabilities of primers in different countries, the SARS-CoV-2 nucleotide variation may only affect molecular detection of very few strains. The differences in the transmissibility, pathogenicity and clinical manifestations of different types of strains require further investigations.

Title: The origin and underlying driving forces of the SARS-CoV-2 outbreak Content: BACKGROUND: SARS-CoV-2 began spreading in December 2019 and has since become a pandemic that has impacted many aspects of human society. Several issues concerning the origin, time of introduction to humans, evolutionary patterns, and underlying force driving the SARS-CoV-2 outbreak remain unclear. METHOD: Genetic variation in 137 SARS-CoV-2 genomes and related coronaviruses as of 2/23/2020 was analyzed. RESULT: After correcting for mutational bias, the excess of low frequency mutations on both synonymous and nonsynonymous sites was revealed which is consistent with the recent outbreak of the virus. In contrast to adaptive evolution previously reported for SARS-CoV during its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation. The sequence similarity in the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression that occurred approximately 40 years ago. The current outbreak of SARS-CoV-2 was estimated to have originated on 12/11/2019 (95% HPD 11/13/2019–12/23/2019). The effective population size of the virus showed an approximately 20-fold increase from the onset of the outbreak to the lockdown of Wuhan (1/23/2020) and ceased to increase afterwards, demonstrating the effectiveness of social distancing in preventing its spread. Two mutations, 84S in orf8 protein and 251 V in orf3 protein, occurred coincidentally with human intervention. The former first appeared on 1/5/2020 and plateaued around 1/23/2020. The latter rapidly increased in frequency after 1/23/2020. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China is two times higher than those derived from the rest of the world. A network analysis found that haplotypes collected from Wuhan were interior and had more mutational connections, both of which are consistent with the observation that the SARS-CoV-2 outbreak originated in China. CONCLUSION: SARS-CoV-2 might have cryptically circulated within humans for years before being discovered. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and determine the critical steps required for effective spreading.

Title: Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine Content: Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15â¯140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

Title: Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis, Vaccine, and Medicine Content: [Image: see text] Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

Title: Coronavirus3D: 3D structural visualization of COVID-19 genomic divergence Content: MOTIVATION: As the COVID-19 pandemics is spreading around the world, the SARS-CoV-2 virus is evolving with mutations that potentially change and fine-tune functions of the proteins coded in its genome. RESULTS: Coronavirus3D website integrates data on the SARS-CoV-2 virus mutations with information about 3D structures of its proteins, allowing users to visually analyze the mutations in their 3D context. AVAILABILITY: Coronavirus3D server is freely available at https://coronavirus3d.org.

Title: Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 Content: Abstract SARS-CoV-2 is a SARS-like coronavirus of likely zoonotic origin first identified in December 2019 in Wuhan, the capital of China's Hubei province. The virus has since spread globally, resulting in the currently ongoing COVID-19 pandemic. The first whole genome sequence was published on January 52,020, and thousands of genomes have been sequenced since this date. This resource allows unprecedented insights into the past demography of SARS-CoV-2 but also monitoring of how the virus is adapting to its novel human host, providing information to direct drug and vaccine design. We curated a dataset of 7666 public genome assemblies and analysed the emergence of genomic diversity over time. Our results are in line with previous estimates and point to all sequences sharing a common ancestor towards the end of 2019, supporting this as the period when SARS-CoV-2 jumped into its human host. Due to extensive transmission, the genetic diversity of the virus in several countries recapitulates a large fraction of its worldwide genetic diversity. We identify regions of the SARS-CoV-2 genome that have remained largely invariant to date, and others that have already accumulated diversity. By focusing on mutations which have emerged independently multiple times (homoplasies), we identify 198 filtered recurrent mutations in the SARS-CoV-2 genome. Nearly 80% of the recurrent mutations produced non-synonymous changes at the protein level, suggesting possible ongoing adaptation of SARS-CoV-2. Three sites in Orf1ab in the regions encoding Nsp6, Nsp11, Nsp13, and one in the Spike protein are characterised by a particularly large number of recurrent mutations (>15 events) which may signpost convergent evolution and are of particular interest in the context of adaptation of SARS-CoV-2 to the human host. We additionally provide an interactive user-friendly web-application to query the alignment of the 7666 SARS-CoV-2 genomes.

Title: Evolutionary analysis of SARS-CoV-2: how mutation of Non-Structural Protein 6 (NSP6) could affect viral autophagy Content: Abstract Background SARS-CoV-2 is a new coronavirus that has spread globally, infecting more than 150000 people, and being declared pandemic by the WHO. We provide here bio-informatic, evolutionary analysis of 351 available sequences of its genome with the aim of mapping genome structural variations and the patterns of selection. Methods A Maximum likelihood tree has been built and selective pressure has been investigated in order to find any mutation developed during the SARS-CoV-2 epidemic that could potentially affect clinical evolution of the infection. Finding We have found in more recent isolates the presence of two mutations affecting the Non-Structural Protein 6 (NSP6) and the Open Reding Frame10 (ORF 10) adjacent regions. Amino acidic change stability analysis suggests both mutations could confer lower stability of the protein structures. Interpretation One of the two mutations, likely developed within the genome during virus spread, could affect virus intracellular survival. Genome follow-up of SARS-CoV-2 spread is urgently needed in order to identify mutations that could significantly modify virus pathogenicity.

Title: Intra-host Variation and Evolutionary Dynamics of SARS-CoV-2 Population in COVID-19 Patients Content: As of middle May 2020, the causative agent of COVID-19, SARS-CoV-2, has infected over 4 million people with more than 300 thousand death as official reports1,2. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. Here, using deep sequencing data, we achieved and characterized consensus genomes and intra-host genomic variants from 32 serial samples collected from eight patients with COVID-19. The 32 consensus genomes revealed the coexistence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparison of allele frequencies of the iSNVs revealed genetic divergence between intra-host populations of the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events among intra-host transmissions. Nonetheless, we observed a maintained viral genetic diversity within GIT, showing an increased population with accumulated mutations developed in the tissue-specific environments. The iSNVs identified here not only show spatial divergence of intra-host viral populations, but also provide new insights into the complex virus-host interactions.

Title: The origin and underlying driving forces of the SARS-CoV-2 outbreak Content: The spread of SARS-CoV-2 since December 2019 has become a pandemic and impacted many aspects of human society. Here, we analyzed genetic variation of SARS-CoV-2 and its related coronavirus and found the evidence of intergenomic recombination. After correction for mutational bias, analysis of 137 SARS-CoV-2 genomes as of 2/23/2020 revealed the excess of low frequency mutations on both synonymous and nonsynonymous sites which is consistent with recent origin of the virus. In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic in 2003, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated. Genetic diversity of SARS-CoV-2 collected from China was two time higher than those derived from the rest of the world. In addition, in network analysis, haplotypes collected from Wuhan city were at interior and have more mutational connections, both of which are consistent with the observation that the outbreak of cov-19 was originated from China. SUMMARY In contrast to adaptive evolution previously reported for SARS-CoV in its brief epidemic, our analysis of SARS-CoV-2 genomes shows signs of relaxation of selection. The sequence similarity of the spike receptor binding domain between SARS-CoV-2 and a sequence from pangolin is probably due to an ancient intergenomic introgression. Therefore, SARS-CoV-2 might have cryptically circulated within humans for years before being recently noticed. Data from the early outbreak and hospital archives are needed to trace its evolutionary path and reveal critical steps required for effective spreading. Two mutations, 84S in orf8 protein and 251V in orf3 protein, occurred coincidentally with human intervention. The 84S first appeared on 1/5/2020 and reached a plateau around 1/23/2020, the lockdown of Wuhan. 251V emerged on 1/21/2020 and rapidly increased its frequency. Thus, the roles of these mutations on infectivity need to be elucidated.

Title: Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions Content: The COVID-19 pandemic has spread across the globe at an alarming rate in the last four months. However, unlike any of the previous global outbreaks the availability of hundreds of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 480 full-length (>29000 nt) sequences from the 1575 SARS-CoV-2 sequences available and identified 37 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, two of the 37 substitutions occur within highly conserved secondary structures in the 5’ and 3’ untranslated regions that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define 3 mutually exclusive lineages (A1, B1 and C1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2.

Title: How fast does the SARS-Cov-2 virus really mutate in heterogeneous populations? Content: We introduce the problem of determining the mutational support of genes in the SARS-Cov-2 virus and estimating the distribution of mutations within different genes using small sample sizes that do not allow for accurate maximum likelihood estimation. The mutational support refers to the unknown number of sites mutated across all strains and individual samples of the SARS-Cov-2 genome; given the high cost and limited availability of real-time polymerase chain reaction (RT-PCR) test kits, especially in early stages of infections when only a small number of genomic samples (∼ 1000s) are available that do not allow for determining the exact degree of mutations in an RNA virus that comprises roughly 30, 000 nucleotides. Nevertheless, working with small sample sets is required in order to quickly predict the mutation rate of this and other viruses and get an insight into their transformational power. Furthermore, with the small number of samples available, it is hard to estimate the mutational landscape across different age/gender groups and geographical locations which may be of great importance in assessing different risk categories and factors influencing susceptibility to infection. To this end, we use our state-of-the art polynomial estimator techniques and the Good-Turing estimator to obtain estimates based on only roughly 1, 000 samples per category. Our analysis reveals an interesting finding: the mutational support appears to be statistically more significant in patients which appear to have lower infection rates and handle the exposure with milder symptoms, such as women and people of relatively young age (≤ 55).

Title: Overwhelming mutations or SNPs of SARS-CoV-2: A point of caution Content: The morbidity of SARS-CoV-2 (COVID-19) is reaching 3 Million landmark causing and a serious public health concern globally and it is enigmatic how several antiviral and antibody treatments were not effective in the different period across the globe. With the drastic increasing number of positive cases around the world WHO raised the importance in the assessment of the risk of spread and understanding genetic modifications that could have occurred in the SARS-CoV-2. Using all available deep sequencing data of complete genome from all over the world (NCBI repository), we identified several hundreds of point mutations or SNPs in SARS-CoV-2 all across the genome. This could be the cause for the constant change and differed virulence with an increase in mortality and morbidity. Among the 12 different countries (one sequence from each country) with complete genome sequencing data, we noted the 47 key point mutations or SNPs located along the entire genome that might have impact in the virulence and response to different antivirals against SARS-CoV-2. In this regard, key viral proteins of spike glycoprotein, Nsp1, RdRp and the ORF8 region got heavily mutated within these 3 months via person-to-person passage. We also discuss what could be the possible cause of this rapid mutation in the SARS-CoV-2.

Title: Structural and Functional Implications of Spike Protein Mutational Landscape in SARS-CoV-2 Content: SARS-CoV-2, the causative agent of COVID-19 pandemic, is an RNA virus prone to mutations. Interaction of SARS-CoV-2 Spike (S) protein with the host cell receptor, Angiotensin-I Converting Enzyme 2 (ACE2) is pivotal for attachment and entry of the virus. Yet, natural mutations acquired on S protein during the pandemic and their impact on viral infectivity, transmission dynamics and disease pathogenesis remains poorly understood. Here, we analysed 2952 SARS-CoV-2 genomes across the globe, and identified a total of 1815 non-synonymous mutations in the S-protein that fall into 54 different types. We observed that six of these distinct mutations were located in the Receptor Binding Domain (RBD) region that directly engages host ACE2. Molecular phylogenetic analysis revealed that these RBD mutations cluster into distinct phyletic clades among global subtypes of SARS-CoV-2 implying possible emergence of novel sublineages of the strain. Structure-guided homology modelling and docking analysis predicted key molecular rearrangements in the ACE2 binding interface of RBD mutants that could result in altered virus-host interactions. We propose that our findings could be significant in understanding disease dynamics and in developing vaccines, antibodies and therapeutics for COVID-19. Importance COVID-19 pandemic shows considerable variations in disease transmission and pathogenesis globally, yet reasons remain unknown. Our study identifies key S-protein mutations prevailing in SARS-CoV-2 strain that could alter viral attachment and infectivity. We propose that the interplay of these mutations could be one of the factors driving global variations in COVID-19 spread. In addition, the mutations identified in this study could be an important indicator in predicting efficacies of vaccines, antibodies and therapeutics that target SARS-CoV-2 RBD-ACE2 interface.

Title: Decoding SARS-CoV-2 transmission, evolution and ramification on COVID-19 diagnosis, vaccine, and medicine Content: Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 6156 genome samples collected up to April 24, 2020, we report that SARS-CoV-2 has had 4459 alarmingly mutations which can be clustered into five subtypes. We introduce mutation ratio and mutation $h$-index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively non-conservative. In particular, the nucleocapsid protein has more than half its genes changed in the past few months, signaling devastating impacts on the ongoing development of COVID-19 diagnosis, vaccines, and drugs.

Title: Comprehensive annotations of the mutational spectra of SARS-CoV-2 spike protein: a fast and accurate pipeline Content: In order to explore nonsynonymous mutations and deletions in the spike (S) protein of SARS-CoV-2, we comprehensively analyzed 35,750 complete S protein gene sequences from across six continents and five climate zones around the world, as documented in the GISAID database as of June 24th, 2020. Through a custom Python-based pipeline for analyzing mutations, we identified 27,801 (77.77 % of spike sequences) mutated strains compared to Wuhan-Hu-1 strain. 84.40% of these strains had only single amino-acid (aa) substitution mutations, but an outlier strain from Bosnia and Herzegovina (EPI_ISL_463893) was found to possess six aa substitutions. The D614G variant of the major G clade was found to be predominant across circulating strains in all climates. We also identified 988 unique aa substitution mutations distributed across 660 positions within the spike protein, with eleven sites showing high variability – these sites had four types of aa variations at each position. Besides, 17 in-frame deletions at four major regions (three in N-terminal domain and one just downstream of the RBD) may have possible impact on attenuation. Moreover, the mutational frequency differed significantly (p= 0.003, Kruskal–Wallis test) among the SARS-CoV-2 strains worldwide. This study presents a fast and accurate pipeline for identifying nonsynonymous mutations and deletions from large dataset for any particular protein coding sequence and presents this S protein data as representative analysis. By using separate multi-sequence alignment with MAFFT, removing ambiguous sequences and in-frame stop codons, and utilizing pairwise alignment, this method can derive nonsynonymus mutations (Reference:Position:Strain). We believe this will aid in the surveillance of any proteins encoded by SARS-CoV-2, and will prove to be crucial in tracking the ever-increasing variation of many other divergent RNA viruses in the future.

Title: SARS-CoV-2: virus mutations in specific European populations Content: Abstract The SARS-CoV-2 virus is being intensively studied, particularly, its evolution in the increasingly available sequences between countries/continents with classical phylogenetic tree representation. More recently, certain protein mutations are correlated with specific functional impacts. Our clinical data from patients suggest different clinical symptoms between European countries. Among others, SARS-CoV-2 mutations could explain these disparities. Our analyses point out an association of diverse mutations, including co-evolving ones, in a few SARS-CoV-2 proteins, with specific countries. We therefore suggest combining clinical information from patients and the determination of the associated SARS-CoV-2 genome to better understand the specific symptoms.

Title: [Genetic variation analysis of SARS coronavirus]. Content: Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been verified as the causative agent of a worldwide outbreak of SARS. Since the observed per-base replication error-rate of RNA polymerase is about 3 x 10(-5), RNA virus populations typically contain genetic variants that form a heterogeneous virus pool. This feature confers great adaptability on viruses and is partly responsible for current difficulties of viral disease prevention and control, such as of HIV and HCV infections. SARS-CoV, a newly-identified single-strand RNA virus, has been identified with common variable property of RNA viruses. In this study, genetic variation of SARS-CoV is analyzed based on available documented data and our study. It suggests that SARS-CoV is of similar mutation frequency and dynamic mutant distributions like other RNA viruses, though relatively less identified variation number than those causing chronic infections. In contrast, the viral spike (S) gene exhibits high variation, while nucleocapsid (N) gene shows relative sequence conservation. No obvious correlation is observed between viral variation and clinical disease manifestation from present analyzed data.

Title: Failure of the cobas(R) SARS-CoV-2 (Roche) E-gene assay is associated with a C-to-T transition at position 26340 of the SARS-CoV-2 genome Content: Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals, while also clearing essential staff to continue work. At the current time a number of RT-PCR tests have been developed to identify SARS-CoV-2, targeting multiple regions in the viral genome. In comparison to other RNA viruses the mutation rate of SARS-CoV-2 is moderate, however given the large number of transmission chains it is prudent to monitor circulating viruses for mutations that might compromise these tests. Here we report the identification of a C-to-T transition at position 26340 of the SARS-CoV-2 genome which is associated with failure of the cobas(R) SARS-CoV-2 E-gene assay. This variant was detected in four health care workers from the same team. Whole genome sequencing of SARS-CoV-2 showed all four to carry genetically identical viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently on three occasions. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of SNPs which might adversely affect the RT-PCRs used in diagnostics. Additionally, it argues that two regions in the SARS-CoV-2 should be targeted in RT-PCRs to avoid false negatives.

Title: COVID-19 Variants Database: A repository for Human SARS-CoV-2 Polymorphism Data Content: COVID-19 is a newly communicable disease with a catastrophe outbreak that affects all over the world. We retrieved about 8,781 nucleotide fragments and complete genomes of SARS-CoV-2 reported from sixty-four countries. The CoV-2 reference genome was obtained from the National Genomics Data Center (NGDC), GISAID, and NCBI Genbank. All the sequences were aligned against reference genomes using Clustal Omega and variants were called using in-house built Python script. We intend to establish a user-friendly online resource to visualize the variants in the viral genome along with the Primer Infopedia. After analyzing and filtering the data globally, it was made available to the public. The detail of data available to the public includes mutations from 5688 SARS-CoV-2 sequences curated from 91 regions. This database incorporated 39920 mutations over 3990 unique positions. According to the translational impact, these mutations include 11829 synonymous mutations including 681 synonymous frameshifts and 21701 nonsynonymous mutations including 10 nonsynonymous frameshifts. Development of SARS-CoV-2 mutation genome browsers is a fundamental step obliging towards the virus surveillance, viral detection, and development of vaccine and therapeutic drugs. The SARS-COV-2 mutation browser is available at http://covid-19.dnageography.com.

Title: Identification of unique mutations in SARS-CoV-2 strains isolated from India suggests its attenuated pathotype Content: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first reported in Wuhan, China in November 2019 has developed into a pandemic since March 2020, causing substantial human casualties and economic losses. Studies on SARS-CoV-2 are being carried out at an unprecedented rate to tackle this threat. Genomics studies, in particular, are indispensable to elucidate the dynamic nature of the RNA genome of SARS-CoV-2. RNA viruses are marked by their unique ability to undergo high rates of mutation in their genome, much more frequently than their hosts, which diversifies their strengths qualifying them to elude host immune response and amplify drug resistance. In this study, we sequenced and analyzed the genomic information of the SARS-CoV-2 isolates from two infected Indian patients and explored the possible implications of point mutations in its biology. In addition to multiple point mutations, we found a remarkable similarity between relatively common mutations of 36-nucleotide deletion in ORF8 of SARS-CoV-2. Our results corroborate with the earlier reported 29-nucleotide deletion in SARS, which was frequent during the early stage of human-to-human transmission. The results will be useful to understand the biology of SARS-CoV-2 and itsattenuation for vaccine development.

Title: Mutational Signatures and Heterogeneous Host Response Revealed Via Large-Scale Characterization of SARS-COV-2 Genomic Diversity Content: To dissect the mechanisms underlying the observed inflation of variants in SARS-CoV-2 genome, we present the largest up-to-date analysis of intra-host genomic diversity, which reveals that the majority of samples present a complex sublineage architecture, due to the interplay between host-related mutational processes and transmission dynamics. Strikingly, the deconvolution of the entire set of intra-host variants reveals the existence of mutually exclusive viral mutational signatures, which prove that distinct hosts differently respond to SARS-CoV-2 infections. In particular, two signatures are likely ruled by APOBEC and Reactive Oxygen Species (ROS), which induce hypermutation in a significant number of samples, and appear to be affected by severe purifying selection. Conversely, several mutations linked to low-rate mutational processes appear to transit to clonality in the population, eventually leading to the definition of new viral genotypes and to an increase of overall genomic diversity. Finally, we demonstrate that a high number of variants are observed in samples associated to independent lineages, likely due to signature-related mutational hotspots or to positive selection.

Title: Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2 Content: We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focusing initially on the Spike (S) protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics. To date we have identified thirteen mutations in Spike that are accumulating. Mutations are considered in a broader phylogenetic context, geographically, and over time, to provide an early warning system to reveal mutations that may confer selective advantages in transmission or resistance to interventions. Each one is evaluated for evidence of positive selection, and the implications of the mutation are explored through structural modeling. The mutation Spike D614G is of urgent concern; it began spreading in Europe in early February, and when introduced to new regions it rapidly becomes the dominant form. Also, we present evidence of recombination between locally circulating strains, indicative of multiple strain infections. These finding have important implications for SARS-CoV-2 transmission, pathogenesis and immune interventions.

Title: Whole-Genome Sequences of the Severe Acute Respiratory Syndrome Coronavirus-2 obtained from Romanian patients between March and June of 2020 Content: Impact of mutations on the evolution of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) are needed for ongoing global efforts to track and trace the current pandemic, in order to enact effective prevention and treatment options. SARS-Co-V-2 viral genomes were detected and sequenced from 18 Romanian patients suffering from coronavirus disease-2019. Viral Spike S glycoprotein sequences were used to generate model structures and assess the role of mutations on protein stability. We integrated the phylogenetic tree within the available European SARS-Co-V-2 genomic sequences. We further provide an epidemiological overview of the pre-existing conditions that are lethal in relevant Romanian patients. Non-synonymous mutations in the viral Spike glycoprotein relating to infectivity are constructed in models of protein structures. Continuing search to limit and treat SARS-CoV-2 benefit from our contribution in delineating the viral Spike glycoprotein mutations, as well as from assessment of their role on protein stability or complex formation with human receptor angiotensin-converting enzyme 2. Our results help implement and extend worldwide genomic surveillance of coronavirus disease-2019.

Title: No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2 Content: The COVID-19 pandemic is caused by the coronavirus SARS-CoV-2, which jumped into the human population in late 2019 from a currently uncharacterised reservoir. Due to this extremely recent association with humans, SARS-CoV-2 may not yet be fully adapted to its human host. This has led to speculations that some lineages of SARS-CoV-2 may be evolving towards higher transmissibility. The most plausible candidate mutations under putative natural selection are those which have emerged repeatedly and independently (homoplasies). Here, we formally test whether any of the recurrent mutations that have been observed in SARS-CoV-2 to date are significantly associated with increased viral transmission. To do so, we developed a phylogenetic index to quantify the relative number of descendants in sister clades with and without a specific allele. We apply this index to a carefully curated set of recurrent mutations identified within a dataset of over 23,000 SARS-CoV-2 genomes isolated from patients worldwide. We do not identify a single recurrent mutation in this set convincingly associated with increased viral transmission. Instead, recurrent SARS-CoV-2 mutations currently in circulation appear to be evolutionary neutral. Recurrent mutations also seem primarily induced by the human immune system via host RNA editing, rather than being signatures of adaption to the novel human host. We find no evidence at this stage for the emergence of more transmissible lineages of SARS-CoV-2 due to recurrent mutations.

Title: SARS-CoV-2 amino acid substitutions widely spread in the human population are mainly located in highly conserved segments of the structural proteins Content: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic offers a unique opportunity to study the introduction and evolution of a pathogen into a completely naïve human population. We identified and analysed the amino acid mutations that gained prominence worldwide in the early months of the pandemic. Eight mutations have been identified along the viral genome, mostly located in conserved segments of the structural proteins and showing low variability among coronavirus, which indicated that they might have a functional impact. At the moment of writing this paper, these mutations present a varied success in the SARS-CoV-2 virus population; ranging from a change in the spike protein that becomes absolutely prevalent, two mutations in the nucleocapsid protein showing frequencies around 25%, to a mutation in the matrix protein that nearly fades out after reaching a frequency of 20%.

Title: Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction Content: The emergence of SARS-CoV-2 has led to the current global coronavirus pandemic and more than one million infections since December 2019. The exact origin of SARS-CoV-2 remains elusive, but the presence of a distinct motif in the S1/S2 junction region suggests the possible acquisition of cleavage site(s) in the spike protein that promoted cross-species transmission. Through plaque purification of Vero-E6 cultured SARS-CoV-2, we found a series of variants which contain 15-30-bp deletions (Del-mut) or point mutations respectively at the S1/S2 junction. Examination of the original clinical specimen from which the isolate was derived, and 26 additional SARS-CoV-2 positive clinical specimens, failed to detect these variants. Infection of hamsters shows that one of the variants (Del-mut-1) which carries deletion of 10 amino acids (30bp) does not cause the body weight loss or more severe pathological changes in the lungs that is associated with wild type virus infection. We suggest that the unique cleavage motif promoting SARS-CoV-2 infection in humans may be under strong selective pressure, given that replication in permissive Vero-E6 cells leads to the loss of this adaptive function. It would be important to screen the prevalence of these variants in asymptomatic infected cases. The potential of the Del-mut variants as an attenuated vaccine or laboratory tool should be evaluated.

Title: Genome Sequencing of a Severe Acute Respiratory Syndrome Coronavirus 2 Isolate Obtained from a South African Patient with Coronavirus Disease 2019 Content: As a contribution to the global efforts to track and trace the ongoing coronavirus pandemic, here we present the sequence, phylogenetic analysis, and modeling of nonsynonymous mutations for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome that was detected in a South African patient with coronavirus disease 2019 (COVID-19).

Title: Stability of SARS-CoV-2 Phylogenies Content: The SARS-CoV-2 pandemic has led to unprecedented, nearly real-time genetic tracing due to the rapid community sequencing response. Researchers immediately leveraged these data to infer the evolutionary relationships among viral samples and to study key biological questions, including whether host viral genome editing and recombination are features of SARS-CoV-2 evolution. This global sequencing effort is inherently decentralized and must rely on data collected by many labs using a wide variety of molecular and bioinformatic techniques. There is thus a strong possibility that systematic errors associated with lab-specific practices affect some sequences in the repositories. We find that some recurrent mutations in reported SARS-CoV-2 genome sequences have been observed predominantly or exclusively by single labs, co-localize with commonly used primer binding sites and are more likely to affect the protein coding sequences than other similarly recurrent mutations. We show that their inclusion can affect phylogenetic inference on scales relevant to local lineage tracing, and make it appear as though there has been an excess of recurrent mutation and/or recombination among viral lineages. We suggest how samples can be screened and problematic mutations removed. We also develop tools for comparing and visualizing differences among phylogenies and we show that consistent clade- and tree-based comparisons can be made between phylogenies produced by different groups. These will facilitate evolutionary inferences and comparisons among phylogenies produced for a wide array of purposes. Building on the SARS-CoV-2 Genome Browser at UCSC, we present a toolkit to compare, analyze and combine SARS-CoV-2 phylogenies, find and remove potential sequencing errors and establish a widely shared, stable clade structure for a more accurate scientific inference and discourse. Foreword We wish to thank all groups that responded rapidly by producing these invaluable and essential sequence data. Their contributions have enabled an unprecedented, lightning-fast process of scientific discovery---truly an incredible benefit for humanity and for the scientific community. We emphasize that most lab groups with whom we associate specific suspicious alleles are also those who have produced the most sequence data at a time when it was urgently needed. We commend their efforts. We have already contacted each group and many have updated their sequences. Our goal with this work is not to highlight potential errors, but to understand the impacts of these and other kinds of highly recurrent mutations so as to identify commonalities among the suspicious examples that can improve sequence quality and analysis going forward.

Title: Virus strain from a mild COVID-19 patient in Hangzhou represents a new trend in SARS-CoV-2 evolution potentially related to Furin cleavage site. Content: The mutations in the SARS-CoV-2 virus genome during the spread of COVID-19 have been unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases, increased liver/kidney damage, and prolonged period of nuclear acid positivity, compared with patients in Wuhan, China. To investigate the underlying mechanisms, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified by gene alignment. Further phylogenetic analysis and relative synonymous codon usage heat map results suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 virus strains collected globally based on the base (C or T) at positions 8824 and 28247. ZJ01 has T at both sites and is currently the only known TT type. The prediction of the Furin cleavage site (FCS) and sequence alignment of the virus family indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was higher in the whole body, especially in glands, liver, kidneys, and colon. Thus, FCS may help SARS-CoV-2 infect these organs. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 (the source of FCS sequence-PRRA) caused mild flu-like symptoms, further showing potential in differentiating into mild COVID-19 subtypes.

Title: Quality control of low-frequency variants in SARS-CoV-2 genomes Content: During the current outbreak of COVID-19, research labs around the globe submit sequences of the local SARS-CoV-2 genomes to the GISAID database to provide a comprehensive analysis of the variability and spread of the virus during the outbreak. We explored the variations in the submitted genomes and found a significant number of variants that can be seen only in one submission (singletons). While it is not completely clear whether these variants are erroneous or not, these variants show lower transition/transversion ratio. These singleton variants may influence the estimations of the viral mutation rate and tree topology. We suggest that genomes with multiple singletons even marked as high-covered should be considered with caution. We also provide a simple script for checking variant frequency against the database before submission.

Title: Time-series analyses of directional sequence changes in SARS-CoV-2 genomes and an efficient search method for advantageous mutations for growth in human cells Content: We first conducted time-series analysis of mono- and dinucleotide composition for over 10,000 SARS-CoV-2 genomes, as well as over 1500 Zaire ebolavirus genomes, and found clear time-series changes in the compositions on a monthly basis, which should reflect viral adaptations for efficient growth in human cells. We next developed a sequence alignment free method that extensively searches for advantageous mutations and rank them in an increase level for their intrapopulation frequency. Time-series analysis of occurrences of oligonucleotides of diverse lengths for SARS-CoV-2 genomes revealed seven distinctive mutations that rapidly expanded their intrapopulation frequency and are thought to be candidates of advantageous mutations for the efficient growth in human cells.

Title: Virus strain from a mild COVID-19 patient in Hangzhou represents a new trend in SARS-CoV-2 evolution potentially related to Furin cleavage site Content: The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.

Title: Mutational patterns correlate with genome organization in SARS and other coronaviruses Content: Abstract Focused efforts by several international laboratories have resulted in the sequencing of the genome of the causative agent of severe acute respiratory syndrome (SARS), novel coronavirus SARS-CoV, in record time. Using cumulative skew diagrams, I found that mutational patterns in the SARS-CoV genome were strikingly different from other coronaviruses in terms of mutation rates, although they were in general agreement with the model of the coronavirus lifecycle. These findings might be relevant for the development of sequence-based diagnostics and the design of agents to treat SARS.

Title: Genomic Mutations and Changes in Protein Secondary Structure and Solvent Accessibility of SARS-CoV-2 (COVID-19 Virus) Content: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic virus that has caused the global COVID-19 pandemic. Tracing the evolution and transmission of the virus is crucial to respond to and control the pandemic through appropriate intervention strategies. This paper reports and analyses genomic mutations in the coding regions of SARS-CoV-2 and their probable protein secondary structure and solvent accessibility changes, which are predicted using deep learning models. Prediction results suggest that mutation D614G in the virus spike protein, which has attracted much attention from researchers, is unlikely to make changes in protein secondary structure and relative solvent accessibility. Based on 6,324 viral genome sequences, we create a spreadsheet dataset of point mutations that can facilitate the investigation of SARS-CoV-2 in many perspectives, especially in tracing the evolution and worldwide spread of the virus. Our analysis results also show that coding genes E, M, ORF6, ORF7a, ORF7b and ORF10 are most stable, potentially suitable to be targeted for vaccine and drug development.

Title: Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia. Content: The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine-tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3' end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3' end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Title: Patient-derived mutations impact pathogenicity of SARS-CoV-2 Content: The sudden outbreak of the severe acute respiratory syndrome-coronavirus (SARS-CoV-2) has spread globally with more than 1,300,000 patients diagnosed and a death toll of 70,000. Current genomic survey data suggest that single nucleotide variants (SNVs) are abundant. However, no mutation has been directly linked with functional changes in viral pathogenicity. Here we report functional characterizations of 11 patient-derived viral isolates, all of which have at least one mutation. Importantly, these viral isolates show significant variation in cytopathic effects and viral load, up to 270-fold differences, when infecting Vero-E6 cells. We observed intrapersonal variation and 6 different mutations in the spike glycoprotein (S protein), including 2 different SNVs that led to the same missense mutation. Therefore, we provide direct evidence that the SARS-CoV-2 has acquired mutations capable of substantially changing its pathogenicity.

Title: Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia Content: The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3' end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3' end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Title: Molecular analysis of several in-house rRT-PCR protocols for SARS-CoV-2 detection in the context of genetic variability of the virus in Colombia Content: The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem unprecedented in the recent history of humanity. Different in-house real-time RT-PCR (rRT-PCR) methods for SARS-CoV-2 diagnosis and the appearance of genomes with mutations in primer regions have been reported. Hence, whole-genome data from locally-circulating SARS-CoV-2 strains contribute to the knowledge of its global variability and the development and fine tuning of diagnostic protocols. To describe the genetic variability of Colombian SARS-CoV-2 genomes in hybridization regions of oligonucleotides of the main in-house methods for SARS-CoV-2 detection, RNA samples with confirmed SARS-CoV-2 molecular diagnosis were processed through next-generation sequencing. Primers/probes sequences from 13 target regions for SARS-CoV-2 detection suggested by 7 institutions and consolidated by WHO during the early stage of the pandemic were aligned with Muscle tool to assess the genetic variability potentially affecting their performance. Finally, the corresponding codon positions at the 3′ end of each primer, the open reading frame inspection was identified for each gene/protein product. Complete SARS-CoV-2 genomes were obtained from 30 COVID-19 cases, representative of the current epidemiology in the country. Mismatches between at least one Colombian sequence and five oligonucleotides targeting the RdRP and N genes were observed. The 3′ end of 4 primers aligned to the third codon position, showed high risk of nucleotide substitution and potential mismatches at this critical position. Genetic variability was detected in Colombian SARS-CoV-2 sequences in some of the primer/probe regions for in-house rRT-PCR diagnostic tests available at WHO COVID-19 technical guidelines; its impact on the performance and rates of false-negative results should be experimentally evaluated. The genomic surveillance of SARS-CoV-2 is highly recommended for the early identification of mutations in critical regions and to issue recommendations on specific diagnostic tests to ensure the coverage of locally-circulating genetic variants.

Title: coronapp: A Web Application to Annotate and Monitor SARS-CoV-2 Mutations Content: The avalanche of genomic data generated from the SARS-CoV-2 virus requires the development of tools to detect and monitor its mutations across the World. Here, we present a webtool, coronapp, dedicated to easily processing user-provided SARS-CoV-2 genomic sequences, in order to detect and annotate protein-changing mutations. This results in an up-to-date status of SARS-CoV-2 mutations, both worldwide and in user-selected countries. The tool allows users to highlight and prioritize the most frequent mutations in specific protein regions, and to monitor their frequency in the population over time. The tool is available at http://giorgilab.dyndns.org/coronapp/ and the full code is freely shared at https://github.com/federicogiorgi/giorgilab/tree/master/coronapp

Title: Molecular biological analysis of genotyping and phylogeny of severe acute respiratory syndrome associated coronavirus. Content: BACKGROUND SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS) which has been associated with outbreaks of SARS in Guangdong, Hong Kong and Beijing of China, and other regions worldwide. SARS-CoV from human has shown some variations but its origin is still unknown. The genotyping and phylogeny of SARS-CoV were analyzed and reported in this paper. METHODS Full or partial genomes of 44 SARS-CoV strains were collected from GenBank. The genotype, single nucleotide polymorphism and phylogeny of these SARS-CoV strains were analyzed by molecular biological, bioinformatic and epidemiological methods. RESULTS There were 188 point mutations in the 33 virus full genomes with the counts of mutation mounting to 297. Further analysis was carried out among 36 of 188 loci with more than two times of mutation. All the 36 mutation loci occurred in coding sequences and 22 loci were non-synonymous. The gene mutation rates of replicase 1AB, S2 domain of spike glycoprotein and nucleocapsid protein were lower (0.079% - 0.103%). There were 4 mutation loci in S1 domain of spike glycoprotein. The gene mutation rate of ORF10 was the highest (3.333%) with 4 mutation loci in this small domain (120 bp) and 3 of 4 loci related to deletion mutation. By bioinformatics processing and analysis, the nucleotides at 7 loci of genome (T:T:A:G:T:C:T/C:G:G:A:C:T:C) can classify SARS-CoV into two types. Therefore a novel definition is put forward that according to these 7 loci of mutation, 40 strains of SARS-CoV in GenBank can be grouped into two genotypes, T:T:A:G:T:C:T and C:G:G:A:C:T:C, and named as SARS-CoV Yexin genotype and Xiaohong genotype. The two genotypes can be further divided into some sub-genotypes. These genotypes can also be approved by phylogenetic tree of three levels of 44 loci of mutation, spike glycoprotein gene and complete genome sequence. Compared to various strains among SARS-CoV Yexin genotype and Xiaohong genotype, GD01 strain of Yexin genotype is more closely related to SARS-CoV like-virus from animals. CONCLUSION The results mentioned above suggest that SARS-CoV is responding to host immunological pressures and experiencing variation which provide clues, information and evidence of molecular biology for the clinical pathology, vaccine developing and epidemic investigation.

Title: Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility Content: Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S(2) domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in S(D) domain) and G1124V (in S(2) subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird’s eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12038-020-00046-1) contains supplementary material, which is available to authorized users.

Title: Genetic Diversity and Genomic Epidemiology of SARS-COV-2 in Morocco Content: COVID-A9 is an infection disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), declared as a pandemic due to its rapid expansion worldwide. In this study we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 using 22 virus genome sequences reported by three different laboratories in Morocco till the date 07/06/2020 as well as (40366) virus genomes from all around the world. The SARS-CoV-2 genomes from Moroccan patients revealed 62 mutations of which 30 were missense mutations. The mutations Spike_D614G and NSP12_P323L were present in all the 22 analyzed sequences, followed by N_G204R and N_R203K which occurred in 9 among the 22 sequences. The mutations NSP10_R134S, NSP15_D335N, NSP16_I169L, NSP3_L431H, NSP3_P1292L and Spike_V6F occurred one time in our sequences with no record in other sequence worldwide. These mutations should be investigated to figure out their potential effects on all around the world virulence. Phylogenetic analyses revealed that Moroccan SARS-CoV-2 genomes included 9 viruses pertaining to clade 20A, 9 to clade 20B and 2 to clade 20C. This finding suggest that the epidemic spread in Morocco did not show a predominant SARS-CoV-2 route. For multiple and unrelated introductions of SARS-CoV-2 into Morocco via different routes have occurred, giving rise to the diversity of virus genomes in the country. Furthermore, very likely, the SARS-CoV-2 virus circulated in cryptic way in Morocco starting from the fifteen January before the discovering of the first case the second of March.

Title: Morphology, Genome Organization, Replication, and Pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Content: SARS-CoV-2 is a single-stranded RNA virus of ~30 kb genome size which belongs to genus Coronavirus and family Coronaviridae. SARS-CoV-2 has recently emerged and has been declared as a pandemic by the World Health Organization. Genomic characterization of SARS-CoV-2 has shown that it is of zoonotic origin. The structure of SARS-CoV-2 is found to be similar to SARS-CoV with virion size ranging from 70 to 90 nm. Spike, membrane, and envelope surface viral proteins of coronavirus are embedded in host membrane-derived lipid bilayer encapsulating the helical nucleocapsid comprising viral RNA. The genome comprises of 6–11 open reading frames (ORFs) with 5′ and 3′ flanking untranslated regions (UTRs). Sequence variation among SARS-CoV-2 and SARS-CoV revealed no significant difference in ORFs and nsps. The nsps includes two viral cysteine proteases including papain-like protease (nsp3), chymotrypsin-like, 3C-like, or main protease (nsp5), RNA-dependent RNA polymerase (nsp12), helicase (nsp13), and others likely to be involved in the transcription and replication of SARS-CoV-2. The structure of spike glycoprotein structure of SARS-CoV-2 resembles that of the spike protein of SARS-CoV with an root-mean-square deviation (RMSD) of 3.8 Å. Like SARS-CoV, SARS-CoV-2 uses the ACE2 receptor for internalization and TMPRSS2 serine proteases for S protein priming. Histopathological investigation of tissues from SARS-CoV-2 infected patients showed virus-induced cytopathic effect with signs of acute respiratory distress syndrome in lung cells. This chapter discusses about the morphology, genome organization, replication, and pathogenesis of SARS-CoV-2 that may help us understand the disease that may leads to identification of effective antiviral drugs and vaccines.

Title: Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility. Content: Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

Title: Time Series Prediction of COVID-19 by Mutation Rate Analysis using Recurrent Neural Network-based LSTM Model Content: SARS-CoV-2, a novel coronavirus mostly known as COVID-19 has created a global pandemic. The world is now immobilized by this infectious RNA virus. As of May 18, already more than 4.8 million people have been infected and 316k people died. This RNA virus has the ability to do the mutation in the human body. Accurate determination of mutation rates is essential to comprehend the evolution of this virus and to determine the risk of emergent infectious disease. This study explores the mutation rate of the whole genomic sequence gathered from the patient's dataset of different countries. The collected dataset is processed to determine the nucleotide mutation and codon mutation separately. Furthermore, based on the size of the dataset, the determined mutation rate is categorized for four different regions: China, Australia, The United States, and the rest of the World. It has been found that a huge amount of Thymine (T) and Adenine (A) are mutated to other nucleotides for all regions, but codons are not frequently mutating like nucleotides. A recurrent neural network-based Long Short Term Memory (LSTM) model has been applied to predict the future mutation rate of this virus. The LSTM model gives Root Mean Square Error (RMSE) of 0.06 in testing and 0.04 in training, which is an optimized value. Using this train and testing process, the nucleotide mutation rate of 400(th) patient in future time has been predicted. About 0.1% increment in mutation rate is found for mutating of nucleotides from T to C and G, C to G and G to T. While a decrement of 0.1% is seen for mutating of T to A, and A to C. It is found that this model can be used to predict day basis mutation rates if more patient data is available in updated time.

Title: Discovery of a 382-nt deletion during the early evolution of SARS-CoV-2 Content: To date, the SARS-CoV-2 genome has been considered genetically more stable than SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence (TRS), which in turn enhances the downstream transcription of the N gene. We also found that viruses with the deletion have been circulating for at least four weeks. During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in the human population [1], and similar variation has since been observed across SARS-related CoVs in humans and bats. Overwhelmingly these viruses had mutations or deletions in ORF8, that have been associated with reduced replicative fitness of the virus [2]. This is also consistent with the observation that towards the end of the outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that may be associated with host adaptation [1]. We therefore hypothesise that the major deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2.

Title: [The genome comparison of SARS-CoV and other coronaviruses]. Content: The genome comparison of inter-species and intra-species can give us much information about the origin and evolution of viruses. There are 137 mutation sites in the 17 genomes of SARS-CoV,and the mutation rate is about 8.04 x 10(-3) substitution/site/year. The distribution of the segregating sites is not steady,the most variable region appears in S1 protein,and the nucleotide sequence of RNA-dependent RNA polymerase has very few mutation sites. The substitution bias of nucleotide acids and amino acids indicates the non-random drift products. The comparison of genome structures of SARS-CoV and other coronaviruses shows that SARS-CoV and IBV share the same genome structure. Phylogenetic analyses of conserved genes of coronaviruses indicate that SARS-CoV is a new branch of coronaviruses and appears more close to the group II coronaviruses. Interestingly,SARS-CoV shares some different features with different groups of coronaviruses. Additional analyses show that the first ORFs between S and E genes of some coronaviruses are transmembrane proteins and share the common motif,indicating the possible common ancestor. From the host distribution of different groups of coronaviruses and the phylogeny of s2m,we can deduce that avian is the probable natural host of SARS-CoV.

Title: Rampant C→U Hypermutation in the Genomes of SARS-CoV-2 and Other Coronaviruses: Causes and Consequences for Their Short- and Long-Term Evolutionary Trajectories Content: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has motivated an intensive analysis of its molecular epidemiology following its worldwide spread. To understand the early evolutionary events following its emergence, a data set of 985 complete SARS-CoV-2 sequences was assembled. Variants showed a mean of 5.5 to 9.5 nucleotide differences from each other, consistent with a midrange coronavirus substitution rate of 3 × 10(−4) substitutions/site/year. Almost one-half of sequence changes were C→U transitions, with an 8-fold base frequency normalized directional asymmetry between C→U and U→C substitutions. Elevated ratios were observed in other recently emerged coronaviruses (SARS-CoV, Middle East respiratory syndrome [MERS]-CoV), and decreasing ratios were observed in other human coronaviruses (HCoV-NL63, -OC43, -229E, and -HKU1) proportionate to their increasing divergence. C→U transitions underpinned almost one-half of the amino acid differences between SARS-CoV-2 variants and occurred preferentially in both 5′ U/A and 3′ U/A flanking sequence contexts comparable to favored motifs of human APOBEC3 proteins. Marked base asymmetries observed in nonpandemic human coronaviruses (U ≫ A > G ≫ C) and low G+C contents may represent long-term effects of prolonged C→U hypermutation in their hosts. The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short- and long-term evolution. Repeated cycles of mutation and reversion in favored mutational hot spots and the widespread occurrence of amino acid changes with no adaptive value for the virus represent a quite different paradigm of virus sequence change from neutral and Darwinian evolutionary frameworks and are not incorporated by standard models used in molecular epidemiology investigations. IMPORTANCE The wealth of accurately curated sequence data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), its long genome, and its low substitution rate provides a relatively blank canvas with which to investigate effects of mutational and editing processes imposed by the host cell. The finding that a large proportion of sequence change in SARS-CoV-2 in the initial months of the pandemic comprised C→U mutations in a host APOBEC-like context provides evidence for a potent host-driven antiviral editing mechanism against coronaviruses more often associated with antiretroviral defense. In evolutionary terms, the contribution of biased, convergent, and context-dependent mutations to sequence change in SARS-CoV-2 is substantial, and these processes are not incorporated by standard models used in molecular epidemiology investigations.

Title: Making sense of mutation: what D614G means for the COVID-19 pandemic remains unclear Content: Abstract Korber et al. (2020) found that a SARS-CoV-2 variant in the spike protein, D614G, rapidly became dominant around the world. While clinical and in vitro data suggest that D614G changes the virus phenotype, the impact of the mutation on transmission, disease, and vaccine and therapeutic development are largely unknown.

Title: Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly Biased Towards C>U Transitions, Indicating Rapid Evolution in Their Hosts Content: The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.

Title: Mutation Patterns of Human SARS-CoV-2 and Bat RaTG13 Coronavirus Genomes Are Strongly Biased Towards C>U Transitions, Indicating Rapid Evolution in Their Hosts. Content: The pandemic caused by the spread of SARS-CoV-2 has led to considerable interest in its evolutionary origin and genome structure. Here, we analyzed mutation patterns in 34 human SARS-CoV-2 isolates and a closely related RaTG13 isolated from Rhinolophus affinis (a horseshoe bat). We also evaluated the CpG dinucleotide contents in SARS-CoV-2 and other human and animal coronavirus genomes. Out of 1136 single nucleotide variations (~4% divergence) between human SARS-CoV-2 and bat RaTG13, 682 (60%) can be attributed to C>U and U>C substitutions, far exceeding other types of substitutions. An accumulation of C>U mutations was also observed in SARS-CoV2 variants that arose within the human population. Globally, the C>U substitutions increased the frequency of codons for hydrophobic amino acids in SARS-CoV-2 peptides, while U>C substitutions decreased it. In contrast to most other coronaviruses, both SARS-CoV-2 and RaTG13 exhibited CpG depletion in their genomes. The data suggest that C-to-U conversion mediated by C deamination played a significant role in the evolution of the SARS-CoV-2 coronavirus. We hypothesize that the high frequency C>U transitions reflect virus adaptation processes in their hosts, and that SARS-CoV-2 could have been evolving for a relatively long period in humans following the transfer from animals before spreading worldwide.

Title: Multi-Omics and Integrated Network Approach to Unveil Evolutionary Patterns, Mutational Hotspots, Functional Crosstalk and Regulatory Interactions in SARS-CoV-2 Content: SARS-CoV-2 responsible for the pandemic of the Severe Acute Respiratory Syndrome resulting in infections and death of millions worldwide with maximum cases and mortality in USA. The current study focuses on understanding the population specific variations attributing its high rate of infections in specific geographical regions which may help in developing appropriate treatment strategies for COVID-19 pandemic. Rigorous phylogenetic network analysis of 245 complete SARS-CoV-2 genomes inferred five central clades named a (ancestral), b, c, d and e (subtype e1 & e2) showing both divergent and linear evolution types. The clade d & e2 were found exclusively comprising of USA strains with highest known mutations. Clades were distinguished by ten co-mutational combinations in proteins; Nsp3, ORF8, Nsp13, S, Nsp12, Nsp2 and Nsp6 generated by Amino Acid Variations (AAV). Our analysis revealed that only 67.46 % of SNP mutations were carried by amino acid at phenotypic level. T1103P mutation in Nsp3 was predicted to increase the protein stability in 238 strains except six strains which were marked as ancestral type; whereas com (P5731L & Y5768C) in Nsp13 were found in 64 genomes of USA highlighting its 100% co-occurrence. Docking study highlighted mutation (D7611G) caused reduction in binding of Spike proteins with ACE2, but it also showed better interaction with TMPRSS2 receptor which may contribute to its high transmissibility in USA strains. In addition, we found host proteins, MYO5A, MYO5B & MYO5C had maximum interaction with viral hub proteins (Nucleocapsid, Spike & Membrane). Thus, blocking the internalization pathway by inhibiting MYO-5 proteins which could be an effective target for COVID-19 treatment. The functional annotations of the Host-Pathogen Interaction (HPI) network were found to be highly associated with hypoxia and thrombotic conditions confirming the vulnerability and severity of infection in the patients. We also considered the presence of CpG islands in Nsp1 and N proteins which may confers the ability of SARS-CoV-2 to enter and trigger methyltransferase activity inside host cell.

Title: Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003 Content: BACKGROUND: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. METHODS: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. RESULTS: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14(th )October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10(-6 )nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. CONCLUSIONS: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

Title: Coding variants in ACE2 and TMPRSS2 are not major drivers of COVID-19 severity in UK Biobank subjects. Content: There is wide individual variation in response to infection with SARS-CoV-2 and this may be influenced by ethnicity. It is plausible that coding variants in the ACE2 and TMPRSS2 genes might contribute to this variation. Exome sequence data was obtained for 49,953 UK Biobank subjects of whom 74 had tested positive for SARS-CoV-2 and could be presumed to have severe disease because mildly affected subjects were not tested in the relevant period. A weighted burden analysis was carried out using SCOREASSOC to determine whether there were differences between these cases and the other sequenced subjects in the overall burden of rare, damaging variants in ACE2 or TMPRSS2. There were no significant differences in weighted burden scores between cases and controls for either gene. There were no individual DNA sequence variants with a markedly different frequency between cases and controls. Genetic variants affecting the structure and function of the ACE2 and TMPRSS2 proteins are not a major determinant of whether infection with SARS-CoV-2 results in severe symptoms. This research has been conducted using the UK Biobank Resource.

Title: The establishment of reference sequence for SARS-CoV-2 and variation analysis Content: Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.

Title: MicroGMT: A Mutation Tracker for SARS-CoV-2 and Other Microbial Genome Sequences Content: With the continued spread of SARS-CoV-2 virus around the world, researchers often need to quickly identify novel mutations in newly sequenced SARS-CoV-2 genomes for studying the molecular evolution and epidemiology of the virus. We have developed a Python package, MicroGMT, which takes either raw sequence reads or assembled genome sequences as input and compares against database sequences to identify and characterize indels and point mutations. Although our default setting is optimized for SARS-CoV-2 virus, the package can be also applied to any other microbial genomes. The software is freely available at Github URL https://github.com/qunfengdong/MicroGMT.

Title: Genetic diversity and evolution of SARS-CoV-2 Content: Abstract COVID-19 is a viral respiratory illness caused by a new coronavirus called SARS-CoV-2. The World Health Organization declared the SARS-CoV-2 outbreak a global public health emergency. We performed genetic analyses of eighty-six complete or near-complete genomes of SARS-CoV-2 and revealed many mutations and deletions on coding and non-coding regions. These observations provided evidence of the genetic diversity and rapid evolution of this novel coronavirus.

Title: A Global Effort to Define the Human Genetics of Protective Immunity to SARS-CoV-2 Infection Content: SARS-CoV-2 infection displays immense inter-individual clinical variability, ranging from silent infection to lethal disease. The role of human genetics in determining clinical response to the virus remains unclear. Studies of outliers-individuals remaining uninfected despite viral exposure and healthy young patients with life-threatening disease-present a unique opportunity to reveal human genetic determinants of infection and disease.

Title: Implications of SARS-CoV-2 Genetic Diversity and Mutations on Pathogenicity of COVID-19 and Biomedical Interventions Content: ABSTRACT Objective Coronavirus disease 2019 (COVID-19) has caused an unprecedented global health emergency. The COVID-19 pandemic has claimed over 350,000 human lives within five months of its emergence, especially in the USA and the European continent. This study analysed the implications of the genetic diversity and mutations in SARS-CoV-2 on its virulence diversity and investigated how these factors could affect the successful development and application of antiviral chemotherapy, immunotherapy, serodiagnosis, and vaccination. Methods All the suitable and eligible full text articles published between 31st December 2019 and 31st May 2020 were filtered and extracted from “PubMed”, “Scopus”, “Web of Science”, and “Hinari” and were critically reviewed. We used the Medical Subject Headings (MeSH) terms “COVID-19, “Mutation”, “Genetic diversity”, “SARS-CoV-2”, “Virulence”, “Pathogenicity”, “Evolution” and “SARS-CoV-2 transmission” for this search. Results Our search showed that SARS-CoV-2 has persistently undergone significant mutations in various parts of its non-structural proteins (NSPs), including NSP2 and NSP3, S protein, and RNA-dependent RNA polymerase (RdRp). In particular, the S protein was found to be the key determinant of evolution, transmission, and virulence of SARS-CoV-2, and could be a potential target for vaccine development. Additionally, RdRp could be a major target in the development of antivirals for the treatment of COVID-19. Conclusion Given the critical importance of mutations in the pathogenicity of SARS-CoV-2 and in the development of sero-diagnostics, antivirals, and vaccines, this study recommends continuous molecular surveillance of SARS-CoV-2. This approach would potentially prompt identification of new mutants and their impact on ongoing biomedical interventions and COVID-19 control measures.

Title: SARS-CoV-2 genome evolution exposes early human adaptations Content: The set of mutations observed at the outset of the SARS-CoV-2 pandemic may illuminate how the virus will adapt to humans as it continues to spread. Viruses are expected to quickly acquire beneficial mutations upon jumping to a new host species. Advantageous nucleotide substitutions can be identified by their parallel occurrence in multiple independent lineages and are likely to result in changes to protein sequences. Here we show that SARS-CoV-2 is acquiring mutations more slowly than expected for neutral evolution, suggesting purifying selection is the dominant mode of evolution during the initial phase of the pandemic. However, several parallel mutations arose in multiple independent lineages and may provide a fitness advantage over the ancestral genome. We propose plausible reasons for several of the most frequent mutations. The absence of mutations in other genome regions suggests essential components of SARS-CoV-2 that could be the target of drug development. Overall this study provides genomic insights into how SARS-CoV-2 has adapted and will continue to adapt to humans. SUMMARY In this study we sought signals of evolution to identify how the SARS-CoV-2 genome has adapted at the outset of the COVID-19 pandemic. We find that the genome is largely undergoing purifying selection that maintains its ancestral sequence. However, we identified multiple positions on the genome that appear to confer an adaptive advantage based on their repeated evolution in independent lineages. This information indicates how SARS-CoV-2 will evolve as it diversifies in an increasing number of hosts.

Title: Snapshot of the evolution and mutation patterns of SARS-CoV-2 Content: The COVID-19 pandemic is the most important public health threat in recent history. Here we study how its causal agent, SARS-CoV-2, has diversified genetically since its first emergence in December 2019. We have created a pipeline combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in a data set consisting of 4,894 SARS-CoV-2 complete genome sequences. Although the phylogenetic diversity of SARS-CoV-2 is low, the whole genome phylogenetic tree can be divided into five clusters/clades based on the tree topology and clustering of specific mutations, but its branches exhibit low genetic distance and bootstrap support values. We also identified 11 residues that are high-frequency substitutions, with four of them currently showing some signal for potential positive selection. These fast-evolving sites are in the non-structural proteins nsp2, nsp5 (3CL-protease), nsp6, nsp12 (polymerase) and nsp13 (helicase), in accessory proteins (ORF3a, ORF8) and in the structural proteins N and S. Temporal and spatial analysis of these potentially adaptive mutations revealed that the incidence of some of these sites was declining after having reached an (often local) peak, whereas the frequency of other sites is continually increasing and now exhibit a worldwide distribution. Structural analysis revealed that the mutations are located on the surface of the proteins that modulate biochemical properties. We speculate that this improves binding to cellular proteins and hence represents fine-tuning of adaptation to human cells. Our study has implications for the design of biochemical and clinical experiments to assess whether important properties of SARS-CoV-2 have changed during the epidemic.

Title: SARS‐CoV‐2 variants: Relevance for symptom granularity, epidemiology, immunity (herd, vaccines), virus origin and containment? Content: The origin of the SARS‐CoV‐2 virus remains enigmatic. It is likely to be a continuum resulting from inevitable mutations and recombination events. These genetic changes keep developing in the present epidemic. Mutations tending to deplete the genome in its cytosine content will progressively lead to attenuation as a consequence of Muller's ratchet, but this is counteracted by recombination when different mutants co‐infect the same host, in particular, in clusters of infection. Monitoring as a function of time the genome sequences in closely related cases is critical to anticipate the future of SARS‐CoV‐2 and hence of COVID‐19.

Title: Genetic lesions within the 3a gene of SARS-CoV Content: A series of frameshift mutations within the 3a gene has been observed in culture-derived severe acute respiratory syndrome coronavirus (SARS-CoV). We report here that viral RNA from clinical samples obtained from SARS-CoV infected patients also contains a heterogeneous population of wild-type and mutant 3a transcripts.

Title: Genetic diversity and evolution of SARS-CoV-2 Content: COVID-19 is a viral respiratory illness caused by a new coronavirus called SARS-CoV-2. The World Health Organization declared the SARS-CoV-2 outbreak a global public health emergency. We performed genetic analyses of eighty-six complete or near-complete genomes of SARS-CoV-2 and revealed many mutations and deletions on coding and non-coding regions. These observations provided evidence of the genetic diversity and rapid evolution of this novel coronavirus.

Title: RdRp mutations are associated with SARS-CoV-2 genome evolution Content: COVID-19, caused by the novel SARS-CoV-2 virus, started in China in late 2019, and soon became a global pandemic. With the help of thousands of viral genome sequences that have been accumulating, it has become possible to track the evolution of viral genome over time as it spread across the world. An important question that still needs to be answered is whether any of the common mutations affect the viral properties, and therefore the disease characteristics. Therefore, we sought to understand the effects of mutations in RNA-dependent RNA polymerase (RdRp), particularly the common 14408C>T mutation, on mutation rate and viral spread. By focusing on mutations in the slowly evolving M or E genes, we aimed to minimize the effects of selective pressure. Our results indicate that 14408C>T mutation increases the mutation rate, while the third-most common RdRp mutation, 15324C>T, has the opposite effect. It is possible that 14408C>T mutation may have contributed to the dominance of its co-mutations in Europe and elsewhere.

Title: Analysis of SARS-CoV-2 Genomes from Southern California Reveals Community Transmission Pathways in the Early Stage of the US COVID-19 Pandemic Content: Given the higher mortality rate and widespread phenomenon of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) within the United States (US) population, understanding the mutational pattern of SARS CoV-2 has global implications for detection and therapy to prevent further escalation. Los Angeles has become an epicenter of the SARS-CoV-2 pandemic in the US. Efforts to contain the spread of SARS-CoV-2 require identifying its genetic and geographic variation and understanding the drivers of these differences. For the first time, we report genetic characterization of SARS-CoV-2 genome isolates in the Los Angeles population using targeted next generation sequencing (NGS). Samples collected at Cedars Sinai Medical Center were collected from patients with confirmed SARS-CoV-2 infection. We identified and diagnosed 192 patients by our in-house qPCR assay. In this population, the highest frequency variants were in known mutations in the 5'UTR, AA193 protein, RdRp and the spike glycoprotein. SARS-CoV-2 transmission within the local community was tracked by integrating mutation data with patient postal codes with two predominant community spread clusters being identified. Notably, significant viral genomic diversity was identified. Less than 10 percent of the Los Angeles community samples resembled published mutational profiles of SARS-CoV-2 genomes from China, while >50 percent of the isolates shared closely similarities to those from New York State. Based on these findings we conclude SARS-CoV-2 was likely introduced into the Los Angeles community predominantly from New York State but also via multiple other independent transmission routes including but not limited to Washington State and China.

Title: Intra-host site-specific polymorphisms of SARS-CoV-2 is consistent across multiple samples and methodologies Content: Despite the potential relevance to clinical outcome, intra-host dynamics of SARS-CoV-2 are unclear. Here, we quantify and characterize intra-host variation in SARS-CoV-2 raw sequence data uploaded to SRA as of 14 April 2020, and compare results between two sequencing methods (amplicon and RNA-Seq). Raw fastq files were quality filtered and trimmed using Trimmomatic, mapped to the WuhanHu1 reference genome using Bowtie2, and variants called with bcftools mpileup. To ensure sufficient coverage, we only included samples with 10X coverage for >90% of the genome (n=406 samples), and only variants with a depth >=10. Derived (i.e. non-reference) alleles were found at 408 sites. The number of polymorphic sites (i.e. sites with multiple alleles) within samples ranged from 0-13, with 72% of samples (295/406) having at least one polymorphic site. Correlation between number of polymorphic sites and coverage was very low for both sequencing methods (R2 < 0.1, p < 0.05). Polymorphisms were observed >1 sample at 66 sites (range: 2-38 samples). The minor allele frequency (MAF) at each shared polymorphic site was 0.03% - 48.5%. 33/66 sites occurred in ORF1a1b, and 37/66 changes were non-synonymous. At 10/66 sites, derived alleles were found in samples sequenced using both methods. Polymorphic amplicon samples were found at 10/10 positions, while polymorphic RNA-Seq samples were found at 7/10 positions. In conclusion, our results suggest that intra-host variation is prevalent among clinical samples. While mutations resulting from amplification and/or sequencing errors cannot be excluded, the observation of shared polymorphic sites with high MAF across multiple samples and sequencing methods is consistent with true underlying variation. Further investigation into intra-host evolutionary dynamics, particularly with longitudinal sampling, is critical for broader understanding of disease progression.

Title: D936Y and Other Mutations in the Fusion Core of the SARS-Cov-2 Spike Protein Heptad Repeat 1 Undermine the Post-Fusion Assembly Content: The iconic “red crown” of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is made of its spike (S) glycoprotein. The S protein is the Trojan horse of coronaviruses, mediating their entry into the host cells. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On April 21st, the GISAID resource had collected 10,823 SARS-CoV-2 genomic sequences. We extracted from them all the complete S protein sequences and identified point mutations thereof. Six mutations were located on a 14-residue segment (929-943) in the “fusion core” of the heptad repeat 1 (HR1). Our modeling in the pre- and post-fusion S protein conformations revealed, for three of them, the loss of interactions stabilizing the post-fusion assembly. On May 29th, the SARS-CoV-2 genomic sequences in GISAID were 34,805. An analysis of the occurrences of the HR1 mutations in this updated dataset revealed a significant increase for the S929I and S939F mutations and a dramatic increase for the D936Y mutation, which was particularly widespread in Sweden and Wales/England. We notice that this is also the mutation causing the loss of a strong inter-monomer interaction, the D936-R1185 salt bridge, thus clearly weakening the post-fusion assembly.