Identification of Evolutionary Trajectories Shared across Human Betacoronaviruses.

Comparing the evolution of distantly related viruses can provide insights into common adaptive processes related to shared ecological niches. Phylogenetic approaches, coupled with other molecular evolution tools, can help identify mutations informative on adaptation, although the structural contextualization of these to functional sites of proteins may help gain insight into their biological properties. Two zoonotic betacoronaviruses capable of sustained human-to-human transmission have caused pandemics in recent times (SARS-CoV-1 and SARS-CoV-2), although a third virus (MERS-CoV) is responsible for sporadic outbreaks linked to animal infections. Moreover, two other betacoronaviruses have circulated endemically in humans for decades (HKU1 and OC43). To search for evidence of adaptive convergence between established and emerging betacoronaviruses capable of sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1, and SARS-CoV-2), we developed a methodological pipeline to classify shared nonsynonymous mutations as putatively denoting homoplasy (repeated mutations that do not share direct common ancestry) or stepwise evolution (sequential mutations leading towards a novel genotype). In parallel, we look for evidence of positive selection and draw upon protein structure data to identify potential biological implications. We find 30 candidate mutations, from which 4 (codon sites 18121 [nsp14/residue 28], 21623 [spike/21], 21635 [spike/25], and 23948 [spike/796]; SARS-CoV-2 genome numbering) further display evolution under positive selection and proximity to functional protein regions. Our findings shed light on potential mechanisms underlying betacoronavirus adaptation to the human host and pinpoint common mutational pathways that may occur during establishment of human endemicity.

Natural selection differences detected in key protein domains between non-pathogenic and pathogenic feline coronavirus phenotypes.

Feline coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed feline enteric coronavirus [FECV]), with around 12 per cent developing into deadly feline infectious peritonitis (FIP; feline infectious peritonitis virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV- and FECV-specific signals of positive selection. We analyzed full-length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site (FCS) and the other within the fusion domain of Spike. We also found fifteen sites subject to positive selection associated with FIPV within Spike, eleven of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were fourteen sites (twelve novel sites) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 FCS and adjacent to C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype and included twenty-four positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV-wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that it is unlikely to be one singular 'switch' mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

RASCL: Rapid Assessment of Selection in CLades through molecular sequence analysis.

An important unmet need revealed by the COVID-19 pandemic is the near-real-time identification of potentially fitness-altering mutations within rapidly growing SARS-CoV-2 lineages. Although powerful molecular sequence analysis methods are available to detect and characterize patterns of natural selection within modestly sized gene-sequence datasets, the computational complexity of these methods and their sensitivity to sequencing errors render them effectively inapplicable in large-scale genomic surveillance contexts. Motivated by the need to analyze new lineage evolution in near-real time using large numbers of genomes, we developed the Rapid Assessment of Selection within CLades (RASCL) pipeline. RASCL applies state of the art phylogenetic comparative methods to evaluate selective processes acting at individual codon sites and across whole genes. RASCL is scalable and produces automatically updated regular lineage-specific selection analysis reports: even for lineages that include tens or hundreds of thousands of sampled genome sequences. Key to this performance is (i) generation of automatically subsampled high quality datasets of gene/ORF sequences drawn from a selected "query" viral lineage; (ii) contextualization of these query sequences in codon alignments that include high-quality "background" sequences representative of global SARS-CoV-2 diversity; and (iii) the extensive parallelization of a suite of computationally intensive selection analysis tests. Within hours of being deployed to analyze a novel rapidly growing lineage of interest, RASCL will begin yielding JavaScript Object Notation (JSON)-formatted reports that can be either imported into third-party analysis software or explored in standard web-browsers using the premade RASCL interactive data visualization dashboard. By enabling the rapid detection of genome sites evolving under different selective regimes, RASCL is well-suited for near-real-time monitoring of the population-level selective processes that will likely underlie the emergence of future variants of concern in measurably evolving pathogens with extensive genomic surveillance.

Sierra SARS-CoV-2 sequence and antiviral resistance analysis program.

INTRODUCTION: Although most laboratories are capable of employing established protocols to perform full-genome SARS-CoV-2 sequencing, many are unable to assess sequence quality, select appropriate mutation-detection thresholds, or report on the potential clinical significance of mutations in the targets of antiviral therapy METHODS: We describe the technical aspects and benchmark the performance of Sierra SARS-CoV-2, a program designed to perform these functions on user-submitted FASTQ and FASTA sequence files and lists of Spike mutations. Sierra SARS-CoV-2 indicates which sequences contain an unexpectedly large number of unusual mutations and which mutations are associated with reduced susceptibility to clinical stage mAbs, the RdRP inhibitor remdesivir, or the Mpro inhibitor nirmatrelvir RESULTS: To assess the performance of Sierra SARS-CoV-2 on FASTQ files, we applied it to 600 representative FASTQ sequences and compared the results to the COVID-19 EDGE program. To assess its performance on FASTA files, we applied it to nearly one million representative FASTA sequences and compared the results to the GISAID mutation annotation. To assess its performance on mutations lists, we applied it to 13,578 distinct Spike RBD mutation patterns and showed that exactly or partially matching annotations were available for 88% of patterns CONCLUSION: Sierra SARS-CoV-2 leverages previously published data to improve the quality control of submitted viral genomic data and to provide functional annotation on the impact of mutations in the targets of antiviral SARS-CoV-2 therapy. The program can be found at https://covdb.stanford.edu/sierra/sars2/ and its source code at https://github.com/hivdb/sierra-sars2.

Molecular evidence for SARS-CoV-2 in samples collected from patients with morbilliform eruptions since late 2019 in Lombardy, northern Italy.

As a reference laboratory for measles and rubella surveillance in Lombardy, we evaluated the association between SARS-CoV-2 infection and measles-like syndromes, providing preliminary evidence for undetected early circulation of SARS-CoV-2. Overall, 435 samples from 156 cases were investigated. RNA from oropharyngeal swabs (N = 148) and urine (N = 141) was screened with four hemi-nested PCRs and molecular evidence for SARS-CoV-2 infection was found in 13 subjects. Two of the positive patients were from the pandemic period (2/12, 16.7%, March 2020-March 2021) and 11 were from the pre-pandemic period (11/44, 25%, August 2019-February 2020). Sera (N = 146) were tested for anti-SARS-CoV-2 IgG, IgM, and IgA antibodies. Five of the RNA-positive individuals also had detectable anti-SARS-CoV-2 antibodies. No strong evidence of infection was found in samples collected between August 2018 and July 2019 from 100 patients. The earliest sample with evidence of SARS-CoV-2 RNA was from September 12, 2019, and the positive patient was also positive for anti-SARS-CoV-2 antibodies (IgG and IgM). Mutations typical of B.1 strains previously reported to have emerged in January 2020 (C3037T, C14408T, and A23403G), were identified in samples collected as early as October 2019 in Lombardy. One of these mutations (C14408T) was also identified among sequences downloaded from public databases that were obtained by others from samples collected in Brazil in November 2019. We conclude that a SARS-CoV-2 progenitor capable of producing a measles-like syndrome may have emerged in late June-late July 2019 and that viruses with mutations characterizing B.1 strain may have been spreading globally before the first Wuhan outbreak. Our findings should be complemented by high-throughput sequencing to obtain additional sequence information. We highlight the importance of retrospective surveillance studies in understanding the early dynamics of COVID-19 spread and we encourage other groups to perform retrospective investigations to seek confirmatory proofs of early SARS-CoV-2 circulation.

Conserved recombination patterns across coronavirus subgenera.

Recombination contributes to the genetic diversity found in coronaviruses and is known to be a prominent mechanism whereby they evolve. It is apparent, both from controlled experiments and in genome sequences sampled from nature, that patterns of recombination in coronaviruses are non-random and that this is likely attributable to a combination of sequence features that favour the occurrence of recombination break points at specific genomic sites, and selection disfavouring the survival of recombinants within which favourable intra-genome interactions have been disrupted. Here we leverage available whole-genome sequence data for six coronavirus subgenera to identify specific patterns of recombination that are conserved between multiple subgenera and then identify the likely factors that underlie these conserved patterns. Specifically, we confirm the non-randomness of recombination break points across all six tested coronavirus subgenera, locate conserved recombination hot- and cold-spots, and determine that the locations of transcriptional regulatory sequences are likely major determinants of conserved recombination break-point hotspot locations. We find that while the locations of recombination break points are not uniformly associated with degrees of nucleotide sequence conservation, they display significant tendencies in multiple coronavirus subgenera to occur in low guanine-cytosine content genome regions, in non-coding regions, at the edges of genes, and at sites within the Spike gene that are predicted to be minimally disruptive of Spike protein folding. While it is apparent that sequence features such as transcriptional regulatory sequences are likely major determinants of where the template-switching events that yield recombination break points most commonly occur, it is evident that selection against misfolded recombinant proteins also strongly impacts observable recombination break-point distributions in coronavirus genomes sampled from nature.

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa.

Three lineages (BA.1, BA.2 and BA.3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern predominantly drove South Africa's fourth Coronavirus Disease 2019 (COVID-19) wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and similar to BA.2 except for the addition of 69-70 deletion (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild-type amino acid at Q493. The two lineages differ only outside of the spike region. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimated growth advantages for BA.4 and BA.5 of 0.08 (95% confidence interval (CI): 0.08-0.09) and 0.10 (95% CI: 0.09-0.11) per day, respectively, over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus.

Detection of SARS-CoV-2 intra-host recombination during superinfection with Alpha and Epsilon variants in New York City.

Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, detection of recombination is only feasible when genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was infected with two distinct viral variants: Alpha (B.1.1.7) and Epsilon (B.1.429). This superinfection was first noted when an Alpha genome sequence failed to exhibit the classic S gene target failure behavior used to track this variant. Full genome sequencing from four independent extracts reveals that Alpha variant alleles comprise around 75% of the genomes, whereas the Epsilon variant alleles comprise around 20% of the sample. Further investigation reveals the presence of numerous recombinant haplotypes spanning the genome, specifically in the spike, nucleocapsid, and ORF 8 coding regions. These findings support the potential for recombination to reshape SARS-CoV-2 genetic diversity.

Susceptibility of SARS-CoV-2 Omicron Variants to Therapeutic Monoclonal Antibodies: Systematic Review and Meta-analysis.

SARS-CoV-2 Omicron variants contain many mutations in its spike receptor-binding domain, the target of all authorized monoclonal antibodies (MAbs). Determining the extent to which Omicron variants reduced MAb susceptibility is critical to preventing and treating COVID-19. We systematically reviewed PubMed and three preprint servers, last updated 11 April 2022, for the in vitro activity of authorized MAbs against the Omicron variants. Fifty-one studies were eligible, including 50 containing Omicron BA.1 susceptibility data and 17 containing Omicron BA.2 susceptibility data. The first two authorized MAb combinations, bamlanivimab/etesevimab and casirivimab/imdevimab, were largely inactive against the Omicron BA.1 and BA.2 variants. In 34 studies, sotrovimab displayed a median 4.0-fold (interquartile range [IQR]: 2.6 to 6.9) reduction in activity against Omicron BA.1, and in 12 studies, it displayed a median 17-fold (IQR: 13 to 30) reduction in activity against Omicron BA.2. In 15 studies, the combination cilgavimab/tixagevimab displayed a median 86-fold (IQR: 27 to 151) reduction in activity against Omicron BA.1, and in six studies, it displayed a median 5.4-fold (IQR: 3.7 to 6.9) reduction in activity against Omicron BA.2. In eight studies against Omicron BA.1 and six studies against Omicron BA.2, bebtelovimab displayed no reduction in activity. Disparate results between assays were common. For authorized MAbs, 51/268 (19.0%) results for wild-type control variants and 78/348 (22.4%) results for Omicron BA.1 and BA.2 variants were more than 4-fold below or 4-fold above the median result for that MAb. Highly disparate results between published assays indicate a need for improved MAb susceptibility test standardization or interassay calibration. IMPORTANCE Monoclonal antibodies (MAbs) targeting the SARS-CoV-2 spike protein are among the most effective measures for preventing and treating COVID-19. However, SARS-CoV-2 Omicron variants contain many mutations in their spike receptor-binding domains, the target of all authorized MAbs. Therefore, determining the extent to which Omicron variants reduced MAb susceptibility is critical to preventing and treating COVID-19. We identified 51 studies that reported the in vitro susceptibility of the two main Omicron variants BA.1 and BA.2 to therapeutic MAbs in advanced clinical development, including eight authorized individual MAbs and three authorized MAb combinations. We estimated the degree to which different MAbs displayed reduced activity against Omicron variants. The marked loss of activity of many MAbs against Omicron variants underscores the importance of developing MAbs that target conserved regions of spike. Highly disparate results between assays indicate the need for improved MAb susceptibility test standardization.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Exploring the Natural Origins of SARS-CoV-2 in the Light of Recombination.

The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (members of the Sarbecovirus subgenus) have been identified, is fueling speculation on the natural origins of SARS-CoV-2. We performed a comprehensive phylogenetic study on SARS-CoV-2 and all the related bat and pangolin sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Distribution of the inferred recombination events is nonrandom with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift events in the ancestry of bat sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm that horseshoe bats, Rhinolophus, are the likely reservoir species for the SARS-CoV-2 progenitor. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and cocirculation of these viruses' ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years. We confirm that a direct proximal ancestor to SARS-CoV-2 has not yet been sampled, since the closest known relatives collected in Yunnan shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for dramatically more wildlife sampling to: 1) pinpoint the exact origins of SARS-CoV-2's animal progenitor, 2) the intermediate species that facilitated transmission from bats to humans (if there is one), and 3) survey the extent of the diversity in the related sarbecoviruses' phylogeny that present high risk for future spillovers.

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa.

The SARS-CoV-2 epidemic in southern Africa has been characterized by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, while the second and third waves were driven by the Beta (B.1.351) and Delta (B.1.617.2) variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron, B.1.1.529) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, which are predicted to influence antibody neutralization and spike function4. Here we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity.

The biological and clinical significance of emerging SARS-CoV-2 variants.

The past several months have witnessed the emergence of SARS-CoV-2 variants with novel spike protein mutations that are influencing the epidemiological and clinical aspects of the COVID-19 pandemic. These variants can increase rates of virus transmission and/or increase the risk of reinfection and reduce the protection afforded by neutralizing monoclonal antibodies and vaccination. These variants can therefore enable SARS-CoV-2 to continue its spread in the face of rising population immunity while maintaining or increasing its replication fitness. The identification of four rapidly expanding virus lineages since December 2020, designated variants of concern, has ushered in a new stage of the pandemic. The four variants of concern, the Alpha variant (originally identified in the UK), the Beta variant (originally identified in South Africa), the Gamma variant (originally identified in Brazil) and the Delta variant (originally identified in India), share several mutations with one another as well as with an increasing number of other recently identified SARS-CoV-2 variants. Collectively, these SARS-CoV-2 variants complicate the COVID-19 research agenda and necessitate additional avenues of laboratory, epidemiological and clinical research.

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Detection of a SARS-CoV-2 variant of concern in South Africa.

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus1,2. Here we describe a newly arisen lineage of SARS-CoV-2 (designated 501Y.V2; also known as B.1.351 or 20H) that is defined by eight mutations in the spike protein, including three substitutions (K417N, E484K and N501Y) at residues in its receptor-binding domain that may have functional importance3-5. This lineage was identified in South Africa after the first wave of the epidemic in a severely affected metropolitan area (Nelson Mandela Bay) that is located on the coast of the Eastern Cape province. This lineage spread rapidly, and became dominant in Eastern Cape, Western Cape and KwaZulu-Natal provinces within weeks. Although the full import of the mutations is yet to be determined, the genomic data-which show rapid expansion and displacement of other lineages in several regions-suggest that this lineage is associated with a selection advantage that most plausibly results from increased transmissibility or immune escape6-8.

Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen.

Virus host shifts are generally associated with novel adaptations to exploit the cells of the new host species optimally. Surprisingly, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has apparently required little to no significant adaptation to humans since the start of the Coronavirus Disease 2019 (COVID-19) pandemic and to October 2020. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects evidence for significant positive episodic diversifying selection acting at the base of the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in these ancestral bat hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor about 1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. While an undiscovered "facilitating" intermediate species cannot be discounted, collectively, our results support the progenitor of SARS-CoV-2 being capable of efficient human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans, which created a relatively generalist virus.

Multiple Recombination Events and Strong Purifying Selection at the Origin of SARS-CoV-2 Spike Glycoprotein Increased Correlated Dynamic Movements.

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

Multiple Recombination Events and Strong Purifying Selection at the Origin of SARS-CoV-2 Spike Glycoprotein Increased Correlated Dynamic Movements

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N-terminal domain and the C-terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C-terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N-terminal domain and C-terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

Viral CpG Deficiency Provides No Evidence That Dogs Were Intermediate Hosts for SARS-CoV-2.

Due to the scope and impact of the COVID-19 pandemic there exists a strong desire to understand where the SARS-CoV-2 virus came from and how it jumped species boundaries to humans. Molecular evolutionary analyses can trace viral origins by establishing relatedness and divergence times of viruses and identifying past selective pressures. However, we must uphold rigorous standards of inference and interpretation on this topic because of the ramifications of being wrong. Here, we dispute the conclusions of Xia (2020. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol. doi:10.1093/molbev/masa095) that dogs are a likely intermediate host of a SARS-CoV-2 ancestor. We highlight major flaws in Xia's inference process and his analysis of CpG deficiencies, and conclude that there is no direct evidence for the role of dogs as intermediate hosts. Bats and pangolins currently have the greatest support as ancestral hosts of SARS-CoV-2, with the strong caveat that sampling of wildlife species for coronaviruses has been limited.