Viral genome sequence datasets display pervasive evidence of strand-specific substitution biases that are best described using non-reversible nucleotide substitution models (preprint)

Background The vast majority of phylogenetic trees are inferred from molecular sequence data (nucleotides or amino acids) using time-reversible evolutionary models which assume that, for any pair of nucleotide or amino acid characters, the relative rate of X to Y substitution is the same as the relative rate of Y to X substitution. However, this reversibility assumption is unlikely to accurately reflect the actual underlying biochemical and/or evolutionary processes that lead to the fixation of substitutions. Here, we use empirical viral genome sequence data to reveal that evolutionary non-reversibility is pervasive among most groups of viruses. Specifically, we consider two non-reversible nucleotide substitution models: (1) a 6-rate non-reversible model (NREV6) in which Watson-Crick complementary substitutions occur at identical relative rates and which might therefor be most applicable to analyzing the evolution of genomes where both complementary strands are subject to the same mutational processes (such as might be expected for double-stranded (ds) RNA or dsDNA genomes); and (2) a 12-rate non-reversible model (NREV12) in which all relative substitution types are free to occur at different rates and which might therefore be applicable to analyzing the evolution of genomes where the complementary genome strands are subject to different mutational processes (such as might be expected for viruses with single-stranded (ss) RNA or ssDNA genomes).Results Using likelihood ratio and Akaike Information Criterion-based model tests, we show that, surprisingly, NREV12 provided a significantly better fit to 21/31 dsRNA and 20/30 dsDNA datasets than did the general time reversible (GTR) and NREV6 models with NREV6 providing a better fit than NREV12 and GTR in only 5/30 dsDNA and 2/31 dsRNA datasets. As expected, NREV12 provided a significantly better fit to 24/33 ssDNA and 40/47 ssRNA datasets. Next, we used simulations to show that increasing degrees of strand-specific substitution bias decrease the accuracy of phylogenetic inference irrespective of whether GTR or NREV12 is used to describe mutational processes. However, in cases where strand-specific substitution biases are extreme (such as in SARS-CoV-2 and Torque teno sus virus datasets) NREV12 tends to yield more accurate phylogenetic trees than those obtained using GTR.Conclusion We show that NREV12 should, be seriously considered during the model selection phase of phylogenetic analyses involving viral genomic sequences.

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

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 February 22, 2022, of the in vitro activity of authorized mAbs against the Omicron variants. Thirty-three studies were eligible including 33 containing Omicron BA.1 susceptibility data and five that also contained Omicron BA.2 susceptibility data. The first two authorized mAb combinations, bamlanivimab/etesevimab and casirivimab/imdevimab, were inactive against the Omicron BA.1 and BA.2 variants. In 24 studies, sotrovimab (third authorized mAb) displayed a median 4.1-fold (IQR: 2.4-7.6) reduced activity against Omicron BA.1 and, in four studies, a median 26-fold (IQR:16-35) reduced activity against Omicron BA.2. In 18 studies, cilgavimab and tixagevimab independently displayed median reductions in activity of >300-fold against Omicron BA.1, while in ten studies, the cilgavimab/tixagevimab combination (fourth authorized mAb preparation) displayed a median 63-fold (IQR: 26-145) reduced activity against Omicron BA.1. In two studies, cilgavimab was approximately 100-fold more susceptible to BA.2 than to BA.1. In two studies, bebtelovimab, the most recently authorized mAb, was fully active against both the Omicron variants. Disparate results between assays were common as evidenced by a median 42-fold range (IQR: 25-625) in IC50 between assays for the eight authorized individual mAbs and three authorized mAb combinations. Highly disparate results between published assays indicates a need for improved mAb susceptibility test standardization or inter-assay calibration.

Capturing intrahost recombination of SARS-CoV-2 during superinfection with Alpha and Epsilon variants in New York City (preprint)

Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, recombination can only be detected when two genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was simultaneously 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 revealed that Alpha variant alleles comprised between 70-80% of the genomes, whereas the Epsilon variant alleles comprised between 20-30% of the sample. Further investigation revealed 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.

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function (preprint)

Among the 30 non-synonymous 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 (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) 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 over all 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.

TopHap: Rapid inference of key phylogenetic structures from common haplotypes in large genome collections with limited diversity (preprint)

Motivation: Building reliable phylogenies from very large collections of sequences with a limited number of phylogenetically informative sites is challenging because sequencing errors and recurrent/backward mutations interfere with the phylogenetic signal, confounding true evolutionary relationships. Massive global efforts of sequencing genomes and reconstructing the phylogeny of SARS-CoV-2 strains exemplify these difficulties since there are only hundreds of phylogenetically informative sites and millions of genomes. For such datasets, we set out to develop a method for building the phylogenetic tree of genomic haplotypes consisting of positions harboring common variants to improve the signal-to-noise ratio for more accurate phylogenetic inference of resolvable phylogenetic features. Results: We present the TopHap approach that determines spatiotemporally common haplotypes of common variants and builds their phylogeny at a fraction of the computational time of traditional methods. To assess topological robustness, we develop a bootstrap resampling strategy that resamples genomes spatiotemporally. The application of TopHap to build a phylogeny of 68,057 genomes (68KG) produced an evolutionary tree of major SARS-CoV-2 haplotypes. This phylogeny is concordant with the mutation tree inferred using the co-occurrence pattern of mutations and recovers key phylogenetic relationships from more traditional analyses. We also evaluated alternative roots of the SARS-CoV-2 phylogeny and found that the earliest sampled genomes in 2019 likely evolved by four mutations of the most recent common ancestor of all SARS-CoV-2 genomes. An application of TopHap to more than 1 million genomes reconstructed the most comprehensive evolutionary relationships of major variants, which confirmed the 68KG phylogeny and provided evolutionary origins of major variants of concern. Availability: TopHap is available on the web at https://github.com/SayakaMiura/TopHap.

Coronavirus Resistance Database (CoV-RDB): SARS-CoV-2 susceptibility to monoclonal antibodies, convalescent plasma, and plasma from vaccinated persons (preprint)

As novel SARS-CoV-2 variants with different patterns of spike mutations have emerged, the susceptibility of these variants to neutralization by antibodies has been rapidly assessed. However, neutralization data are generated using different approaches and are scattered across different publications making it difficult for these data to be located and synthesized. The Stanford Coronavirus Resistance Database (CoV-RDB; https://covdb.stanford.edu) is designed to house comprehensively curated published data on the neutralizing susceptibility of SARS-CoV-2 variants and spike mutations to monoclonal antibodies (mAbs), convalescent plasma (CP), and vaccinee plasma (VP). As of October 2021, CoV-RDB contains 186 publications including 64 (34%) containing 7,328 neutralizing mAb susceptibility results, 96 (52%) containing 11,390 neutralizing CP susceptibility results, and 125 (68%) containing 20,872 neutralizing VP results. The database also records which spike mutations are selected during in vitro passage of SARS-CoV-2 in the presence of mAbs and which emerge in persons receiving mAbs as treatment. The CoV-RDB interface interactively displays neutralizing susceptibility data at different levels of granularity by filtering and/or aggregating query results according to one or more experimental conditions. The CoV-RDB website provides a companion sequence analysis program that outputs information about mutations present in a submitted sequence and that also assists users in determining the appropriate mutation-detection thresholds for identifying non-consensus amino acids. The most recent data underlying the CoV-RDB can be downloaded in its entirety from a Github repository in a documented machine-readable format.

Conserved recombination patterns across coronavirus subgenera (preprint)

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 breakpoints 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 breakpoints 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 breakpoint hot-spot locations. We find that while the locations of recombination breakpoints 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 breakpoints most commonly occur, it is evident that selection against misfolded recombinant proteins also strongly impacts observable recombination breakpoint distributions in coronavirus genomes sampled from nature.

Recent zoonotic spillover and tropism shift of a Canine Coronavirus is associated with relaxed selection and putative loss of function in NTD subdomain of spike protein. (preprint)

A recent study reported the occurrence of Canine Coronavirus (CCoV) in nasopharyngeal swabs from a small number of patients hospitalized with pneumonia during a 2017-18 period in Sarawak, Malaysia. Because the genome sequence for one of these isolates is available, we conducted comparative evolutionary analyses of the spike gene of this strain (CCoV-HuPn-2018), with other available Alphacoronavirus 1 spike sequences. The most N-terminus subdomain (0-domain) of the CCoV-HuPn-2018 spike protein has sequence similarity to Transmissible Gastroenteritis Virus (TGEV) and CCoV2b strains, but not to other members of the type II Alphacoronaviruses (i.e., CCoV2a and Feline CoV2-FCoV2). This 0-domain in CCoV-HuPn-2018 has evidence for relaxed selection pressure, an increased rate of molecular evolution, and a number of unique amino acid substitutions relative to CCoV2b and TGEV sequences. A region of the 0-domain determined to be key to sialic acid binding and pathogenesis in TGEV had clear differences in amino acid sequences in CCoV-HuPn-2018 relative to both CCoV2b (enteric) and TGEV (enteric and respiratory). The 0-domain of CCoV-HuPn-2018 also had several sites inferred to be under positive diversifying selection, including sites within the signal peptide. Downstream of the 0-domain, FCoV2 shared sequence similarity to the CCoV2b and TGEV sequences, with analyses of this larger alignment identifying positively selected sites in the putative Receptor Binding Domain (RBD) and Connector Domain (CD). Recombination analyses strongly implicated a particular FCoV2 strain in the recombinant history of CCoV-HuPn-2018 with molecular divergence times estimated at around 60 years ago. We hypothesize that CCoV-HuPn-2018 had an enteric origin, but that it has lost that particular tropism, because of mutations in the sialic acid binding region of the spike 0-domain. As selection pressure on this region was reduced, the virus evolved a respiratory tropism, analogous to other Alphacoronavirus 1, such as Porcine Respiratory Coronavirus (PRCV), that have lost this region entirely. We also suggest that signals of positive selection in the signal peptide as well as other changes in the 0-domain of CCoV-HuPn-2018 could represent an adaptive role in this new host and that this could be in part due to the different spatial distribution of the N-linked glycan repertoire for this strain.

Identification of site-specific evolutionary trajectories shared across human betacoronaviruses (preprint)

Comparison of evolution among related viruses can provide insights into shared adaptive processes, for example following host switching to a mutual host species. Whilst phylogenetic methods can help identify mutations that may be important for evolutionary processes such as adaptation to a new host, these can be enhanced by positioning candidate mutations to known functional sites on protein structures. Over the past two decades, three zoonotic betacoronaviruses have significantly impacted human public health: SARS-CoV-1, MERS-CoV and SARS-CoV-2, whilst two other betacoronaviruses, HKU1 and OC43, have circulated endemically in the human population for over 100 years. In this study, we use a comparative approach to prospectively search for potentially evolutionarily-relevant mutations within the Orf1ab and S genes across betacoronavirus species that have demonstrated sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1 and SARS-CoV-2). We used a combination of molecular evolution methods to identify 30 sites that display evidence of homoplasy and/or stepwise evolution, that may be suggestive of adaptation across emerging and endemic betacoronaviruses. Of these, seven sites also display evidence of being selectively relevant. Drawing upon known protein structure data, we find that four of the identified mutations [18121 (exonuclease/27), 21623 (spike/21), 21635 (spike/25) and 23948 (spike/796), in SARS-CoV-2 genome coordinates] are proximal to regions of known functionality. Our results provide a molecular-level context for common evolutionary pathways that betacoronaviruses may undergo during adaptation to the human host.

The emergence and ongoing convergent evolution of the N501Y lineages coincides with a major global shift in the SARS-CoV-2 selective landscape (preprint)

The emergence and rapid rise in prevalence of three independent SARS-CoV-2 '501Y lineages', B.1.1.7, B.1.351 and P.1, in the last three months of 2020 has prompted renewed concerns about the evolutionarily capacity of SARS-CoV-2 to adapt to both rising population immunity and public health interventions such as vaccines and social distancing. Viruses giving rise to the different 501Y lineages have, presumably under intense natural selection following a shift in host environment, independently acquired multiple unique and convergent mutations. As a consequence all have gained epidemiological and immunological properties that will likely complicate the control of COVID-19. Here, by examining patterns of mutations that arose in SARS-CoV-2 genomes during the pandemic we find evidence of a major change in the selective forces acting on immunologically important SARS-CoV-2 genes (such as N and S) that likely coincided with the emergence of the 501Y lineages. In addition to involving continuing sequence diversification, we find evidence that a significant portion of the ongoing adaptive evolution of the 501Y lineages also involves further convergence between the lineages. Our findings highlight the importance of monitoring how members of these known 501Y lineages, and others still undiscovered, are convergently evolving similar strategies to ensure their persistence in the face of mounting infection and vaccine induced host immune recognition.

Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa (preprint)

Continued uncontrolled transmission of the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in many parts of the world is creating the conditions for significant virus evolution. Here, we describe a new SARS-CoV-2 lineage (501Y.V2) characterised by eight lineage-defining mutations in the spike protein, including three at important residues in the receptor-binding domain (K417N, E484K and N501Y) that may have functional significance. This lineage emerged in South Africa after the first epidemic wave in a severely affected metropolitan area, Nelson Mandela Bay, located on the coast of the Eastern Cape Province. This lineage spread rapidly, becoming within weeks the dominant lineage in the Eastern Cape and Western Cape Provinces. Whilst the full significance of the mutations is yet to be determined, the genomic data, showing the rapid displacement of other lineages, suggest that this lineage may be associated with increased transmissibility.

An evolutionary portrait of the progenitor SARS-CoV-2 and its dominant offshoots in COVID-19 pandemic (preprint)

We report the likely most recent common ancestor of SARS-CoV-2 - the coronavirus that causes COVID-19. This progenitor SARS-CoV-2 genome was recovered through a novel application and advancement of computational methods initially developed to reconstruct the mutational history of tumor cells in a patient. The progenitor differs from the earliest coronaviruses sampled in China by three variants, implying that none of the earliest patients represent the index case or gave rise to all the human infections. However, multiple coronavirus infections in China and the USA harbored the progenitor genetic fingerprint in January 2020 and later, suggesting that the progenitor was spreading worldwide as soon as weeks after the first reported cases of COVID-19. Mutations of the progenitor and its offshoots have produced many dominant coronavirus strains, which have spread episodically over time. Fingerprinting based on common mutations reveals that the same coronavirus lineage has dominated North America for most of the pandemic. There have been multiple replacements of predominant coronavirus strains in Europe and Asia and the continued presence of multiple high-frequency strains in Asia and North America. We provide a continually updating dashboard of global evolution and spatiotemporal trends of SARS-CoV-2 spread (http://sars2evo.datamonkey.org/).

Evidence of significant natural selection in the evolution of SARS-CoV-2 in bats, not humans (preprint)

RNA viruses are proficient at switching host species, and evolving adaptations to exploit the new hosts cells efficiently. Surprisingly, SARS-CoV-2 has apparently required no significant adaptation to humans since the start of the COVID-19 pandemic, with no observed selective sweeps since genome sampling began. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus 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 significant positive episodic diversifying selection acting on 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 ancestral hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor [~]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. Collectively our results demonstrate the progenitor of SARS-CoV-2 was capable of near immediate human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans.

Re-insights into origin and adaptation of SARS-CoV-2 (preprint)

In depth evolutionary and structural analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from bats, pangolins, and humans are necessary to assess the role of natural selection and recombination in the emergence of the current pandemic strain. The SARS-CoV-2 S glycoprotein unique features have been associated with efficient viral spread in the human population. Phylogeny-based and genetic algorithm methods clearly show that recombination events between viral progenitors infecting animal hosts led to a mosaic structure in the S gene. We identified recombination coldspots in the S glycoprotein and strong purifying selection. Moreover, although there is little evidence of diversifying positive selection during host-switching, structural analysis suggests that some of the residues emerged along the ancestral lineage of current pandemic strains may contribute to enhanced ability to infect human cells. Interestingly, recombination did not affect the long-range covariant movements of SARS-CoV-2 S glycoprotein monomer in pre-fusion conformation but, on the contrary, could contribute to the observed overall viral efficiency. Our dynamic simulations revealed that the movements between the host cell receptor binding domain (RBD) and the novel furin-like cleavage site are correlated. We identified threonine 333 (under purifying selection), at the beginning of the RBD, as the hinge of the opening/closing mechanism of the SARS-CoV-2 S glycoprotein monomer functional to hACE2 binding. Our findings support a scenario where ancestral recombination and fixation of amino acid residues in the RBD of the S glycoprotein generated a virus with unique features, capable of extremely efficient infection of the human host.