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.

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.

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.

Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

Evolutionary Shortcuts via Multinucleotide Substitutions and Their Impact on Natural Selection Analyses.

Inference and interpretation of evolutionary processes, in particular of the types and targets of natural selection affecting coding sequences, are critically influenced by the assumptions built into statistical models and tests. If certain aspects of the substitution process (even when they are not of direct interest) are presumed absent or are modeled with too crude of a simplification, estimates of key model parameters can become biased, often systematically, and lead to poor statistical performance. Previous work established that failing to accommodate multinucleotide (or multihit, MH) substitutions strongly biases dN/dS-based inference towards false-positive inferences of diversifying episodic selection, as does failing to model variation in the rate of synonymous substitution (SRV) among sites. Here, we develop an integrated analytical framework and software tools to simultaneously incorporate these sources of evolutionary complexity into selection analyses. We found that both MH and SRV are ubiquitous in empirical alignments, and incorporating them has a strong effect on whether or not positive selection is detected (1.4-fold reduction) and on the distributions of inferred evolutionary rates. With simulation studies, we show that this effect is not attributable to reduced statistical power caused by using a more complex model. After a detailed examination of 21 benchmark alignments and a new high-resolution analysis showing which parts of the alignment provide support for positive selection, we show that MH substitutions occurring along shorter branches in the tree explain a significant fraction of discrepant results in selection detection. Our results add to the growing body of literature which examines decades-old modeling assumptions (including MH) and finds them to be problematic for comparative genomic data analysis. Because multinucleotide substitutions have a significant impact on natural selection detection even at the level of an entire gene, we recommend that selection analyses of this type consider their inclusion as a matter of routine. To facilitate this procedure, we developed, implemented, and benchmarked a simple and well-performing model testing selection detection framework able to screen an alignment for positive selection with two biologically important confounding processes: site-to-site synonymous rate variation, and multinucleotide instantaneous substitutions.

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.

Weak selection on synonymous codons substantially inflates dN/dS estimates in bacteria.

Synonymous codon substitutions are not always selectively neutral as revealed by several types of analyses, including studies of codon usage patterns among genes. We analyzed codon usage in 13 bacterial genomes sampled from across a large order of bacteria, Enterobacterales, and identified presumptively neutral and selected classes of synonymous substitutions. To estimate substitution rates, given a neutral/selected classification of synonymous substitutions, we developed a flexible [Formula: see text] substitution model that allows multiple classes of synonymous substitutions. Under this multiclass synonymous substitution (MSS) model, the denominator of [Formula: see text] includes only the strictly neutral class of synonymous substitutions. On average, the value of [Formula: see text] under the MSS model was 80% of that under the standard codon model in which all synonymous substitutions are assumed to be neutral. The indication is that conventional [Formula: see text] analyses overestimate these values and thus overestimate the frequency of positive diversifying selection and underestimate the strength of purifying selection. To quantify the strength of selection necessary to explain this reduction, we developed a model of selected compensatory codon substitutions. The reduction in synonymous substitution rate, and thus the contribution that selection makes to codon bias variation among genes, can be adequately explained by very weak selection, with a mean product of population size and selection coefficient, [Formula: see text].

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.

Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Stepwise Evolution and Exceptional Conservation of ORF1a/b Overlap in Coronaviruses.

The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for the propagation of coronaviruses. The combination of genomic features that make up PFE-the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements-places severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess the evolutionary dynamics of PFE in great detail. Here, we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and ORF1b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well-defined phylogenetic specificity. We further examined sequencing data from over 1,500,000 complete genomes and 55,000 raw read data sets to demonstrate exceptional conservation and detect signatures of selection within the PFE region.

Contrast-FEL-A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches.

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.

No more business as usual: Agile and effective responses to emerging pathogen threats require open data and open analytics.

The current state of much of the Wuhan pneumonia virus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) research shows a regrettable lack of data sharing and considerable analytical obfuscation. This impedes global research cooperation, which is essential for tackling public health emergencies and requires unimpeded access to data, analysis tools, and computational infrastructure. Here, we show that community efforts in developing open analytical software tools over the past 10 years, combined with national investments into scientific computational infrastructure, can overcome these deficiencies and provide an accessible platform for tackling global health emergencies in an open and transparent manner. Specifically, we use all SARS-CoV-2 genomic data available in the public domain so far to (1) underscore the importance of access to raw data and (2) demonstrate that existing community efforts in curation and deployment of biomedical software can reliably support rapid, reproducible research during global health crises. All our analyses are fully documented at https://github.com/galaxyproject/SARS-CoV-2.

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.

A structural basis for antibody-mediated neutralization of Nipah virus reveals a site of vulnerability at the fusion glycoprotein apex.

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F-specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F-nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.

Synonymous Site-to-Site Substitution Rate Variation Dramatically Inflates False Positive Rates of Selection Analyses: Ignore at Your Own Peril.

Most molecular evolutionary studies of natural selection maintain the decades-old assumption that synonymous substitution rate variation (SRV) across sites within genes occurs at levels that are either nonexistent or negligible. However, numerous studies challenge this assumption from a biological perspective and show that SRV is comparable in magnitude to that of nonsynonymous substitution rate variation. We evaluated the impact of this assumption on methods for inferring selection at the molecular level by incorporating SRV into an existing method (BUSTED) for detecting signatures of episodic diversifying selection in genes. Using simulated data we found that failing to account for even moderate levels of SRV in selection testing is likely to produce intolerably high false positive rates. To evaluate the effect of the SRV assumption on actual inferences we compared results of tests with and without the assumption in an empirical analysis of over 13,000 Euteleostomi (bony vertebrate) gene alignments from the Selectome database. This exercise reveals that close to 50% of positive results (i.e., evidence for selection) in empirical analyses disappear when SRV is modeled as part of the statistical analysis and are thus candidates for being false positives. The results from this work add to a growing literature establishing that tests of selection are much more sensitive to certain model assumptions than previously believed.

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.