Dynamics of severe acute respiratory syndrome coronavirus 2 genome variants in the feces during convalescence

In response to the current coronavirus disease 2019 (COVID-19) pandemic, it is crucial to understand the origin, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which relies on close surveillance of genomic diversity in clinical samples. Although the mutation at the population level had been extensively investigated, how the mutations evolve at the individual level is largely unknown. Eighteen time-series fecal samples were collected from nine patients with COVID-19 during the convalescent phase. The nucleic acids of SARS-CoV-2 were enriched by the hybrid capture method. First, we demonstrated the outstanding performance of the hybrid capture method in detecting intra-host variants. We identified 229 intra-host variants at 182 sites in 18 fecal samples. Among them, nineteen variants presented frequency changes > 0.3 within 1–5 days, reflecting highly dynamic intra-host viral populations. Moreover, the evolution of the viral genome demonstrated that the virus was probably viable in the gastrointestinal tract during the convalescent period. Meanwhile, we also found that the same mutation showed a distinct pattern of frequency changes in different individuals, indicating a strong random drift. In summary, dramatic changes of the SARS-CoV-2 genome were detected in fecal samples during the convalescent period; whether the viral load in feces is sufficient to establish an infection warranted further investigation.     

High-throughput,single-copy sequencing reveals SARS-CoV-2 spike variants coincident with mounting humoral immunity during acute COVID-19

Tracking evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within infected individuals will help elucidate coronavirus disease 2019 (COVID-19) pathogenesis and inform use of antiviral interventions. In this study, we developed an approach for sequencing the region encoding the SARS-CoV-2 virion surface proteins from large numbers of individual virus RNA genomes per sample. We applied this approach to the WA-1 reference clinical isolate of SARS-CoV-2 passaged in vitro and to upper respiratory samples from 7 study participants with COVID-19. SARS-CoV-2 genomes from cell culture were diverse, including 18 haplotypes with non-synonymous mutations clustered in the spike NH₂-terminal domain (NTD) and furin cleavage site regions. By contrast, cross-sectional analysis of samples from participants with COVID-19 showed fewer virus variants, without structural clustering of mutations. However, longitudinal analysis in one individual revealed 4 virus haplotypes bearing 3 independent mutations in a spike NTD epitope targeted by autologous antibodies. These mutations arose coincident with a 6.2-fold rise in serum binding to spike and a transient increase in virus burden. We conclude that SARS-CoV-2 exhibits a capacity for rapid genetic adaptation that becomes detectable in vivo with the onset of humoral immunity, with the potential to contribute to delayed virologic clearance in the acute setting.     

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the “class 3” epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.     

Genomic Perspectives on the Emerging SARS-CoV-2 Omicron Variant

A new variant of concern for SARS-CoV-2,Omicron(B.1.1.529),was designated by the World Health Organization on November 26,2021.This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron.First,we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to.Second,the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the sam...     

Survey of SARS-CoV-2 genetic diversity in two major Brazilian cities using a fast and affordable Sanger sequencing strategy

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the detection of the P1 variant, which became dominant in less than one month after it was first detected.     

Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69–70, would cause a “spike gene target failure” (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69–70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675–3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675–3677 as the primary target and spike Δ69–70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.

Surveillance for SARS-CoV-2 variants is very important, but sequencing is not always practical or affordable. This study presents a multiplex qPCR that is able to distinguish among different SARS-CoV-2 variants of concern that are currently circulating.     

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.     

Analysis of the docking property of host variants of hACE2 for SARS-CoV-2 in a large cohort

The recent novel coronavirus disease (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is threatening global health. However, an understanding of the interaction of SARS-CoV-2 with human cells, including the physical docking property influenced by the host’s genetic diversity, is still lacking. Here, based on germline variants in the UK Biobank covering 502,543 individuals, we revealed the molecular interactions between human angiotensin-converting enzyme 2 (hACE2), which is the representative receptor for SARS-CoV-2 entry, and COVID-19 infection. We identified six nonsense and missense variants of hACE2 from 2585 subjects in the UK Biobank covering 500000 individuals. Using our molecular dynamics simulations, three hACE2 variants from 2585 individuals we selected showed higher levels of binding free energy for docking in the range of 1.44–3.69 kcal/mol. Although there are diverse contributors to SARS-CoV-2 infections, including the mobility of individuals, we analyzed the diagnosis records of individuals with these three variants of hACE2. Our molecular dynamics simulations combined with population-based genomic data provided an atomistic understanding of the interaction between hACE2 and the spike protein of SARS-CoV-2.     

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242–244 deletion (242–244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242–244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.Subject terms: Immunology, Cryoelectron microscopy, X-ray crystallography     

Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system’s response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.     

Detection of R.1 lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with spike protein W152L/E484K/G769V mutations in Japan

We aimed to investigate novel emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages in Japan that harbor variants in the spike protein receptor-binding domain (RBD). The total nucleic acid contents of samples from 159 patients with coronavirus disease 2019 (COVID-19) were subjected to whole genome sequencing. The SARS-CoV-2 genome sequences from these patients were examined for variants in spike protein RBD. In January 2021, three family members (one aged in their 40s and two aged under 10 years old) were found to be infected with SARS-CoV-2 harboring W152L/E484K/G769V mutations. These three patients were living in Japan and had no history of traveling abroad. After identifying these cases, we developed a TaqMan assay to screen for the above hallmark mutations and identified an additional 14 patients with the same mutations. The associated virus strain was classified into the GR clade (Global Initiative on Sharing Avian Influenza Data [GISAID]), 20B clade (Nextstrain), and R.1 lineage (Phylogenetic Assignment of Named Global Outbreak [PANGO] Lineages). As of April 22, 2021, R.1 lineage SARS-CoV-2 has been identified in 2,388 SARS-CoV-2 entries in the GISAID database, many of which were from Japan (38.2%; 913/2,388) and the United States (47.1%; 1,125/2,388). Compared with that in the United States, the percentage of SARS-CoV-2 isolates belonging to the R.1 lineage in Japan increased more rapidly over the period from October 24, 2020 to April 18, 2021. R.1 lineage SARS-CoV-2 has potential escape mutations in the spike protein RBD (E484K) and N-terminal domain (W152L); therefore, it will be necessary to continue to monitor the R.1 lineage as it spreads around the world.     

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.     

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally, from B.1.17 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) to B.1.1.529 (Omicron). The emerging or reemerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics, pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 cells. We utilized both animal models of human ACE2 (hACE2) transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.     

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus

Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model.

This study explores the immune response induced by wild type and variant SARS-CoV-2 spike proteins, and the protection that these immune responses provide against challenge with wild type virus in the mouse model.     

Infection,recovery and re-infection of farmed mink with SARS-CoV-2

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene as well as the A22920T mutation, resulting in the Y453F substitution within this protein, seen previously in mink. The infected mink recovered and after free-testing of 300 mink (a level giving 93% confidence of detecting a 1% prevalence), the animals remained seropositive. During further follow-up studies, after a period of more than 2 months without any virus detection, over 75% of tested animals again scored positive for SARS-CoV-2 RNA. Whole genome sequencing showed that the viruses circulating during this re-infection were most closely related to those identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies in serum samples after the second round of infection than at free-testing or during recovery from initial infection, consistent with a boosted immune response. Thus, it was concluded that following recovery from an initial infection, seropositive mink were readily re-infected by SARS-CoV-2.     

Genetic and Structural Analysis of SARS-CoV-2 Spike Protein for Universal Epitope Selection

Evaluation of immunogenic epitopes for universal vaccine development in the face of ongoing SARS-CoV-2 evolution remains a challenge. Herein, we investigate the genetic and structural conservation of an immunogenically relevant epitope (C662–C671) of spike (S) protein across SARS-CoV-2 variants to determine its potential utility as a broad-spectrum vaccine candidate against coronavirus diseases. Comparative sequence analysis, structural assessment, and molecular dynamics simulations of C662–C671 epitope were performed. Mathematical tools were employed to determine its mutational cost. We found that the amino acid sequence of C662–C671 epitope is entirely conserved across the observed major variants of SARS-CoV-2 in addition to SARS-CoV. Its conformation and accessibility are predicted to be conserved, even in the highly mutated Omicron variant. Costly mutational rate in the context of energy expenditure in genome replication and translation can explain this strict conservation. These observations may herald an approach to developing vaccine candidates for universal protection against emergent variants of coronavirus.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Genomic surveillance of Nevada patients revealed prevalence of unique SARS-CoV-2 variants bearing mutations in the RdRp gene

Patients with signs of COVID-19 were tested through diagnostic RT-PCR for SARS-CoV-2 using RNA extracted from the nasopharyngeal/nasal swabs.To determine the variants of SARS-CoV-2 circulating in the state of Nevada,specimens from 200 COVID-19 patients were sequenced through our robust sequencing platform,which enabled sequencing of SARS-CoV-2 from specimens with even very low viral loads,without the need of culture-based amplification.High genome coverage allowed the identification of single and multi-nucleotide variants in SARS-CoV-2 in the community and their phylogenetic relationships with other variants present during the same period of the outbreak.We report the occurrence of a novel mutation at 323aa (314aa of orf1b) of nsp12 (RNA-dependent RNA polymerase) changed to phenylalanine(F) from proline (P),in the first reported isolate of SARS-CoV-2,Wuhan-Hu-1.This 323F variant was present at a very high frequency in Northern Nevada.Structural modeling determined this mutation in the interface domain,which is important for the association of accessory proteins required for the polymerase.In conclusion,we report the introduction of specific SARS-CoV-2 variants at very high frequency in distinct geographic locations,which is important for understanding the evolution and circulation of SARS-CoV-2variants of public health importance,while it circulates in humans.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.