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Evolution of viral quasispecies during SARS-CoV-2 infection.

ABSTRACT: OBJECTIVES:Studies are needed to better understand the genomic evolution of the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe genomic diversity of SARS-CoV-2 by next-generation sequencing (NGS) in a patient with longitudinal follow-up for SARS-CoV-2 infection. METHODS:Sequential samples collected between January 29th and February 4th, 2020, from a patient infected by SARS-CoV-2 were used to perform amplification of two genome fragments-including genes encoding spike, envelope, membrane and nucleocapsid proteins-and NGS was carried out with Illumina® technology. Phylogenetic analysis was performed with PhyML and viral variant identification with VarScan. RESULTS:Majority consensus sequences were identical in most of the samples (5/7) and differed in one synonymous mutation from the Wuhan reference sequence. We identified 233 variants; each sample harboured in median 38 different minority variants, and only four were shared by different samples. The frequency of mutation was similar between genes and correlated with the length of the gene (r = 0.93, p = 0.0002). Most of mutations were substitution variations (n = 217, 93.1%) and about 50% had moderate or high impact on gene expression. Viral variants also differed between lower and upper respiratory tract samples collected on the same day, suggesting independent sites of replication of SARS-CoV-2. CONCLUSIONS:We report for the first time minority viral populations representing up to 1% during the course of SARS-CoV-2 infection. Quasispecies were different from one day to the next, as well as between anatomical sites, suggesting that in vivo this new coronavirus appears as a complex and dynamic distributions of variants.

SUBMITTER: Jary A

PROVIDER: S-EPMC7378485 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Evolution of viral quasispecies during SARS-CoV-2 infection.

Jary Aude A Leducq Valentin V Malet Isabelle I Marot Stéphane S Klement-Frutos Elise E Teyssou Elisa E Soulié Cathia C Abdi Basma B Wirden Marc M Pourcher Valérie V Caumes Eric E Calvez Vincent V Burrel Sonia S Marcelin Anne-Geneviève AG Boutolleau David D

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 20200724 11

<h4>Objectives</h4>Studies are needed to better understand the genomic evolution of the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe genomic diversity of SARS-CoV-2 by next-generation sequencing (NGS) in a patient with longitudinal follow-up for SARS-CoV-2 infection.<h4>Methods</h4>Sequential samples collected between January 29th and February 4th, 2020, from a patient infected by SARS-CoV-2 were used to perform amplification of two ...[more]

PMID: 32717416

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Project description:BackgroundThe genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contact tracing.MethodsLeveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies variants, and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints.ResultsWe built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and a combination of mutations that appear in only a small number of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of 100 SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome.ConclusionsWe conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients and determined their general prevalence when compared to over 70,000 other strains. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Project description:BackgroundThe genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contract tracing.MethodsLeveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints.ResultsWe built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and rare ones that occur in only small fraction of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome.ConclusionsWe conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients, mutations demarcating dominant species and the prevalence of mutation signatures, of which a significant number were relatively unique. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Project description:The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

Project description:Long-term infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a challenge to virus dispersion and the control of coronavirus disease 2019 (COVID-19) pandemic. The reason why some people have prolonged infection and how the virus persists for so long are still not fully understood. Recent studies suggested that the accumulation of intra-host single nucleotide variants (iSNVs) over the course of the infection might play an important role in persistence as well as emergence of mutations of concern. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during prolonged infection. Thirty-three patients who remained reverse transcription polymerase chain reaction (RT-PCR) positive in the nasopharynx for on average 18 days from the symptoms onset were included in this study. Whole-genome sequences were obtained for each patient at two different time points. Phylogenetic, populational, and computational analyses of viral sequences were consistent with prolonged infection without evidence of coinfection in our cohort. We observed an elevated within-host genomic diversity at the second time point samples positively correlated with cycle threshold (Ct) values (lower viral load). Direct transmission was also confirmed in a small cluster of healthcare professionals that shared the same workplace by the presence of common iSNVs. A differential accumulation of missense variants between the time points was detected targeting crucial structural and non-structural proteins such as Spike and helicase. Interestingly, longitudinal acquisition of iSNVs in Spike protein coincided in many cases with SARS-CoV-2 reactive and predicted T cell epitopes. We observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures. G→A mutations may be associated with RNA-editing enzyme activities; therefore, the mutational profiles observed in our analysis were suggestive of innate immune mechanisms of the host cell defense. Therefore, we unveiled a dynamic and complex landscape of host and pathogen interaction during prolonged infection of SARS-CoV-2, suggesting that the host's innate immunity shapes the increase of intra-host diversity. Our findings may also shed light on possible mechanisms underlying the emergence and spread of new variants resistant to the host immune response as recently observed in COVID-19 pandemic.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB610, was replaced by a minor quasispecies (MB61222) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB610. Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61222 over MB610 replication. This finding may be explained by a faster replicative activity of MB61222 as compared to MB610 as well as by the capability of MB61222 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB610 cannot escape this host antiviral response during MB61222 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

Dataset Information

Evolution of viral quasispecies during SARS-CoV-2 infection.

Publications

Evolution of viral quasispecies during SARS-CoV-2 infection.

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