SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing.

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Traceability of SARS-CoV-2 transmission through quasispecies analysis.

During COVID-19 pandemic, consensus genomic sequences were used for rapidly monitor the spread of the virus worldwide. However, less attention was paid to intrahost genetic diversity. In fact, in the infected host, SARS-CoV-2 consists in an ensemble of replicating and closely related viral variants so-called quasispecies. Here we show that intrahost single nucleotide variants (iSNVs) represent a target for contact tracing analysis. Our data indicate that in the acute phase of infection, in highly likely transmission links, the number of viral particles transmitted from one host to another (bottleneck size) is large enough to propagate iSNVs among individuals. Furthermore, we demonstrate that, during SARS-CoV-2 outbreaks when the consensus sequences are identical, it is possible to reconstruct the transmission chains by genomic investigations of iSNVs. Specifically, we found that it is possible to identify transmission chains by limiting the analysis of iSNVs to only three well-conserved genes, namely nsp2, ORF3, and ORF7.

High Mutation Rate Leads to Fitness Loss for Coronavirus Quasispecies

Background: RNA viruses evolve very fast, with a mutation rate of 103 to 105 base sub-stitution per nucleotides per copy. The mutation is a survival strategy for the viruses, which leads them to survive in the new host. Fitness is defined as the replication capacity of the virus in an ex-perimental setup. Generally, the large population passage of the virus leads to fitness gain, but the world data of the coronavirus infection and death shows the flattened curve with time. It is contra-dictory to the principle of fitness gain due to large population passage. The coronavirus is losing its potency but remains infectious as it is passaging into millions that leads to a decline in the death of COVID patients and high recovery rates. Fitness loss of coronaviruses attributed to a high level of mutation in the RNA genome as well as host immune response. The current outbreak of SARS CoV-2 is surfaced in December 2019 in Hubei province of China and considered as bats/pangolin origin, spreading 235 countries of the world, infecting nearly 31,664,104 people, and claimed nearly 972,221 lives as of September 24, 2020 (Death rate approximately 3%). This coronavirus has passaged into 31,664,104 people from the beginning of this pandemic until September 24, 2020. Now the virus is losing potency rather than being monotonous and continuous in producing virus-related complications. The population is still getting infected at the same rate, but the severity of the disease is reduced due to the potency of the virus diminished due to the passage effect as well as fitness loss of the virus due to high mutation rates. The death rate is reduced to 3% as compared to 6% in June 2020, when this paper was first submitted. Objective(s): The purpose of the study is to prove the fact that the coronavirus loses its potency with time but, they remain infective. It becomes more infectious due to mutation of the gene but loses the capacity to kill the host. Method(s): Since the WHO announces the COVID-19 outbreak is an emergency of international con-cern, every country in the world is taking many measures to mitigate the viral load to their popula-tion. Simultaneously, the WHO, CDC USA, CDC Europe, and much other organization is updating the COVID cases and death online daily as reported by the respective country. With the help of the COVID-19 outbreak data published by the European CDC and ourworldindata.org, we correlate the total cases of coronavirus and total death in the top ten affected countries in the world. We also link the trends of total cases vs. total death and total new cases vs. total new death related to COVID-19 in Germany, Spain, the United Kingdom, Italy, and New Zealand from January 30, 2020, until September 24, 2020. The reason to select these countries for the study is that these countries updating the COVID cases and deaths regularly and said to achieve the peak of COVID related infections and recovering from the pandemic. Result(s): We have tried to correlate the high mutation rate of the virus that leads to losing its potency to severe infection and death in the human. Viral extinction through high mutation could be considered as the new anti-viral strategies. Conclusion(s): Coronavirus is losing its potency to causing death to the human. The new infection is still being reported from every corner of the world, but the death rate is significantly decreasing.Copyright © 2021 Bentham Science Publishers.

Cellular senescence as a source of SARS-CoV-2 quasispecies.

In-depth analysis of SARS-CoV-2 biology and pathogenesis is rapidly unraveling the mechanisms through which the virus induces all aspects of COVID-19 pathology. Emergence of hundreds of variants and several important variants of concern has focused research on the mechanistic elucidation of virus mutagenesis. RNA viruses evolve quickly either through the error-prone polymerase or the RNA-editing machinery of the cell. In this review, we are discussing the links between cellular senescence, a natural aging process that has been recently linked to SARS-CoV-2 infection, and virus mutagenesis through the RNA-editing enzymes APOBEC. The action of APOBEC, enhanced by cellular senescence, is hypothesized to assist the emergence of novel variants, called quasispecies, within a cell or organism. These variants when introduced to the community may lead to the generation of a variant of concern, depending on fitness and transmissibility of the new genome. Such a mechanism of virus evolution may highlight the importance of inhibitors of cellular senescence during SARS-CoV-2 clinical treatment.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Convergent Evolution Dynamics of SARS-CoV-2 and HIV Surface Envelope Glycoproteins Driven by Host Cell Surface Receptors and Lipid Rafts: Lessons for the Future.

Although very different, in terms of their genomic organization, their enzymatic proteins, and their structural proteins, HIV and SARS-CoV-2 have an extraordinary evolutionary potential in common. Faced with various selection pressures that may be generated by treatments or immune responses, these RNA viruses demonstrate very high adaptive capacities, which result in the continuous emergence of variants and quasi-species. In this retrospective analysis of viral proteins, ensuring the adhesion of these viruses to the plasma membrane of host cells, we highlight many common points that suggest the convergent mechanisms of evolution. HIV and SARS-CoV-2 first recognize a lipid raft microdomain that acts as a landing strip for viral particles on the host cell surface. In the case of mucosal cells, which are the primary targets of both viruses, these microdomains are enriched in anionic glycolipids (gangliosides) forming a global electronegative field. Both viruses use lipid rafts to surf on the cell surface in search of a protein receptor able to trigger the fusion process. This implies that viral envelope proteins are both geometrically and electrically compatible to the biomolecules they select to invade host cells. In the present study, we identify the surface electrostatic potential as a critical parameter controlling the convergent evolution dynamics of HIV-1 and SARS-CoV-2 surface envelope proteins, and we discuss the impact of this parameter on the phenotypic properties of both viruses. The virological data accumulated since the emergence of HIV in the early 1980s should help us to face present and future virus pandemics.

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by three to four different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced coassembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

A Glimpse on the Evolution of RNA Viruses: Implications and Lessons from SARS-CoV-2.

RNA viruses are characterised by extremely high genetic variability due to fast replication, large population size, low fidelity, and (usually) a lack of proofreading mechanisms of RNA polymerases leading to high mutation rates. Furthermore, viral recombination and reassortment may act as a significant evolutionary force among viruses contributing to greater genetic diversity than obtainable by mutation alone. The above-mentioned properties allow for the rapid evolution of RNA viruses, which may result in difficulties in viral eradication, changes in virulence and pathogenicity, and lead to events such as cross-species transmissions, which are matters of great interest in the light of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemics. In this review, we aim to explore the molecular mechanisms of the variability of viral RNA genomes, emphasising the evolutionary trajectory of SARS-CoV-2 and its variants. Furthermore, the causes and consequences of coronavirus variation are explored, along with theories on the origin of human coronaviruses and features of emergent RNA viruses in general. Finally, we summarise the current knowledge on the circulating variants of concern and highlight the many unknowns regarding SARS-CoV-2 pathogenesis.

Multiple Mutations Associated with Emergent Variants Can Be Detected as Low-Frequency Mutations in Early SARS-CoV-2 Pandemic Clinical Samples.

Genetic analysis of intra-host viral populations provides unique insight into pre-emergent mutations that may contribute to the genotype of future variants. Clinical samples positive for SARS-CoV-2 collected in California during the first months of the pandemic were sequenced to define the dynamics of mutation emergence as the virus became established in the state. Deep sequencing of 90 nasopharyngeal samples showed that many mutations associated with the establishment of SARS-CoV-2 globally were present at varying frequencies in a majority of the samples, even those collected as the virus was first detected in the US. A subset of mutations that emerged months later in consensus sequences were detected as subconsensus members of intra-host populations. Spike mutations P681H, H655Y, and V1104L were detected prior to emergence in variant genotypes, mutations were detected at multiple positions within the furin cleavage site, and pre-emergent mutations were identified in the nucleocapsid and the envelope genes. Because many of the samples had a very high depth of coverage, a bioinformatics pipeline, "Mappgene", was established that uses both iVar and LoFreq variant calling to enable identification of very low-frequency variants. This enabled detection of a spike protein deletion present in many samples at low frequency and associated with a variant of concern.

Quasispecies Analysis of SARS-CoV-2 of 15 Different Lineages during the First Year of the Pandemic Prompts Scratching under the Surface of Consensus Genome Sequences.

The tremendous majority of SARS-CoV-2 genomic data so far neglected intra-host genetic diversity. Here, we studied SARS-CoV-2 quasispecies based on data generated by next-generation sequencing (NGS) of complete genomes. SARS-CoV-2 raw NGS data had been generated for nasopharyngeal samples collected between March 2020 and February 2021 by the Illumina technology on a MiSeq instrument, without prior PCR amplification. To analyze viral quasispecies, we designed and implemented an in-house Excel file ("QuasiS") that can characterize intra-sample nucleotide diversity along the genomes using data of the mapping of NGS reads. We compared intra-sample genetic diversity and global genetic diversity available from Nextstrain. Hierarchical clustering of all samples based on the intra-sample genetic diversity was performed and visualized with the Morpheus web application. NGS mapping data from 110 SARS-CoV-2-positive respiratory samples characterized by a mean depth of 169 NGS reads/nucleotide position and for which consensus genomes that had been obtained were classified into 15 viral lineages were analyzed. Mean intra-sample nucleotide diversity was 0.21 ± 0.65%, and 5357 positions (17.9%) exhibited significant (>4%) diversity, in ≥2 genomes for 1730 (5.8%) of them. ORF10, spike, and N genes had the highest number of positions exhibiting diversity (0.56%, 0.34%, and 0.24%, respectively). Nine hot spots of intra-sample diversity were identified in the SARS-CoV-2 NSP6, NSP12, ORF8, and N genes. Hierarchical clustering delineated a set of six genomes of different lineages characterized by 920 positions exhibiting intra-sample diversity. In addition, 118 nucleotide positions (0.4%) exhibited diversity at both intra- and inter-patient levels. Overall, the present study illustrates that the SARS-CoV-2 consensus genome sequences are only an incomplete and imperfect representation of the entire viral population infecting a patient, and that quasispecies analysis may allow deciphering more accurately the viral evolutionary pathways.

Quasispecies Fitness Partition to Characterize the Molecular Status of a Viral Population. Negative Effect of Early Ribavirin Discontinuation in a Chronically Infected HEV Patient.

The changes occurring in viral quasispecies populations during infection have been monitored using diversity indices, nucleotide diversity, and several other indices to summarize the quasispecies structure in a single value. In this study, we present a method to partition quasispecies haplotypes into four fractions according to their fitness: the master haplotype, rare haplotypes at two levels (those present at <0.1%, and those at 0.1−1%), and a fourth fraction that we term emerging haplotypes, present at frequencies >1%, but less than that of the master haplotype. We propose that by determining the changes occurring in the volume of the four quasispecies fitness fractions together with those of the Hill number profile we will be able to visualize and analyze the molecular changes in the composition of a quasispecies with time. To develop this concept, we used three data sets: a technical clone of the complete SARS-CoV-2 spike gene, a subset of data previously used in a study of rare haplotypes, and data from a clinical follow-up study of a patient chronically infected with HEV and treated with ribavirin. The viral response to ribavirin mutagenic treatment was selection of a rich set of synonymous haplotypes. The mutation spectrum was very complex at the nucleotide level, but at the protein (phenotypic/functional) level the pattern differed, showing a highly prevalent master phenotype. We discuss the putative implications of this observation in relation to mutagenic antiviral treatment.

Competition for dominance within replicating quasispecies during prolonged SARS-CoV-2 infection in an immunocompromised host.

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.

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.

MOLNUPIRAVIR INCREASES RANDOM SARS-CoV-2 RNA ERRORS WITHOUT SELECTION for RESISTANCE

Background: Molnupiravir (MOV), the orally administered prodrug of the antiviral ribonucleoside analogue, N-hydroxycytidine (NHC) has received emergency use authorization for treatment of COVID-19. NHC inhibits viral replication by introduction of random transition errors across the viral genome, resulting in non-infectious virus. In the Phase II/III (MOVe-OUT) study, non-hospitalized participants received MOV or placebo (PBO) for 5 days and followed to Day 29. Viral RNA was sequenced to determine the rate, distribution and type of viral errors observed. Methods: SARS-CoV-2 RNA isolated from nasopharyngeal swabs was quantified by RT-PCR followed by complete genome NGS using the Ion AmpliSeq SARS-CoV-2 Research panel and Ion Torrent sequencing. To distinguish between nucleotide errors resulting from the mechanism of action of MOV and those potentially associated with reduced susceptibility to NHC, two different analyses were used. To measure impact of MOV on accumulation of low-frequency errors in the viral quasispecies, nucleotide variants were identified using VarScan 2.4 mutation caller with 0.4% minimum variant allele frequency cut-off. Resistance-associated changes were identified as amino acid substitutions occurring in D3 or D5 samples from ≥2 participants with a frequency of ≥5% of NGS reads. Phenotypic analysis of selected amino acid substitutions was performed using a replicon model. Results: NGS results showed a relationship between the number of random errors across the viral genome with increasing MOV dose. By Day 5 the mean number of viral genome errors were 21, 83, 129 and 223 in the PBO, 200, 400 and 800 mg groups, respectively. Among the sequence changes observed, the majority were transitions errors, consistent with MOV's mechanism of action. After MOV treatment, few treatment-emergent amino acid substitutions were identified in the viral replicase genes. These included nsp12 (T731I) and nsp14 (A220S/T/V, V466I, S503L/P);none associated with loss of susceptibility to MOV. Changes in spike protein in both PBO and MOV groups were at sites previously described in circulating variants. Conclusion: Consistent with the mechanism of action, MOV treatment resulted in a dose-dependent increase in transition errors across the SARS-CoV-2 genome. No resistance-associated mutations were identified in the viral replicase and no evidence that MOV treatment selected for unique mutations in spike protein not previously observed in circulating variants.

Whole-Genome Sequencing of SARS-CoV-2 Infection in a Cluster of Immunocompromised Children in Indonesia.

Background: Thus far, Indonesia has recorded over 4,000,000 confirmed COVID-19 cases and 144,000 fatalities; 12.8% of cases have been in children under 18 years. Whole-genome viral sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been demonstrated to help differentiate hospital-acquired infection from community-acquired coronavirus disease 2019 (COVID-19) infection. Our study highlighted the use of WGS to investigate the origin of infection among pediatric oncology patients in Jakarta. The aim of our study was to evaluate clinical and laboratory characteristics and also the efficacy of using WGS to confirm hospital-acquired COVID-19 infection in a cluster of immunocompromised children within a single ward of a tertiary hospital in metropolitan Jakarta based on quasispecies, viral load, and admission dates. Method: Real-time reverse-transcription polymerase chain reaction (RT-PCR) from nasopharyngeal (NP) swabs was used to diagnose the patients and also guardians and healthcare workers (HCWs) in the ward, followed by WGS of RT-PCR positive cases to establish their phylogenetic relationships. Result: Using WGS, we showed that SARS-CoV-2 transmission in a cluster of children with underlying malignancy was characterized by high similarity of whole virus genome, which suggests nosocomial transmission.

Impact of Casirivimab-Imdevimab on Severe Acute Respiratory Syndrome Coronavirus 2 Delta Variant Nasopharyngeal Virus Load and Spike Quasispecies.

Background: The increasing use of monoclonal antibodies (mAbs) to treat coronavirus disease 2019 raises questions about their impact on the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mAb-resistant variants. We assessed the impact of Casirivimab-Imdevimab on SARS-CoV-2 mutations associated with reduced mAb activity in treated patients. Methods: We measured the nasopharyngeal (NP) viral load and sequenced the haplotypes of spike gene of 50 patients infected with the SARS-CoV-2 delta variant and treated with Casirivimab-Imdevimab using single-molecule real-time sequencing. Results: The NP SARS-CoV-2 viral load of patients treated with Casirivimab-Imdevimab decreased from 8.13 (interquartile range [IQR], 7.06-8.59) log10 copies/mL pretreatment to 3.67 (IQR, 3.07-5.15) log10 copies/mL 7 days later (P < .001). Of the 36 patients for whom follow-up timepoints Spike sequencing were available, none of the Spike mutations that reduced mAb activity were detected. Conclusions: Casirivimab-Imdevimab is an effective treatment for patients infected with the SARS-CoV-2 delta variant. Despite selective pressure on SARS-CoV-2 Spike quasispecies, we detected no key mutations that reduced mAb activity in our patients.

Multiorgan and Vascular Tropism of SARS-CoV-2.

Although the respiratory tract is the main target of SARS-CoV-2, other tissues and organs are permissive to the infection. In this report, we investigated this wide-spectrum tropism by studying the SARS-CoV-2 genetic intra-host variability in multiple tissues. The virological and histological investigation of multiple specimens from a post-mortem COVID-19 patient was performed. SARS-CoV-2 genome was detected in several tissues, including the lower respiratory system, cardio-vascular biopsies, stomach, pancreas, adrenal gland, mediastinal ganglion and testicles. Subgenomic RNA transcripts were also detected, in favor of an active viral replication, especially in testicles. Ultra-deep sequencing allowed us to highlight several SARS-CoV-2 mutations according to tissue distribution. More specifically, mutations of the spike protein, i.e., V341A (18.3%), E654 (44%) and H655R (30.8%), were detected in the inferior vena cava. SARS-CoV-2 variability can contribute to heterogeneous distributions of viral quasispecies, which may affect the COVID-19 pathogeny.

Diversity due to mutations in circulating virus strains of SARS-CoV-2 may delay control of COVID-19

Severe acute respiratory syndrome (SARS)-coronavirus-2 (CoV-2) is a beta-coronavirus (beta-CoV;sarbecovirus), like its predecessors SARS and MERS CoVs. Of the structural proteins of the virus, the Spike (S) protein on the virion envelope binds to the host cell ACE2 through viral epitopes in the receptor-binding domain (RBD). Deletions in the ORF8 as well as mutations in the S gene of SARS-CoV of 2003 were related to adaptation of the virus to humans. The emergence of novel variants of SARS-CoV-2, viz., B.1.1.7, B.1.427 and B.1.429, B.1.617 and its Kappa and Delta strains/ variants, B.1.351, and P.1 in the United Kingdom, Americas, India, South Africa and Brazil, respectively, has been found be associated with the current waves of the COVID-19 pandemic. These variants are antigenically dissimilar, whereas the current COVID-19 vaccines are monovalent. This is a handicap in the control program. The Delta variant has been reported in 74 countries as of 14 June 2021 and the anticipated third wave involving this variant is of concern to the countries (www.gavi.org). Of late, on 17 June 2021, Delta Plus variant was identified in India (AIIMS, Bhopal, India). Circulation of virus in vaccinated population may lead to endemicity, and this can be monitored by regular serosurveillance for antibodies against select non-structural proteins (NSPs) of the virus;antibodies to NSPs will indicate virus replication in the host. Endemic areas will have higher NSP reactors. It is predicted that the Delta B.1 variant may ignite the third wave of the disease in many countries. As it has been difficult to achieve uniformity in time and density of the vaccination even in the districts, circulation of the virus in partially immune population may lead to the selection of newer variants of SARS-CoV-2. The presence of monoclonal antibody resistant mutants and neutralization—escape mutants in quasispecies structure of another + sense RNA virus, i.e., Aphthovirus (FMD virus;foot and mouth disease virus) in the family Picornaviridae is well documented. The situation could be similar in the Coronaviridae member SARS-CoV-2. Previous immunity may not protect against current/ future mutants thereby pro-longing the COVID-19 control Programme. © 2022 Sharanagouda S. Patil et al.

Emergence of Avian coronavirus Escape Mutants Under Suboptimal Antibody Titers.

To perform a quasispecies assessment of the effect of vaccine combinations and antibody titers on the emergence of Avian coronavirus (AvCoV) escape mutants, 5-week-old males from a commercial chicken breeder lineage were vaccinated intramuscularly with one dose of a monovalent (genotype GI-1) or a bivalent (genotypes GI-1 and GI-11 (n = 40 birds/group) AvCoV vaccine. Seven birds were kept as controls. Six weeks later, pools of sera of each group were prepared and incubated at virus neutralization doses of 10 and 10-1 with the Beaudette strain (GI-1) of AvCoV in VERO cells. Rescued viruses were then submitted to genome-wide deep sequencing for subconsensus variant detection. After treatment with serum from birds vaccinated with the bivalent vaccine at a titer of 10-1, an F307I variant was detected in the spike glycoprotein that mapped to an important neutralizing region, which indicated an escape mutant derived from natural selection. Further variants were detected in nonstructural proteins and non-coding regions that are not targets of neutralizing antibodies and might be indicators of genetic drift. These results indicate that the evolution of AvCoV escape mutants after vaccination depends on the type of vaccine strain and the antibody titer and must be assessed based on quasispecies rather than consensus dominant sequences only because quasispecies may be otherwise undetected.

Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.