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.

Viral Fitness, Population Complexity, Host Interactions, and Resistance to Antiviral Agents.

Fitness of viruses has become a standard parameter to quantify their adaptation to a biological environment. Fitness determinations for RNA viruses (and some highly variable DNA viruses) meet with several uncertainties. Of particular interest are those that arise from mutant spectrum complexity, absence of population equilibrium, and internal interactions among components of a mutant spectrum. Here, concepts, fitness measurements, limitations, and current views on experimental viral fitness landscapes are discussed. The effect of viral fitness on resistance to antiviral agents is covered in some detail since it constitutes a widespread problem in antiviral pharmacology, and a challenge for the design of effective antiviral treatments. Recent evidence with hepatitis C virus suggests the operation of mechanisms of antiviral resistance additional to the standard selection of drug-escape mutants. The possibility that high replicative fitness may be the driver of such alternative mechanisms is considered. New broad-spectrum antiviral designs that target viral fitness may curtail the impact of drug-escape mutants in treatment failures. We consider to what extent fitness-related concepts apply to coronaviruses and how they may affect strategies for COVID-19 prevention and treatment.

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.

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.

Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron.

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.

The International Virus Bioinformatics Meeting 2022.

The International Virus Bioinformatics Meeting 2022 took place online, on 23-25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus-host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting.

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

Mutation Rates, Mutation Frequencies, and Proofreading-Repair Activities in RNA Virus Genetics.

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus-host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10-3 to 10-6 copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3' to 5' exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses.

Population Disequilibrium as Promoter of Adaptive Explorations in Hepatitis C Virus.

Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium-revealed by the changing composition of the mutant spectrum-may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed.

The time for COVID-19 vaccination.

The composition and dynamics of viral mutant spectra in infected individuals advice that to avoid selection of SARS-CoV-2 escape mutants, vaccination campaigns for COVID-19 should be launched when disease incidence is low.