The frequency of defective genomes in Omicron differs from that of the Alpha, Beta and Delta variants.

The SARS-CoV-2 Omicron variant emerged showing higher transmissibility and possibly higher resistance to current COVID-19 vaccines than other variants dominating the global pandemic. In March 2020 we performed a study in clinical samples, where we found that a portion of genomes in the SARS-CoV-2 viral population accumulated deletions immediately before the S1/S2 cleavage site (furin-like cleavage site, PRRAR/S) of the spike gene, generating a frameshift and appearance of a premature stop codon. The main aim of this study was to determine the frequency of defective deletions in prevalent variants from the first to sixth pandemic waves in our setting and discuss whether the differences observed might support epidemiological proposals. The complete SARS-CoV-2 spike gene was deeply studied by next-generation sequencing using the MiSeq platform. More than 90 million reads were obtained from respiratory swab specimens of 78 COVID-19 patients with mild infection caused by the predominant variants circulating in the Barcelona city area during the six pandemic waves: B.1.5, B.1.1, B.1.177, Alpha, Beta, Delta, and Omicron. The frequency of defective genomes found in variants dominating the first and second waves was similar to that seen in Omicron, but differed from the frequencies seen in the Alpha, Beta and Delta variants. The changing pattern of mutations seen in the various SARS-CoV-2 variants driving the pandemic waves over time can affect viral transmission and immune escape. Here we discuss the putative biological effects of defective deletions naturally occurring before the S1/S2 cleavage site during adaption of the virus to human infection.

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.

Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach.

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified.

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.

Host-dependent editing of SARS-CoV-2 in COVID-19 patients.

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients.

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site (PRRAR/S), generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We suggest that natural selection has favoured a "Don't burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.