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Background: Defective hepatitis C virus (HCV) genomes with deletion of the envelope region have been occasionally reported by short-read sequencing analyses. However, the clinical and virological details of such deletion HCV have not been fully elucidated. Methods: We developed a highly accurate single-molecule sequencing system for full-length HCV genes by combining the third-generation nanopore sequencing with rolling circle amplification (RCA) and investigated the characteristics of deletion HCV through the analysis of 21 patients chronically infected with genotype-1b HCV. Result: In 5 of the 21 patients, a defective HCV genome with approximately 2000 bp deletion from the E1 to NS2 region was detected, with the read frequencies of 34–77%, suggesting the trans-complementation of the co-infecting complete HCV. Deletion HCV was found exclusively in decompensated cirrhosis (5/12 patients), and no deletion HCV was observed in nine compensated patients. Comparing the amino acid substitutions between the deletion and complete HCV (DAS, deletion-associated substitutions), the deletion HCV showed higher amino acid mutations in the ISDR (interferon sensitivity-determining region) in NS5A, and also in the TMS (transmembrane segment) 3 to H (helix) 2 region of NS2. Conclusions: Defective HCV genome with deletion of envelope genes is associated with decompensated cirrhosis. The deletion HCV seems susceptible to innate immunity, such as endogenous interferon with NS5A mutations, escaping from acquired immunity with deletion of envelope proteins with potential modulation of replication capabilities with NS2 mutations. The relationship between these mutations and liver damage caused by HCV deletion is worth investigating. [ABSTRACT FROM AUTHOR]

Next generation sequencing (NGS) provides an accurate analysis of hepatitis B virus (HBV) as compared to Sanger sequencing. In 2019, samples from HBV infected family of mother and her two sons samples were collected from Lahore, Pakistan. Sequencing of S gene, Geno2pheno (HBV) 2.0 tool and GHOST platform were used to analyze the genotype, mutations (natural and escape) and quasispecies analysis of the HBV. All samples in family were infected with genotype HBV/D1 and had high viral load (10e8 IU). An vaccine escape mutation "C137W" was identified in mother and a son. Mother sample were shown more number of haplotypes and total diversity as compared to sons. The quasispecies analysis showed the mother and sons were infected with HBV having similar genetic makeup, indicating the vertical transfer of infection. This case study provides knowledge about genetic variations that contributes to the improved diagnosis of the infection, treatment therapy, follow-up, and future treatment strategies. [ABSTRACT FROM AUTHOR]

RNA viruses adapt rapidly to new host environments by generating highly diverse genome sets, so-called “quasispecies.” Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistance or immune-escape mutants. Understanding these adaptation processes is highly relevant to assessing the risk of cross-species transmission and the safety and efficacy of vaccines and antivirals. We hypothesized that genetic memory within a viral genome population facilitates rapid adaptation. To test this, we investigated the adaptation of the Morbillivirus canine distemper virus to ferrets and compared an attenuated, Vero cell-adapted virus isolate with its recombinant derivative over consecutive ferret passages. Although both viruses adapted to the new host, the reduced initial genetic diversity of the recombinant virus resulted in delayed disease onset. The non-recombinant virus gradually increased the frequencies of beneficial mutations already present at very low frequencies in the input virus. In contrast, the recombinant virus first evolved de novo mutations to compensate for the initial fitness impairments. Importantly, while both viruses evolved different sets of mutations, most mutations found in the adapted non-recombinant virus were identical to those found in a previous ferret adaptation experiment with the same isolate, indicating that mutations present at low frequency in the original virus stock serve as genetic memory. An arginine residue at position 519 in the carboxy terminus of the nucleoprotein shared by all adapted viruses was found to contribute to pathogenesis in ferrets. Our work illustrates the importance of genetic diversity for adaptation to new environments and identifies regions with functional relevance. IMPORTANCE When viruses encounter a new host, they can rapidly adapt to this host and cause disease. How these adaptation processes occur remains understudied. Morbilliviruses have high clinical and veterinary relevance and are attractive model systems to study these adaptation processes. The canine distemper virus is of particular interest, as it exhibits a broader host range than other morbilliviruses and frequently crosses species barriers. Here, we compared the adaptation of an attenuated virus and its recombinant derivative to that of ferrets. Pre-existing mutations present at low frequency allowed faster adaptation of the non-recombinant virus compared to the recombinant virus. We identified a common point mutation in the nucleoprotein that affected the pathogenesis of both viruses. Our study shows that genetic memory facilitates environmental adaptation and that erasing this genetic memory by genetic engineering results in delayed and different adaptation to new environments, providing an important safety aspect for the generation of live-attenuated vaccines. [ABSTRACT FROM AUTHOR]

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants. [ABSTRACT FROM AUTHOR]

ABSTRACT Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1–HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis.IMPORTANCEAdaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

The evolution of SARS-CoV-2, the agent of COVID-19, has been remarkable for its high mutation potential, leading to the appearance of variants. Some mutations have never appeared in the published genomes, which represent consensus, or bona fide genomes. Here we tested the hypothesis that mutations that did not appear in consensus genomes were, in fact, as frequent as the mutations that appeared during the various epidemic episodes, but were not expressed because lethal. To identify these mutations, we analysed the genomes of 90 nasopharyngeal samples and the quasispecies determined by next-generation sequencing. Mutations observed in the quasispecies and not in the consensus genomes were considered to be lethal, what we called “outlaw” mutations. Among these mutations, we analysed the 21 most frequent. Eight of these “outlaws” were in the RNA polymerase and we were able to use a structural biology model and molecular dynamics simulations to demonstrate the functional incapacity of these mutated RNA polymerases. Three other mutations affected the spike, a major protein involved in the pathogenesis of COVID-19. Overall, by analysing the SARS-CoV-2 quasispecies obtained during sequencing, this method made it possible to identify “outlaws,” showing areas that could potentially become the target of treatments.

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.

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5′ end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A–H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies. [ABSTRACT FROM AUTHOR]

In quasispecies diversity studies, the comparison of two samples of varying sizes is a common necessity. However, the sensitivity of certain diversity indices to sample size variations poses a challenge. To address this issue, rarefaction emerges as a crucial tool, serving to normalize and create fairly comparable samples. This study emphasizes the imperative nature of sample size normalization in quasispecies diversity studies using next-generation sequencing (NGS) data. We present a thorough examination of resampling schemes using various simple hypothetical cases of quasispecies showing different quasispecies structures in the sense of haplotype genomic composition, offering a comprehensive understanding of their implications in general cases. Despite the big numbers implied in this sort of study, often involving coverages exceeding 100,000 reads per sample and amplicon, the rarefaction process for normalization should be performed with repeated resampling without replacement, especially when rare haplotypes constitute a significant fraction of interest. However, it is noteworthy that different diversity indices exhibit distinct sensitivities to sample size. Consequently, some diversity indicators may be compared directly without normalization, or instead may be resampled safely with replacement. [ABSTRACT FROM AUTHOR]

Molnupiravir, an oral direct‐acting antiviral effective in vitro against SARS‐CoV‐2, has been largely employed during the COVID‐19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS‐CoV‐2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS‐CoV‐2 whole‐genome sequencing on 38 molnupiravir‐treated persistently positive COVID‐19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab‐treated outpatients served as controls. Mutational analyses confirmed that SARS‐CoV‐2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G‐>A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide‐like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies. [ABSTRACT FROM AUTHOR]

Characterizing the mutational diversity of viruses can shed light on both intra-host and between-host evolutionary processes. In this study, we analyzed the transcriptome profiles of five different strains of Influenza A virus H1N1 isolated from humans, ducks, and pigs. We detected positive selection that led to the evolution and fixation of virus variants at the intra-host level. Among the strains studied, A/mallard/Balkhash/6304/2014 was the most affected by intra-host dynamics, with 143 substitutions, while A/Puerto Rico/8/34 (PR8) had the least, with only one substitution. A/California/04/09 had the highest frequency of distribution in the HA epitope sites. We also detected amino acid substitutions in immunodominant antigenic regions of A/WSN/1933, mallard, and swine influenza strains. We detected a total of 19 shared substitutions across both the inter-host and intra-host levels. The sequences of the different strains showed different patterns of gene expression. Phylogenetic trees constructed based on the HA and NA segments of the 26 GenBank isolates showed incongruences with other phylogenetic trees based on other IAV segments. We also identified amino acid changes in critical regions of the studied isolates, which could provide valuable information on the contingency of novel recombinants with different morphogenesis, infectivity, and behavior against antivirals. Our results show that influenza A viruses (IAVs) could potentially adapt more efficiently within their host organisms, mostly in wild birds. This underlines the importance of certain population characteristics, such as the elimination of genetic bottlenecks, as a means to expand their ability to infect new hosts. [ABSTRACT FROM AUTHOR]

Background: The successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. Method: From a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. Results: Sequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). Conclusion: The study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Background: To analysis of quasispecies (QS) changes and high-frequency mutations in the BCP/PreC/C region of patients at different phases of hepatitis B virus (HBV) infection and provides novel biomarkers for the diagnosis of chronic hepatitis B (CHB) patients. Methods: With the application of next-generation sequencing technology, we were able to sequence the HBV BCP/PreC/C regions in 40 patients, each at different phases of the HBV infection. The heterogeneity of QS and the frequency of mutations were calculated using MEGA 7 software. Results: Our results show that the complexity and diversity of the BCP/PreC/C QS in HBeAg-positive CHB patients are significantly higher than those in HBeAg-positive chronic infection patients, while HBeAg-negative chronic infection patients had significantly higher QS complexity and diversity than HBeAg-negative CHB patients. In addition, HBeAg-negative patients showed reduced complexity but increased diversity compared with HBeAg-positive patients. Receiver operating characteristic curves showed that G1764A, C2102T, dN and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-positive CHB, while the A2189C, dS and complexity of QS could be used as potential biomarkers for diagnosing HBeAg-negative chronic hepatitis. Finally, our study also found that G1896A and A2159G may be hotspot mutations affecting HBeAg seroconversion. Conclusion: Our research elucidates the evolution of HBV by analyzing QS heterogeneity and mutation patterns, offering novel serum biomarkers for enhancing clinical diagnosis and disease prognosis. This comprehensive approach sheds light on the intricate dynamics of HBV progression and paves the way for more precise medical interventions.

Mutations associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) resistance to antiprotease nirmatrelvir were reported. We aimed to detect them in SARS‐CoV‐2 genomes and quasispecies retrieved in our institute before drug availability in January 2022 and to analyze the impact of mutations on protease (3CLpro) structure. We sought for 38 3CLpro nirmatrelvir resistance mutations in a set of 62 673 SARS‐CoV‐2 genomes obtained in our institute from respiratory samples collected between 2020 and 2023 and for these mutations in SARS‐CoV‐2 quasispecies for 90 samples collected in 2020, using Python. SARS‐CoV‐2 protease with major mutation E166V was generated with Swiss Pdb Viewer and Molegro Molecular Viewer. We detected 22 (58%) of the resistance‐associated mutations in 417 (0.67%) of the genomes analyzed; 325 (78%) of these genomes had been obtained from samples collected in 2020−2021. APOBEC signatures were found for 12/22 mutations. We also detected among viral quasispecies from 90 samples some minority reads harboring any of 15 nirmatrelvir resistance mutations, including E166V. Also, we predicted that E166V has a very limited effect on 3CLpro structure but may prevent drug attachment. Thus, we evidenced that mutations associated with nirmatrelvir resistance pre‐existed in SARS‐CoV‐2 before drug availability. These findings further warrant SARS‐CoV‐2 genomic surveillance and SARS‐CoV‐2 quasispecies characterization. [ABSTRACT FROM AUTHOR]

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible. [ABSTRACT FROM AUTHOR]

To find the predictors of early HCC based on the dynamic changes of HBV quasispecies, this study utilizing the second-generation sequencing (NGS) and high-order multiplex droplet digital PCR (ddPCR) technology to examine the HBV quasispecies in serum of total 247 subjects recruited from high-incidence area of HCC. In the discovery stage, 15 non-synonymous Single Nucleotide Polymorphisms (SNPs) with higher variant proportion in HCC case group were founded (all P

A strong association between rubella virus (RuV) and chronic granulomas, in individuals with inborn errors of immunity, has been recently established. Both the RA27/3 vaccine and wild-type RuV strains were highly sensitive to a broad-spectrum antiviral drug, nitazoxanide (NTZ), in vitro. However, NTZ treatment, used as a salvage therapy, resulted in little or no improvements of RuV-associated cutaneous granulomas in patients. Here, we report investigations of possible causes of treatment failures in two ataxia-telangiectasia patients. Although a reduction in RuV RNA in skin lesions was detected by real-time RT-PCR, live immunodeficiency-related vaccine-derived rubella viruses (iVDRV) were recovered from granulomas, before and after the treatments. Tizoxanide, an active NTZ metabolite, inhibited replications of all iVDRVs in cultured A549 cells, but the 50% and 90% inhibitory concentrations were 10-40 times higher than those for the RA27/3 strain. There were no substantial differences in iVDRV sensitivities, neither before nor after treatments. Analysis of quasispecies in the E1 gene, a suspected NTZ target, showed no effect of NTZ treatments on quasispecies' complexity in lesions. Thus, failures of NTZ therapies were likely due to low sensitivities of iVDRVs to the drug, and not related to the emergence of resistance, following long-term NTZ treatments.

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants.

ABSTRACTMolecular mimicry of short linear interaction motifs has emerged as a key mechanism for viral proteins binding host domains and hijacking host cell processes. Here, we examine the role of RNA virus sequence diversity in the dynamics of the virus-host interface by analyzing the uniquely vast sequence record of viable SARS-CoV-2 species with focus on the multi-functional nucleocapsid protein. We observe the abundant presentation of motifs encoding several essential host protein interactions, alongside a majority of possibly non-functional and randomly occurring motif sequences absent in subsets of viable virus species. A large number of motifs emerge ex nihilo through transient mutations relative to the ancestral consensus sequence. The observed mutational landscape implies an accessible motif space that spans at least 25% of known eukaryotic motifs. This reveals motif mimicry as a highly dynamic process with the capacity to broadly explore host motifs, allowing the virus to rapidly evolve the virus-host interface.IMPORTANCEShort linear motifs (SLiMs) are 3–10 amino acid long binding motifs in intrinsically disordered protein regions (IDRs) that serve as ubiquitous protein-protein interaction modules in eukaryotic cells. Through molecular mimicry, viruses hijack these sequence motifs to control host cellular processes. It is thought that the small size of SLiMs and the high mutation frequencies of viral IDRs allow rapid host adaptation. However, a salient characteristic of RNA viruses, due to high replication errors, is their obligate existence as mutant swarms. Taking advantage of the uniquely large genomic database of SARS-CoV-2, here, we analyze the role of sequence diversity in the presentation of SLiMs, focusing on the highly abundant, multi-functional nucleocapsid protein. We find that motif mimicry is a highly dynamic process that produces an abundance of motifs transiently present in subsets of mutant species. This diversity allows the virus to efficiently explore eukaryotic motifs and evolve the host-virus interface.

The tremendous majority of RNA genomes from pathogenic viruses analyzed and deposited in databases are consensus or "democratic" genomes. They represent the genomes most frequently found in the clinical samples of patients but do not account for the huge genetic diversity of coexisting genomes, which is better described as quasispecies. A viral quasispecies is defined as the dynamic distribution of nonidentical but closely related mutants, variants, recombinant, or reassortant viral genomes. Viral quasispecies have collective behavior and dynamics and are the subject of internal interactions that comprise interference, complementation, or cooperation. In the setting of SARS‐CoV‐2 infection, intrahost SARS‐CoV‐2 genetic diversity was recently notably reported for immunocompromised, chronically infected patients, for patients treated with monoclonal antibodies targeting the viral spike protein, and for different body compartments of a single patient. A question that deserves attention is whether such diversity is generated postinfection from a clonal genome in response to selection pressure or is already present at the time of infection as a quasispecies. In the present review, we summarize the data supporting that hosts are infected by a "wild bunch" of viruses rather than by multiple virions sharing the same genome. Each virion in the "wild bunch" may have different virulence and tissue tropisms. As the number of viruses replicated during host infections is huge, a viral quasispecies at any time of infection is wide and is also influenced by host‐specific selection pressure after infection, which accounts for the difficulty in deciphering and predicting the appearance of more fit variants and the evolution of epidemics of novel RNA viruses. [ABSTRACT FROM AUTHOR]

Mutations in the SARS-CoV-2 Spike glycoprotein can affect monoclonal antibody efficacy. Recent findings report the occurrence of resistant mutations in immunocompromised patients after tixagevimab/cilgavimab treatment. More recently, the Food and Drug Agency revoked the authorization for tixagevimab/cilgavimab, while this monoclonal antibody cocktail is currently recommended by the European Medical Agency. We retrospectively reviewed 22 immunocompetent patients at high risk for disease progression who received intramuscular tixagevimab/cilgavimab as early COVID-19 treatment and presented a prolonged high viral load. Complete SARS-CoV-2 genome sequences were obtained for a deep investigation of mutation frequencies in Spike protein before and during treatment. At seven days, only one patient showed evidence of treatment-emergent cilgavimab resistance. Quasispecies analysis revealed two different deletions on the Spike protein (S:del138–144 or S:del141–145) in combination with the resistance S:K444N mutation. The structural and dynamic impact of the two quasispecies was characterized by using molecular dynamics simulations, showing the conservation of the principal functional movements in the mutated systems and their capabilities to alter the structure and dynamics of the RBD, responsible for the interaction with the ACE2 human receptor. Our study underlines the importance of prompting an early virological investigation to prevent drug resistance or clinical failures in immunocompetent patients. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian "avian-like" (EA) swine H1N1 and "human-like" H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals. [ABSTRACT FROM AUTHOR]

Background: It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Materials and methods: Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). Results: The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). Conclusions: The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC. [ABSTRACT FROM AUTHOR]

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5′ end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A–H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

In quasispecies diversity studies, the comparison of two samples of varying sizes is a common necessity. However, the sensitivity of certain diversity indices to sample size variations poses a challenge. To address this issue, rarefaction emerges as a crucial tool, serving to normalize and create fairly comparable samples. This study emphasizes the imperative nature of sample size normalization in quasispecies diversity studies using next-generation sequencing (NGS) data. We present a thorough examination of resampling schemes using various simple hypothetical cases of quasispecies showing different quasispecies structures in the sense of haplotype genomic composition, offering a comprehensive understanding of their implications in general cases. Despite the big numbers implied in this sort of study, often involving coverages exceeding 100,000 reads per sample and amplicon, the rarefaction process for normalization should be performed with repeated resampling without replacement, especially when rare haplotypes constitute a significant fraction of interest. However, it is noteworthy that different diversity indices exhibit distinct sensitivities to sample size. Consequently, some diversity indicators may be compared directly without normalization, or instead may be resampled safely with replacement.

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian “avian-like” (EA) swine H1N1 and “human-like” H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wildtype, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

Hepatitis B virus (HBV) exists as quasispecies (QS). However, the evolutionary characteristics of haplotypes of HBV X gene in the hepatocellular carcinoma (HCC) microenvironment remain unclear. Mutations across X gene are essential for the tumorigenicity of HBV X protein (HBx). However, the functional phenotypes of many mutant HBx remain unknown. This study aims to compare the characteristics of X gene evolution between tumour and non-tumour tissues in HCC patients and investigate the tumorigenic phenotype of HBx harbouring mutation T81P/S101P/L123S. This study included 24 HCC patients. Molecular cloning of X gene was performed to analyse characteristics of haplotypes in liver tissues. HCC cell lines stably expressing wild-type or mutant HBx and subcutaneous tumour xenograft mouse model were used to assess HBx-T81P/S101P/L123S tumorigenicity. The mean heterogeneity of HBV QS across X gene in tumour tissues was lower than that in non-tumour tissues. A location bias was observed in X gene clones with genotype C or D in tumour tissues compared to those with genotype B. Mutations in genotype-C or – D clones were mainly clustered in the dimerization region and aa110-aa140 within the transactivation region. A novel mutation combination at residues 81, 101 and 123 was identified in tumour tissues. Further, HBx-T81P/S101P/L123S promotes cell proliferation and increases genomic instability, which was mediated by MYC. This study elucidates the compartmentalized evolution patterns of HBV X gene between intra tumour and non-tumour tissues in HCC patients and provides a new mechanism underlying HBV-driven hepatocarcinogenesis, suggesting a potential viral marker for monitoring HCC.

Prions are proteinaceous pathogens responsible for a wide range of neurodegenerative diseases in animal and human. Prions are formed from misfolded, ß-sheet rich, and aggregated conformers (PrPSc) of the host-encoded prion protein (PrPC). Prion replication stems from the capacity of PrPSc to self-replicate by templating PrPC conversion and polymerization. The question then arises about the molecular mechanisms of prion replication, host invasion, and capacity to contaminate other species. Studying these mechanisms has gained in recent years further complexity with evidence that PrPSc is a pleiomorphic protein. There is indeed compelling evidence for PrPSc structural heterogeneity at different scales: (i) within prion susceptible host populations with the existence of different strains with specific biological features due to different PrPSc conformers, (ii) within a single infected host with the co-propagation of different strains, and (iii) within a single strain with evidence for co-propagation of PrPSc assemblies differing in their secondary to quaternary structure. This review summarizes current knowledge of prion assembly heterogeneity, potential mechanisms of formation during the replication process, and importance when crossing the species barrier. [ABSTRACT FROM AUTHOR]

EV-A71 is a common viral pathogen that causes hand, foot and mouth disease. It is a single-stranded RNA virus that has a low fidelity RNA polymerase and, as a result, spontaneous mutations frequently occur in the EV-A71 genome. The mutations within the genome give rise to quasispecies within the viral population that could be further defined by haplotypes. In vitro virulence of EV-A71 was shown by plaque size in Rhabdomyosarcoma (RD) cells, which was substantiated by in vitro characterizations of growth, RNA replication, binding, attachment and host cell internalization. Viruses could exhibit different host cell adaptations in different cell lines during viral passaging. The EV-A71/WT (derived from EV-A71 subgenotype B4) was shown to comprise six haplotypes through next-generation sequencing, where only EV-A71/Hap2 was found to be cultivable in RD cells, while EV-A71/Hap4 was the only cultivable haplotype in Vero cells. The EV-A71/WT produced plaques of four different sizes (small, medium, big, huge) in RD cells, while only two plaque variants (small, medium) were present in Vero cells. The small plaque variant isolated from RD cells displayed lower RNA replication rates, slower in vitro growth kinetics, higher TCID50 and lower attachment, binding and entry ability when compared against EV-A71/WT due to the mutation at 3D-S228P that disrupted the active site of the RNA polymerase, resulting in low replication and growth of the variant. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

All the conserved detailed results of evolution stored in DNA must be read, transcribed, and translated via an RNA-mediated process. This is required for the development and growth of each individual cell. Thus, all known living organisms fundamentally depend on these RNA-mediated processes. In most cases, they are interconnected with other RNAs and their associated protein complexes and function in a strictly coordinated hierarchy of temporal and spatial steps (i.e., an RNA network). Clearly, all cellular life as we know it could not function without these key agents of DNA replication, namely rRNA, tRNA, and mRNA. Thus, any definition of life that lacks RNA functions and their networks misses an essential requirement for RNA agents that inherently regulate and coordinate (communicate to) cells, tissues, organs, and organisms. The precellular evolution of RNAs occurred at the core of the emergence of cellular life and the question remained of how both precellular and cellular levels are interconnected historically and functionally. RNA networks and RNA communication can interconnect these levels. With the reemergence of virology in evolution, it became clear that communicating viruses and subviral infectious genetic parasites are bridging these two levels by invading, integrating, coadapting, exapting, and recombining constituent parts in host genomes for cellular requirements in gene regulation and coordination aims. Therefore, a 21st century understanding of life is of an inherently social process based on communicating RNA networks, in which viruses and cells continuously interact.

Simple Summary: The deformed wing virus (DWV) has been detected in Apis florea and its ectoparasite Euvarroa sinhai since they invaded Taiwan. Using a reverse transcription-PCR with specific primers enabled us to ascertain that DWV type A was prevalent among A. florea samples. Homology analysis and maximum-likelihood phylogenetic tree between different genotypes disclosed a complete polyprotein nucleotide sequence identity of 88% between isolates from A. florea, E. sinhai, and DWV type A strains from GenBank. The results demonstrated that a novel variant of deformed wing viruses exists in the invasive species A. florea and E. sinhai. The invasion of Apis florea in Taiwan was first recorded in 2017. The deformed wing virus (DWV) has been identified as a common bee virus in apiculture around the world. Ectoparasitic mites are the main DWV vector for horizontal transmission. However, there are few studies about the ectoparasitic mite of Euvarroa sinhai, which has been found in A. florea. In this study, the prevalence of DWV among four hosts, including A. florea, Apis mellifera, E. sinhai, and Varroa destructor, was determined. The results showed that a high DWV-A prevalence rate in A. florea, ranging from 69.2% to 94.4%, was detected. Additionally, the genome of DWV isolates was sequenced and subjected to phylogenetic analysis based on the complete polyprotein sequence. Furthermore, isolates from A. florea and E. sinhai both formed a monophyletic group for the DWV-A lineage, and the sequence identity was 88% between the isolates and DWV-A reference strains. As noted above, two isolates could be the novel DWV strain. It cannot be excluded that novel DWV strains could pose an indirect threat to sympatric species, such as A. mellifera and Apis cerana. [ABSTRACT FROM AUTHOR]

Viral quasispecies are distinct but closely related mutants formed by the disparity in viral genomes due to recombination, mutations, competition, and selection pressure. Theoretical derivation for the origin of a quasispecies is owed to the error-prone replication by polymerase and mutants of RNA replicators. Here, we briefly addressed the theoretical and mathematical origin of quasispecies and their dynamics. The impact of quasispecies for major salient human pathogens is reviewed. In the current global scenario, rapid changes in geographical landscapes favor the origin and selection of mutants. It comes as no surprise that a cauldron of mutants poses a significant risk to public health, capable of causing pandemics. Mutation rates in RNA viruses are magnitudes higher than in DNA organisms, explaining their enhanced virulence and evolvability. RNA viruses cause the most devastating pandemics; for example, members of the Orthomyxoviridae family caused the great influenza pandemic (1918 flu or Spanish flu), the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) outbreak, and the human immunodeficiency viruses (HIV), lentiviruses of the Retroviridae family, caused worldwide devastation. Rapidly evolving RNA virus populations are a daunting challenge for the designing of effective control measures like vaccines. Developing awareness of the evolutionary dispositions of RNA viral mutant spectra and what influences their adaptation and virulence will help curtail outbreaks of past and future pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Lyssavirus rabies (RABV) is an RNA virus and, therefore, is subject to mutations due to low RNA polymerase replication fidelity, forming a population structure known as a viral quasispecies, which is the core of RNA viruses' adaptive strategy. Under new microenvironmental conditions, the fittest populations are selected, and the study of this process on the molecular level can help determine molecular signatures related to virulence. Our aim was to survey gene signatures on nucleoprotein and glycoprotein genes that might be involved in virulence modulation during the in vitro evolution of RABV lineages after serial passages in a neuronal cell system with or without the presence of neutralizing antibodies based on replicative fitness, in vivo neurotropism and protein structure and dynamics. The experiments revealed that amino acids at positions 186 and 188 of the glycoprotein are virulence factors of Lyssavirus rabies, and site 186 specifically might allow the attachment to heparan as a secondary cell receptor, while polymorphism at position 333 might allow the selection of escape mutants under suboptimal neutralizing antibodies titers. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection remains a significant global health burden. The genetic variation of HBV is complex. HBV can be divided into nine genotypes, which show significant differences in geographical distribution, clinical manifestations, transmission routes and treatment response. In recent years, substantial progress has been made through various research methods in understanding the development, pathogenesis, and antiviral treatment response of clinical disease associated with HBV genetic variants. This progress provides important theoretical support for a deeper understanding of the natural history of HBV infection, virus detection, drug treatment, vaccine development, mother-to-child transmission, and surveillance management. This review summarizes the mechanisms of HBV diversity, discusses methods used to detect viral diversity in current studies, and the impact of viral genome variation during infection on the development of clinical disease. • The evolution and diversity of HBV genome remain ambiguous. • Current detection techniques have flaws. • HBV diversity is closely related to clinical practice. [ABSTRACT FROM AUTHOR]

Abstract Introduction Transplantation among HIV positive patients may be a valuable therapeutic intervention. This study involves an HIV D+/R+ kidney–liver transplantation, where PBMC-associated HIV quasispecies were analyzed in donor and transplant recipients (TR) prior to transplantation and thereafter, together with standard viral monitoring. Methods The donor was a 54 year of age HIV infected woman: kidney and liver recipients were two HIV infected men, aged 49 and 61. HIV quasispecies in PBMC was analyzed by ultra-deep sequencing of V3 env region. During TR follow-up, plasma HIV-1 RNA, HIV-1 DNA in PBMC, analysis of proviral integration sites and drug-resistance genotyping were performed. Other virological and immunological monitoring included CMV and EBV DNA quantification in blood and CD4 T cell counts. Results Donor and TR were all ART-HIV suppressed at transplantation. Thereafter, TR maintained a nearly suppressed HIV-1 viremia, but HIV-1 RNA blips and the increase of proviral integration sites in PBMC attested some residual HIV replication. A transient peak in HIV-1 DNA occurred in the liver recipient. No major changes of drug-resistance genotype were detected after transplantation. CMV and EBV transient reactivations were observed only in the kidney recipient, but did not require specific treatment. CD4 counts remained stable. No intermixed quasispecies between donor and TR was observed at transplantation or thereafter. Despite signs of viral evolution in TR, HIV genetic heterogeneity did not increase over the course of the months of follow up. Conclusions No evidence of HIV superinfection was observed in the donor nor in the recipients. The immunosuppressive treatment administrated to TR did not result in clinical relevant viral reactivations.

Recombination is one of the determinants of genetic diversity in the foot-and-mouth disease virus (FMDV). FMDV sequences have a mosaic structure caused by extensive intra- and inter-serotype recombination, with the exception of the capsid-encoding region. While these genome-wide patterns of broad-scale recombination are well studied, not much is known about the patterns of recombination that may exist within infected hosts. In addition, detection of recombination among viruses evolving at the within-host level is challenging due to the similarity of the sequences and the limitations in differentiating recombination from point mutations. Here, we present the first analysis of recombination events between closely related FMDV sequences occurring within buffalo hosts. The detection of these events was made possible by the occurrence of co-infection of two viral swarms with about 1% nucleotide divergence. We found more than 15 recombination events, unequally distributed across eight samples from different animals. The distribution of these events along the FMDV genome was neither uniform nor related to the phylogenetic distribution of recombination breakpoints, suggesting a mismatch between within-host evolutionary pressures and long-term selection for infectivity and transmissibility.

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies’ composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual’s respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies’ profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.

The viral genome quasispecies composition of hepatitis C virus (HCV) could have important implications to viral pathogenesis and resistance to anti-viral treatment. The purpose of the present study was to profile the HCV RNA quasispecies. We developed a strategy to determine the full-length HCV genome sequences co-existing within a single patient serum by using next-generation sequencing technologies. The isolated viral clones were divided into the groups that can be distinguished by core amino acid 70 substitution. Subsequently, we determined HCV full-length genome sequences of three independent dominant species co-existing in the sequential serum with a 7-year interval. From phylogenetic analysis, these dominant species evolved independently. Our study demonstrated that multiple dominant species co-existed in patient sera and evolved independently.

Viruses are the most abundant biological entities on Earth, and yet, they have not received enough consideration in astrobiology. Viruses are also extraordinarily diverse, which is evident in the types of relationships they establish with their host, their strategies to store and replicate their genetic information and the enormous diversity of genes they contain. A viral population, especially if it corresponds to a virus with an RNA genome, can contain an array of sequence variants that greatly exceeds what is present in most cell populations. The fact that viruses always need cellular resources to multiply means that they establish very close interactions with cells. Although in the short term these relationships may appear to be negative for life, it is evident that they can be beneficial in the long term. Viruses are one of the most powerful selective pressures that exist, accelerating the evolution of defense mechanisms in the cellular world. They can also exchange genetic material with the host during the infection process, providing organisms with capacities that favor the colonization of new ecological niches or confer an advantage over competitors, just to cite a few examples. In addition, viruses have a relevant participation in the biogeochemical cycles of our planet, contributing to the recycling of the matter necessary for the maintenance of life. Therefore, although viruses have traditionally been excluded from the tree of life, the structure of this tree is largely the result of the interactions that have been established throughout the intertwined history of the cellular and the viral worlds. We do not know how other possible biospheres outside our planet could be, but it is clear that viruses play an essential role in the terrestrial one. Therefore, they must be taken into account both to improve our understanding of life that we know, and to understand other possible lives that might exist in the cosmos. [ABSTRACT FROM AUTHOR]