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Viral diversity and disease progression in chronic infections, and particularly how quasispecies structure affects antiviral treatment, remain key unresolved issues. Previous studies show that advanced liver fibrosis in long-term viral infections is linked to higher rates of antiviral treatment failures. Additionally, treatment failure is associated with high quasispecies fitness, which indicates greater viral diversity and adaptability. As a result, resistant variants may emerge, reducing retreatment effectiveness and increasing the chances of viral relapse. Additionally, using a mutagenic agent in monotherapy can accelerate virus evolution towards a flat-like quasispecies structure. This study examines 19 chronic HCV patients who failed direct-acting antiviral (DAA) treatments, using NGS to analyze quasispecies structure in relation to fibrosis as a marker of infection duration. Results show that HCV evolves towards a flat-like quasispecies structure over time, leading also to advanced liver damage (fibrosis F3 and F4/cirrhosis). Based on our findings and previous research, we propose that the flat-like fitness quasispecies structure is the final stage of any quasispecies in chronic infections unless eradicated. The longer the infection persists, the lower the chances of achieving a cure. Interestingly, this finding may also be applicable to other chronic infection and drug resistance in cancer.

The repeated failure to treat patients chronically infected with hepatitis E (HEV) and C (HCV) viruses, despite the absence of resistance-associated substitutions (RAS), particularly in response to prolonged treatments with the mutagenic agents of HEV, suggests that quasispecies structure may play a crucial role beyond single point mutations. Quasispecies structured in a flat-like manner (referred to as flat-like) are considered to possess high average fitness, occupy a significant fraction of the functional genetic space of the virus, and exhibit a high capacity to evade specific or mutagenic treatments. In this paper, we studied HEV and HCV samples using high-depth next-generation sequencing (NGS), with indices scoring the different properties describing flat-like quasispecies. The significance of these indices was demonstrated by comparing the values obtained from these samples with those from acute infections caused by respiratory viruses (betacoronaviruses, enterovirus, respiratory syncytial viruses, and metapneumovirus). Our results revealed that flat-like quasispecies in HEV and HCV chronic infections without RAS are characterized by numerous low-frequency haplotypes with no dominant one. Surprisingly, these low-frequency haplotypes (at the nucleotide level) exhibited a high level of synonymity, resulting in much lower diversity at the phenotypic level. Currently, clinical approaches for managing flat-like quasispecies are lacking. Here, we propose methods to identifying flat-like quasispecies, which represents an essential initial step towards exploring alternative treatment protocols for viruses resistant to conventional 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.

Abstract 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.

Herein, we describe the genetic diversity of circulating SARS-CoV-2 viruses by whole-genome sequencing (WGS) in Barcelona city (Catalonia, Spain) throughout the first four pandemic waves. From weeks 11/2020–24/2021, SARS-CoV-2-positive respiratory samples were randomly selected per clinical setting (80% from primary care or 20% from the hospital), age group, and week. WGS was performed following the ARTICv3 protocol on MiSeq or NextSeq2000 Illumina platforms. Nearly complete consensus sequences were used for genetic characterization based on GISAID and PANGOLIN nomenclatures. From 2475 samples, 2166 (87%) were fully sequenced (78% from primary care and 22% from hospital settings). Multiple genetic lineages were co-circulating, but four were predominant at different periods. While B.1.5 (50.68%) and B.1.1 (32.88%) were the major lineages during the first pandemic wave, B.1.177 (66.85%) and B.1.1.7 (83.80%) were predominant during the second, third, and fourth waves, respectively. Almost all (96.4%) were carrying D614G mutation in the S protein, with additional mutations that define lineages or variants. But some mutations of concern, such as E484K from B.1.351 and P.1 lineages are currently under monitoring, together with those observed in the receptor-binding domain or N-terminal domain, such as L452R and T478K from B.1.617.2 lineage. The fact that a predominant lineage was observed in each pandemic wave suggests advantageous properties over other contemporary co-circulating variants. This genetic variability should be monitored, especially when a massive vaccination campaign is ongoing because the potential selection and emergence of novel antigenic SARS-CoV-2 strains related to immunological escapement events.

Hepatitis is an inflammation of the liver whose etiology is very heterogeneous. The most common cause of hepatitis is viral infections from hepatotropic viruses, including hepatitis A, B, C, D and E. However, other factors such as infections from other agents, metabolic disorders, or autoimmune reactions can also contribute to hepatitis, albeit to a lesser extent. On April 5, 2022, the United Kingdom Health Security Agency alerted the World Health Organization (WHO) on the increased incidence of severe acute hepatitis of unknown causes (not A-E) in previously healthy young children, with symptoms of liver failure that in some cases required liver transplantation. By July 2022, 1,296 cases were reported in 37 countries. Acute hepatitis of unknown causes is not an exceptional phenomenon: in fact, it represents more than 30% of cases of acute hepatitis in children, however in the present instance the large proportion of severe cases was surprising and alarming (6% of liver transplants and almost 3% mortality). Multiple hypotheses have been proposed to explain the etiology of such higher proportion of acute hepatitis, including their co-occurrence in the context of COVID-19 pandemic. This is a review of the history of a clinical threat that has put in check a world health care system highly sensitized by the current COVID-19 pandemics, and that it looks like has ended with the arguments that the severe acute pediatric hepatitis is caused by Adeno-associated virus 2 (AAV2) infection associated with a coinfection with a helper virus (human Adenovirus HAdV or human herpesvirus 6) in susceptible children carrying HLA-class II antigen HLA-DRB1*04:01.

Phage therapy is still in its infancy, but it is increasingly promising as a future alternative for treating antibiotic-resistant bacteria. To investigate the effect of phages on Mycobacterium abscessus complex (MABC), we isolated 113 environmental phages, grown them to high titres, and assayed them on MABC clinical strains through the spot test. Of all the phages, only 16 showed killing activity. Their activity was so temperate to MABC that they could not generate any plaque-forming units (PFUs). The Appelmans method of directed evolution was carried out to evolve these 16 phages into more lytic ones. After only 11 of 30 rounds of evolution, every single clinical strain in our collection, including those that were unsusceptible up to this point, could be lysed by at least one phage. The evolved phages were able to form PFUs on the clinical strains tested. Still, they are temperate at best and require further training. The genomes of one random parental phage and three random evolved phages from Round 13 were sequenced, revealing a diversity of clusters and genes of a variety of evolutionary origins, mostly of unknown function. These complete annotated genomes will be key for future molecular characterisations.

Hepatitis E virus (HEV) is the major cause of acute viral hepatitis in several countries in Europe. HEV is acquired mainly by consumption of contaminated pork but can also be transmitted through blood transfusion. HEV infection is usually self-limited but can become persistent in immunocompromised persons. During the first 30 months of HEV RNA universal screening of blood donations in Catalonia, Spain, we identified 151 HEV RNA–positive donations (1/4,341 blood donations). Most infected donors reported consumption of pates and sausages, and 58% were negative for HEV IgM and IgG. All HEV isolates belonged to genotype 3. All infected donors spontaneously resolved the infection, and no neurologic symptoms and reinfections were observed after 1 year of follow-up. Since the implementation of HEV RNA universal screening, no new cases of transfusion-transmitted HEV infection were reported. Our data indicate HEV screening of blood donations provides safer blood for all recipients, especially for immunosuppressed persons.

Abstract Patients with HBeAg-negative chronic infection (CI) have not been extensively studied because of low viremia. The HBx protein, encoded by HBX, has a key role in viral replication. Here, we analyzed the viral quasispecies at the 5′ end of HBX in CI patients and compared it with that of patients in other clinical stages. Fifty-eight HBeAg-negative patients were included: 16 CI, 19 chronic hepatitis B, 16 hepatocellular carcinoma and 6 liver cirrhosis. Quasispecies complexity and conservation were determined in the region between nucleotides 1255 and 1611. Amino acid changes detected were tested in vitro. CI patients showed higher complexity in terms of mutation frequency and nucleotide diversity and higher quasispecies conservation (p

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.

BackgroundSince the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main concern is whether reinfections are possible, and which are the associated risk factors. This study aims to describe the clinical and molecular characteristics of 24 sequence-confirmed reinfection SARS-CoV-2 cases over 1 year in Barcelona (Catalonia, Spain).MethodsPatients with > 45 days between two positive PCR tests regardless of symptoms and negative tests between episodes were initially considered as suspected reinfection cases from November 2020 to May 2021. Whole-genome sequencing (WGS) was performed to confirm genetic differences between consensus sequences and for phylogenetic studies based on PANGOLIN nomenclature. Reinfections were confirmed by the number of mutations, change in lineage, or epidemiological criteria.ResultsFrom 39 reported suspected reinfection cases, complete viral genomes could be sequenced from both episodes of 24 patients, all were confirmed as true reinfections. With a median age of 44 years (interquartile range [IQR] 32–65), 66% were women and 58% were healthcare workers (HCWs). The median days between episodes were 122 (IQR 72–199), occurring one-third within 3 months. Reinfection episodes were frequently asymptomatic and less severe than primary infections. The absence of seroconversion was associated with symptomatic reinfections. Only one case was reinfected with a variant of concern (VOC).ConclusionSevere acute respiratory syndrome coronavirus 2 reinfections can occur in a shorter time than previously reported and are mainly found in immunocompetent patients. Surveillance through WGS is useful to identify viral mutations associated with immune evasion.

Background & Aims: Direct-acting antiviral (DAA) regimens provide a cure in >95% of patients with chronic HCV infection. However, in some patients in whom therapy fails, resistance-associated substitutions (RASs) can develop, limiting retreatment options and risking onward resistant virus transmission. In this study, we evaluated RAS prevalence and distribution, including novel NS5A RASs and clinical factors associated with RAS selection, among patients who experienced DAA treatment failure. Methods: SHARED is an international consortium of clinicians and scientists studying HCV drug resistance. HCV sequence linked metadata from 3,355 patients were collected from 22 countries. NS3, NS5A, and NS5B RASs in virologic failures, including novel NS5A substitutions, were examined. Associations of clinical and demographic characteristics with RAS selection were investigated. Results: The frequency of RASs increased from its natural prevalence following DAA exposure: 37% to 60% in NS3, 29% to 80% in NS5A, 15% to 22% in NS5B for sofosbuvir, and 24% to 37% in NS5B for dasabuvir. Among 730 virologic failures, most were treated with first-generation DAAs, 94% had drug resistance in ≥1 DAA class: 31% single-class resistance, 42% dual-class resistance (predominantly against protease and NS5A inhibitors), and 21% triple-class resistance. Distinct patterns containing ≥2 highly resistant RASs were common. New potential NS5A RASs and adaptive changes were identified in genotypes 1a, 3, and 4. Following DAA failure, RAS selection was more frequent in older people with cirrhosis and those infected with genotypes 1b and 4. Conclusions: Drug resistance in HCV is frequent after DAA treatment failure. Previously unrecognized substitutions continue to emerge and remain uncharacterized. Lay summary: Although direct-acting antiviral medications effectively cure hepatitis C in most patients, sometimes treatment selects for resistant viruses, causing antiviral drugs to be either ineffective or only partially effective. Multidrug resistance is common in patients for whom DAA treatment fails. Older patients and patients with advanced liver diseases are more likely to select drug-resistant viruses. Collective efforts from international communities and governments are needed to develop an optimal approach to managing drug resistance and preventing the transmission of resistant viruses.

ABSTRACT The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log10 higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.

ABSTRACTThe 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.

Who is a native signer? Since around 95% of deaf infants are born into a hearing family, deaf signers are exposed to a sign language at various moments of their life, and not only from birth. Moreover, the linguistic input they are exposed to is not always a fully fledged natural sign language. In this situation, is the notion of native signer as someone exposed to language from birth of any use? We review the results of the first large-scale cross-linguistic investigation on the effects of age of exposure to sign language. This research involved about 45 Deaf adult signers in each of three sign languages (Catalan Sign Language, French Sign Language, and Italian Sign Language). Across the three languages, participants were divided into three groups – those exposed from birth, those between 1 and 5 years of age, and those exposed between 6 and 15 years of age – and received a battery of tests designed for each language targeting various aspects of morphosyntactic competence. In particular, the tests focused on both those morphosyntactic phenomena that are known from the spoken language literature to be good detectors of language impairment or delay (i.e., wh-interrogatives and relative clauses) and on morphosyntactic phenomena that are sign language specific (i.e., role shift and directional verbs). The results showed a clear effect of being native, with significant differences across languages and tests between signers exposed to sign language from birth and those exposed in the 1st years of life. This confirms the life-long importance of language exposure from birth and the reliability of the notion of “nativeness”, at least for syntax. On the other hand, while in most domains the differences observed between populations might be differences in performance, for some specific constructions, signers belonging to the three groups may have different grammars. This latter finding challenges the generalized use of native signers’ grammar as the baseline for language description and language assessment.

Epidemics and pandemics have occurred since the beginning of time, resulting in millions of deaths. Many such disease outbreaks are caused by viruses. Some viruses, particularly RNA viruses, are characterized by their high genetic variability, and this can affect certain phenotypic features: tropism, antigenicity, and susceptibility to antiviral drugs, vaccines, and the host immune response. The best strategy to face the emergence of new infectious genomes is prompt identification. However, currently available diagnostic tests are often limited for detecting new agents. High-throughput next-generation sequencing technologies based on metagenomics may be the solution to detect new infectious genomes and properly diagnose certain diseases. Metagenomic techniques enable the identification and characterization of disease-causing agents, but they require a large amount of genetic material and involve complex bioinformatic analyses. A wide variety of analytical tools can be used in the quality control and pre-processing of metagenomic data, filtering of untargeted sequences, assembly and quality control of reads, and taxonomic profiling of sequences to identify new viruses and ones that have been sequenced and uploaded to dedicated databases. Although there have been huge advances in the field of metagenomics, there is still a lack of consensus about which of the various approaches should be used for specific data analysis tasks. In this review, we provide some background on the study of viral infections, describe the contribution of metagenomics to this field, and place special emphasis on the bioinformatic tools (with their capabilities and limitations) available for use in metagenomic analyses of viral pathogens.

Background: A huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016–2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure. Methods: We performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines. Findings: HAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation. Interpretation: Our data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group. Keywords: Vaccine escape-mutants, MSM, Quasispecies, Deep-sequencing

Background & aimsHCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV.MethodsWe analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays.ResultsWe observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies.ConclusionsOur results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA's therapies.

Deletions in the 3′ end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3–8.4%), and 25% (IQR, 13.1–40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7–12) years. In conclusion, we observed variants with Indels in the HBX 3′ end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

Virus pandemics have happened, are happening and will happen again. In recent decades, the rate of zoonotic viral spillover into humans has accelerated, mirroring the expansion of our global footprint and travel network, including the expansion of viral vectors and the destruction of natural spaces, bringing humans closer to wild animals. Once viral cross-species transmission to humans occurs, transmission cannot be stopped by cement walls but by developing barriers based on knowledge that can prevent or reduce the effects of any pandemic. Controlling a local transmission affecting few individuals is more efficient that confronting a community outbreak in which infections cannot be traced. Genetic detection, identification, and characterization of infectious agents using next-generation sequencing (NGS) has been proven to be a powerful tool allowing for the development of fast PCR-based molecular assays, the rapid development of vaccines based on mRNA and DNA, the identification of outbreaks, transmission dynamics and spill-over events, the detection of new variants and treatment of vaccine resistance mutations, the development of direct-acting antiviral drugs, the discovery of relevant minority variants to improve knowledge of the viral life cycle, strengths and weaknesses, the potential for becoming dominant to take appropriate preventive measures, and the discovery of new routes of viral transmission.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688–771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715–745. No difference in QS was observed over time. The most variable region was between nt 739–769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Abstract Background Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 5–10% of treated patients do not respond to current antiviral therapies, and basal resistance to DAAs is increasingly detected among treatment-naïve infected individuals. Identification of amino acid substitutions (including those in minority variants) associated with treatment failure requires analytical designs that take into account the high diversification of HCV in more than 86 subtypes according to the ICTV website (June 2017). Methods The methodology has involved five sequential steps: (i) to design 280 oligonucleotide primers (some including a maximum of three degenerate positions), and of which 120 were tested to amplify NS3, NS5A-, and NS5B-coding regions in a subtype-specific manner, (ii) to define a reference sequence for each subtype, (iii) to perform experimental controls to define a cut-off value for detection of minority amino acids, (iv) to establish bioinformatics’ tools to quantify amino acid replacements, and (v) to validate the procedure with patient samples. Results A robust ultra-deep sequencing procedure to analyze HCV circulating in serum samples from patients infected with virus that belongs to the ten most prevalent subtypes worldwide: 1a, 1b, 2a, 2b, 2c, 2j, 3a, 4d, 4e, 4f has been developed. Oligonucleotide primers are subtype-specific. A cut-off value of 1% mutant frequency has been established for individual mutations and haplotypes. Conclusion The methodological pipeline described here is adequate to characterize in-depth mutant spectra of HCV populations, and it provides a tool to understand HCV diversification and treatment failures. The pipeline can be periodically extended in the event of HCV diversification into new genotypes or subtypes, and provides a framework applicable to other RNA viral pathogens, with potential to couple detection of drug-resistant mutations with treatment planning.

Background: The effectiveness of the new generation of hepatitis C treatments named direct-acting antiviral agents (DAAs) depends on the genotype, subtype, and resistance-associated substitutions present in individual patients. The aim of this study was to evaluate a massive sequencing platform for the analysis of genotypes and subtypes of hepatitis C virus (HCV) in order to optimize therapy. Methods: A total of 84 patients with hepatitis C were analyzed. The routine genotyping methodology for HCV used at the study institution (Versant HCV Assay, LiPA) was compared with a deep sequencing platform (454/GS-Junior and Illumina MiSeq). Results: The mean viral load in these HCV patients was 6.89 × 106 ± 7.02 × 105. Viral genotypes analyzed by LiPA were distributed as follows: 26% genotype 1a (22/84), 55% genotype 1b (46/84), 1% genotype 1 (1/84), 2.5% genotype 3 (2/84), 6% genotype 3a (5/84), 6% genotype 4a/c/d (5/84). When analyzed by deep sequencing, the samples were distributed as follows: 27% genotype 1a (23/84), 56% genotype 1b (47/84), 8% genotype 3a (7/84), 5% genotype 4d (4/84), 2.5% genotype 4f (2/84). Six of the 84 patients (7%) were infected with more than one subtype. Among these, 33% (2/6) failed DAA-based triple therapy. Conclusions: The detection of mixed infection could explain some treatment failures. Accurate determination of viral genotypes and subtypes would allow optimal patient management and improve the effectiveness of DAA therapy.

Universal history is characterized by continuous evolution, in which civilizations are born and die. This evolution is associated with multiple factors, among which the role of microorganisms is often overlooked. Viruses and bacteria have written or decisively contributed to terrible episodes of history, such as the Black Death in 14th century Europe, the annihilation of pre-Columbian American civilizations, and pandemics such as the 1918 Spanish flu or the current COVID-19 pandemic caused by the coronavirus SARS-CoV-2. Nevertheless, it is clear that we could not live in a world without these tiny beings. Endogenous retroviruses have been key to our evolution and for the regulation of gene expression, and the gut microbiota helps us digest compounds that we could not otherwise process. In addition, we have used microorganisms to preserve or prepare food for millennia and more recently to obtain drugs such as antibiotics or to develop recombinant DNA technologies. Due to the enormous importance of microorganisms for our survival, they have significantly influenced the population genetics of different human groups. This paper will review the role of microorganisms as “villains” who have been responsible for tremendous mortality throughout history but also as “friends” who help us survive and evolve.

An interview on linguistic variation with Josep Quer

Natural languages come in two different modalities. The impact of modality on the grammatical structure and linguistic theory has been discussed at great length in the last 20 years. By contrast, the impact of modality on linguistic data elicitation and collection, corpus studies, and experimental (psycholinguistic) studies is still underinvestigated. In this article, we address specific challenges that arise in judgment data elicitation and experimental studies of sign languages. These challenges are related to the socio-linguistic status of the Deaf community and the larger variability across signers within the same community, to the social status of sign languages, to properties of the visual-gestural modality and its interface with gesture, to methodological aspects of handling sign language data, and to specific linguistic features of sign languages. While some of these challenges also pertain to (some varieties of) spoken languages, other challenges are more modality-specific. The special combination of the challenges discussed in this article seems to be a specific facet empirical research on sign languages is faced with. In addition, we discuss the complementarity of theoretical approaches and experimental studies and show how the interaction of both approaches contributes to a better understanding of sign languages in particular and linguistic structures in general.

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.

Diagnosis and clinical management of people infected with hepatitis C virus (HCV) relies on results from a combination of serological and virological tests. The aim of this study was to compare the performance of dried plasma spots (DPS), prepared using the cobas® Plasma Separation Card (PSC), to plasma and serum from venipuncture, for HCV diagnosis. We carried out a prospective study using DPS and paired plasma or serum samples. Serum and DPS samples were analyzed by immunoassay using Elecsys® Anti-HCV II (Roche). Plasma and DPS samples were analyzed using the cobas® HCV viral load and cobas® HCV genotyping tests (Roche). All DPS samples that had high anti-HCV antibody titers in serum were also antibody-positive, as were five of eight samples with moderate titers. Eight samples with low titers in serum were negative with DPS. Among 80 samples with plasma HCV viral loads between 61.5 and 2.2 × 108 IU/mL, 74 were RNA-positive in DPS. The mean viral load difference between plasma and DPS was 2.65 log10 IU/mL. The performance of DPS for detection of serological and virological markers of hepatitis C virus infection was comparable to that of the conventional specimen types. However, the limits of detection were higher for DPS.

[This corrects the article DOI: 10.1371/journal.pone.0201850.].

Noroviruses are the main cause of epidemics of acute gastroenteritis at a global scale. Although chronically infected immunocompromised individuals are regarded as potential reservoirs for the emergence of new viral variants, viral quasispecies distribution and evolution patterns in acute symptomatic and asymptomatic infections has not been extensively studied. Amplicons of 450 nts from the P2 coding capsid domain were studied using next-generation sequencing (454/GS-Junior) platform. Inter-host diversity between symptomatic and asymptomatic acutely infected individuals linked to the same outbreak as well as their viral intra-host diversity over time were characterized. With an average of 2848 reads per sample and a cutoff frequency of 0.1%, minor variant haplotypes were detected in 5 out of 8 specimens. Transmitted variants could not be confirmed in all infected individuals in one outbreak. The observed initial intra-host viral diversity in asymptomatically infected subjects was higher than in symptomatic ones. Viral quasispecies evolution over time within individuals was host-specific, with an average of 2.8 nt changes per day (0.0062 changes per nucleotide per day) in a given symptomatic case. Nucleotide polymorphisms were detected in 28 out of 450 analyzed nucleotide positions, 32.14% of which were synonymous and 67.86% were non-synonymous. Most observed amino acid changes emerged at or near blockade epitopes A, B, D and E. Our results suggest that acutely infected individuals, even in the absence of symptoms, which go underreported and may enhance transmission, may contribute to norovirus genetic variability and evolution.

RNA viruses replicate with a template-copying fidelity, which lies close to an extinction threshold. Increases of mutation rate by nucleotide analogues can drive viruses towards extinction. This transition is the basis of an antiviral strategy termed lethal mutagenesis. We have introduced a new diversity index, the rare haplotype load (RHL), to describe NS5B (polymerase) mutant spectra of hepatitis C virus (HCV) populations passaged in absence or presence of the mutagenic agents favipiravir or ribavirin. The increase in RHL is more prominent in mutant spectra whose expansions were due to nucleotide analogues than to multiple passages in absence of mutagens. Statistical tests for paired mutagenized versus non-mutagenized samples with 14 diversity indices show that RHL provides consistently the highest standardized effect of mutagenic treatment difference for ribavirin and favipiravir. The results indicate that the enrichment of viral quasispecies in very low frequency minority genomes can serve as a robust marker for lethal mutagenesis. The diagnostic value of RHL from deep sequencing data is relevant to experimental studies on enhanced mutagenesis of viruses, and to pharmacological evaluations of inhibitors suspected to have a mutagenic activity.

Selection of inhibitor-resistant viral mutants is universal for viruses that display quasi-species dynamics, and hepatitis C virus (HCV) is no exception. Here we review recent results on drug resistance in HCV, with emphasis on resistance to the newly-developed, directly-acting antiviral agents, as they are increasingly employed in the clinic. We put the experimental observations in the context of quasi-species dynamics, in particular what the genetic and phenotypic barriers to resistance mean in terms of exploration of sequence space while HCV replicates in the liver of infected patients or in cell culture. Strategies to diminish the probability of viral breakthrough during treatment are briefly outlined.

The aims of this study were to describe the evolution of acute hepatitis C virus (HCV) infections since 2004 and to determine its associated factors. Acute HCV infections diagnosed in Barcelona from 2004 to 2015 were included. Incidence ratios (IR) were then estimated for sex and age groups. Cases were grouped between 2004-2005, 2006-2011 and 2012-2015, and their incidence rate ratios (IRR) were calculated. In addition, risk factors for acute HCV infection were identified using multinomial logistic regression for complete, available and multiple imputed data. 204 new HCV cases were identified. Two peaks of higher IR of acute HCV infection in 2005 and 2013 were observed. Men and those aged 35-54 had higher IR. IRR for men was 2.9 times greater than in women (95% confidence intervals (CI): 1.8 ‒ 4.7). Factors related to the period 2012-2015 (versus 2006-2011) were: a) sexual risk factor for transmission versus nosocomial (relative-risk ratio (RRR): 13.0; 95% CI: 2.3 ‒ 72.1), b) higher educated versus lower (RRR: 5.4; 95% CI: 1.6 ‒ 18.7), and c) HIV co-infected versus not HIV-infected (RRR: 53.1; 95% CI: 5.7 ‒ 492.6). This is one of the few studies showing IR and RRRs of acute HCV infections and the first focused on a large city in Spain. Sexual risk for transmission between men, higher educational level and HIV co-infection are important factors for understanding current HCV epidemic. There has been a partial shift in the pattern of the risk factor for transmission from nosocomial to sexual.

This article offers an overview of research in sign language acquisition by children, mostly in American Sign Language. The results indicate that the developmental milestones and the linguistic errors made by children acquiring a sign language coincide with those observed in hearing children acquiring spoken languages. The visual-gestural modality is shown not to have the expected impact on the acquisition processes, which is arguably regulated by strictly linguistic principles.

Aquest article presenta la creació del projecte pilot del primer corpus de referència de la llengua de signes catalana (LSC). El corpus d’una llengua consisteix en una col·lecció representativa d’exemples en format llegible per una màquina i té com a objectiu oferir una visió del conjunt d’un domini lingüístic, ja que constitueix una representació àmplia de la llengua i de les seves varietats geogràfiques, de registre, generacionals i discursives. Pel fet de tractar-se d’un corpus de llengua de signes, les col·leccions de vídeos anotats contenen material sincronitzat amb les dades visuals enregistrades. Aquestes dades contribueixen a millorar les descripcions gramaticals i sociolingüístiques de la LSC, tot proporcionant una base sòlida per a l’anàlisi posterior, així com a fer possible una anàlisi basada en dades tant espontànies com induïdes. Aquí presentem la feina feta durant el procés d’elaboració d’aquest projecte, tant pel que fa a la metodologia d’obtenció de dades, a les filmacions i als materials que s’han utilitzat, com a l’anotació que s’ha dut a terme de les dades signades. Aquesta feina constitueix el pas previ necessari per al projecte de constitució del corpus de referència de la LSC. Paraules clau: anotació, documentació, corpus, llengua de signes catalana (LSC), modalitat gestovisual, obtenció de dades.

Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens.

Josep Quer1–3, Maria Buti1–3, Maria Cubero1–3, Jaume Guardia1–3, Rafael Esteban1–3, Juan Ignacio Esteban1–31Liver Unit, Internal Medicine Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Barcelona, Spain; 2Universitat Autònoma de Barcelona, Barcelona, Spain; 3Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, SpainAbstract: Persistent hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma and the major indication for liver transplantation in adults. Current standard of care treatment (SOC) with pegylated-interferon-a 2 and ribavirin (RBV) has a limited efficacy and is associated with significant side effects frequently associated with poor compliance or treatment discontinuation, requiring specialized and frequent monitoring. To overcome the limited efficacy of SOC, more than 50 direct-acting antiviral agents (DAA) designed to target viral-encoded proteins essential in the HCV life cycle are currently under development. The rapid selection of resistant mutants associated with the quasispecies nature of HCV with high mutation and replication rates is one of the main challenges for the new HCV therapies. Predictive host and viral factors together with combination of DAAs with or without IFN and/or RBV need to be accurately evaluated to design the most effective individualized treatment strategy within the shortest time interval and with minimum side effects.Keywords: HCV, treatment, quasispecies, resistance

This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p

AimTo evaluate HBV quasispecies (QA) complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUC) treatment.MethodsUltra-deep pyrosequencing of HBV preCore/Core regions in 30 sequential samples (baseline [diagnosis], treatment-free, and treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status over time: HBeAg+ (N = 4), HBeAg- (N = 2), and fluctuating HBeAg (transient seroreversion/seroconversion pattern) (N = 4). QA complexity was defined by Shannon entropy, mutation frequency, nucleotide diversity, and mutation frequency of amino acids (MfAA) in preCore and Core.ResultsThe QA was less complex in HBeAg+ than in HBeAg- or fluctuating HBeAg. High complexity in preCore was associated with decreased viral replication (preCore MfAA negatively correlated with HBV-DNA, p = 0.005). QA complexity in the treatment-free period negatively correlated with values seen during treatment. Specific variants were mainly selected in the Core region in HBeAg- and fluctuating HBeAg patients, suggesting higher immune pressure than in HBeAg+.ConclusionsThe negative correlation between QA natural evolution and on-treatment evolution indicates the importance of pre-treatment QA study to predict QA changes in NUC nonresponders. Study of QA complexity could be useful for managing HBV infection.

Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited.Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients.Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls.IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

BACKGROUND: Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. CONCLUSIONS: UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.