Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation.

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.

Fitness-Dependent, Mild Mutagenic Activity of Sofosbuvir for Hepatitis C Virus.

The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC50) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.

Quantifying In-Host Quasispecies Evolution.

What takes decades, centuries or millennia to happen with a natural ecosystem, it takes only days, weeks or months with a replicating viral quasispecies in a host, especially when under treatment. Some methods to quantify the evolution of a quasispecies are introduced and discussed, along with simple simulated examples to help in the interpretation and understanding of the results. The proposed methods treat the molecules in a quasispecies as individuals of competing species in an ecosystem, where the haplotypes are the competing species, and the ecosystem is the quasispecies in a host, and the evolution of the system is quantified by monitoring changes in haplotype frequencies. The correlation between the proposed indices is also discussed, and the R code used to generate the simulations, the data and the plots is provided. The virtues of the proposed indices are finally shown on a clinical case.

Comparison of Nanopore and Synthesis-Based Next-Generation Sequencing Platforms for SARS-CoV-2 Variant Monitoring in Wastewater.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

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.

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

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

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

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

Broad and Dynamic Diversification of Infectious Hepatitis C Virus in a Cell Culture Environment.

Previous studies documented that long-term hepatitis C virus (HCV) replication in human hepatoma Huh-7.5 cells resulted in viral fitness gain, expansion of the mutant spectrum, and several phenotypic alterations. In the present work, we show that mutational waves (changes in frequency of individual mutations) occurred continuously and became more prominent as the virus gained fitness. They were accompanied by an increasing proportion of heterogeneous genomic sites that affected 1 position in the initial HCV population and 19 and 69 positions at passages 100 and 200, respectively. Analysis of biological clones of HCV showed that these dynamic events affected infectious genomes, since part of the fluctuating mutations became incorporated into viable genomes. While 17 mutations were scored in 3 biological clones isolated from the initial population, the number reached 72 in 3 biological clones from the population at passage 200. Biological clones differed in their responses to antiviral inhibitors, indicating a phenotypic impact of viral dynamics. Thus, HCV adaptation to a specific constant environment (cell culture without external influences) broadens the mutant repertoire and does not focus the population toward a limited number of dominant genomes. A retrospective examination of mutant spectra of foot-and-mouth disease virus passaged in cell cultures suggests a parallel behavior here described for HCV. We propose that virus diversification in a constant environment has its basis in the availability of multiple alternative mutational pathways for fitness gain. This mechanism of broad diversification should also apply to other replicative systems characterized by high mutation rates and large population sizes.IMPORTANCE The study shows that extensive replication of an RNA virus in a constant biological environment does not limit exploration of sequence space and adaptive options. There was no convergence toward a restricted set of adapted genomes. Mutational waves and mutant spectrum broadening affected infectious genomes. Therefore, profound modifications of mutant spectrum composition and consensus sequence diversification are not exclusively dependent on environmental alterations or the intervention of population bottlenecks.

Resistance-associated substitutions after sofosbuvir/velpatasvir/voxilaprevir triple therapy failure.

Direct-acting antivirals (DAAs) resolve chronic HCV infection in >95% of patients, but a small percentage do not respond to DAA-based therapy. These may be difficult to treat because of resistance-associated substitutions (RAS) emerging after treatment failure. Triple therapy with sofosbuvir (SOF)/velpatasvir (VEL)/voxilaprevir (VOX) is the recommended retreatment after DAA-based failure. However, in rare cases, failure to triple therapy occurs, and there is little information characterizing the viruses that relapse. To determine the RAS profile after failing SOF/VEL/VOX, and seek suitable alternatives for retreatment, samples from 5 patients were analysed using MiSeq Illumina deep sequencing before and after triple therapy. All patients were men, aged 59-78 years, 2 HCV genotype (G) 1b and 3 G3a. The most prevalent NS3 substitutions after SOF/VEL/VOX failure were Y56F and A166T. Four patients had the NS5A RAS, Y93H, after triple failure, and Y93H was observed in both G1b patients before retreatment and after SOF/ledipasvir failure. In 2 G3a patients, Y93H appeared at triple failure, and on the other G3a, A30K persisted in 100% of viral genomes. Finally, G1b patients showed C316N in NS5B, associated with SOF failure, but G3a patients had no known NS5B substitutions. HCV RAS analysis identified the following substitutions present at higher rates after triple failure: Y56F in NS3 (G1b), A166T in NS3 (G3a), A30K or Y93H in NS5A, and C316N in NS5B (G1b). A RAS-based salvage treatment (SOF + glecaprevir/pibrentasvir + RBV) was successfully used in one G3a patient.

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

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

Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection.

Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.

Synergistic lethal mutagenesis of hepatitis C virus.

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 mutagenic agent, often a nucleotide analogue. One of its advantages is its broad spectrum nature that renders the strategy potentially effective against emergent RNA viral infections. Here we describe synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method as implemented in the CompuSyn graphics, with average dose reduction index (DRI) above 1 (68.02±101.6 for favipiravir, and 5.83±6.07 for ribavirin), and average combination indices (CI) below 1 (0.52±0.28). Furthermore, analogue concentrations that individually did not extinguish high fitness HCV in ten serial infections, when used in combination they extinguished high fitness HCV in one to two passages. Although both analogues display a preference for G→A and C→U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. Prospects of synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.

Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation.

Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence analysis of the complete non-structural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC.

HIV-1 Protease Evolvability Is Affected by Synonymous Nucleotide Recoding.

One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the levels of development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair-reengineered protease sequence including 38 (13%) synonymous mutations. The WT and MAX viruses showed indistinguishable replication in MT-4 cells or peripheral blood mononuclear cells (PBMCs). Both viruses were subjected to serial passages in MT-4 cells, with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (50% inhibitory concentrations [IC50s] of 14.6 ± 5.3 and 21.2 ± 9 nM, respectively, for ATV and 5.9 ± 1.0 and 9.3 ± 1.9, respectively, for DRV). Ultradeep sequence clonal analysis revealed that both viruses harbored previously described mutations conferring resistance to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in the WT and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in the MAX virus. Remarkably, the G48L and L89I substitutions are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P < 0.0001), suggesting a higher selective pressure for change in this recoded virus. Our results indicate that the HIV-1 protease position in sequence space delineates the evolution of its mutant spectrum. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to those of the WT virus.IMPORTANCE Large-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a virus's position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease affects the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and a virus with recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotypes were indistinguishable between the recoded virus and the WT virus, suggesting that the HIV-1 strain with synonymously recoded protease and the WT virus are equally robust and evolvable.

New hepatitis C virus genotype 1 subtype naturally harbouring resistance-associated mutations to NS5A inhibitors.

Hepatitis C virus (HCV) is a highly divergent virus currently classified into seven major genotypes and 86 subtypes (ICTV, June 2017), which can have differing responses to therapy. Accurate genotyping/subtyping using high-resolution HCV subtyping enables confident subtype identification, identifies mixed infections and allows detection of new subtypes. During routine genotyping/subtyping, one sample from an Equatorial Guinea patient could not be classified into any of the subtypes. The complete genomic sequence was compared to reference sequences by phylogenetic and sliding window analysis. Resistance-associated substitutions (RASs) were assessed by deep sequencing. The unclassified HCV genome did not belong to any of the existing genotype 1 (G1) subtypes. Sliding window analysis along the complete genome ruled out recombination phenomena suggesting that it belongs to a new HCV G1 subtype. Two NS5A RASs (L31V+Y93H) were found to be naturally combined in the genome which could limit treatment possibilities in patients infected with this subtype.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment.IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

Hepatitis C virus early kinetics and resistance-associated substitution dynamics during antiviral therapy with direct-acting antivirals.

The emergence of resistance-associated substitutions (RASs) can compromise the high efficacy of direct-acting antivirals (DAAs). Little is known about RASs selection at very early time points during DAA treatment. Therefore, we analyzed the potential emergence of RASs immediately after therapy initiation. Samples of 71 patients treated with different DAAs were collected at baseline, during therapy (hours 4 and 8; days 1-7; weeks 2-4) or until target not detected. HCV-RNA levels were determined by qPCR, and RASs were detected by deep sequencing. Sixty-three (89%) patients achieved a sustained virological response (SVR), 7 (10%) relapsed, and 1 (1%) experienced a breakthrough. Almost all non-SVR (7/8, 88%) showed RASs either at baseline or relapse. High-frequency RASs detected at baseline (Y93H and L159F+C316N) remained detectable at early time points during therapy and reappeared as most prevalent substitutions at relapse. Conversely, emergent RASs at relapse (Q80R, D168E/V, R155K and L31V) were not observed during the first hours-days, before HCV-RNA became undetectable. HCV-RNA decay and genetic evolution of the quasispecies followed a similar pattern during the first hours of therapy in SVR and non-SVR patients. In conclusion, the absence of early RASs selection and the similar dynamics of HCV kinetics and quasispecies in SVR and non-SVR patients after therapy initiation suggest that RASs selection may occur at later stages in the remaining reservoir, where viral populations persist hidden at very low replication levels. Nevertheless, we cannot completely exclude very early selection, when RASs are present below the sensitivity limit of deep sequencing.

Thrombotic thrombocytopenic purpura relapse induced by acute hepatitis E transmitted by cryosupernatant plasma and successfully controlled with ribavirin.

BACKGROUND: Hepatitis E virus (HEV) can be transmitted by transfusion of any type of blood component, but there are few data on the potential risk of transmitting this virus and the associated complications. We provide evidence that HEV can be transmitted by cryosupernatant plasma, and that HEV infection can act as a trigger for thrombotic thrombocytopenic purpura (TTP). STUDY DESIGN AND METHODS: A patient with a history of TTP treated with plasmapheresis 2 months previously developed jaundice and a TTP exacerbation with purpura, thrombocytopenia, schistocytes, and undetectable ADAMTS-13 activity. He was diagnosed with acute hepatitis E and treated with ribavirin. TTP remitted with remission of HEV infection. RESULTS: Traceback to determine the source of the infection showed that 1 cryosupernatant plasma among the 99 different blood components used for the patient's last plasmapheresis was positive for HEV RNA, with an estimated viral load of 5000 to 10,000 IU/mL. Phylogenetic analysis proved the transfusion-transmitted route of acute hepatitis E. CONCLUSION: In a patient with TTP, acute HEV infection transmitted by cryosupernatant plasma may trigger exacerbation of TTP, which can be controlled on remission of HEV infection with ribavirin.

Pipeline for specific subtype amplification and drug resistance detection in hepatitis C virus.

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.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy.

BACKGROUND & AIMS: Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC). METHODS: Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes). RESULTS: Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion. CONCLUSIONS: The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects. LAY SUMMARY: Long-term treatment with antiviral drugs carries the risk of selecting mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals.