HCV Subtype Research Articles

An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains.

The objective was to develop a method of HCV genome sequencing that allowed simultaneous genotyping and NS5A inhibitor resistance profiling. In order to validate the use of a unique RT-PCR for genotypes 1–5, 142 plasma samples from patients infected with HCV were analysed. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. In parallel, partial NS3 sequences were analyzed obtained for genotyping. Phylogenetic analysis showed concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and compared to the list of known Resistant Associated Substitutions recently published. In conclusion, this tool allows determination of HCV genotypes, subtypes and identification of NS5A resistance mutations. This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5) in the EU and the US

Abstract 2585: viGEN: An open source bioinformatics pipeline for viral RNA detection and quantification in human tumor samples

Abstract We present a novel pipeline for viral RNA detection and quantification in human RNA-seq data. Our pipeline has been tested on the TCGA liver cancer cohort, can not only detect the presence of a viral species, but also provide gene level read counts for individual viral species and extract viral-variants. Introduction Approximately 20% of human cancer types are associated with viral infection that is routinely detected in blood samples. However the extent and biological significance of viral presence/infection in actual tumor samples is generally unknown but could be measured using existing Human RNA-seq data from tumor samples. We have developed a bioinformatics pipeline viGEN combining existing and novel RNAseq tools that allows for detection and quantification of viral RNA in human RNAseq data. Methods The pipeline includes 4 major modules: The first module allows to align and filter out human RNA sequences; second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module calculates quantitate read counts at the individual viral genes level thus allowing for downstream differential expression analysis of viral genes between experimental and controls groups. The fourth module calls variants in these viruses. To the best of our knowledge there are no publicly available pipelines or packages that would provide this type of complete analysis in one package. Customized solutions have been reported in the literature however were not made public. Results We used this pipeline to examine viruses present in RNA-seq data from 75 liver cancer patients in the TCGA data collection. Our pipeline allows conducting quantitative analysis at the gene level for visualization and detection of statistically significant differentially expressed viral genes between groups of patients known to be infected with both HBV and various subtypes of HCV. Once the viral genomes are detected at the genome level, we examine the differences between “Dead” and “Alive” samples at the viral-transcript level, and at the viral-variant level. Conclusion Our results show that it is possible to detect viral sequences from whole-transcriptome (RNA-seq) data in humans. We were able to not only quantify them at a viral-gene/CDS level, but also extract variants from the 75-sample dataset from TCGA. The results presented here are in correspondence with published literature and are a proof of concept of our pipeline. This pipeline can be used on cancer and non-cancer human RNA-seq or other NGS data to provide additional insights into the biological significance of viral infection in complex diseases, tumorigeneses and cancer immunology. Citation Format: Krithika Bhuvaneshwar, Lei Song, Yuriy Gusev. viGEN: An open source bioinformatics pipeline for viral RNA detection and quantification in human tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2585. doi:10.1158/1538-7445.AM2017-2585

Molecular Modeling and Docking Revealed Superiority of IDX-184 As Hcv Polymerase Inhibitor

Aim: IDX-184 is a nonstructural 5b nucleoside inhibitor (NI) that was under clinical trials against HCV. This work adopts a molecular modeling approach in order to study the interaction between IDX-184 and HCV polymerase from four different genotypes. Methods: Comparisons to the native nucleotide (Guanosine triphosphate) and other NIs were performed using interaction descriptors, calculated using semiempirical quantum mechanics and molecular docking. Results: IDX-184 shows potent binding to the active site of the polymerases. In addition, IDX-184 was better than Sofosbuvir and Ribavirin when docked into polymerase active site (even with experimentally solved structure). Conclusion: Analysis of the interaction descriptors and docking complexes suggests IDX-184 as a superior NI against the studied HCV subtypes.

A pharmacokinetic/viral kinetic model to evaluate treatment of chronic HCV infection with a non-nucleoside polymerase inhibitor.

Viral kinetic models have proven useful in characterizing treatment effectiveness during HCV therapy with interferon (IFN) as well as with direct-acting antivirals (DAAs). Here we use a pharmacokinetic/viral kinetic (PK/VK) model to describe HCV RNA kinetics during treatment with setrobuvir, a non-nucleosidic inhibitor of the HCV NS5B polymerase enzyme. Using PK data from three studies in healthy volunteers and PK and VK data from a Phase I study, where setrobuvir was administered for 3 days, we fitted a two-compartment PK model with first-order absorption and lag-time, an Emax pharmacodynamics model and a standard biphasic VK model. Setrobuvir's EC50 and Hill coefficient and the viral clearance rate were significantly different (P=0.014, P<0.001 and P=0.004, respectively) between patients infected with HCV subtypes 1b and 1a, leading to an increased viral load decline in patients infected with genotype 1b virus. Understanding the combined effects of PK/VK on the performance of a non-nucleoside polymerase inhibitor such as setrobuvir could provide valuable insights into their use in combination with other DAAs as well as to optimize future therapy. Further, our work suggests that patients infected with subtype 1a would need higher doses than those infected with subtype 1b to achieve the same effectiveness. Whether this is true for other non-nucleoside polymerase inhibitors needs to be examined.

High Prevalence of Q80K Among NS3 Resistance-Associated Substitutions in Subtype 1a Patients with Chronic Hepatitis C Prior to Treatment with Direct Acting Antivirals: The Croatian Data

Background: Therapy for chronic hepatitis C is based on direct-acting antiviral drugs (DAA) that include protease inhibitors, polymerase inhibitors, as well as inhibitors of NS5A protein. Resistance-associated substitutions (RAS) can be associated with inadequate treatment outcomes with DAA. People with HCV subtype 1a infection carrying Q80K polymorphism could have a reduced treatment response to a protease inhibitor simeprevir. The data on the prevalence of Q80K polymorphism and other RAS worldwide are quite variable. Objectives: The study goal was to analyze the frequency of Q80K polymorphism and other substitutions associated with HCV resistance to NS3 inhibitors in patients previously not treated with simeprevir infected with HCV subtype 1a from Croatia. Methods: The study included 136 people with chronic hepatitis C and infected with HCV subtype 1a receiving clinical care at the department of viral hepatitis of the University hospital for infectious diseases, Zagreb and Croatian reference center for viral hepatitis from July 2015 to April 2016. All participants were not previously treated with simeprevir and not co-infected with HIV. Detection of Q80K polymorphism and other substitutions associated with resistance to NS3 inhibitors was performed by population-based sequencing on ABI PRISM® 3100 genetic analyzer. Phylogenetic tree was constructed using the Maximum Likelihood method and supported with a bootstrap test of 1000 replicates. Geno2Pheno algorithm was used for the interpretation of sequences, detection of resistance associated substitutions and determination of the clade of the sequence. Results: The prevalence of Q80K polymorphism was observed in 42.6% of patients while resistance to simeprevir (mediated by other RAS as well) was detected in 46.3% of patients. Phylogenetic analysis of subtype 1a sequences showed the separation into 2 clades and Q80K polymorphism was exclusively present in clade I isolates. Other RAS detected in the patients included V36L, T54S, V55A, S122R, and I170T. Conclusions: In conclusion, the prevalence of Q80K polymorphism was found to be rather high in patients with chronic hepatitis C infected with clade I subtype 1a while other substitutions associated with resistance to protease inhibitors were rarely found in Croatian cohort. The results of this study confirm the need for pre-treatment screening for Q80K in subtype 1a patients considered for simeprevir treatment.

Ombitasvir, paritaprevir, and ritonavir plus dasabuvir for 8 weeks in previously untreated patients with hepatitis C virus genotype 1b infection without cirrhosis (GARNET): a single-arm, open-label, phase 3b trial

Clinical studies have shown high rates of sustained virological response (hepatitis C virus [HCV] RNA <15 IU/mL) at post-treatment week 12 (SVR12) in patients with genotype 1b infection with and without cirrhosis who received coformulated ombitasvir, paritaprevir, and ritonavir, plus dasabuvir, without ribavirin, for 12 weeks. In this study, we aimed to assess 8-week treatment with ombitasvir, paritaprevir, and ritonavir, plus dasabuvir, without ribavirin in patients infected with HCV genotype 1b without cirrhosis. We did a multicentre, open-label, single-arm, phase 3b study (GARNET) in 20 hospitals or clinics in Australia, Canada, France, Germany, Israel, Italy, Spain, and the UK, to assess the safety and efficacy of an 8-week treatment duration of once-daily oral ombitasvir 25 mg, paritaprevir 150 mg, and ritonavir 100 mg, plus twice-daily oral dasabuvir 250 mg in previously untreated patients with chronic HCV genotype 1b infection without cirrhosis (as assessed by liver biopsy, transient elastography, or serum markers). Eligible patients were aged at least 18 years, with more than 1000 IU/mL HCV RNA, and a laboratory result at screening indicating infection with HCV genotype 1b subtype only. Patients were excluded if they had evidence of HCV genotype or subtype other than genotype 1b, if they tested positive for hepatitis B surface antigen or anti-HIV antibody at screening, or if they had previously been treated for HCV. The primary endpoint was the proportion of patients with SVR12; the primary endpoint and safety were assessed in all patients who received at least one dose of study drugs. This trial is registered with ClinicalTrials.gov, number NCT02582632. Patients were screened between Nov 24, 2015, and March 1, 2016, and 166 patients were enrolled. 163 (98%) of 166 enrolled patients had HCV genotype 1b infection, and three (2%) of 166 had other genotypes or subtypes (genotype 1a, genotype 1d, and genotype 6). All enrolled patients received at least one dose of study drugs. 162 (98% [95% CI 95·3-99·9]) of 166 patients achieved SVR12. One patient discontinued treatment on day 45 due to adverse events. Most adverse events were mild in severity, and the most common adverse events were headache (35 [21%] of 166) and fatigue (28 [17%] of 166). Two (1%) of 166 patients had serious adverse events; neither were considered related to study drug treatment. Treatment with ombitasvir, paritaprevir, and ritonavir, plus dasabuvir, without ribavirin, for 8 weeks was efficacious and well tolerated. 8-week treatment options for previously untreated patients with HCV genotype 1b infection without cirrhosis are limited; shortening the treatment duration might reduce the burden associated with medical visits and procedures, thereby improving access to care and enabling the treatment of more patients. AbbVie.

Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000-2015 period (the GEHEP 005 study).

We report the largest study on the prevalence and distribution of HCV genotypes in Spain (2000-2015), and we relate them with clinical, epidemiological and virological factors. Patients from 29 hospitals in 10 autonomous communities (Andalusia, Aragon, Castilla-Leon, Catalonia, Galicia, Canary Islands, Madrid Community, Valencian Community, Murcia Region and Basque Country) have been studied. Annual distribution of HCV genotypes and subtypes, as well as gender, age, transmission route, HIV and/or HBV coinfection, and treatment details were recorded. We included 48595 chronically HCV-infected patients with the following characteristics: median age 51 years (IQR, 44-58), 67.9% male, 19.1% HIV-coinfected, 23.5% HBV-coinfected. Parenteral transmission route was the most frequent (58.7%). Genotype distribution was 66.9% GT1 (24.9% subtype 1a and 37.9% subtype 1b), 2.8% GT2, 17.3% GT3, 11.4% GT4 and 0.1% GT5 and 0.02% GT6. LiPA was the most widely HCV genotyping test used (52.4%). HCV subtype 1a and genotypes 3 and 4 were closely associated with male gender, parenteral route of infection and HIV and HBV coinfection; in contrast, subtype 1b and genotype 2 were associated with female gender, nonparenteral route and mono-infection. Age was related to genotype distribution, and different patterns of distribution and biodiversity index were observed between different geographical areas. Finally, we describe how treatment and changes in transmission routes may have affected HCV genotype prevalence and distribution patterns. We present the most recent data on molecular epidemiology of hepatitis C virus in Spain. This study confirms that genotype distributions vary with age, sex, HIV and HBV coinfection and within geographical areas and epidemiological groups.

Implications of hepatitis C virus subtype 1a migration patterns for virus genetic sequencing policies in Italy

BackgroundIn-depth phylogeographic analysis can reveal migration patterns relevant for public health planning. Here, as a model, we focused on the provenance, in the current Italian HCV subtype 1a epidemic, of the NS3 resistance-associated variant (RAV) Q80K, known to interfere with the action of NS3/4A protease inhibitor simeprevir. HCV1a migration patterns were analysed using Bayesian phylodynamic tools, capitalising on newly generated and publicly available time and geo-referenced NS3 encoding virus genetic sequence data.ResultsOur results showed that both immigration and local circulation fuel the current Italian HCV1a epidemic. The United States and European continental lineages dominate import into Italy, with the latter taking the lead from the 1970s onwards. Since similar migration patterns were found for Q80K and other lineages, no clear differentiation of the risk for failing simeprevir can be made between patients based on their migration and travel history. Importantly, since HCV only occasionally recombines, these results are readily transferable to the genetic sequencing policy concerning NS5A RAVs.ConclusionsThe patient migration and travel history cannot be used to target only part of the HCV1a infected population for drug resistance testing before start of antiviral therapy. Consequently, it may be cost-effective to expand genotyping efforts to all HCV1a infected patients eligible for simeprevir-based therapies.

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The encoded portion of the complete genomes of 46 strains of the genotype 6 of hepatitis C virus through bioinformat-ics RDP programs complex group of 6 recombinants strains was identified, in which 7 recombination sites were fixed. Strains correspond to the three-recombinant HCV subtypes: 6a, 6b and 6I. For each of the identified recombinant we defined parent strains from which they can be obtained. Three recombinants were obtained from parent strains of the same subtype (homologous inside subgenotypic recombination). For the remaining three recombinants parent strains were members of three different subtypes (between subgenotypic recombination). In one strain we identified a unique recombination site in a highly conservative NS3 gene. Most of the recombination sites occurred in the region of the structural genes C, E1 and E2, and in the area of non-structural genes NS5a and NS5b. In the recombinant strain DQ480518-6a two recombination site were identified. One site is located in the structural and nonstructural genes (E2 + NS1 + NS2), and a second one in non-structural region. Dimensions of recombination sites can vary from 86 to 1072 nucleotide bases. The study identified “hot spots” of recombination in the strains of genotype 6 of hepatitis C virus. The recombinants were found in the population of the three countries: the United States (from the serum of an immigrant), Hong Kong and China.The encoded portion of the complete genomes of 46 strains of the genotype 6 of hepatitis C virus through bioinformat-ics RDP programs complex group of 6 recombinants strains was identified, in which 7 recombination sites were fixed. Strains correspond to the three-recombinant HCV subtypes: 6a, 6b and 6I. For each of the identified recombinant we defined parent strains from which they can be obtained. Three recombinants were obtained from parent strains of the same subtype (homologous inside subgenotypic recombination). For the remaining three recombinants parent strains were members of three different subtypes (between subgenotypic recombination). In one strain we identified a unique recombination site in a highly conservative NS3 gene. Most of the recombination sites occurred in the region of the structural genes C, E1 and E2, and in the area of non-structural genes NS5a and NS5b. In the recombinant strain DQ480518-6a two recombination site were identified. One site is located in the structural and nonstructural genes (E2 + NS1 + NS2), and a second one in non-structural region. Dimensions of recombination sites can vary from 86 to 1072 nucleotide bases. The study identified “hot spots” of recombination in the strains of genotype 6 of hepatitis C virus. The recombinants were found in the population of the three countries: the United States (from the serum of an immigrant), Hong Kong and China.

Correlates of infection and molecular characterization of blood-borne HIV, HCV, and HBV infections in HIV-1 infected inmates in Italy: An observational cross-sectional study.

Coinfection of blood-borne hepatitis B and hepatitis C viruses (HBV and HCV, respectively) in human immunodeficiency virus type 1 (HIV-1)-positive individuals frequently occurs in inmate population and peculiar viral strains and patterns of virological markers may be observed.Plasma from 69 HIV-1-positive inmates was obtained from 7 clinical centers connected with correctional centers in different towns in Italy. HIV, HBV, and HCV markers were tested by commercial assays. Virus genotyping was carried out by sequencing the protease and reverse transcriptase-encoding region (PR-RT region) for HIV and a region encompassing the NS5B gene for HCV and subsequent phylogenetic analysis.Twelve over 14 HIV-subtyped inmates were infected with HIV-1 subtype B strains. The 2 non-B strains belonged to subtype G and CRF02_AG, in an Italian and a Gambian patient, respectively. Variants carrying the K103N and Y181C resistance mutations to non-nucleoside reverse transcriptase inhibitors (NNRTIs) were found in 2 out of 9 patients naive for combined antiretroviral therapy (cART) (22.2%). Most HIV-positive patients (92.8%) showed evidence of past or present HBV and/or HCV infection. Prevalence of HBV and HCV was 81.2% for both viruses, whereas prevalence of HBV/HCV coinfection was 69.6%. A significantly higher presence of HCV infection was found in Italians [odds ratio (OR) 11.0; interval 1.7–80.9] and in drug users (OR 27.8; interval 4.9–186.0). HCV subtypes were determined in 42 HCV or HBV/HCV-coinfected individuals. HCV subtypes 1a, 3a, 4d, and 1b were found in 42.9%, 40.5%, 14.3%, and 2.4% of inmates, respectively. Low titers of HBV DNA in HBV DNA positive subjects precluded HBV subtyping.The high prevalence of HBV and HCV coinfections in HIV-infected inmates, as well as the heterogeneity of HIV and HCV subtypes suggest the need to adopt systematic controls in prisons to monitor both the burden and the genetic forms of blood-borne viral infections, in order to apply targeted therapeutic interventions.

HCV dispersal patterns among intravenous drug users (IDUs) in Athens metropolitan area

BackgroundMost of the HCV transmission the recent years in Greece was among IDUs. Our aim was to estimate the prevalence of HCV genotypes and to investigate the patterns of HCV dispersal among IDUs in Athens using current state of the art molecular epidemiology methods. MethodsHCV sequences were determined from 238 HIV-negative IDUs collected on the basis of the “ARISTOTLE” prevention program carried out in Athens between 2012 and 2013. Phylogenetic trees were inferred on HCV sequences isolated from IDUs in Athens for the most prevalent HCV clades (subtypes 1a and 3a). Phylogenetic analysis was performed by Neighbor-Joining and Bayesian methods using GTR+G as nucleotide substitution model. HCV dispersal patterns were estimated using as references, all globally available HCV sequences for subtypes 1a and 3a. ResultsThe prevalence of HCV subtypes was: 3a (59.2%), 1a (21.9%), 4 (13.0%), 1b (5.4%) and 2 (0.5%). Phylogenetic analyses revealed that most sequences (63.5%) οf subtypes 1a and 3a fell within IDU-specific monophyletic groups. The proportion of sequences in monophyletic clades was similar for subtype 3a (62.9%) and 1a (65.3%). For the latter group, monophyletic clades were smaller in size. Multivariable logistic regression analyses showed that monophyletic clustering was marginally associated recent onset of injecting ([AOR]=1.44; 95% CI (0.97–2.13), p=0.068). ConclusionsThe high proportions of HCV sequences within IDU-specific monophyletic clusters suggest that transmissions occurred locally among IDUs in Greece. The numerous clusters for both 1a and 3a provide evidence that both sub-epidemics were the result of multiple introductions among the IDUs. Higher regional clustering was probably associated with a more recent onset of drug use.

HCV Diversity among Chinese and Burmese IDUs in Dehong, Yunnan, China

HCV transmission is closely associated with drug-trafficking routes in China. Dehong, a prefecture of Yunnan, is the important trade transfer station linking Southeast Asia and China, as well as the drug-trafficking channel linking “Golden triangle” and other regions of China and surrounding countries. In this study, we investigated the HCV genotype diversity among IDUs in Dehong based on 259 HCV positive samples from 118 Chinese and 141 Burmese IDUs. HCV genotypes were determined based on the phylogenies of C/E2 and NS5B genomic sequences. Six HCV subtypes, including 1a, 1b, 3a, 3b, 6n and 6u, were detected. Interestingly, 4 HCV sequences from Burmese IDUs did not cluster with any known HCV subtypes, but formed a well-supported independent clade in the phylogenetic trees of both C/E2 and NS5B, suggesting a potential new HCV subtype circulating in Dehong. Subtype 3b was the predominant subtype, followed by subtypes 6n and 6u. Comparison showed that Dehong had a unique pattern of HCV subtype distribution, obviously different from other regions of China. In particular, HCV subtypes 6u and the potential new HCV subtype had a relatively high prevalence in Dehong, but were rarely detected in other regions of China. There was no significant difference in HCV subtype distribution between Burmese and Chinese IDUs. Few HCV sequences from Burmese and Chinese IDUs clustered together to form transmission clusters. Furthermore, about half of HCV sequences from Burmese IDUs formed small transmission clusters, significantly higher than that from Chinese IDUs (p<0.01). These suggest that the Chinese and Burmese IDUs were relatively isolated from each other in injection drug use behavior and the Burmese IDUs might prefer to inject drugs themselves together. The unique genotype distribution and complex diversity of genotype 6 among IDUs may be associated with the special geographical position of Dehong.

Prevalence, determinants and genetic diversity of hepatitis C virus in the multi-ethnic population living in Suriname

Little is known about the epidemiology of HCV in Suriname, a former Dutch colony in South America. To study the prevalence, determinants and genetic diversity of HCV, a one-month survey was conducted at the only Emergency Department in the capital Paramaribo. Participants (≥18 years) completed an interviewer-led standardized HCV risk-factor questionnaire, were tested for HCV-antibodies, and if positive also for HCV RNA. The overall HCV prevalence was 1.0% (22/2128 participants; 95%CI 0.7–1.5). Male sex (OR=4.11; 95%CI 1.30–13.01), older age (OR=1.06 per year increase; 95%CI 1.04–1.09), Javanese ethnicity (OR=7.84; 95%CI 3.25–18.89) and cosmetic tattooing (OR=31.7; 95%CI 3.25–323.87) were independently associated with HCV-infection. Phylogenetic analysis revealed six distinct HCV subtypes, all HCV-genotype 2 (HCV-2): subtype 2f (also circulating in Indonesia) plus five yet unassigned HCV-2 subtypes exclusively linked to Suriname.

MULTI-YEAR DYNAMICS OF SPREAD OF HEPATITIS C VIRUS GENOTYPES IN MOSCOW REGION

Aim. Determine spread of hepatitis C vims genotypes, circulating in Moscow Region over the last decade. Materials and methods. The presence of HCV RNA, genotype and subtype of the vims were determined in blood sera samples obtained in 2006 - 2014 from 2847 individuals with the presence of HCV infection, who had or did not have injectable dmg administration in anamnesis. Results, lb and 3a remain the main subtypes, circulating in the population over the last decade. A notable reduction of la subtype circulation took place among injectable dmg users (IDU). Recombinant form RFl_2k/lb of hepatitis vims is present only among this risk group and constitutes 2% of the overall amount of subtypes in both 2007 and 2014. Genotype 4d was detected in 2014, that is not typical for Russian Federation. Genotype 3a is dominant in IDU age groups of 20 - 29 and older than 40, and in the 30 - 39 group the main - subtype lb. Phylogenetic analysis has shown the lack of certain genetic variants of subtypes lb and 3a, characteristic for IDU, that gives evidence on a wide circulation of the main subtypes of the vims in the whole population of individuals, infected by HCV. Conclusion. Spread of main genotypes/subtypes of HCV in the overall population and among IDU in Moscow remains stable over the last decade.

In-depth phylogenetic analysis of hepatitis C virus subtype 1a and occurrence of 80K and associated polymorphisms in the NS3 protease.

HCV genetic diversity is high and impacts disease progression, treatment and drug resistance. HCV subtype 1a is divided in two clades (I and II), and the 80 K natural polymorphism in the viral NS3 protease is prevalent in clade I. Paradoxically, countries dominated by this clade have contrasting frequencies of 80 K. Over 2,000 HCV 1a NS3 sequences were retrieved from public databases representing Europe, Oceania and the Americas. Sequences were aligned with HCV reference sequences and subjected to phylogenetic analysis to investigate the relative presence of different subtype 1a clades and NS3 protease mutations. HCV-1a sequences split into clades I and II. Clade I was further structured into three subclades, IA to C. Sub-clade IA prevailed in the U.S., while subclade IC was major in Brazil. The NS3 80 K polymorphism was associated with subclade IA, but nearly absent in subclades IB and IC, a pattern similarly seen for the 91S/T compensatory mutation. Three HCV-1a-I sub-clades have been identified, with different frequencies in distinct regions. The 80 K and 91A/S mutations were associated with subclade IA, which provide an explanation for the disparities seen in simeprevir resistance profiles of countries dominated by HCV 1a-I, like the U.S. and Brazil.

Mapping the genomic diversity of HCV subtypes 1a and 1b: Implications of structural and immunological constraints for vaccine and drug development.

Despite significant progress in hepatitis C (HCV) treatment, global viral eradication remains a challenge. An in-depth map of its genome diversity within the context of structural and immunological constraints could contribute to the design of pan-genotypic antivirals and preventive vaccines. For such analyses, extensive information is only available for the highly prevalent HCV genotypes (GT) 1a and 1b. Using 647 GT1a and 408 GT1b full-genome sequences obtained from the Los Alamos database, we found that respectively 3 per cent and 82 per cent of all codon positions are under positive and negative selective pressure, suggesting variation mainly accumulates due to random genetic drift. An association between conservation and both structured RNA and secondary protein structures confirmed the important role of structural elements at nucleotide and at amino acid level. Remarkably, CD8+ T-cell epitopes in HCV GT1a were significantly more conserved, while at the same time containing more sites under positive selection. Similarly, CD4+ T-cell epitopes were significantly more conserved in both HCV subtypes, but under less positive selective pressure in GT1b and more negative selective pressure in GT1a. In contrast, B-cell epitopes in both subtypes were less conserved and under less stringent negative selection. These findings argue against immune selective pressure as the main force of between-host diversifying evolution. Despite its high variability, HCV is under strict evolutionary constraints, most probably to keep its genes and proteins functional during the replication cycle. These are encouraging findings for vaccine and drug design, which could consider these newly established genetic diversity profiles.

Prevalence of occult hepatitis C virus infection in Iranian patients with beta thalassemia major.

Occult hepatitis C virus infection (OCI) is a new pathological form of chronic hepatitis virus (HCV) infection characterized by the presence of HCV RNA in liver biopsy and/or peripheral blood mononuclear cell (PBMC) specimens and the absence of HCV RNA and anti-HCV antibodies (Abs) in plasma samples. β-thalassemia major is a hereditary recessive blood disease with deficiency in the hemoglobin beta chain. Thalassemic patients need blood transfusion therapy; repeated blood transfusion increases the risk of viral blood-borne infection. The aim of this study was to determine the prevalence of OCI in Iranian patients with β-thalassemia major. From February 2015 to November 2015, a total of 147 Iranian patients with β-thalassemia major were enrolled in this cross-sectional study. After extraction of viral RNA from the plasma and PBMC samples, HCV genomic RNA in the specimens was amplified by RT-nested PCR using primers from the 5'-UTR. The HCV genotypes of the positive specimens were tested using the RFLP assay. To confirm the HCV genotypes, the 5'-UTR fragment was amplified and cloned into the pJET1.2/blunt cloning vector and then sequenced. Out of 147 patients, 106 (72.1%) were negative for anti-HCV Abs and HCV RNA. HCV RNA was found in PBMC specimens of six (5.7%) patients, from a total of 106 patients with undetectable plasma HCV RNA and anti-HCV Abs. Therefore, six out of 106 patients had OCI. HCV genotyping revealed that three patients were infected with HCV subtype 1b, two patients were infected with HCV subtype 3a, and one patient was infected with HCV subtype 1a. These results revealed that Iranian patients with beta-thalassemia major might have OCI. Therefore, it seems that the design of a study to identify this infection in patients with β-thalassemia major would provide valuable information.

The Structure of Hepatitis C Virus Genotypes/Subtypes in Adult Patients with Chronic Hepatitis C in the Moscow Region

The structure of HCV genotypes/subtypes and dynamics of its changes in a cohort of adult patients with chronic hepatitis C (n = 17229) was studied during 2008-2015 in the Moscow region. The prevalence of subtypes 1b and 3A HCV, whose relative density have made 47.5% (95%CI 46.8 - 48.3%) and 39.4% (95% CI 38.7 - 40.2 per cent) respectively was revealed. The average proportion of subtype 1A HCV was 5.4% (95%CI of 5.1 - 5.8%) and genotype 2 - 7.2% (95% CI 6,8 - 7,6%). It was established that the dynamics of 1b subtype HCV relative density was characterized by a moderate decline rate of 1.3% per year, while the proportion of subtype 3A HCV increased (+2.9% per year). The differences in the ratio of subtypes 1b and 3A HCV were revealed when dividing the patients by gender. The subtype 1b HCV was more frequently identified in women during the observation period. In the cohort of male patients a shift of the leading HCV subtype was detected - since 2010, the 3A subtype HCV was identified with a higher frequency than subtype 1b HCV. It was shown that in patients under 30 years the proportion of subtype 3A HCV was higher than in the age group older than 30 years, regardless of gender.

Using NS5B Sequencing for Hepatitis C Virus Genotyping Reveals Discordances with Commercial Platforms.

We aimed to evaluate the correct assignment of HCV genotypes by three commercial methods—Trugene HCV genotyping kit (Siemens), VERSANT HCV Genotype 2.0 assay (Siemens), and Real-Time HCV genotype II (Abbott)—compared to NS5B sequencing. We studied 327 clinical samples that carried representative HCV genotypes of the most frequent geno/subtypes in Spain. After commercial genotyping, the sequencing of a 367 bp fragment in the NS5B gene was used to assign genotypes. Major discrepancies were defined, e.g. differences in the assigned genotype by one of the three methods and NS5B sequencing, including misclassification of subtypes 1a and 1b. Minor discrepancies were considered when differences at subtype levels, other than 1a and 1b, were observed. The overall discordance with the reference method was 34% for Trugene and 15% for VERSANT HCV2.0. The Abbott assay correctly identified all 1a and 1b subtypes, but did not subtype all the 2, 3, 4 and 5 (34%) genotypes. Major discordances were found in 16% of cases for Trugene HCV, and the majority were 1b- to 1a-related discordances; major discordances were found for VERSANT HCV 2.0 in 6% of cases, which were all but one 1b to 1a cases. These results indicated that the Trugene assay especially, and to a lesser extent, Versant HCV 2.0, can fail to differentiate HCV subtypes 1a and 1b, and lead to critical errors in clinical practice for correctly using directly acting antiviral agents.

Frequency of Natural Resistance within NS5a Replication Complex Domain in Hepatitis C Genotypes 1a, 1b: Possible Implication of Subtype-Specific Resistance Selection in Multiple Direct Acting Antivirals Drugs Combination Treatment.

Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs) were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV) or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV) was higher in G1b compared to G1a (37/1552 (2.4%) in 1b sequences and 15/1209 (1.2%) in 1a isolates, p = 0.040). Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1%) G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5%) harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001). Finally, 28 (2.3%) G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001). In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs) targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs.