NS5B Region Research Articles

Unveiling the dynamics of hepatitisC virus transmission among injection drug users and men who have sex with men: A comprehensive study in Japan.

In Japan, despite low nationwide hepatitisC (HCV) incidence, new infections among people who inject drugs (PWID) and men who have sex with men (MSM) hinder HCV elimination. We explored HCV transmission dynamics and screened HCV recombination within these populations. This cross-sectional study recruited HCV-infected patients from Osaka National Hospital, Osaka, Japan, from January 2010 to September 2023. Data from questionnaires and medical records were analyzed. Serum samples collected before anti-HCV treatment underwent HCV RNA extraction, and sequencing of full core (576bp) and NS5B (267bp) regions using the Sanger method. Genotype distribution was determined by phylogenetic analysis, and recombinant screening was conducted. A total of 115 patients were categorized into non-MSM PWID (31), MSM PWID (15), MSM non-PWID (25), and non-MSM non-PWID (44). Positive amplification rates were 99.1% (114/115) for the full-core region, and 96.5% (111/115) for NS5B. No intergenotypic recombination was detected. The predominant genotype in non-MSM PWID was 2a (58%), whereas genotype 1b was most common in MSM PWID, MSM non-PWID, and non-MSM non-PWID groups (79%, 64%, and 68%, respectively). Nucleotide sequence similarity of 94.75%-100% was found in HCV strains from MSM PWID and MSM non-PWID in both full-core and NS5B regions, whereas strains from non-MSM PWID and non-MSM non-PWID were distinct. The findings suggest that the transmission route in PWID is determined by MSM status, whereas MSM groups showed the same transmission route regardless of PWID. HCV control measures should be focused not only on PWID, but also on MSM to achieve HCV elimination in Japan.

Hepatitis C virus genotypes among population with reported risk factors in Assam, north-east India: Emergence of genotype-8.

Background & objectives Hepatitis C virus (HCV) exhibits extensive genetic diversity in infected hosts. There are few published reports of HCV genotype (GT) distribution from the north-east Indian States lying close to the 'Golden Triangle' known for illicit drug trafficking. Real-time knowledge of HCVGT distribution is important for studies on epidemiologic aspects and virus evolution and for the development of new target-specific, direct-acting antiviral drugs. This study aims to examine the distribution of HCVGTs and their subtypes in different risk groups from Assam, north-east India. Methods It is a hospital-based cross-sectional study. Plasma samples reactive for anti-HCV antibody in enzyme-linked immunosorbent assay (ELISA) were subjected to viral load test and genotyping by real-time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) or characterization of non-structural protein NS5B region by nested PCR. Nucleotide sequences were subjected to phylogenetic analysis. Results The most common HCVGT detected was GT-3 (95.89%), followed by GT-1 (3.42%), GT-6xa (0.34%) and GT-8 (0.34%). The mean age of subjects was 30.24 yr, and males outnumbered females. The most commonly associated risk factor was injecting drug use (IDU) (74.31%), followed by tattooing and/or piercing (33.22%), transfusion of blood/blood products (10.27%), and haemodialysis (9.25%). Co-infection with human immunodeficiency virus (HIV) was found in 17.8 per cent, and with Hepatitis B virus (HBV) in 3.42 per cent of the cases. Interpretation & conclusions The detection of HCVGT-8 makes this the first report from Assam and the second from India as per the authors' knowledge. This may indicate strain's endemic nature in India. The increasing trend of HCV infection among young IDUs and HCV-HIV co-infection indicates the need for enhancing surveillance and intensified prevention efforts among young adults.

Comprehensive survey of conserved RNA secondary structures in full-genome alignment of Hepatitis C virus

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large ‘cloud’ of RNA genomes (quasispecies) which—by trial and error—comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

HCV drug resistance mutations in HIV-infected patients

The aim of our work was to assess prevalence in the HCV drug resistance mutations in the NS3, NS5A, NS5B genes in HIV-infected patients. The material of the study was 157 blood plasma samples collected from HIV patients living in the Leningrad Region, with virological inefficiency of antiretroviral therapy. Samples were examined for the presence of anti-HCV antibodies and HCV RNA using commercial test systems in accordance with the manufacturer’s recommendations. In the case of detecting HCV RNA, amplification reaction was carried out using a set of primers co-flanking the NS3, NS5A, and NS5B genes. After sequencing the nucleotide sequences of the above genes, the virus subtype was determined. To assess HCV drug resistance mutations to direct-acting antiviral agents (DAAs), the Geno2pheno HCV resistance software (https:// hcv.geno2pheno.org) was used. The age of the patients varied from 18 to 65 and averaged 34.3±7.27 years. The number of males vs females prevailed comprising 71% and 28%, respectively. Antibodies against HCV were detected in 94.2% cases, whereas HCV RNA — in 98 (61.7%) HIV-infected persons. The distribution of HCV genotypes in this group was as follows: 1a — 7% (n = 7), 1b — 53% (n = 52), 2 — 2% (n = 2), 3a — 38% (n = 37). The results of determining the viral load varied from 5.3 × 103 to 2.3 × 108 IU/ml. The nucleotide sequence of all three regions NS3, NS5A, NS5B was determined in 73 samples. Analysis of drug resistance mutations in the NS3 region was performed for 82 HCV isolates of genotypes 1b (n = 46), 3a (n = 31), 1a (n = 4), 2 (n = 1). In total, the proportion of strains containing mutations in the NS3 region was 9.8%. The presence of significant amino acid substitutions in the NS5A region was tested for 83 HCV isolates of genotypes 1b (n = 47), 3a (n = 30), 1a (n = 6). The percentage of strains containing mutations in the NS5A region was 19.3%. Resistance mutations in the NS5B region were analysed for 87 HCV isolates of genotypes 1b (n = 48), 3a (n = 32), 2 (n = 2), 1a (n = 5). The proportion of strains containing mutations in the NS5B region was 11.5%. In the study group, mutations associated with hepatitis C virus resistance to DAAs in all regions were found in 16.6% (95% CI: 11.11–23.32%) cases (n = 26). In the NS3, NS5A and NS5B, there have been identified 6, 15 and 10 significant amino acid substitutions, respectively.

Distribution of hepatitis С virus drug resistance mutations among patients with recurrence of the disease during therapy with direct antiviral drugs

The aim of the study was to identify the prevalence of drug resistance mutations in the hepatitis C virus among patients with relapse of the disease on therapy with direct antiviral drugs.Materials and methods. The study material included 31 blood plasma samples from patients with chronic hepatitis C with relapse of the disease on therapy with direct antiviral drugs. Samples were screened for the presence of HCV RNA. In case of detection of HCV RNA, amplification was carried out using a set of primers jointly flanking the NS3, NS5A, NS5B genes. After sequencing the nucleotide sequences of these genes, the subtype of the virus was determined and drug resistance mutations were identified.Results and discussion. The age of the patients ranged from 33 to 62 and averaged 45.8±8.38 years. The number of men in the group prevailed compared to women — 21 (67%) and 10 (33%), respectively. Viral load determination results ranged from 3.1×103 to 4.2×107 IU/ml. The distribution of genotypes was as follows: 1a — 26% (n=8), 1b — 29% (n=9), 3a — 45% (n=14). The nucleotide sequence of the NS3, NS5A, NS5B regions was determined in all samples. Mutations associated with drug resistance were detected in 87% (n=27). In all identified cases, the mutations resulted in viral resistance to at least one drug included in the patient’s current treatment regimen. In one patient, amino acid substitutions were found in three regions at once, which led to the emergence of resistance to two drugs in the regimen.Conclusion. Conducting a preliminary examination of patients to identify mutations of drug resistance to direct antiviral drugs can affect the effectiveness of the planned treatment and the choice of the optimal regimen.

Hepatitis C: A Review on Current and Emerging Genotyping Assays

Hepatitis C is a global public health concern that infects millions of people worldwide. The continual discovery of new genotypes and subtypes of hepatitis C virus (HCV) is an indication of a persistent molecular evolution of the virus. This remains a concern in the efforts towards hepatitis C elimination, as effective management of the disease is, in part, dependent on the HCV genotype responsible for the infection. Accurate HCV screening and quantification using rapid but highly sensitive and reliable methods are crucial for the diagnosis and subsequent management of HCV-related diseases. Thus, this article discusses HCV and the common methods employed for HCV detection and genotyping. While nucleotide sequencing and phylogenetic analysis of core/E1 and NS5B region are regarded as the gold standard and the most recommended method used for HCV genotyping, electrochemical sensors are being explored for their rapidity.

Molecular characterization of patients with chronic hepatitis C virus infection in Jordan: implications on response to direct-acting antiviral therapy

ObjectivesTo investigate the molecular characteristics of Hepatitis C Virus (HCV) detected in patients with chronic HCV infection in Jordan. MethodsThe study included 48 Jordanian treatment-naïve patients with active chronic HCV recruited from seven governorates. HCV genotype and the resistance-associated substitutions (RAS) profile were investigated by next-generation sequencing of the NS5B, NS5A, and NS3 regions of HCV. Results“Unusual genotype 4 subtypes” were detected in four (8.3%) patients (4n-n = 1, 4o-n = 2, 4v-n = 1); one patient (2.1%) was co-infected by genotypes 1b+4a. Overall prevalence of NS5A RASs was 38.3% (3% cutoff); genotype 4a showed the highest NS5A RAS prevalence (n = 11, 55.0%). Overall prevalence of NS3 RASs was 21.8% (7/32), all genotype 1a-infected patients. ConclusionsWe report, for the first time in Jordanian patients with chronic HCV infection, the detection of unusual genotype 4 subtypes 4n, 4o, and 4v. Baseline RASs in NS5A are frequent, with complex RASs patterns in some of the unusual subtypes. Our data support the need for sequencing surveillance programs in sub-Saharan Africa, Asia, and the Middle East and North African region to monitor response to treatment in these subtypes and to facilitate the World Health Organization's 2030 elimination strategy.

Study of drug resistance-associated genetic mutations, and phylo-genetic analysis of HCV in the Province of Sindh, Pakistan

Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. The aim of the current study was to identify such mutations in the NS3, and NS5B genes in DAAs treatment-naïve Pakistani chronic HCV 3a patients. Peripheral blood samples were collected from 233 chronic HCV 3a patients at different tertiary care hospitals in Karachi, Pakistan, between August 2020 to September 2021. PCR-amplified target regions of the NS3/NS5B gene were subjected to Sanger sequencing to identify resistance-associated mutations. Phylogenetic analysis of the identified amino acid sequences was performed using HCV3a sequences of the global population in the virus pathogen resource (VIPR) database. Sequence analysis identified five amino acid mutations, Leu36Pro, Gln41His, Gln80Lys/Arg, Ala156Tyr, and Gln168Arg in the NS3 region, and two mutations Leu159Phe and Cys316Arg in the NS5B region. Phylogenetic analysis revealed a high genetic diversity in the studied isolates. Overall, the prevalence of resistance-associated substitutions was almost similar to other geographic regions worldwide. This data could be helpful in selecting the most effective treatment regimen for HCV chronically infected people in Pakistan.

High-Resolution Genomic Profiling of a Genotype 3b Hepatitis C Virus from a Flare of an Occult Hepatitis Patient with Acute-on-Chronic Liver Failure

Acute-on-chronic liver failure (ACLF) is defined as a syndrome of acutely decompensated cirrhosis in patients with chronic liver disease (CLD). Here we report an ACLF case caused by a flare of occult hepatitis C infection. This patient was infected with hepatitis C virus (HCV) more than a decade ago and hospitalized due to alcohol-associated CLD. Upon admission, the HCV RNA in the serum was negative and the anti-HCV antibody was positive, whereas the viral RNA in the plasma dramatically increased during hospitalization, which suggests an occult hepatitis C infection. Overlapped fragments encompassing the nearly whole HCV viral genome were amplified, cloned, and sequenced. Phylogenetic analysis indicated an HCV genotype 3b strain. Sanger sequencing to 10-fold coverage of the 9.4-kb nearly whole genome reveals high diversity of viral quasispecies, an indicator of chronic infection. Inherent resistance-associated substitutions (RASs) in the NS3 and NS5A but not in the NS5B regions were identified. The patient developed liver failure and accepted liver transplantation, followed by direct-acting antiviral (DAA) treatment. The hepatitis C was cured by the DAA treatment despite the existence of RASs. Thus, care should be taken for occult hepatitis C in patients with alcoholic cirrhosis. The analysis of viral genetic diversity may help to identify an occult hepatitis C virus infection and predict the efficacy of anti-viral treatment.

Healthcare-Related HCV Genotype 4d Infections in Kayseri, Turkey.

The frequency of genotype 4 hepatitis C virus infection is significantly higher in a city compared to other provinces in Turkey. In this study, we aimed to investigate the epidemiology and risk factors of hepatitis C virus genotype 4 infection in Kayseri province of Turkey. A case-control study was conducted with 61 hepatitis C virus genotype 4-infected patients and 71 controls. A questionnaire was administered to the patients and controls, asking for information about the risk factors of hepatitis C virus transmission. Core/ E1 and NS5B regions of hepatitis C virus genome were amplified and sequenced by Sanger method. Phylogenetic analysis and molecular clock analysis were performed. The risk was determined by calculating the odds ratio and 95% CI. Logistic regression analysis was performed to determine the effect of risk factors by controlling for confounding variables. Kayseri isolates were closely related to type 4d sequences but formed a separate cluster. According to the molecular clock analysis, hepatitis C virus genotype 4d entered Kayseri province probably between 1941 and 1988. Blood transfusion and surgical intervention were found to be significant risk factors for the infection. Epidemiological data showed that hepatitis C virus genotype 4d infections are significantly associated with unsafe medical procedures.

Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction(PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia.

Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia.

Transmission networks of hepatitis C virus among HIV/HCV-coinfected patients in Guangdong, China

BackgroundCoinfection with hepatitis C virus (HCV) is common in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients due to shared routes of transmission. We aimed to investigate the characteristics of HCV subgenotypes among HIV/HCV-coinfected patients in Guangdong and explore the molecular transmission networks and related risk factors for HCV strains.MethodsPlasma samples were obtained from 356 HIV/HCV-coinfected patients for HCV NS5B region sequencing. A neighbor-joining phylogenetic tree was constructed to affirm HCV subgenotypes. The transmission networks based on maximum likelihood phylogenetic tree were determined by Cluster Picker, and visualized using Cytoscape 3.2.1.ResultsA total of 302 HCV NS5B sequences were successfully amplified and sequenced from the 356 plasma samples. A neighbor-joining phylogenetic tree based on the 302 NS5B sequences revealed the profile of HCV subgenotypes circulating among HIV/HCV coinfection patients in Guangdong. Two predominant strains were found to be 6a (58.28%, 176/302) and 1b (18.54%, 56/302), followed by 3a (10.93%, 33/302), 3b (6.95%, 21/302), 1a (3.64%, 11/302), 2a (0.99%, 3/302) and 6n (0.66%, 2/302). A molecular transmission network of five major HCV genotypes was constructed, with a clustering rate of 44.04%. The clustering rates of subgenotypes 1a, 3a, 3b, 1b, and 6a were 18.18% (2/11), 42.42%, 52.38%, 48.21%, and 44.89%, respectively. Multivariate logistic regression analysis showed no significant effects from sex, age, transmission route, geographical region, baseline CD4 + T cell count or subgenotype (P > 0.05), except marital status. Married or cohabiting people (compared with unmarried people) had more difficulty forming transmission networks.ConclusionsIn summary, this study, based on HCV NS5B subgenotypes, revealed the HCV subtype diversity and distribution among HIV/HCV-coinfected patients in Guangdong. Marital status inclined to be the factor influencing HCV transmission networks formation.

Genetic diversity of hepatitis C virus and resistance associated substitutions to direct-acting antiviral treatment in Colombia

Hepatitis C virus (HCV) infection is one of the leading risk factors for end-stage liver disease development worldwide. This RNA virus displays high genetic diversity with 8 genotypes and 96 subgenotypes with heterogeneous geographical distribution around the world. In this study, we carried out an active case finding of individuals with a history of transfusion events before 1996 in three cities in Colombia. Then, the characterization of the HCV genotypes, subgenotypes, and resistance associate substitutions (RAS) was performed in samples positives for antibodies anti-HCV + from this study population. In addition, samples from PWID and patients with end-stage liver disease submitted to liver transplantation were included in the phylogenetic and RAS analysis. The 5′UTR, NS5A, and NS5B regions of the HCV genome were amplified in serum or liver explants samples. After the edition, assembly, and alignment of the sequences, genotyping through phylogenetic analysis was performed using IQTREE V2.0.5 based on the maximum likelihood approach. The identification of RAS was carried out by alignments based on the reference sequence (GenBank NC_004102). Two hundred sixty individuals with blood transfusion events before 1996 were recruited. The seroprevalence of antibodies anti-HCV was 2.69% in this population. The HCV genotypes 1, 2, and 4 and subgenotypes 1a, 1b, 2a, 4a and 4d were characterized in samples of the study populations. Three RAS (Q30R, C316N, and Y93H) were identified in samples obtained from 2 individuals who received blood transfusion before 1996 and without previous antiviral treatment and 6 samples obtained from patients with end-stage liver disease. Among the 20 samples analyzed, the HCV genotype 1, subgenotype 1b, was the most frequent (60%). We report the first characterization of HCV subgenotypes 4a and 4d and the first RAS identification in patients in Colombia.

Treatment Response and Drug Resistance Profiling of Genotype 6 of Hepatitis C Virus in HCV/HIV Co-Infected Patients: A Pilot Study from INDIA.

Hepatitis C Virus (HCV) genotype (GT) 6 demonstrates maximum genomic diversity out of all the known genotypes of HCV, attributable to its inherent intra-genotype and inter-genotype recombination property. This is the most common genotype seen in HCV/HIV co-infected cases. HIV/HCV co-infection is linked with increased genetic diversity in HCV structural genes. The detailed information on the distribution of HCV GT6, its subtypes, and resistance to currently available antiviral drugs is limited in the Indian subcontinent. Therefore, in this single-center retrospective cross-sectional study, we aimed to map the occurrence of HCV GT6, its subtypes and resistance-associated substitution (RAS), and its correlation with antiviral treatment response in HCV-infected patients. From a cohort of 2052 HCV-infected patients, the overall prevalence of GT6 was 2.5% (n = 53), with a maximum of 81.1% (n = 43) seen in HCV/HIV co-infected patients. Nine different subtypes, 6a, 6b, 6f, 6i, 6n, 6u, 6v, 6w, and 6xa, were detected in the Indian population for the first time, with a predominance of 6xa (41.5%), a rare subtype, followed by 6n (39.6%). The phylogenetic analysis by the neighbor-joining method revealed three prominent viral clades, 6v, 6n, and 6xa–6u. The baseline (before treatment initiation) plasma samples of all GT6-infected patients were retrieved from −80 °C and a part of the NS5a and NS5b region of the viral genome was analyzed for the presence of RAS. No RASs were seen in the NS5b region, while in two patients (3.7%) RASs were seen at baseline in the NS5a region of the virus. Sustained viral response (SVR) was attained in 81% (n = 43) of patients. No difference in GT6 subtype distribution or occurrence of RAS was seen between mono-infected HCV and HIV/HCV co-infected cases. Our study revealed that RAS at baseline did not influence the attainment of SVR and the currently available antiviral therapy is effective against GT6 mono-infected and HIV/HCV co-infected patients.

Emergence and Persistence of Resistance-Associated Substitutions in HCV GT3 Patients Failing Direct-Acting Antivirals.

Background: The hepatitis C virus has a high mutation rate, which results in the emergence of resistance-associated substitutions (RASs). Despite direct-acting antivirals (DAAs) efforts to treat chronically infected HCV genotype 3 (GT3) patients, there are concerns about the emergence and persistence of RASs in DAA failures. The objective of this study was to determine the prevalence of clinically relevant RASs in HCV NS5A and NS5B regions before and after treatment to better understand the role of RASs in treatment failures. Methods: Viral RNA was extracted before and after treatment from serum samples. NS5A and NS5B regions of HCV were amplified by nested PCR, followed by Sanger sequencing. The nucleotide sequences were aligned against HCV GT3 reference sequences, and amino acid substitutions were analyzed using the geno2pheno [hcv] webserver. Results: A total of 76 patients failing DAA therapy were stratified from the cohort of 1388. RASs were detected at the baseline in 15/76 patients and at relapse in 20/76 patients with cirrhosis and previously treated with interferons. The most prevalent NS5A RAS was Y93H found in all treatment-failing patients (14/54 in DCV vs. 6/22 in VEL), followed by A62S/T and A30K. No RASs were identified in NS5B. RASs that were present at the baseline persisted through the 24-week follow-up period and were enriched with emerging RASs during the treatment. The presence of RASs may be one of the causes of treatment failures in 26.3% of patients. Amino acid substitutions were present at the baseline in most of the patients with RASs against NS5A inhibitors. Patients with the baseline Y93H and/or A30K relapse more frequently than patients harboring A62S/T. Conclusion: Treatment-failing patients harbored NS5A RASs, and the most frequent were A30K (5/20), A62S/T (20/20), and Y93H (20/20). Direct resistance testing is recommended for optimizing re-treatment strategies in treatment-failing patients.

Phylogenetic Analysis of Spread of Hepatitis C Virus Identified during HIV Outbreak Investigation, Unnao, India.

An HIV outbreak investigation during 2017–2018 in Unnao District, Uttar Pradesh, India, unearthed high prevalence of hepatitis C virus (HCV) antibodies among the study participants. We investigated these HCV infections by analyzing NS5B and core regions. We observed no correlation between HIV–HCV viral loads and clustering of HCV sequences, regardless of HIV serostatus. All HCV isolates belonged to genotype 3a. Monophyletic clustering of isolates in NS5B phylogeny indicates emergence of the outbreak from a single isolate or its closely related descendants. The nucleotide substitution rate for NS5B was 6 × 10−3 and for core was 2 × 10−3 substitutions/site/year. Estimated time to most recent common ancestor of these isolates was 2012, aligning with the timeline of this outbreak, which might be attributable to unsafe injection practices while seeking healthcare. HIV–HCV co-infection underlines the need for integrated testing, surveillance, strengthening of healthcare systems, community empowerment, and molecular analyses as pragmatic public health tools.

Simultaneous determination of HCV genotype and NS5B resistance associated substitutions using dried serum spots from São Paulo state, Brazil.

Hepatitis C virus (HCV) is responsible for more than 180 million infections worldwide, and about 80 % of infections are reported in Low and Middle-income countries (LMICs). Therapy is based on the administration of interferon (INF), ribavirin (RBV) or more recently Direct-Acting Antivirals (DAAs). However, amino acid substitutions associated with resistance (RAS) have been extensively described and can contribute to treatment failure, and diagnosis of RAS requires considerable infrastructure, not always locally available. Dried serum spots (DSS) sampling is an alternative specimen collection method, which embeds drops of serum onto filter paper to be transported by posting to a centralized laboratory. Here, we assessed feasibility of genotypic analysis of HCV from DSS in a cohort of 80 patients from São Paulo state Brazil. HCV RNA was detected on DSS specimens in 83 % of samples of HCV infected patients. HCV genotypes 1a, 1b, 2a, 2c and 3a were determined using the sequence of the palm domain of NS5B region, and RAS C316N/Y, Q309R and V321I were identified in HCV 1b samples. Concerning therapy outcome, 75 % of the patients who used INF +RBV as a previous protocol of treatment did not respond to DAAs, and 25 % were end-of-treatment responders. It suggests that therapy with INF plus RBV may contribute for non-response to a second therapeutic protocol with DAAs. One patient that presented RAS (V321I) was classified as non-responder, and combination of RAS C316N and Q309R does not necessarily imply in resistance to treatment in this cohort of patients. Data presented herein highlights the relevance of studying circulating variants for a better understanding of HCV variability and resistance to the therapy. Furthermore, the feasibility of carrying out genotyping and RAS phenotyping analysis by using DSS card for the potential of informing future treatment interventions could be relevant to overcome the limitations of processing samples in several location worldwide, especially in LMICs.

Characterization of primary direct-acting antiviral (DAA) drugs resistance mutations in NS5A/NS5B regions of hepatitis C virus with genotype 1a and 1b from patients with chronic hepatitis.

ABSTRACTThe Hepatitis C virus (HCV) infection is a public health problem. The high level of HCV replication and its lack of post-transcriptional correction mechanisms results in the emergence of viral variants and the difficulty in determining polymorphisms and variants that contain the substitutions associated with resistance towards new antivirals. The main focus of this study was to map the NS5A and NS5B polymorphisms and resistance mutations to new antiviral drugs in HCV strains genotype 1 from patients with chronic hepatitis C infection. Serum samples were collected from patients who underwent routine viral load tests at the Instituto Adolfo Lutz, Sao Paulo city, Brazil. A total of 698 and 853 samples were used for the characterization of NS5A and NS5B regions, respectively, which comprise the HCV genotypes 1a and 1b. The prevalence of resistance mutations found in the NS5A region was 6.4%, with Y93H, L31M, Q30R, and Y93N as the main resistance-associated substitutions (RAS). No NS5B-associated RAS was observed for any of the analyzed drugs. These findings support that the RAS test should be offered to individuals with poor response to double combination regimens prior to treatment initiation, thereby assisting strain vigilance and selection of effective treatment or retreatment options using DAA regimens.

Frequency distribution of HCV resistance-associated variants in infected patients treated with direct-acting antivirals

Hepatitis C virus (HCV) is a global public health problem. Second-generation direct-acting antivirals targeting non-structural regions on the viral genome are the cornerstone for treatment of chronic infection. However, resistance-associated variants (RAVs) have been reported to be associated with therapeutic failure. The aim of this study was to assess the frequency of variants, including RAVs, in the NS3, NS5A and NS5B regions at baseline in Brazilian patients with chronic hepatitis C with HCV genotypes 1a, 1b and 3a. Serum samples from 13 patients were used to obtain viral RNA. Massively parallel sequencing was performed using genotype-specific amplicons and a panel of Ampliseq technology for all genotypes. Several non-synonymous substitutions were detected at baseline for 11 responders and pre-/post-treatment for two non-responders. HCV genotype 3a was found to have significantly more non-synonymous substitutions than HCV genotype 1 in the NS3 and NS5A regions. Analyses were conducted using quantitative and qualitative inter- and intrapatient comparisons. Variants that confer resistance to the treatment used by the patients were found in both responders and non-responders. A wide frequency distribution of RAVs was found at baseline, and this did not interfere with the achievement of a sustained response. Evaluation of the presence of RAVs requires additional study in order to determine clinical relevance.