Hepatitis C Virus Isolates Research Articles

Predominant amino acid substitutions in NS5B gene of hepatitis C virus in blood donors and treatment-naïve hepatitis and HIV patients in Nigeria

Background: Hepatitis C virus (HCV) genome undergoes high rate of mutation, which results in generation of genetically diverse HCV isolates. There is paucity of data on mutations in the nonstructural 5b (NS5b) gene of circulating HCV and their implications in the Nigerian population. Here, we identified clinically-important mutations in HCV isolates, which may influence response to therapy and disease prognosis.
 Methodology: HCV RNA was extracted from a total of 301 blood samples collected from 99 symptomatic treatment-naïve hepatitis patients, 125 HIV-infected individuals and 77 asymptomatic blood donors in Ibadan, Nigeria. The RNA was reverse–transcribed to complimentary DNA and HCV NS5B gene amplified by nested PCR. The amplified products of 42 HCV were sequenced and sequences were aligned with those from GenBank and HCV databases in MEGA 7.0. Nucleotide sequences were translated to amino acids while substitutions in the amino acids were analyzed with reference to H77 prototype strain of HCV.
 Results: A total of 10 amino acid polymorphisms were observed from the 42 sequenced NS5B gene, with the major clinically-important amino acid mutations being S15G in 28 (66.7%) participants, T7N (24, 57.1%), G61R (23, 54.8%), S54L (22, 52.4%), G89E (14, 33.3%), T79M (12, 28.6%), and T711 (11, 26.2%). Others were Q67R (7, 16.7%), Q47H (7, 16.7%) and S84F (2, 4.8%). S15G/A/V mutations were more predominant in patients with HIV (76.9%, 10/13) followed by patients with clinical hepatitis (75.0%, 12/16) and blood donors (46.1%, 6/13). Q67R and T71I mutations were not predominant in patients with clinical hepatitis as they were detected in only 31.3% (5/16) and 43.8% (7/16) participants respectively, compared to S15G (75.0%, 12/16), S54L (68.8%, 11/16), G61R/E (68.8%, 11/16) and T7N/S (56.3%, 9/16). There was no statistically significant difference in the distribution of each of the 10 amino acid polymorphisms detected within patients with symptomatic clinical hepatitis (x2=9.311, p=0.409), HIV-infected patients (x2=13.431, p=0.1440) and asymp- tomatic blood donors (x2=3.775, p=0.9256). Similarly, there was no significant difference in the distribution between the 3 categories of the study participants except for T79M mutation, which was significantly higher in HIV-infected patients (61.5%, 8/13) compared to patients with clinical hepatitis (18.8%, 3/16) and asymp- tomatic blood donors (7.7%, 1/13) (x2=10.456, p=0.0054).
 Conclusion: Mutations in the NS5B gene could be associated with worse prognosis of the disease or antiviral failure due to viral resistance in patients undergoing therapy. The absence of Q47H mutations in majority of the study participants in our study implies that they will not respond well to daprevir and mericitabine. Screening of patients for pre-existing resistant mutations before commencement of therapy and monitoring during and after therapy are recommended.

Hepatitis C virus infects and perturbs liver stem cells.

The hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression.

Incidence and molecular epidemiology of hepatitis C virus reinfection in prisons in Catalonia, Spain (Re-HCV study)

Hepatitis C virus (HCV) reinfection may hamper HCV elimination in prisons. We aimed to (i) determine the reinfection rate in people treated for HCV in Catalan prisons, (ii) measure reinfection in people entering prisons, and (iii) characterize the molecular epidemiology of HCV in prisons and people who inject drugs (PWID) in the community. Re-HCV was a prospective study in eight prisons (2019–2020) including two groups: (1) people cured with treatment in prison and followed-up every 6 months, and (2) people testing HCV-RNA positive at incarceration. Bio-behavioral data were collected. HCV isolates were sequenced and phylogenetically analyzed with those of PWID in the community. Reinfection follow-up after treatment was achieved in 97 individuals (103.05 person-years). Two reinfections were detected, resulting in an incidence ≤ 10/100 person-years. Among people entering prison, 2% (359/17,732) were viremic, of which 334 (93.0%) were included, and 44 (13.5%) presented with reinfection (84.7% being PWID). Frequently, HCV isolates in prisons and PWID in the community were phylogenetically related. Although HCV reinfection is low after treatment, it is common in people entering Catalan prisons. To maintain a low HCV prevalence in prisons, harm-reduction services and test-and-treat programs for PWID should be strengthened both inside and outside prisons.

Optimization of the pseudoparticle system for standardized assessments of neutralizing antibodies against hepatitis C virus.

A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.

A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

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.

Safety and efficacy of sofosbuvir-velpatasvir to treat chronic hepatitis C virus infection in treatment-naive patients in Rwanda (SHARED-3): a single-arm trial

Hepatitis C virus (HCV) genotype 4 is the predominant type of HCV found in sub-Saharan Africa. Various genotype 4 subtypes, such as 4r, frequently have resistance-associated substitutions that can increase rates of treatment failure with common direct-acting antiviral regimens. In-vitro studies suggest that the NS5A inhibitor velpatasvir is effective against viral isolates containing such resistance-associated substitutions, but its clinical efficacy against genotype 4 non-a/d subtypes in sub-Saharan Africa remains to be confirmed. We aimed to evaluate the safety and efficacy of sofosbuvir-velpatasvir among adults chronically infected with HCV and naive to direct-acting antiviral treatment in Rwanda, where genotype 4 non-a/d subtypes predominate. In this single-arm prospective trial, we enrolled adults (age ≥18 years) in Rwanda who had chronic HCV infection and a plasma HCV RNA titre of at least 1000 IU/mL. Patients were referred from hospitals with HCV treatment programmes throughout Rwanda and were sequentially enrolled and assessed for eligibility at a single study site. Individuals with decompensated liver disease or hepatitis B virus co-infection were excluded. Participants were given an oral fixed-dose combination tablet of sofosbuvir (400 mg) and velpatasvir (100 mg) once-daily for 12 weeks. The primary endpoint was the proportion of participants with a sustained virological response 12 weeks after completion of treatment (SVR12) in the intention-to-treat population. Viral sequencing of the NS5A and NS5B genes was done at baseline for all participants and end of follow-up (week 24) for participants who did not have SVR12. This study is registered with ClinicalTrials.gov (NCT03888729) and is completed. Between Sept 23, 2019, and Jan 10, 2020, 73 individuals were screened for eligibility, of whom 12 (16%) were excluded and 61 (84%) were enrolled. 40 (66%) participants were female, 21 (34%) were male, median age was 64 years (IQR 51-74), and median baseline HCV viral load was 5·7 log10 IU/mL (5·2-6·2). The genotypes identified among the participants were 4k (28 [46%] participants), 4r (11 [18%]), 4v (eight [13%]), 4q (five [8%]), 4l (three [5%]), 4b (one [2%]), 4c (one [2%]), and one undetermined genotype 4 subtype. Three isolates could not be sequenced and were of indeterminate genotype. Of the 55 HCV isolates that were successfully sequenced, all had at least two NS5A resistance-associated substitutions. 59 (97% [95% CI 89-99]) participants had SVR12. 18 (30%) participants had grade 3 adverse events (including 12 [20%] with hypertension), and none had grade 4 adverse events. Four (7%) participants had serious adverse events, including one asthma exacerbation, one abscess, one uterine myoma, and one pelvic fracture related to a motor vehicle accident. No serious adverse events were attributed to the study drug and no adverse event resulted in discontinuation of the study drug. A 12-week regimen of sofosbuvir-velpatasvir is safe and efficacious in treating chronic HCV genotype 4 infection in patients in Rwanda. This regimen could be an effective treatment option in regions known to have a high prevalence of HCV genotype 4 of diverse non-a/d subtypes. Gilead Sciences.

Simultaneous Hepatitis C Virus Genotyping and Variant Detection in Patients with Thalassemia: A Single-Center Phylogenetic Study.

BACKGROUND: Hepatitis C virus (HCV) genotype distribution is different in various regions. A variety of strategies could be used to detect HCV genotypes and subtypes. The aim of the present study was to introduce a genotyping method by an in-house protocol that could be used to determine HCV drug-resistant variants and phylogeny studies. METHODS: Samples from 91 patients with thalassemia were used for HCV genotyping by Cobas 4800 platform, and 50 cases of 1a, 1b, and 3a genotypes underwent amplification and sequencing of NS5A and NS5B by using consensus primers via conventional reverse transcription-polymerase chain reaction (RT-PCR) method. An ABI 3730xl system used for direct sequencing. Raw sequences were analyzed by popular bioinformatics software MEGA6 and CLC workbench 5. Phylogenetic construction was drawn using 1000 replicates bootstrap by the neighbor-joining method. Multiple sequence alignment (MSA) was performed for mutation detection. RESULTS: Sequencing results of 50 HCV isolates subtypes 1a (31/45), 3a (15/22) and 1b (4/8) NS5A and NS5B genes showed there were 72 NS5A and 105 NS5B mutations. Moreover, 8 resistant associated substitutions (RASs) were identified in nine thalassemia cases by multiple sequence alignment (MSA) protein analysis. The phylogenetic tree construct drew confirmed by the Cobas HCV genotyping results. CONCLUSION: The phylogenetic analysis could be a useful tool for HCV genotyping in case of determining the drug-resistant substitutions; however, it is time-consuming and needs expert analysis and interpretation. This preliminary study in Iranian patients with thalassemia introduces specific conventional RT-PCR to find RASs to direct acting antivirals (DAAs) and subtype determination at the same time.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development

Background & AimsDevelopment of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays.MethodsWe chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps.ResultsBoth the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones.ConclusionsNeutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Haplotype Network Analysis and Phylogenetic Tree Construction of Hepatitis C Virus (HCV) Isolated from Tuban, Indonesia

This study aimed to analyzed the haplotype network and constructed the phylogenetic tree topology of hepatitis C virus (HCV) in Tuban, Indonesia and those from other countries, to determine the visualize genealogical relationship and inference about gene connected. The HCV isolates were collected from blood transfusion center of Tuban, East Java, Indonesia in 2015 and as a comparison, also the sequences of HCV isolates were retrieved from the GenBank®, National Center of Biotechnology Information (NCBI), USA database. To constructed of distribution map was performed through median joining analysis using Haplotype Network v4.6, whereas to constructed and phylogenetic model analyses were constructed based on the NS5B and 5’UTR regions using MEGA X, maximum-likelihood method based on the Tamura-Nei model. In the HCV NS5B region haplotype network showed high haplotype diversity (Hd=1.00) in 22 haplotypes. Based on phylogenetic analysis 3 sample isolates Tuban (BDT-55-p23, BDT-79-p23 and BDT-112-p23) were identified one group and closely related to isolates from Indonesia. However, isolates Tuban also have the same sequence characters as the isolates from Thailand. It was also confirmed in haplotype network that the three isolates are connected to each other. The identification of HCV genotypes circulating in blood donors in the Tuban of East Java confirmed were closely related to HCV isolates from Indonesia and Thailand. Therefore, this result might contribute in a better medical management towards HCV.

Development of cell culture infectious clones for hepatitis C virus genotype 1b and transcription analysis of 1b-infected hepatoma cells

Globally, hepatitis C virus (HCV) genotype 1b is the most prevalent, and its infection has been found to associate with a higher risk of hepatocellular carcinoma (HCC) than other genotype viruses. However, an efficient infectious HCV genotype 1b culture system is unavailable, which has largely hampered the study of this important genotype virus. In this study, by using a systematic approach combining the sequences of infectious 1a TNcc clone and adaptive mutations, we succeeded in culture adaption of two full-length 1b clones for the reference strain Con1 and a clinical isolate A6, and designated as Con1cc and A6cc, respectively. Con1cc and A6cc replicated efficiently in hepatoma Huh7.5.1 cells, released HCV infectivity titers of 104.1 and 103.72 focus forming units per milliliter, respectively, and maintained the engineered mutations after passages. Both viruses responded to sofosbuvir and velpatasvir in a dose-dependent manner. With culture infectious 1b clones, we characterized the transcriptomes of 1b Con1cc-infected cells, in comparison with 2a-infected and uninfected cells. In conclusion, we have developed two infectious clones for genotype 1b and shown a novel strategy for culture adaptation of HCV isolates by using a genetically close backbone sequence. Furthermore, this study provides transcriptional landscape of HCV 1b-infected hepatoma cells facilitating the study of genotype 1b infection.

Development of Cell Culture Infectious Hepatitis C Virus Genotype 1b Clones and 1b-Induced Angiogenesis and Cholesterol Disorders

Globally, hepatitis C virus (HCV) genotype 1b is most prevalent, and its infection has been found to associate with a higher risk of hepatocellular carcinoma (HCC) than other genotype viruses. However, an efficient infectious HCV genotype 1b culture system is unavailable, which has largely hampered the study of this important genotype virus. In this study, by using a systematic approach combining adaptive mutations and infectious 1a TNcc sequences, we succeeded in culture adaption of two full-length 1b clones for the reference strain Con1 and a clinical isolate A6, and designated as Con1cc and A6cc, respectively. Con1cc and A6cc replicated efficiently in hepatoma Huh7.5.1 cells, released HCV infectivity titers of 104.1 and 103.72 focus forming units per milliliter, respectively, and maintained the engineered mutations after passages. Both viruses responded to sofosbuvir and velpatasvir in a dose-dependent manner. With culture infectious 1b clones, we characterized the transcriptomes of 1b Con1cc-infected cells. Con1cc infection activated angiogenesis and disturbed cholesterol homeostasis compared with uninfected cells. In conclusion, we have developed infectious clones for genotype 1b and shown a novel strategy for culture adaptation of HCV isolates by using a genetically close backbone sequence. Besides, this study provides transcriptional landscape of HCV 1b-infected hepatoma cells facilitating the study of genotype 1b infection. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 81971938 for Y.-P.L.; 81772923 for Y.Z.), The Innovation Research Team for Basic and Clinical Studies on Chronic Liver Diseases of 2018 High-Level Health Teams of Zhuhai (for Y.-P.L. and F.X.), National Key Basic Research Program of China (No. 2015CB554301 for Y.-P.L.), Entrepreneurial Talent Team Award of Guangdong Province (No. 2016ZT06S252 for Y.-P.L.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The use of patient serum was approved by the Medical Ethics Committee at the Zhongshan School of Medicine, Sun Yat-sen University (No. 2014-072).

Virological surveillance, molecular phylogeny, and evolutionary dynamics of hepatitis C virus subtypes 1a and 4a isolates in patients from Saudi Arabia.

Hepatitis C virus (HCV) subtypes are pre-requisite to predict endemicity, epidemiology, clinical pathogenesis, diagnosis, and treatment of chronic hepatitis C infection. HCV genotypes 4 and 1 are the most prevalent in Saudi Arabia, however; less consensus data exist on circulating HCV subtypes in infected individuals. This study was aimed to demonstrate the virological surveillance, phylogenetic analysis, and evolutionary relationship of HCV genotypes 4 and 1 subtypes in the Saudi population with the rest of the world. Fifty-five clinical specimens from different parts of the country were analyzed based on 5′ untranslated region (5′ UTR) amplification, direct sequencing, and for molecular evolutionary genetic analysis. Pair-wise comparison and multiple sequence alignment were performed to determine the nucleotide conservation, nucleotide variation, and positional mutations within the sequenced isolates. The evolutionary relationship of sequenced HCV isolates with referenced HCV strains from the rest of the world was established by computing pairwise genetic distances and generating phylogenetic trees. Twelve new sequences were submitted to GenBank, NCBI database. The results revealed that HCV subtype 4a is more prevalent preceded by 1a in the Saudi population. Molecular phylogeny predicts the descendants’ relationship of subtype 4a isolates very close to Egyptian prototype HCV strains, while 1a isolates were homogeneous and clustering to the European and North American genetic lineages. The implications of this study highlight the importance of HCV subtyping as an indispensable tool to monitor the distribution of viral strains, to determine the risk factors of infection prevalence, and to investigate clinical differences of treatment outcomes among intergenotypic and intragenotypic isolates in the treated population.

Antibody Responses to Immunization With HCV Envelope Glycoproteins as a Baseline for B-Cell–Based Vaccine Development

We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice. We analyzed 519 serum samples from participants in a phase 1 vaccine trial(ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n= 28) and rhesus macaques (n= 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized. Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human VH1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs. In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.

High Prevalence of Hepatitis C Virus Infection in Primitive Tribes of Eastern India and Associated Sociobehavioral Risks for Transmission: A Retrospective Analysis.

Purpose: The primitive tribal groups (PTGs) need special attention because of their low population growth: declining population size with high mortality rates. Scanty reports are available on the prevalence of hepatitis C virus (HCV) infection in primitive tribes of the country emphasizing their cultural and social practices associated with transmission of the disease.Methods: The study was conducted on 1765 tribal individuals covering 5 PTGs, namely Lodha, Saora, Khadia, Juanga, and Mankidia, from 6 districts of Odisha. Serum samples were tested for the anti-HCV antibody using commercially available enzyme immunoassays. HCV RNA was detected based on the 5′ NCR region and genotyping was done by direct sequencing of the core region. Potential risk factors for HCV transmission were collected using a questionnaire and subjected to regression analysis through SPSS, version 17.0.Results: Antibody to HCV was detected in 0%, 3.3%, 5.7%, 8.5%, and 13.4% in Saora, Lodha, Khadia, Juanga, and Mankidia tribes, respectively. HCV RNA was detected in 8.6% (11/127) of the samples tested. Genotyping of HCV isolates in all HCV RNA-positive samples revealed genotype 1b. Sharing of razors and shaving by the village barber were found to be significantly (p<0.05) associated with HCV transmission in males, whereas tattooing and multiple injections were found to be significant risk factors for females.Conclusion: This study indicated a high prevalence of HCV infection in Mankidia and Juanga tribes in comparison with the national scenario, which needs public health attention.

Core encoding sequences of Hepatitis C virus in Ghanaian blood donors are predominantly mosaics of different genotype 2 strains and cannot distinguish subtypes

BackgroundDistribution of Hepatitis C virus (HCV) genotypes varies significantly worldwide. Genomic diversity between genotypes has implications for treatment, vaccine development and optimal design of HCV diagnostic assays. Molecular characterization of HCV in different geographical areas is therefore very essential for management and public health control of HCV infection. This study investigated the molecular epidemiology and characteristics of HCV genotypes in healthy individuals in Accra, Ghana.MethodsAn experimental study was carried out on blood samples obtained from voluntary blood donors. Two hundred samples were initially screened for HCV antibodies and infection was confirmed by RNA detection through RT-PCR of the 5′-untranslated region (5’UTR). The core gene sequences were analysed for HCV genotype determination by genotype-specific PCR; and then by cloning and direct sequencing followed by phylogenetic analysis. The sequences were further analysed in detail by similarity plotting.ResultsMolecular diagnosis confirmed the presence of HCV RNA in 2 out of 200 (1%) blood donors. Initial genotyping by genotype-specific PCR identified all two infections as subtypes 2a and 2b of genotype 2. Extensive evolutionary and genetic analyses indicated two epidemiological profiles. First, phylogenetic tree topologies clearly showed that, collectively, the core sequences of the Ghanaian HCV isolates belong to a single, distinct genetic group within HCV genotype 2 cluster, with high genetic similarity and rapid sequence variation in a single individual. Second, the sequences are mosaics comprising 2e and other genotype 2 subtype fragments. The analyses underscore a unique and complex HCV genotype 2 core sequence profile of the Ghanaian isolates.ConclusionsAnalysis of HCV core encoding sequences from Ghanaian blood donors in Accra confirmed predominance of genotype 2 HCV among healthy individuals. However, the isolates could not be classified into subtypes, possibly due to their complex sequence pattern that might suggest high mutability of the prevailing genotype. The core region of Ghanaian HCV therefore may not be suitable for distinguishing subtypes. These findings extend those from previous studies and thus underscore the need to search for subtype-informative region of Ghanaian HCV to elucidate the genetic diversity and factors determining outcome of HCV infections in Ghana.

Phylogenetic analysis of hepatitis C virus among HIV/ HCV co-infected patients in Nigeria.

Hepatitis C virus (HCV) infection has been associated with liver disease including liver cirrhosis and hepatocellular carcinoma (HCC) in chronically-infected persons. However, in HIV/HCV co-infected patients, increased rate of progression to cirrhosis and HCC has been reported. Limited information exists regarding genetic variants of HCV circulating among co-infected patients, which could be important in the design of broadly protective vaccine and management of the disease. Here, we determined the genotypes of HCV isolates circulating among HIV/HCV co-infected patients in Ibadan, southwestern Nigeria. One hundred and twenty-five HIV/HCV IgM positive samples obtained from HIV laboratory, University of Ibadan were used for this study. HCV NS5B gene was amplified using polymerase chain reaction (PCR). The amplified NS5B gene was sequenced using gene specific primers. Twenty isolates were amplified, out of which 13 were successfully sequenced. Phylogenetic analysis of the 13 sequenced isolates showed three HCV subtypes 1a, 3a and 5a belonging to genotypes 1, 3 and 5 respectively. Ten isolates (77%) belong to subtype 5a, followed by 2 isolates (15%) subtype 1a and 1 isolate (8%) was subtype 3a. The predominant HCV genotype was 5, followed by genotype 1 (subtype 1a). The findings, as well as the observed mutations in NS5B gene, indicate the need for screening and monitoring of HIV/HCV co-infected patients. Further study to determine the phylogeny of isolates circulating in other parts of Nigeria will be carried out.

In Vitro Neutralization Assay Using Cultured Hepatitis C Virus.

The method outlined here enables evaluation of the neutralization potency of monoclonal and polyclonal antibodies against in vitro cultured hepatitis C virus (HCV). The high variation in envelope protein sequence among HCV isolates necessitates the inclusion of several isolates, spanning the major genotypes of HCV, in order to make strong conclusions concerning the cross-reactive neutralization potential of a given antibody. This would be particularly relevant for any neutralization experiments aimed at uncovering novel therapeutic- or vaccine-relevant antibodies. In addition, these assays can also be used to compare neutralization sensitivity of novel cultured HCV to that of previously characterized isolates.

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5′UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5′UTR and NS3-3′UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5′UTR and 3′UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated “CH6acc”), releasing HCV of 104.3–104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.

SEC14L2, a lipid-binding protein, regulates HCV replication in culture with inter- and intra-genotype variations

The lipid-binding protein, SEC14L2, is crucial for the efficient viral replication of clinical hepatitis C virus (HCV) isolates in cell culture. Given the role of SEC14L2 in HCV replication, we aimed to study a large number of HCV positive sera carrying genotypes 1-4, to identify viral factors associated with efficient replication in culture. Additionally, we investigated whether 13 single nucleotide polymorphisms (SNPs) of SEC14L2 have an impact on RNA replication of naturally occurring HCV isolates. We generated Huh-7.5 cell lines overexpressing SEC14L2 or 13 coding SNPs and tested 73 different HCV positive sera for in vitro replication. Furthermore, we genotyped a cohort of 262 patients with chronic HCV for the common SNP (rs757660) and investigated its effect on the clinical phenotype. HCV isolates from genotype 1, 2, 3 and 4 replicate in Huh-7.5 cells overexpressing SEC14L2. Interestingly, only subgenomic replicons from genotypes 1 and 3 showed enhanced replication whereas genotypes 2 and 4 remained unaffected. Furthermore, replication was independent of viral load. Importantly, all tested SNPs supported HCV RNA replication in vitro, while 1 SNP was associated with decreased SEC1L2 expression and viral RNA. All SNPs exhibited comparable cellular cholesterol and vitamin E abundance in naïve Huh-7.5 cells. This large screen of natural HCV isolates of 4 genotypes underscores the relevance of SEC14L2 as an in vitro HCV host factor. Additionally, SEC14L2 variants appear to recapitulate the wild-type enhancement of HCV replication. Variant rs191341134 showed a decreased effect due to lowered stability, whereas variant rs757660, a high prevalence mutant, showed a similar phenotype to the wild-type. Until the year 2015, consistent replication of patient-derived isolates of hepatitis C virus (HCV) in an in vitro model remained a limitation in HCV research. In 2015 a group of authors identified a protein named SEC14L2 that enabled the replication of HCV isolates in cell culture. We performed a large screen encompassing 73 isolates of 4 different HCV genotypes. Additionally, we replaced the natural SEC14L2 with 13 different mutants to test if the protein variation significantly altered its HCV replication enhancing functions. We showed that different genotypes of HCV react differently to the presence of this protein and the variants of the protein mimic the behavior of the wild-type.