Hepatitis C Virus Sequences Research Articles

Distribution pattern, molecular transmission networks, and phylodynamic of hepatitis C virus in China.

In China, few molecular epidemiological data on hepatitis C virus (HCV) are available and all previous studies were limited by small sample sizes or specific population characteristics. Here, we report characterization of the epidemic history and transmission dynamics of HCV strains in China. We included HCV sequences of individuals belonging to three HCV surveillance programs: 1) patients diagnosed with HIV infection at the Beijing HIV laboratory network, most of whom were people who inject drugs and former paid blood donors, 2) men who have sex with men, and 3) the general population. We also used publicly available HCV sequences sampled in China in our study. In total, we obtained 1,603 Ns5b and 865 C/E2 sequences from 1,811 individuals. The most common HCV strains were subtypes 1b (29.1%), 3b (25.5%) and 3a (15.1%). In transmission network analysis, factors independently associated with clustering included the region (OR: 0.37, 95% CI: 0.19-0.71), infection subtype (OR: 0.23, 95% CI: 0.1-0.52), and sampling period (OR: 0.43, 95% CI: 0.27-0.68). The history of the major HCV subtypes was complex, which coincided with some important sociomedical events in China. Of note, five of eight HCV subtype (1a, 1b, 2a, 3a, and 3b), which constituted 81.8% HCV strains genotyped in our study, showed a tendency towards decline in the effective population size during the past decade until present, which is a good omen for the goal of eliminating HCV by 2030 in China.

Hepatitis C Virus in people with experience of injection drug use following their displacement to Southern Ukraine before 2020

BackgroundDue to practical challenges associated with genetic sequencing in low-resource environments, the burden of hepatitis C virus (HCV) in forcibly displaced people is understudied. We examined the use of field applicable HCV sequencing methods and phylogenetic analysis to determine HCV transmission dynamics in internally displaced people who inject drugs (IDPWID) in Ukraine.MethodsIn this cross-sectional study, we used modified respondent-driven sampling to recruit IDPWID who were displaced to Odesa, Ukraine, before 2020. We generated partial and near full length genome (NFLG) HCV sequences using Oxford Nanopore Technology (ONT) MinION in a simulated field environment. Maximum likelihood and Bayesian methods were used to establish phylodynamic relationships.ResultsBetween June and September 2020, we collected epidemiological data and whole blood samples from 164 IDPWID (PNAS Nexus.2023;2(3):pgad008). Rapid testing (Wondfo® One Step HCV; Wondfo® One Step HIV1/2) identified an anti-HCV seroprevalence of 67.7%, and 31.1% of participants tested positive for both anti-HCV and HIV. We generated 57 partial or NFLG HCV sequences and identified eight transmission clusters, of which at least two originated within a year and a half post-displacement.ConclusionsLocally generated genomic data and phylogenetic analysis in rapidly changing low-resource environments, such as those faced by forcibly displaced people, can help inform effective public health strategies. For example, evidence of HCV transmission clusters originating soon after displacement highlights the importance of implementing urgent preventive interventions in ongoing situations of forced displacement.

Hepatitis C virus transmission between eight high-income countries among men who have sex with men: a whole-genome analysis

Microelimination of the hepatitis C virus (HCV) among men who have sex with men (MSM) could be complicated by continuous external introductions and the emergence of phylogenetic clusters harbouring clinically significant resistance-associated substitutions (RAS). To investigate international clustering and the prevalence and transmission of RAS, we aimed to analyse whole-genome HCV sequences from MSM with a recently acquired infection who participated in a large, international HCV treatment trial. For this whole-genome analysis, we obtained HCV sequences from 128 MSM who had acquired HCV within the past 12 months and were participating in the REACT trial. The participants from whom sequences were obtained were recruited at 24 sites in eight countries. We inferred maximum-likelihood phylogenies and identified transmission clusters for HCV genotypes separately. We constructed time-scaled phylogenies to estimate cluster introduction dates and used a Bayesian Skygrid approach to estimate the effective population size over the past 50 years. We calculated the prevalence of RAS and the extent of RAS transmission in the study population. The majority of recent HCV infections were part of international networks that arose in the late 1990s and early 2000s. Sequences obtained in the same country clustered frequently, and in 36% of subclusters since 2015 we found evidence of international transmission. European MSM were more likely than non-European MSM to be in a cluster (odds ratio 11·9 [95% CI 3·6-43·4], p<0·0001). The effective population size decreased rapidly since around 2015 in Europe. RAS associated with substantially diminished cure rates were infrequently detected and transmission of highly resistant viruses was not observed. Despite antiviral treatment becoming widely available, international transmission of HCV among MSM has still occurred over the past 8 years, which could complicate microelimination of the virus in this population. RAS-enriched clusters and widespread RAS transmission are currently not a threat to elimination goals. These findings support an international approach for HCV microelimination among MSM. National Institutes of Health and Dr. C.J. Vaillant Fonds.

Sub-genotypes of hepatitis C virus in the Middle East and North Africa: Patterns of distribution and temporal changes.

Infection with the hepatitis C virus (HCV) remains a considerable public health concern in the Middle East and North Africa (MENA). The objectives of this study were to analyze the HCV genotype (GT) and sub-genotype (SGT) distribution in the MENA region and to assess the temporal change in the number of sequences within the MENA region. All HCV molecular sequences collected in the MENA region had been retrieved from GenBank as of 1 August 2022. The number of HCV sequences retrieved was 6740 representing sequences from a total of 17 MENA countries with a majority from Iran (n=1969, 29.2%), Egypt (n=1591, 23.6%), Tunisia (n=1305, 19.4%) and Saudi Arabia (n=1085, 16.1%). The determination of GT/SGT was based on the NCBI genotyping and Blast tool. Genotype 1 (GT1) dominated infections in the MENA (n=2777, 41.2%), followed by GT4 (n=2566, 39.0%). Additionally, SGT4a (1515/6393, 23.7%) was the most common SGT in the MENA, and SGT4a was dominant in Egypt and Saudi Arabia, followed by SGT1b (n=1308, 20.5%), which was dominant in Morocco and Tunisia, while SGT1a (n=1275, 19.9%) was common in Iran, Iraq and Palestine. Furthermore, significant temporal increase in the number of HCV MENA sequences was observed. On the SGT level, specific patterns of HCV genetic diversity were seen in the MENA region, with the most common SGT being 4a, in addition to increasing the availability of HCV sequences in the MENA region.

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.

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.

A clinical and molecular epidemiological survey of hepatitis C in Blantyre, Malawi, suggests a historic mechanism of transmission.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. There are no previous representative community HCV prevalence studies from Southern Africa, and limited genotypic data. Epidemiological data are required to inform an effective public health response. We conducted a household census‐based random sampling serological survey, and a prospective hospital‐based study of patients with cirrhosis and hepatocellular carcinoma (HCC) in Blantyre, Malawi. We tested participants with an HCV antigen/antibody ELISA (Monolisa, Bio‐Rad), confirmed with PCR (GeneXpert, Cepheid) and used line immunoassay (Inno‐LIA, Fujiribio) for RNA‐negative participants. We did target‐enrichment whole‐genome HCV sequencing (NextSeq, Illumina). Among 96,386 censused individuals, we randomly selected 1661 people aged ≥16 years. Population‐standardized HCV RNA prevalence was 0.2% (95% CI 0.1–0.5). Among 236 patients with cirrhosis and HCC, HCV RNA prevalence was 1.9% and 5.0%, respectively. Mapping showed that HCV RNA+ patients were from peri‐urban areas surrounding Blantyre. Community and hospital HCV RNA+ participants were older than comparator HCV RNA‐negative populations (median 53 vs 30 years for community, p = 0.01 and 68 vs 40 years for cirrhosis/HCC, p < 0.001). Endemic HCV genotypes (n = 10) were 4v (50%), 4r (30%) and 4w (10%). In this first census‐based community serological study in Southern Africa, HCV was uncommon in the general population, was centred on peri‐urban regions and was attributable for <5% of liver disease. HCV infection was observed only among older people, suggesting a historic mechanism of transmission. Genotype 4r, which has been associated with treatment failure with ledipasvir and daclatasvir, is endemic.

Do patients at high risk for Hepatitis C receive recommended testing? A retrospective cohort study of statewide Medicaid claims linked with OneFlorida clinical data.

Hepatitis C virus (HCV) infection is a leading risk factor for hepatocellular carcinoma.We employed a retrospective cohort study design and analyzed 2012–2018 Medicaid claims linked with electronic health records data from the OneFlorida Data Trust, a statewide data repository containing electronic health records data for 15.07 million Floridians from 11 health care systems. Only adult patients at high-risk for HCV (n = 30,113), defined by diagnosis of: HIV/AIDS (20%), substance use disorder (64%), or sexually transmitted infections (22%) were included. Logistic regression examined factors associated with meeting the recommended sequence of HCV testing.Overall, 44.1% received an HCV test. The odds of receiving an initial test were significantly higher for pregnant females (odds ratio [OR]1.99; 95% confidence interval [CI] 1.86–2.12; P < .001) and increased with age (OR 1.01; 95% CI 1.00–1.01; P < .001).Among patients with low Charlson comorbidity index (CCI = 1), non-Hispanic (NH) black patients (OR 0.86; 95% CI 0.81–0.9; P < .001) had lower odds of getting an HCV test; however, NH black patients with CCI = 10 had higher odds (OR 1.41; 95% CI 1.21–1.66; P < .001) of receiving a test. Of those who tested negative during initial testing, 17% received a second recommended test after 6 to 24 months. Medicaid-Medicare dual eligible patients, those with high CCI (OR 1.14; 95% CI 1.11–1.17; P < .001), NH blacks (OR 1.93; 95% CI 1.61–2.32; P < .001), and Hispanics (OR 1.49; 95% CI 1.08–2.06; P = .02) were significantly more likely to have received a second HCV test, while pregnant females (OR 0.71; 95% CI 0.57–0.89; P = .003), had lower odds of receiving it. The majority of patients who tested positive during the initial test (97%) received subsequent testing.We observed suboptimal adherence to the recommended HCV testing among high-risk patients underscoring the need for tailored interventions aimed at successfully navigating high-risk individuals through the HCV screening process. Future interventional studies targeting multilevel factors, including patients, clinicians and health systems are needed to increase HCV screening rates for high-risk populations.

Longer Poly(U) Stretches in the 3'UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo.

The 3′ untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5′ and 3′ RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5′UTR and 177 nucleotides of the 3′UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3′UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3′-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45–4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.

Prevalence and Predominant Genotype of Hepatitis C Virus Infection and Associated Risk Factors among Pregnant Women in Iran.

Objective Knowledge regarding the prevalence and risk factors of hepatitis C virus (HCV) infection among pregnant women can give clue to health care providers regarding the appropriate management of HCV infection. Therefore, this study was conducted to determine the prevalence, genotypic pattern, and risk factors of HCV infection among pregnant women in the northern shores of the Persian Gulf, south of Iran. Methods From January 2018 to June 2019, serum samples were obtained from 1425 pregnant women, ages ranging from 14 to 46 years (28.1 ± 5.99). Serum samples were tested for detection of anti-HCV antibodies using an enzyme-linked immunosorbent assay (ELISA) (HCV Ab ELISA kit, Dia.Pro, Milan, Italy). Following the extraction of nucleic acid, the molecular evaluation of HCV infection was performed by seminested reverse transcriptase-polymerase chain reaction assay (RT-PCR), targeting the 5′ untranslated region (5′UTR) and core of HCV genome and sequencing. Results Of the 1425 pregnant women, 19 women (1.33%, 95% CI: 0.85%–2.07%) were positive for anti-HCV antibodies. The majority of HCV-seropositive women were in the third trimester of pregnancy, educated, and had a history of blood transfusion, abortion, surgery, or dentistry. Moreover, Arab and Fars pregnant women and those aged >39 years had the highest rate of HCV seroprevalence. Nevertheless, none of these variables were significantly associated with HCV seropositivity. In contrast, HCV seropositivity was associated with place of residency, so that residents of Khormuj city had significantly higher HCV seroprevalence compared to the residents of other cities (OR: 7.05; 95% CI: 1.75–28.39; P = 0.006). According to the molecular evaluation, 9 of the 19 HCV-seropositive pregnant women (47.37%) had HCV viremia with genotype 3a. Conclusion This study reports the HCV prevalence of 1.33% for anti-HCV antibodies and 0.63% for HCV RNA among pregnant women in the south of Iran. Considering the asymptomatic nature of chronic HCV infection and the fact that vertical transmission is possible in women with detectable viremia, therefore, screening of women before pregnancy is recommended to reduce the risk of HCV infection and its complications during pregnancy.

Assessing hepatitis C virus distribution among vulnerable populations in London using whole genome sequencing: results from the TB-REACH study

Background: Injecting drugs substantially increases the risk of hepatitis C virus (HCV) infection and is common in vulnerable population groups, such as the homeless and prisoners. Capturing accurate data on relative genotype distribution within these groups is essential to inform strategies to reduce HCV transmission. The aim of this study was to utilise a next-generation whole-genome sequencing method recently validated by Public Health England, in order to produce near complete HCV genomes. Methods: In total, 98 HCV positive patients were recruited from homeless hostels and drug treatment services through the National Health Services (NHS) Find and Treat (F&amp;T) Service between May 2011 and June 2013 in London, UK. Samples were sequenced by Next-generation sequencing, with 88 complete HCV genomes constructed by a de novo assembly pipeline. They were analysed phylogenetically for an estimate of their genetic distance. Results: Of the 88 complete HCV genomes, 50/88 (56.8%) were genotype 1; 32/88 (36.4%) genotype 3; 4/88 (4.5%) genotype 2; and 1/88 (1.1%) for genotypes 4 and 6 each. Subtype 1a had the highest number of samples (51.1%), followed by subtype 3a (35.2%), 1b (5.7%), and 2b (3.4%). Samples collected from drug treatment services had the highest number of genotype 1 (69%); genotypes 4 and 6 were only found from samples collected in homeless shelters. Small clusters of highly related genomic sequences were observed both across and within the vulnerable groups sampled. Conclusions: Subsequent phylogenetic analysis provides a first indication that there are related HCV sequences amongst the three vulnerable population groups, reflecting their overlapping social behaviours. This study is the first presentation of whole genome HCV sequences from such vulnerable groups in London and paves the way for similar research in the future.

Identification of Genetically Related HCV Infections Among Self-Described Injecting Partnerships.

The current opioid epidemic across the United States has fueled a surge in the rate of new hepatitis C virus (HCV) infections among young persons who inject drugs (PWIDs). Paramount to interrupting transmission is targeting these high-risk populations and understanding the underlying network structures facilitating transmission within these communities. Deep sequencing data were obtained for 52 participants from 32 injecting partnerships enrolled in the U-Find-Out (UFO) Partner Study, which is a prospective study of self-described injecting dyad partnerships from a large community-based study of HCV infection in young adult PWIDs from San Francisco. Phylogenetically linked transmission events were identified using traditional genetic-distance measures and viral deep sequence phylogenies reconstructed to determine the statistical support of inferences and the direction of transmission within partnerships. Using deep sequencing data, we found that 12 of 32 partnerships were genetically similar and clustered. Three additional phylogenetic clusters were found describing novel putative transmission links outside of the injecting relationship. Transmission direction was inferred correctly for 5 partnerships with the incorrect transmission direction inferred in more than 50% of cases. Notably, we observed that phylogenetic linkage was most often associated with a lower number of network partners and involvement in a sexual relationship. Deep sequencing of HCV among self-described injecting partnerships demonstrates that the majority of transmission events originate from outside of the injecting partnership. Furthermore, these findings caution that phylogenetic methods may be unable to routinely infer the direction of transmission among PWIDs especially when transmission events occur in rapid succession within high-risk networks.

Preparation of nuclease-resistant RNA standard for hepatitis C virus quantification

Positive control (or standard) is an indispensable ingredient in molecular biology assays widely used for the quantification of nucleic acid. The commonly used standards are plasmid DNA, cDNA, or naked RNA, which are unstable and easily degraded by nucleases in the surrounding environment; this might affect the accuracy of quantitative results. In this study, the authors designed and created a positive control for the hepatitis C virus (HCV) quantification based on armored RNA technology. The 5’UTR non-encoding sequence of HCV was cloned into the BH20 plasmid. Armored RNA HCV (AR-HCV) was induced for expression in the E. coli BL21 (DE3) by the addition of an IPTG inducer. AR-HCV was collected by sucrose density gradient ultracentrifugation followed by gel filtration chromatography using Superdex 75 column. Created AR-HCV was determined the concentration and examined the formation of pseudo viral particles by transmission electron microscopy (TEM). Stability assessment of AR-HCV to DNase and RNase treatment simultaneously has demonstrated its ability to resist these nucleases. Moreover, AR-HCV is stable over time and storage conditions. Strikingly, AR-HCV can be directly added to the specimen, allowing better and more accurate control of the whole quantitative procedure of HCV.

HCV Diagnosis and Sequencing Using Dried Blood Spots from Patients in Kinshasa (DRC): A Tool to Achieve WHO 2030 Targets.

The World Health Organization has established an elimination plan for hepatitis C virus (HCV) by 2030. In Sub-Saharan Africa (SSA) access to diagnostic tools is limited, and a number of genotype 4 subtypes have been shown to be resistant to some direct-acting antivirals (DAAs). This study aims to analyze diagnostic assays for HCV based on dried blood spots (DBS) specimens collected in Kinshasa and to characterize genetic diversity of the virus within a group of mainly HIV positive patients. HCV antibody detection was performed on 107 DBS samples with Vidas® anti-HCV and Elecsys anti-HCV II, and on 31 samples with INNO-LIA HCV. Twenty-six samples were subjected to molecular detection. NS3, NS5A, and NS5B regions from 11 HCV viremic patients were sequenced. HCV seroprevalence was 12.2% (72% with detectable HCV RNA). Both Elecsys Anti-HCV and INNO-LIA HCV were highly sensitive and specific, whereas Vidas® anti-HCV lacked full sensitivity and specificity when DBS sample was used. NS5B/NS5A/NS3 sequencing revealed exclusively GT4 isolates (50% subtype 4r, 30% 4c and 20% 4k). All 4r strains harbored NS5A resistance-associated substitutions (RAS) at positions 28, 30, and 31, but no NS3 RAS was detected. Elecsys Anti-HCV and INNO-LIA HCV are reliable methods to detect HCV antibodies using DBS. HCV subtype 4r was the most prevalent among our patients. RASs found in subtype 4r in NS5A region confer unknown susceptibility to DAA.

Phylogenetic Analysis of NS5B Region of Hepatitis C Genotype 3a Virus among Different Risk Groups in Iran

Background: Hepatitis C virus (HCV) is a major cause of liver diseases. Transmission of HCV, as a blood-borne virus, is a major concern for the safety of blood products. In Iran, intravenous drug abuse is a major risk factor for HCV transmission. Recently a change in the distribution of HCV genotype among Iranian blood donors has been reported, and genotype 3a is reported as the most frequent genotype. Also, genotype 3a is the dominant genotype among IDUs. Objective: To investigate the association between HCV genotype 3a circulating in the community, sequences of HCV genotype 3a were analyzed among different risk groups in Iran. Methods: In this cross-sectional study, a partial sequence of the NS5B region of the HCV genome was isolated from 58 blood donors, 48 intravenous drug users (IDUs), and 31 patients with inherited bleeding disorders (IBDs) infected with HCV genotype 3a were included. The published sequence data were obtained from the Gen Bank database and were compared to construct the phylogenetic trees. Results: Of 58 blood donors infected with subtype 3a, 31 (53.5%) reported the risk of intravenous drug abuse. In the phylogenetic tree, no separate cluster was formed to differentiate between HCV sequences in blood donors and IDUs. A cluster at the middle level was found in the phylogenetic tree formed by sequences from blood donors, IDUs, and patients with inherited bleeding disorders. Conclusions: The phylogenetic tree showed the phylogenetic mixing of genotype 3a sequences in BDs, IBD patients, and IDUs. The results confirmed the extensive transmission of IDU-related genotype 3a towards the general population.

Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

Hepatitis C Virus Transmission Clusters in Public Health and Correctional Settings, Wisconsin, USA, 2016-20171.

Ending the hepatitis C virus (HCV) epidemic requires stopping transmission among networks of persons who inject drugs. Identifying transmission networks by using genomic epidemiology may inform community responses that can quickly interrupt transmission. We retrospectively identified HCV RNA–positive specimens corresponding to 459 persons in settings that use the state laboratory, including correctional facilities and syringe services programs, in Wisconsin, USA, during 2016–2017. We conducted next-generation sequencing of HCV and analyzed it for phylogenetic linkage by using the Centers for Disease Control and Prevention Global Hepatitis Outbreak Surveillance Technology platform. Analysis showed that 126 persons were linked across 42 clusters. Phylogenetic clustering was higher in rural communities and associated with female sex and younger age among rural residents. These data highlight that HCV transmission could be reduced by expanding molecular-based surveillance strategies to rural communities affected by the opioid crisis.

Mutations Identified in the Hepatitis C Virus (HCV) Polymerase of Patients with Chronic HCV Treated with Ribavirin Cause Resistance and Affect Viral Replication Fidelity.

Ribavirin has been used for 25 years to treat patients with chronic hepatitis C virus (HCV) infection; however, its antiviral mechanism of action remains unclear. Here, we studied virus evolution in a subset of samples from a randomized 24-week trial of ribavirin monotherapy versus placebo in chronic HCV patients, as well as the viral resistance mechanisms of the observed ribavirin-associated mutations in cell culture. Thus, we performed next-generation sequencing of the full-length coding sequences of HCV recovered from patients at weeks 0, 12, 20, 32 and 40 and analyzed novel single nucleotide polymorphisms (SNPs), diversity, and mutation-linkage. At week 20, increased genetic diversity was observed in 5 ribavirin-treated compared to 4 placebo-treated HCV patients due to new synonymous SNPs, particularly G-to-A and C-to-U ribavirin-associated transitions. Moreover, emergence of 14 nonsynonymous SNPs in HCV nonstructural 5B (NS5B) occurred in treated patients, but not in placebo controls. Most substitutions located close to the NS5B polymerase nucleotide entry site. Linkage analysis showed that putative resistance mutations were found in the majority of genomes in ribavirin-treated patients. Identified NS5B mutations from genotype 3a patients were further introduced into the genotype 3a cell-culture-adapted DBN strain for studies in Huh7.5 cells. Specific NS5B substitutions, including DBN-D148N+I363V, DBN-A150V+I363V, and DBN-T227S+S183P, conferred resistance to ribavirin in long-term cell culture treatment, possibly by reducing the HCV polymerase error rate. In conclusion, prolonged exposure of HCV to ribavirin in chronic hepatitis C patients induces NS5B resistance mutations leading to increased polymerase fidelity, which could be one mechanism for ribavirin resistance.

Genome-wide capture sequencing to detect hepatitis C virus at the end of antiviral therapy

BackgroundViral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Next-generation sequencing (NGS) provides an alternative option for viral detection in a genome-wide manner. However, owing to the overwhelming dominance of human genetic content in clinical specimens, direct detection of HCV by NGS has a low sensitivity and hence viral enrichment is required.MethodsBased on template-dependent multiple displacement amplification (tdMDA), an improved method for whole genome amplification (Wang et al., 2017. Biotechniques 63, 21–27), we evaluated two strategies to enhance the sensitivity of NGS-based HCV detection: duplex-specific nuclease (DSN)-mediated depletion of human sequences and HCV probe-based capture sequencing.ResultsIn DSN-mediated depletion, human sequences were significantly reduced in the two HCV serum samples tested, 65.3% → 55.6% → 33.7% (#4727) and 68.6% → 56% → 21% (#4970), respectively for no normalization, self- and driver-applied normalization. However, this approach was associated with a loss of HCV sequences perhaps due to its micro-homology with the human genome. In capture sequencing, HCV-mapped sequencing reads occupied 96.8% (#4727) and 22.14% (#4970) in NGS data, equivalent to 1936x and 7380x enrichment, respectively. Capture sequencing was then applied to ten serum samples collected at the end of HCV antiviral therapy. Interestingly, the number of HCV-mapped reads was significantly higher in relapsed patients (n = 5) than those from patients with sustained virological response (SVR) (n = 5), 102.4 ± 72.3 vs. 2.6 ± 0.55, p = 0.014.ConclusionsOur data provides concept evidence for a highly sensitive HCV detection by capture sequencing. The abundance difference of HCV sequencing reads at the end of HCV antiviral therapy could be applied to predict treatment outcomes.