Hepatitis C Virus Variants Research Articles

Polymorphisms associated with resistance to protease inhibitors in naïve patients infected with hepatitis C virus genotype 1 in Argentina: Low prevalence of Q80K

Incorporation of direct acting antivirals (DAA) in the treatment of Hepatitis C Virus (HCV) significantly increases sustained virologic response rates. However, despite the greater potency offered by these antivirals, drug resistance plays a key role in patients with failure to DAA. Nevertheless, there is no information about the prevalence of resistance-associated substitutions (RASs) in Argentina.The aim of this study was to analyze HCV variants resistant to protease inhibitors (PI) in naïve patients infected with HCV genotype 1 from Argentina.In this retrospective cross-sectional study, 103 patients infected with HCV-1 were included. Eighteen positions related with RASs were analyzed by Sanger at baseline and phylogenetic analysis was performed to determine the diversification of this samples.The analyzed RASs were present in 38 out of 103 patients (36.9%) infected with HCV-1. Patients infected with subtype HCV-1b had higher prevalence of baseline RASs than patients infected with HCV-1a [51.6% vs. 12.8%, respectively (p<0.001)]. The Q80K polymorphism was not found in HCV-1a samples, even when 51% of them belonged to cluster 1, which is associated with a high frequency of Q80K. Phylogenetic analysis showed that Argentinean samples were intermingled with sequences from other geographic regions.RASs to PI were highly prevalent and subtype dependent in treatment-naïve Argentinean patients. Surprisingly, Q80K polymorphism was not detected in our study population. The phylogenetic analysis showed no relationship between our samples and other samples from Brazil which also present a low prevalence of Q80K. This study supports the need for surveillance of resistance in patients who will be treated with DAA in each particular country since the observed RASs have very different prevalence worldwide.

HCV replication in gastrointestinal mucosa: Potential extra-hepatic viral reservoir and possible role in HCV infection recurrence after liver transplantation.

PurposeHepatitis C virus (HCV) predominantly infects hepatocytes, although it is known that receptors for viral entry are distributed on a wide array of target cells. Chronic HCV infection is indeed characterized by multiple non-liver manifestations, suggesting a more complex HCV tropism extended to extrahepatic tissues and remains to be fully elucidated. In this study, we investigated the gastrointestinal mucosa (GIM) as a potential extrahepatic viral replication site and its contribution to HCV recurrence.MethodsWe analyzed GIM biopsies from a cohort of 76 patients, 11 of which were HCV-negative and 65 HCV-positive. Of these, 54 biopsies were from liver-transplanted patients. In 29 cases, we were able to investigate gastrointestinal biopsies from the same patient before and after transplant. To evaluate the presence of HCV, we looked for viral antigens and genome RNA, whilst to assess viral replicative activity, we searched for the replicative intermediate minus-strand RNA. We studied the genetic diversity and the phylogenetic relationship of HCV quasispecies from plasma, liver and gastrointestinal mucosa of HCV-liver-transplanted patients in order to assess HCV compartmentalization and possible contribution of gastrointestinal variants to liver re-infection after transplantation.ResultsHere we show that HCV infects and replicates in the cells of the GIM and that the favorite hosts were mostly enteroendocrine cells. Interestingly, we observed compartmentalization of the HCV quasispecies present in the gastrointestinal mucosa compared to other tissues of the same patient. Moreover, the phylogenetic analysis revealed a high similarity between HCV variants detected in gastrointestinal mucosa and those present in the re-infected graft.ConclusionsOur results demonstrated that the gastrointestinal mucosa might be considered as an extrahepatic reservoir of HCV and that could contribute to viral recurrence. Moreover, the finding that HCV infects and replicates in neuroendocrine cells opens new perspectives on the role of these cells in the natural history of HCV infection.

Genetic diversity of hepatitis C virus in Ethiopia.

Hepatitis C virus (HCV) is genetically highly divergent and classified in seven major genotypes and approximately hundred subtypes. These genotypes/subtypes have different geographic distribution and response to antiviral therapy. In Ethiopia, however, little is known about their molecular epidemiology and genetic diversity. The aim of this study was to investigate the distribution and genetic diversity of HCV genotypes/subtypes in Ethiopia, using 49 HCV RNA positive samples. HCV genotypes and subtypes were determined based on the sequences of the core and the nonstructural protein 5B (NS5B) genomic regions. Phylogenetic analysis revealed that the predominant was genotype 4 (77.6%) followed by 2 (12.2%), 1 (8.2%), and 5 (2.0%). Seven subtypes were identified (1b, 1c, 2c, 4d, 4l, 4r and 4v), with 4d (34.7%), 4r (34.7%) and 2c (12.2%) as the most frequent subtypes. Consistent with the presence of these subtypes was the identification of a potential recombinant virus. One strain was typed as genotype 2c in the NS5B region sequence and genotype 4d in the core region. In conclusion, genotype 4 HCV viruses, subtypes 4d and 4r, are most prevalent in Ethiopia. This genotype is considered to be difficult to treat, thus, our finding has an important impact on the development of treatment strategies and patient management in Ethiopia.

SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants.

SB 9200 is a novel, first-in-class oral modulator of innate immunity that is believed to act via the activation of the RIG-I and NOD2 pathways. SB 9200 has broad-spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10 . Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture-fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct-acting antiviral treatment, including viruses containing known NS5A resistance-associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens.

An Accurate Sequence Assembly Algorithm for Livestock, Plants and Microorganism Based on Spark

Sequence Assembly is one of the important topics in bioinformatics research. Sequence assembly algorithm has always met the problems of poor assembling precision and low efficiency. In view of these two problems, this paper designs and implements a precise assembling algorithm under the strategy of finding the source of reads based on the MapReduce (SA-BR-MR) and Eulerian path algorithm. Computational results show that SA-BR-MR is more accurate than other algorithms. At the same time, SA-BR-MR calculates 54 sequences which are randomly selected from animals, plants and microorganisms with base lengths from hundreds to tens of thousands from NCBI. All matching rates of the 54 sequences are 100%. For each species, the algorithm summarizes the range of [Formula: see text] which makes the matching rates to be 100%. In order to verify the range of [Formula: see text] value of hepatitis C virus (HCV) and related variants, the randomly selected eight HCV variants are calculated. The results verify the correctness of [Formula: see text] range of hepatitis C and related variants from NCBI. The experiment results provide the basis for sequencing of other variants of the HCV. In addition, Spark platform is a new computing platform based on memory computation, which is featured by high efficiency and suitable for iterative calculation. Therefore, this paper designs and implements sequence assembling algorithm based on the Spark platform under the strategy of finding the source of reads (SA-BR-Spark). In comparison with SA-BR-MR, SA-BR-Spark shows a superior computational speed.

Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification.

Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus’s but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies.

Humoral immune system targets clonotypic antibody-associated hepatitis C virus

Hypervariable region 1 (HVR1) is one of the potential neutralization domains in the E2 glycoprotein of hepatitis C virus (HCV). Point mutations of the HVR1 can lead to humoral immune escape in HCV-infected patients. In this study, we segregated the chronically infected viraemic sera from HCV-infected patients into populations of antibody-free virus and antibody-associated virus (AAV) and mapped potential epitopes within the E1E2 gene junction of AAV sequences (residues 364-430). Furthermore, we generated HCV pseudoparticles (HCVpp) derived from AAV sequences to assess their infectivity. We studied the neutralization potential of virus-free Fab obtained from antibody-virus complexes, in the HCVpp system. We observed selective targeting of clonotypic HCV variants from the quasispecies pool. Moreover, we identified potential neutralizing epitopes within the HVR1 and an additional epitope that overlapped with a broadly neutralizing AP33 epitope (amino acid 412-423 in E2). We observed a marked difference in the infectivity of HCVpp generated using E1E2 sequences isolated from AAV. We document reduction in the infectivity of HCVpp-H77 and HCVpp derived from AAV sequences when challenged with virus-free Fab. Our results provide novel insights into the complexities of engagement between HCV and the humoral immune system.

Identification of Genotype 2 HCV in Serotype-1 Hepatitis C Patients Unresponsive to Daclatasvir plus Asunaprevir Treatment.

It is important to determine the genotypes or serotypes of hepatitis C virus (HCV) in patients before treatment with direct-acting antiviral agents (DAAs), because the effects of DAAs differ among genotypes. In Japan, two tests for HCV typing are available clinically, but only serotyping, not genotyping, is approved by the public health insurance. Although most serotype-1 Japanese patients are infected with genotype 1b HCV, it is known that a small proportion of patients show different results from two typing methods. This study focused on such patients and the effectiveness of treatment with daclatasvir plus asunaprevir (DCV/ASV) was evaluated. We analyzed 644 DCV/ASV-treated patients with serotype 1 or genotype 1b, and among them, 166 serotype-1 patients received a commercial-based direct sequencing (DS) test for resistant-associated variants of genotype 1b HCV. We found four patients (2.4%) with DS test failure, suggesting that the PCR primers targeting genotype 1b may not match. Importantly, none of the four patients achieved a sustained virological response. Our in-house DS test analyzing the 5'-untranslated region and coding regions for NS4 and NS5B of HCV showed that three of the four patients were infected with genotype 2 HCV, and one patient was infected with genotype 1a HCV. No recombinant virus of different genotypes was found. This study indicates that a subset of serotype-1 hepatitis C patients is infected with HCV of genotype 2 or 1a in Japan and that DCV/ASV is not effective for such patients. Thus, attention should be paid to DAA treatment without HCV genotyping.

Use of whole genome sequencing in the Dutch Acute HCV in HIV study: focus on transmitted antiviral resistance

ObjectiveWithin HIV-positive men having sex with men, the epidemic of hepatitis C virus (HCV) is ongoing. Transmission of resistant variants of HCV after failure of treatment with directly acting antivirals (DAA) could be a major threat to the effectivity of therapy. We determined whether HCV-resistant variants to DAAs were prevalent amongst patients with an acute HCV infection diagnosed in 2013 and 2014 in the Netherlands. MethodsTarget enrichment for viral nucleic acid separation and deep sequencing were used to recover whole HCV genomes of 50 patients with an acute HCV infection. The genomes were assembled by de novo assembly and analysed for known DAA resistance mutations. ResultsIn acute HCV infected treatment-naive patients, the relevant resistance-associated substitutions were Q80K (40%) in NS3/4a, M28V (24%) and Q30H combined with Y93H (2%) in NS5A and M414T (2%) or S556G (2%) in NS5b. Patients whose HCV infection failed to respond to boceprevir, peginterferon and ribavirin therapy developed mutations in NS3 at position T54A and R155K. ConclusionsTarget enrichment and whole genome sequencing were successfully applied directly on clinical samples from patients with an acute HCV infection.

Epidemic History of Hepatitis C Virus among Patients with Inherited Bleeding Disorders in Iran.

The high rate of hepatitis C virus (HCV) infection among transfusion related risk groups such as patients with inherited bleeding disorders highlighting the investigation on prevalent subtypes and their epidemic history among this group. In this study, 166 new HCV NS5B sequences isolated from patients with inherited bleeding disorders together with 29 sequences related to hemophiliacs obtained from a previous study on diversity of HCV in Iran were analyzed. The most prevalent subtype was 1a (65%), followed by 3a (18.7%),1b (14.5%),4(1.2%) and 2k (0.6%). Subtypes 1a and 3a showed exponential expansion during the 20th century. Whereas expansion of 3a started around 20 years earlier than 1a among the study patients, the epidemic growth of 1a revealed a delay of about 10 years compared with that found for this subtype in developed countries. Our results supported the view that the spread of 3a reached the plateau 10 years prior to the screening of blood donors for HCV. Rather, 1a reached the plateau when screening program was implemented. The differences observed in the epidemic behavior of HCV-1a and 3a may be associated with different transmission routes of two subtypes. Indeed, expansion of 1a was more commonly linked to blood transfusion, while 3a was more strongly associated to drug use and specially IDU after 1960. Our findings also showed HCV transmission through blood products has effectively been controlled from late 1990s. In conclusion, the implementation of strategies such as standard surveillance programs and subsiding antiviral treatments seems to be essential to both prevent new HCV infections and to decline the current and future HCV disease among Iranian patients with inherited bleeding disorders.

Compartmentalization of hepatitis C virus variants in patients with hepatocellular carcinoma.

Chronic Hepatitis C Virus (HCV) infection is a major risk for hepatocellular carcinoma (HCC) development. HCV Core protein has been associated with the modulation of potentially oncogenic cellular processes and E2 protein has been useful in evolutive studies to analyze the diversity of HCV. Thus, the aim of this study was to evaluate HCV compartmentalization in tumoral, non-tumoral liver tissue and serum and to identify viral mutations potentially involved in carcinogenesis. Samples were obtained from four patients with HCC who underwent liver transplantation. Core and E2 were amplified, cloned and sequenced. Phylogenies and BaTS Test were performed to analyze viral compartmentalization and a signature sequence analysis was conducted by VESPA. The likelihood and Bayesian phylogenies showed a wide degree of compartmentalization in the different patients, ranging from total clustering to a more scattered pattern with small groups. Nevertheless, the association test showed compartmentalization for the three compartments and both viral regions tested in all the patients. Signature amino acid pattern supported the compartmentalization in three of the cases for E2 protein and in two of them for Core. Changes observed in Core included polymorphism R70Q/H previously associated with HCC. In conclusion, evidence of HCV compartmentalization in the liver of HCC patients was provided and further biological characterization of these variants may contribute to the understanding of carcinogenesis mediated by HCV infection. © 2016 Wiley Periodicals, Inc.

Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5' Untranslated Region (5'UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping.

Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100%. The highest prevalent genotype was 1b (50%) followed by genotypes 1a (16%), 4a (12%), 2a (10%), 2c (8%), and 3a (4%). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations.

Deep sequencing of hepatitis C virus reveals genetic compartmentalization in cerebrospinal fluid from cognitively impaired patients.

Hepatitis C virus (HCV) causes neuropsychiatric impairment and fatigue with recent studies suggesting HCV invasion of the central nervous system (CNS). Our previous finding that endothelial cells from the blood-brain barrier support HCV infection warrants further investigation to elucidate whether the CNS can serve as a reservoir for independent HCV evolution. Cerebrospinal fluid (CSF) and plasma from six HCV-infected patients without liver disease or co-morbidities together with plasma from six healthy subjects were profiled for markers of immune activation and viral quasispecies measured by deep sequencing. Unsupervised data analyses were used to identify any associations between cytokine activation markers and clinical outcomes. Four of six HCV-infected patients showed significant evidence of cognitive dysfunction and fatigue. Deep sequencing revealed independent viral evolution within the CNS of two cognitively impaired patients. Principal component analysis of peripheral cytokines demonstrated that individuals without cognitive impairment clustered together while a distinct cytokine pattern emerged with patients exhibiting cognitive dysfunction and fatigue. Deep sequencing demonstrated unique viral variants in the CSF of two cognitively impaired patients consistent with CNS replication or sequestration. Meanwhile, compartmentalization was absent in infected patients with no neurocognitive impairment. Examination of cytokine profiles in HCV-infected patients with cognitive dysfunction revealed elevated peripheral cytokine levels resulting in a distinct cytokine profile that may be related to cognitive impairment or viral penetration into the CNS. Further studies to determine the significance of unique HCV variants within the CNS are warranted.

Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.

Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation

Hepatitis C virus (HCV) entry inhibitors have been hypothesized to prevent infection of the liver after transplantation. ITX5061 is a scavenger receptor class B type I antagonist that blocks HCV entry and infection in vitro. We assessed the safety and efficacy of ITX5061 to limit HCV infection of the graft. The study included 23 HCV-infected patients undergoing liver transplantation. The first 13 "control" patients did not receive drug. The subsequent 10 patients received 150 mg of ITX5061 immediately before and after transplant and daily for 1 week thereafter. ITX5061 pharmacokinetics and plasma HCV RNA were quantified. Viral genetic diversity was measured by ultradeep pyrosequencing (UDPS). ITX5061 was well tolerated with measurable plasma concentrations during therapy. Although the median HCV RNA reduction was greater in ITX-treated patients at all time points in the first week after transplantation, there was no difference in the overall change in the area over the HCV RNA curve in the 7-day treatment period. However, in genotype (GT) 1-infected patients, treatment was associated with a sustained reduction in HCV RNA levels compared to the control group (area over the HCV RNA curve analysis, P = 0.004). UDPS revealed a complex and evolving pattern of HCV variants infecting the graft during the first week. ITX5061 significantly limited viral evolution where the median divergence between day 0 and day 7 was 3.5% in the control group compared to 0.1% in the treated group. In conclusion, ITX5061 reduces plasma HCV RNA after transplant notably in GT 1-infected patients and slows viral evolution. Following liver transplantation, the likely contribution of extrahepatic reservoirs of HCV necessitates combining entry inhibitors such as ITX5061 with inhibitors of replication in future studies.

Global prevalence of pre-existing HCV variants resistant to direct-acting antiviral agents (DAAs): mining the GenBank HCV genome data.

Direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) proteins open a whole new era for anti-HCV therapy, but DAA resistance associated variants (RAVs) could jeopardize the effectiveness of DAAs. We reported the global prevalence of DAA RAVs using published GenBank data. 58.7% of sequences (854/1455) harbored at least one dominant resistance variant and the highest RAV frequency occurred in Asia (74.1%), followed by Africa (71.9%), America (53.5%) and Europe (51.4%). The highest RAV frequency was observed in genotype (GT) 6 sequences (99%), followed by GT2 (87.9%), GT4 (85.5%), GT1a (56%), GT3 (50.0%) and GT1b (34.3%). Furthermore, 40.0% and 29.6% of sequences were detected RAVs of non-structural (NS) 5A inhibitors and NS3 protease inhibitors, respectively. However, RAVs to NS5B nucleo(t)ide inhibitor (NI) and NI-based combinations were uncommon (<4% of sequences). As expected, combinations of multiple RAVs to the IFN-free regimens recommended by current guidelines were rarely detected (0.2%–2.0%). Our results showed that the overall global prevalence of DAA RAVs was high irrespective of geography or genotype. However, the NI-based multi-DAA regimens had a low RAV prevalence, suggesting that these regimens are the most promising strategies for cure of the long-term HCV infection.

A Hepatitis C Virus Envelope Polymorphism Confers Resistance to Neutralization by Polyclonal Sera and Broadly Neutralizing Monoclonal Antibodies.

Hepatitis C virus (HCV) infection is a global health problem, with millions of chronically infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine development is vital in the effort toward disease control and eradication, an undertaking aided by an increased understanding of the mechanisms of resistance to broadly neutralizing antibodies (bNAbs). In this study, we identified HCV codons that vary deep in a phylogenetic tree of HCV sequences and showed that a polymorphism at one of these positions renders Bole1a, a computationally derived, ancestral genotype 1a HCV strain, resistant to neutralization by both polyclonal-HCV-infected plasma and multiple broadly neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance mutation reduces replicative fitness, which may explain the persistence of both neutralization-sensitive and neutralization-resistant variants in circulating viral strains. This work identifies an important determinant of bNAb resistance in an ancestral, representative HCV genome, which may inform HCV vaccine development. Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the leading cause of hepatocellular carcinoma and liver transplantation in the United States. Despite recent significant advances in HCV treatment, a vaccine is needed. Control of the HCV pandemic with drug treatment alone is likely to fail due to limited access to treatment, reinfections in high-risk individuals, and the potential for resistance to direct-acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV variants and therefore serve as a useful guide for vaccine development, but our understanding of resistance to bNAbs is incomplete. In this report, we identify a viral polymorphism conferring resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies, which may inform HCV vaccine development.

Next Generation Sequencing-Based Investigation of Potential Patient-to-Patient Hepatitis C Virus Transmission during Hemodialytic Treatment.

We investigated potential patient-to-patient transmission of hepatitis C virus (HCV) in two hemodialysis centers in Beijing, China. Approximately 8.25% (32/388) hemodialysis patients were HCV antibody positive, and 4.90% (19/388) were HCV RNA-positive, which consisted of 2a genotype (1/19) and 1b genotypes (18/19). Using next generation sequencing (NGS) approach, MiSeq platform, we sequenced HCV, targeting hypervariable region 1 (263 base-pairs) of genotype 1b specimens and obtained 18 to 243 unique HCV variants. Analysis of phylogenetic tree, viral epidemiology signature pattern (VESP) and Shannon entropy indicated no obvious HCV similarity for most HCV infections but limited HCV variants from Patient 31 (P31) were closer with respect to evolutionary relationship with Patient 24 (P24). However, it was unlikely that HCV was transmitted directly from P24 to P31 in the hemodialysis center. Otherwise, their genetic distance (3.92%-8.92%), would have been much less. Moreover, P31 was infected less than two years before specimen collection, and other external high risk factors existed for these two patients. Thus, our data indicated no evidence of patient-to-patient transmission of HCV in the two hemodialysis centers, suggesting that current HCV infection control measures are effective.

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana.

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3–4 centuries, indicating a long epidemic history of HCV-2 in Ghana.

A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope glycoprotein and their cross-reactivity.

BackgroundHepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses.ResultsStructural characterization of E2 core protein and a broadly neutralizing antibody AR3C together with E1E2 sequence information enabled the analysis of B-cell epitope variability. The E2 binding site by AR3C and its surrounding area were identified from the crystal structure of E2c-AR3C complex. We clustered HCV strains using the concept of "discontinuous motif/peptide" and classified B-cell epitopes based on their similarity.ConclusionsThe assessment of antibody neutralizing coverage provides insights into potential cross-reactivity of the AR3C neutralizing antibody across a large number of HCV variants.