Hepatitis C Virus 3a Research Articles

Eukaryotic expression of the core gene of hepatitis C virus genotype 1a

Background : Worldwide, hepatitis C virus (HCV) infection is a serious public health disease unlike hepatitis A and B, there is currently no vaccine against HCV available. Thus, extensive studies are under way to design new and effective treatments against HCV. Core protein is a component of HCV particle which is the first antigen recognized by the immune system.beside protective properties of core protein, anti –core antibodies can be used to monitor the disease progress. The purpose of the present study was to isolate and clone the core (C) gene from HCV genotype 1a in an attempt to construct a recombinant vector and subsequently evaluate its expression in a cell culture system. Methods: RNA genome of HCV genotype 1a was extracted from the blood of an infected patient. Complementary DNA (cDNA) was synthesized. HCV 1a core gene was amplified by PCR using specific primers and it was cloned into a eukaryotic expression vector. Huh7.5 cells were transfected by the designed recombinant vector and the cellular expression of the core gene was confirmed by RT-PCR. Results : Recombinant pcDNA3.1 (+) vector containing the HCV core gene with approximate size of 576bp was successfully designed. RT-PCR was used to confirm the expression of core antigen in an Huh7.5 cell line. Conclusion : The results showed that the core gene was successfully isolated from HCV genotype 1a and was cloned into the eukaryotic expression vector. This recombinant vector effectively replicated in Huh7.5 cell line. and its protective and therapeutic effects can be examined in further investigations.

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.

Antibody Responses to a Quadrivalent Hepatitis C Viral-Like Particle Vaccine Adjuvanted with Toll-Like Receptor 2 Agonists.

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.

The Rationale for a Preventative HCV Virus-Like Particle (VLP) Vaccine.

HCV represents a global health problem with ~200 million individuals currently infected, worldwide. With the high cost of antiviral therapies, the global burden of chronic hepatitis C infection (CHCV) infection will be substantially reduced by the development of an effective vaccine for HCV. The field of HCV vaccines is generally divided into proponents of strategies to induce neutralizing antibodies (NAb) and those who propose to elicit cell mediated immunity (CMI). However, for a hepatitis C virus (HCV) vaccine to be effective in preventing infection, it must be capable of generating cross-reactive CD4+, CD8+ T cell, and NAb responses that will cover the major viral genotypes. Simulation models of hepatitis C have predicted that a vaccine of even modest efficacy and coverage will significantly reduce the incidence of hepatitis C. A HCV virus like particle (VLP) based vaccine would fulfill the requirement of delivering critical conformational neutralizing epitopes in addition to providing HCV specific CD4+ and CD8+ epitopes. Several approaches have been reported including insect cell-derived genotype 1b HCV VLPs; a human liver-derived quadrivalent genotype 1a, 1b, 2, and 3a vaccine; a genotype 1a HCV E1 and E2 glycoprotein/MLV Gag pseudotype VLP vaccine; and chimeric HBs-HCV VLP vaccines. All to result in the production of cross-NAb and/or T cell responses against HCV. This paper summarizes the evidence supporting the development of a HCV VLP based vaccine.

Pattern of hepatitis C virus genotypes and subtypes circulating in war-stricken Khyber Pakhtunkhwa, Pakistan: Review of published literature.

Infection due to hepatitis C virus (HCV) is a major cause of fibrosis and hepatocellular carcinoma in Pakistan. In the current review, pattern of HCV genotypes and subtypes in Khyber Pakhtunkhwa province was ascertained in light of the available literature. After thorough analysis, genotype 3 (58.27%) was determined to be the leading HCV genotype, followed by genotypes 2 (12.39%), 1 (9.54%) and 4 (0.86%). The proportions of genotypes 5 and 6 were recorded as 0.09% and 0.22% respectively. Subtype wise, 3a accounted for 48.67%, followed by subtype 2a (10.91%), 3b (9.43%), 1a (5.84%), 1b (3.66%), 2b (1.45%) and genotype 4 with its undefined subtypes contributed a portion of 0.86%. The cumulative share of subtypes 1c, 2c, 3c, 5a and 6a was less than 1%. In 11.51% cases, the subtype was untypeable while in 7.17% cases mixed subtypes were recorded. Gender wise, proportions of most HCV subtypes were marginally higher among males as compared to females. On the basis of studied groups, 3a was pervasive among all groups except in intravenous drug users where 2a was the major HCV subtype. Similarly, based on various geographical locations (provincial divisions), subtype 3a revealed a ubiquitous distribution. Conclusively, HCV 3a persists to be the principal subtype across the province of Khyber Pakhtunkhwa. The considerable number of untypeable subtypes in most studies urges for an improved genotyping system on the basis of local sequence data and practice of sequencing for determination of underlying subtype in untypeable cases. Further, studies on identification of subtypes transmission pattern are imperative for assessment of transmission origin and reinforcement of efficient control strategies. In addition, the current review emphasizes the need of attention toward HCV risk groups and ignored southern side of Khyber Pakhtunkhwa province for better holistic understanding of HCV genotype distribution pattern in the province.

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion: mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines.

Searching for synergy: Identifying optimal antiviral combination therapy using Hepatitis C virus (HCV) agents in a replicon system

Direct acting antiviral agents (DAAs) are potent inhibitors of Hepatitis C virus (HCV) that have revolutionized the treatment landscape for this important viral disease. There are currently four classes of DAAs that inhibit HCV replication via distinct mechanisms of action: nonstructural protein (NS) 3/4a protease inhibitors, NS5A inhibitors, NS5B nucleoside polymerase inhibitors, and NS5B non-nucleoside polymerase inhibitors. Combination therapy with two or more DAAs has great potential to further enhance antiviral potency. The purpose of this study was to identify optimal combinations of DAAs against genotype 1 HCV replicons that maximized the inhibition of replicon replication. All possible two-drug combinations were evaluated against genotype 1a and 1b HCV replicons using a 96-well plate luciferase-based assay in triplicate. The Greco Universal Response Surface Area mathematical model was fit to the luciferase data to identify drug-drug interactions (i.e.: synergy, additivity, and antagonism) for antiviral effect against both genotypes. This information was used to rank-order combinations of DAAs based on their ability to inhibit replicon replication against genotype 1a and 1b HCV. These preclinical findings can provide information as to which antiviral regimens should move on in the development process.

Glecaprevir plus pibrentasvir for chronic hepatitis C virus genotype 1, 2, 4, 5, or 6 infection in adults with compensated cirrhosis (EXPEDITION-1): a single-arm, open-label, multicentre phase 3 trial

The once-daily, ribavirin-free, pangenotypic, direct-acting antiviral regimen, glecaprevir coformulated with pibrentasvir, has shown high rates of sustained virological response in phase 2 and 3 studies. We aimed to assess the efficacy and safety of 12 weeks of coformulated glecaprevir and pibrentasvir in patients with hepatitis C virus (HCV) infection and compensated cirrhosis. We did this single-arm, open-label, multicentre phase 3 study at 40 sites in Belgium, Canada, Germany, South Africa, Spain, and the USA. We enrolled patients aged 18 years or older with HCV genotype 1, 2, 4, 5, or 6 infection and compensated cirrhosis. Patients were either HCV treatment-naive or had not responded to treatment with interferon or pegylated interferon with or without ribavirin, or sofosbuvir plus ribavirin with or without pegylated interferon. Oral glecaprevir (300 mg) coformulated with pibrentasvir (120 mg) was administered once daily for 12 weeks. The primary efficacy endpoint was sustained virological response at post-treatment week 12 (HCV RNA <15 IU/mL). We assessed efficacy and safety in all patients who received at least one dose of study drug (intention-to-treat population). This study is registered with ClinicalTrials.gov, number NCT02642432. Between Dec 7, 2015, and May 4, 2016, we enrolled 146 patients with compensated cirrhosis, of whom 48 (33%) had genotype 1a HCV infection, 39 (27%) had genotype 1b infection, 34 (23%) had genotype 2 infection, 16 (11%) had genotype 4 infection, two (1%) had genotype 5 infection, and seven (5%) had genotype 6 infection. 12 weeks after treatment, 145 patients (99%, 95% CI 98-100) achieved sustained virological response, with one (1%) relapse at post-treatment week 8. We recorded 101 (69%) adverse events, of which 65 (64%) were mild. The most common adverse events were fatigue (n=28 [19%]) and headache (n=20 [14%]). 11 (8%) patients had serious adverse events, none of which were deemed related to study drugs. No patients had elevations in alanine aminotransferase and no patients prematurely discontinued treatment because of adverse events. Our results show that 99% of patients treated with once-daily glecaprevir plus pibrentasvir achieved a sustained virological response at 12 weeks. Furthermore, this drug regimen had a favourable safety profile in previously treated or untreated patients with chronic HCV genotype 1, 2, 4, 5, or 6 infection and compensated cirrhosis. These findings could help simplify treatment algorithms and reduce treatment burden. AbbVie.

Effect of hepatitis C virus genotype on antiviral therapy in patients with human immunodeficiency virus/hepatitis C virus coinfection

Objective To investigate the effect of hepatitis C virus (HCV) genotype on antiviral therapy in patients with human immunodeficiency virus (HIV)/HCV coinfection in Henan province. Methods A total of 129 patients were coinfected with HIV and HCV, among whom, 70 were HCV 1b genotype and 57 HCV 2a genotype. And 131 patients were HIV single infection. Immunological failure rate, virological suppression, CD4+ T lymphocyte counts and liver and renal function after antiretroviral therapy (ART) were compared among the three groups. Flow cytometry was used to count CD4+ T lymphocytes and polymerase chain reaction amplification was used to detect HIV RNA. The liver and renal function were tested by automatic biochemical analysis. Statistical analysis was conducted by χ2 test, analysis of variance and LSD-t method. Results Immunological failure rate in HCV 1b group, HCV 2a group and HIV single infection group were 7.14% (5/70), 15.79% (9/57) and 9.92% (13/131), respectively. There was no significant statistical difference among the three groups (χ2=2.59, P>0.05). The CD4+ T lymphocyte counts in three groups were (614±258), (529±245), and (518±243) cells/μL, respectively. The difference was statistically significant (F=3.17, P 0.05). The levels of aspartate transaminase, alanine aminotransferase and total bilirubin in HCV 1b group and HCV 2a group were all significantly higher than those in HIV single infection group (F=27.38, 15.22 and 7.33, respectively, all P 0.05). Conclusions The main HCV genotypes in patients with HIV/HCV coinfection by blood transmission are HCV 1b and HCV 2a in Henan province. HIV/HCV coinfection does not affect the effect of ART, but could aggravate the liver damage in acquired immune deficiency syndrome patients. Key words: Human immunodeficiency virus; Hepacivirus; Genotype; Antiviral therapy; Effect analysis

Population dynamics of hepatitis C virus subtypes in injecting drug users on methadone maintenance treatment in China associated with economic and health reform.

The extensive genetic heterogeneity of hepatitis C virus (HCV) requires in-depth understanding of the population dynamics of different viral subtypes for more effective control of epidemic outbreaks. We analysed HCV sequences data from 125 participants in Wuhan, China. These participants were newly infected by subtype 1b (n=13), 3a (n=15), 3b (n=50) and 6a (n=39) while on methadone maintenance treatment (MMT). Bayesian phylogenies and demographic histories were inferred for these subtypes. Participants infected with HCV-1b and 3a were clustered in well-supported monophyletic clades, indicating local subepidemics. Subtypes 3b and 6a strains were intermixed with other Chinese isolates, as well as isolates from other Asian countries, reflecting ongoing across geographic boundary transmissions. Subtypes 1b and 3a declined continuously during the past ten years, consistent with the health and economic reform in China, while subtype 3b showed ongoing exponential growth and 6a was characterized by several epidemic waves, possibly related to the recently growing number of travellers between China and other Asian countries. In conclusion, results of this study suggest that HCV subtype 3b and 6a subepidemics in China are currently not under control, and new epidemic waves may emerge given the rapid increase in international travelling following substantial economic growth.

NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome

The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV. NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir. Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively. Pretreatment ledipasvir-specific RASs that were present in 8-16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV. The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8-16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV.

The Prevalence of Hepatitis C Virus Genotypes in Mazandaran Province, Iran

Background: Hepatitis C virus (HCV) infection is a major public health concern and the third most common cause of death from cancer worldwide. This infection is the most common risk factor for the progression of chronic liver disease, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Hepatitis C virus infection is classified into six major genotypes and multiple subtypes. The distribution of HCV genotypes seems to vary in different areas around the globe. Objectives: In this study, we aimed to identify HCV RNA and perform genotyping in anti-HCV-positive patients through polymerase chain reaction (PCR). We compared the results of PCR and ELISA tests among patients referred to several healthcare centers of Mazandaran province, situated in North of Iran, during 2015 - 2016. Methods: A total of 157 patients with anti-HCV-positive specimens were enrolled in this study during 2015-2016. Hepatitis C virus-RNA was extracted from the serum samples, using pure link viral RNA Mini kit. Detection of HCV nucleic acids was performed by using the serum samples from anti-HCV positive specimens. The genotypes of the HCV genome were determined by PCR, using the RNA-PCR kit according to the specified protocol. Also, the PCR products (DNA fragments) from these samples were run on 1.5% agarose gel and visualized on a UV analyzer. All the samples were examined at the virology laboratory of Mazandaran University of Medical Sciences, Sari, Iran. Results: The mean age of the patients was 39 ± 14.3 years (range: 20 - 68 years). In total, 110 (70.06%) and 47 (29.94%) anti-HCV-positive patients were male and female, respectively. Hepatitis C virus–RNA was detected in 84 (53.50%) out of 157 patients with positive HCV antibodies. Based on the findings, the majority of HCV-RNA positive patients were intravenous drug users (46.43%). In this study, the 3a HCV genotype was predominant (59.52%), followed by the 1a/b genotype (40.48%). Conclusions: The present findings indicate that the 3a genotype is the most frequent HCV genotype, followed by 1a/b in the study region. The prevalence and incidence of HCV genotypes seem to vary in different areas, with each genotype showing a different response to interferon therapy. Therefore, it is essential to determine the predominant subtypes of HCV.

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.

Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease.

The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.

The Phylogeographic and Spatiotemporal Spread of HCV in Pakistani Population.

Hepatitis C Virus (HCV) is the most prevalent human pathogen in Pakistan and is the major cause of liver cirrhosis and hepatocellular carcinoma in infected patients. It has shifted from being hypo-endemic to being hyper-endemic. There was no information about the origin and evolution of the local variants. Here we use newly developed phyloinformatic methods of sequence analysis to conduct the first comprehensive investigation of the evolutionary and biogeographic history in unprecedented detail and breadth. Considering evolutionary rate and molecular-clock hypothesis in context, we reconstructed the spatiotemporal spread of HCV in the whole territory of its circulation using a combination of Bayesian MCMC methods utilizing all sequences available in GenBank. Comparative analysis were performed and were addressed. Whole genome and individual gene analysis have shown that sub-types 1a, 1b and 3a are recognized as epidemic strains and are distributed globally. Here we confirm that the origin of HCV 3a genotypes is in South Asia and HCV has evolved in the region to become stably adapted to the host environment.

Evaluation of the new cobas® HCV genotyping test based on real-time PCRs of three different HCV genome regions

Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to evaluate the performance of the recently introduced cobas® HCV GT assay (Roche) and to compare it to two comparator assays. The cobas® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE® HCV 5'NC Genotyping Kit (Siemens) and the VERSANT® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included. When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample. The cobas® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.

Phylogeographical Analysis Reveals Distinct Sources of HIV-1 and HCV Transmitted to Former Blood Donors in China.

Historically, coinfection of HIV and hepatitis C virus (HCV) was frequent among Chinese former blood donors (FBDs). This is largely due to ignorance/lack of education regarding appropriate sterilizing techniques and/or the availability of single-use needles and equipment. Although HCV shares identical transmission routes with HIV, the source of HCV in the Chinese blood donor population still remains unknown. In this study, we investigated the evolution and transmission of HCV and HIV in the Chinese FBD group. Similar to previous reports, two HCV subtypes (HCV 1b and 2a) and one HIV subtype (Thai-B) were identified in FBDs. The HCV 1b subtype had a similar evolutionary rate of 1.9 × 10-3 substitutions/site/year to that of HIV (2.06 × 10-3 substitutions/site/year), while the HCV 2a subtype had a faster evolutionary rate of 3.8 × 10-3 substitutions/site/year. Phylogeographical analysis indicated that the introduction of HCV 1b into FBDs was estimated to be earlier than that of HCV 2a and HIV (late 1970s vs. late 1980s). Bayesian Skyline Plot (BSP) analysis further confirmed our findings, showing that HCV 1b infections breached a fast exponential growth from 1991 to 1998, while the HCV 2a infections had a fast exponential growth that occurred in around 1996-2001. Overall, this investigation helps to better understand HCV transmission in China and supports improvements of HCV prevalence control.

Biophysical Studies on HCV 1a NS3/4A Protease and Its Catalytic Triad in Wild Type and Mutants by the In Silico Approach.

The hepatitis C virus (HCV), of the family flaviviridae, is one of the major causes of chronic liver diseases. Until the year 2012, HCV infections were treated using PEG-interferon and ribavirin combinations, which have a low cure rate and severe side effects. Currently, many direct-acting antivirals (DAAs) are available, e.g. protease inhibitors, NS5A and polymerase inhibitors. These drugs have proven to be efficient in interferon-free treatment combinations and capable of enhancing the cure rate to above 90%. Unlike PEG-interferon and ribavirin combinations, DAAs select for resistance in HCV. The R155K mutation in the HCV was found to resist all the currently available protease inhibitors. Here, we studied biophysical parameters like pocket (cavity) geometries and stabilizing residues of HCV 1a NS3/4A protease in wild type and mutants. We also studied HCV 1a NS3/4A protease's catalytic residues: their accessibility, energy, flexibility and binding to Phase II oral protease inhibitor vedroprevir (GS-9451), and compared these parameters between wild type and mutant(s). All these studies were performed using various bioinformatics tools (e.g. Swiss-PdbViewer and Schrödinger's Maestro) and web servers (e.g. DoGSiteScorer, SRide, ASA-View, WHAT IF, elNémo, CABS-flex, PatchDock and PLIP). From our study, we found that introduction of R155K, A156T or D168A mutation to wild-type NS3/4A protease increases the pocket's volume, surface (in the R155K mutant, surface decreases), lipo surface and depth and decreases the number of stabilizing residues. Additionally, differences in catalytic residues' solvent accessibility, energy, root-mean-square deviation (RMSD) and flexibility between wild type and mutantsmight explain changes in the protease activity and the resistance to protease inhibitors.

Hepatitis C virus: The role of N-glycosylation sites of viral genotype 1b proteins for formation of viral particles in insect and mammalian cells

Hepatitis C virus (HCV) is characterized by considerable genetic variability and, as a consequence, it has 6 genotypes and multitude of subtypes. HCV envelope glycoproteins are involved in the virion formation; the correct folding of these proteins plays the key role in virus infectivity. Glycosylation at certain sites of different genotypes HCV glycoproteins shows substantial differences in functions of the individual glycans (Goffard et al., 2005; Helle et al., 2010) [1,2]. In this study, differential glycosylation sites of HCV genotype 1b envelope proteins in insect and mammalian cells was demonstrated. We showed that part of glycosylation sites was important for folding of the proteins involved in the formation of viral particles. Point mutations were introduced in the protein N-glycosylation sites of HCV (genotype 1b) and the mutant proteins were analyzed using baculovirus expression system in mammalian and insect cells. Our data showed that, in contrast to HCV 1a and 2a, the folding of HCV 1b envelope proteins E2 (sites N1, N2, N10) and E1 (sites N1, N5) was disrupted, however that did not prevent the formation of virus-like particles (VLP) with misfolded glycoproteins having densities typical for HCV particles containing RNA fragments. Experimental data are supported by mathematical modeling of the structure of E1 mutant variants.

Genomic Surveillance Elucidates HCV 1a Phylodynamics and Molecular Evolution

The hepatitis C virus (HCV) infects at least 3 % of people worldwide, and continues to cause substantial morbidity and mortality. To better understand the phylodynamics and molecular evolution of the HCV 1a, a phylogenetic analysis of 186 full-length genomic sequences isolated from five countries between 1977 and 2009 was conducted in this study. Nucleotide substitution rates and molecular epidemiology were assessed by Bayesian coalescent analysis using time-stamped entire coding sequences. We showed that the substitution rates of ten genomic regions are diverse and higher than those of previously estimated. The coalescent analysis indicated that the transmission of subtype 1a probably started in the second half of the twentieth century and an explosion of epidemics occurred between 1960s and 1980s. Selection analysis suggested that the HCV 1a evolves under purifying selection. However, a total of 58 positively selected sites were detected and further analysis suggested that these sites may play an important role in adaptive evolution of HCV 1a strains. In addition, the codon usage and the factors accounting for shaping the codon usage pattern of HCV 1a were investigated to evaluate the dynamics of the virus evolution. Surveys of codon usage variation showed that mutational pressure and selection pressure account for HCV 1a codon usage pattern.