Hepatitis C Virus 3a Research Articles

B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood

Abstract Background Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes. Methods We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification. Results The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed. Conclusions Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.

Historical drivers of HCV Subtypes 1b and 3a in Thailand and 6f in Phetchabun, an HCV endemic area of the country.

The World Health Organization has set a target to eliminate viral hepatitis as a public threat by 2030. In pursuit of this goal, Thailand initiated a hepatitis C virus (HCV) microelimination project targeting Phetchabun province, a well-recognized high-burden HCV endemic area. However, the historical transmission dynamics of HCV in Phetchabun, and in Thailand in general, remain unclear. This study investigates the epidemic histories of HCV in Phetchabun, focusing on Subtypes 1b, 3a, and 6f, and their relationship with HCV in other regions of Thailand, using molecular phylogenetic analyses. Our results reveal nationwide the presence of Subtypes 1b and 3a, while Subtype 6f is mainly confined to Phetchabun. The initial spread of Subtype 1b was inferred to coincide with World War II and the period of suboptimal medical and hygienic standards in Thai blood transfusion services, suggesting a correlation between the two. The early expansion of Subtype 3a was, on the other hand, found to correlate with the epidemic of intravenous drug use in Thailand during the time of Vietnam War. The early expansion of Subtype 6f, in contrast, appears to coincide with the period of severe regional political conflict and social and economic instability. All these findings suggest the complex interplay between social determinants of health and HCV transmission. Post the mid-1990s/early 2000s, all subtypes showed significantly reduced population growth rates, aligning with improvements in blood transfusion safety standards, the nationwide "War on Drugs" policy, and enhanced accessibility to public healthcare and HCV treatments. These combined efforts likely have contributed to curbing the spread of HCV in Thailand. Nevertheless, our analyses reveal that the prevalence of HCV in Thailand remains high overall, emphasizing the need for further research and a nationwide approach to more effectively reduce the HCV burden in Thailand.

The distribution of hepatitis C viral genotypes shifted among chronic hepatitis C patients in Yunnan, China, between 2008-2018.

The hepatitis C virus (HCV) is prevalent across China, with a distinctive genotypic distribution that varies by geographical region and mode of transmission. Yunnan is one such geographical region wherein the local population continues to experience a high level of HCV infection, severely straining public health resources. This high prevalence is likely due to the increased incidence of intravenous drug use in that region, as Yunnan is a major point of entry for illegal heroin into China. We investigated 510 individuals with chronic HCV infections in Yunnan Province from 2008 through 2018. Using reverse transcription PCR and Sanger sequencing to amplify and sequence samples. Bayesian analyses was performed to estimate the common ancestors and Bayesian skyline plot to estimate the effective viral population size. Molecular network was conducted to explore the characteristics of HCV transmission. We successfully amplified and sequenced a total of 503 viral samples and genotyped each as either 3b (37.6%), 3a (21.9%), 1b (19.3%), 2a (10.5%), HCV-6 (10.1%), or 1a (0.6%). Over this 11-year period, we observed that the proportion of 3a and 3b subtypes markedly increased and, concomitantly, that the proportion of 1b and 2a subtypes decreased. We also performed Bayesian analyses to estimate the common ancestors of the four major subtypes, 1b, 2a, 3a, and 3b. Finally, we determined that our Bayesian skyline plot and transmission network data correlated well with the changes we observed in the proportions of HCV subtypes over time. Taken together, our results indicate that the prevalence of HCV 3a and 3b subtypes is rapidly increasing in Yunnan, thus demonstrating a steadily growing public health requirement to implement more stringent preventative and therapeutic measures to curb the spread of the virus.

Patterns and correlates of hepatitis C virus phylogenetic clustering among people living with HIV in Australia in the direct-acting antiviral era: A molecular epidemiology study among participants in the CEASE cohort.

Background and AimsIn moving towards the elimination of hepatitis C virus (HCV) infection among people living with HIV, understanding HCV transmission patterns may provide insights to guide and evaluate interventions. In this study, we evaluated patterns of, and factors associated with HCV phylogenetic clustering among people living with HIV/HCV co‐infection in Australia in the direct‐acting antiviral era.MethodsHCV RNA was extracted from dried blood spot (DBS) samples collected between 2014 and 2018 in the CEASE cohort study. The HCV Core‐E2 region was amplified by a polymerase chain reaction and Sanger sequenced. Maximum likelihood phylogenetic trees (1000 bootstrap replicates) were used to identify patterns of clustering (3% genetic distance threshold). Mixed‐effects logistic regression was used to determine correlates of phylogenetic clustering. Factors assessed were sexual risk behavior, education, injecting drug use, housing, employment, HIV viral load, age, sex, and sexuality.ResultsPhylogenetic trees were reconstructed for HCV subtype 1a (n = 139) and 3a (n = 63) sequences, with 29% (58/202) in a pair or cluster. Overall (n = 202), phylogenetic clustering was positively associated with younger age (under 40; adjusted odds ratio [aOR] 2.52, 95% confidence interval [CI] 1.20–5.29), and among gay and bisexual men (n = 168), was positively associated with younger age (aOR 2.61, 95% CI 1.10–6.19), higher education (aOR 2.58, 95% CI 1.09–6.13), and reporting high‐risk sexual behavior (aOR 3.94, 95% CI 1.31–11.84). During follow‐up, five reinfections were observed, but none were in phylogenetic clusters.ConclusionThis study found a high proportion of phylogenetic relatedness, predominantly among younger people and gay and bisexual men reporting high‐risk sexual behavior. Despite this, few reinfections were observed, and reinfections demonstrated little relationship with known clusters. These findings highlight the importance of rapid HCV treatment initiation, together with monitoring of the phylogeny.

Characterisation of a Hepatitis C Virus Subtype 2a Cluster in Scottish PWID with a Suboptimal Response to Glecaprevir/Pibrentasvir Treatment.

Direct-acting antivirals (DAAs) have revolutionised the treatment of Hepatitis C virus (HCV), allowing the World Health Organisation (WHO) to set a target of eliminating HCV by 2030. In this study we aimed to investigate glecaprevir and pibrentasvir (GP) treatment outcomes in a cohort of patients with genotype 2a infection. Methods: Clinical data and plasma samples were collected in NHS Greater Glasgow & Clyde. Next generation whole genome sequencing and replicon assays were carried out at the MRC-University of Glasgow Centre for Virus Research. Results: 132 cases infected with genotype 2a HCV were identified. The SVR rate for this group was 91% (112/123) following treatment with GP. An NS5A polymorphism, L31M, was detected in all cases of g2a infection, and L31M+R353K in individuals that failed treatment. The results showed that R353K was present in 90% of individuals in the Glasgow genotype 2a phylogenetic cluster but in less than 5% of all HCV subtype 2a published sequences. In vitro efficacy of pibrentasvir against sub-genomic replicon constructs containing these mutations showed a 2-fold increase in IC50 compared to wildtype. Conclusion: This study describes a cluster of HCV genotype 2a infection associated with a lower-than-expected SVR rate following GP treatment in association with the NS5A mutations L31M+R353K.

The Glycodiversity of HCV E2 Glycoprotein-Specific Antibodies as a Signature of Hepatic Damage and Virotherapy Efficacy

The HCV E2 glycoprotein-specific Abs (E2-Ab) is an important factor in the host resistance to hepatitis C virus (HCV) infection. There is evidence that the E2-Ab sialylation is associated with liver damage and virotherapy efficacy. The aim of this study was to further profile the E2-Ab glycosylation. The fucosylation and sialylation of E2-Ab in one hundred six (HCV)-infected patients were tested using the lectin-based ELISA platform. Data were analyzed by the stage of hepatic fibrosis, HCV genotype and the response to IF-RBV virotherapy. The changes in the E2–Ab glycosylation were also evaluated by the receiver operator characteristic curve (ROC) and multiple regression analysis. The E2-Ab reactivity to fucose-specific Aleuria aurantia lectin (AAL) and sialo-specific Sambucus nigra agglutinin (SNA) was decreased in the advanced stages of liver fibrosis. The SNA binding analysis was more informative in the discrimination of patients with advanced fibrosis compared to those with earlier fibrosis stages or no fibrosis group. No significant correlation between the reactivities of SNA and AAL lectins was established irrespective of HCV genotype. The patients infected with HCV 3a genotype showed an increased E2-Ab fucosylation, a lower E2-SNA/E2-AAL ratio and a better response to virotherapy. The association of the E2-SNA/E2-AAL ratio with virotherapy outcome was observed in patients infected with HCV 1b genotype. A better response to IF-RBV therapy was found in patients with a higher fucosylated E2 Abs and a lower E2-SNA/E2-AAL ratio. Thus the significance of E2 antibodies in the course of HCV hepatitis was demonstrated to be dependent on their glycosylation, the sialylation/fucosylation ratio, as well as on HCV genotype.

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

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

Complete genome sequencing and evolutionary analysis of hepatitis C virus subtype 6a, including strains from Guangdong Province, China.

We performed an evolutionary analysis using whole genome sequence isolates of hepatitis C virus (HCV) 6a from Guangdong Province and reference sequences from various countries. Less than 5% of the HCV genome was found to be under positive selection. The E1 and E2 proteins had the highest proportion of positively selected sites both within and outside of CD8 T cell epitopes in all of the strains. Regions corresponding to CD8 T cell epitopes were under negative selection except in the isolates from Guangdong. Furthermore, we found evidence of three introductions of the virus into Guangdong from Vietnam and other Southeast Asian countries. Thus, this study provides information about the transmission of HCV 6a by comparison of full-length sequences, indicating the impact of selective constraints in Guangdong and across China.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05358-7.

Evaluation of Epidemiologic Peculiarities of HIV-Infection and Genotype Distribution of Hepatitis C Virus among People Living with HIV in Constituent Entities of the Far Eastern Federal District

Relevance. Monitoring the genotypic diversity of the hepatitis C virus as well as its prevalence in the population, including in certain population groups, is an important public health task.Aim. To analyze main epidemiological indices (incidence and prevalence) among people living with HIV and evaluate abundance of chronic hepatitis C among them, reveal molecular-genetic distribution of hepatitis C virus (HCV) isolated from HIV-positive people living in the Far Eastern Federal district (FEFD) of the Russian Federation.Materials and methods: a retrospective epidemiological analysis including estimation of HIV-infection incidence and prevalence as well as percent of HIV-positive people suffering from chronic viral hepatitis C was conducted for nine constituent entities of the FEFD. Molecular genetic analysis included evaluation of 120 samples of serum positive for HCV RNA that was obtained from people diagnosed with HIV-infection. A type-specific commercial PCR test-kit as well as partial sequencing by Sanger of HCV NS5B gene (241 bp and 350 bp) were utilized in order to specify HCV genotypes.Results and discussion: in total 26.26% of people living with HIV were registered for regular medical examination due to chronic hepatitis C in the FEFD in year 2019. Their percentage has grown over a 5-year period. Dominant HCV subtype among surveyed HIV-positive people was subtype 3a followed by HCV subtype 1b. HCV genotype 2 and subtype 1a were detected less frequently and were identified as minor genotypes. Genotype distribution among people aged under 35 years was more significant in comparison with older citizens. Some regional particularities were discovered. For example, HCV genotype 2 was registered more frequently in the Khabarovsk krai compared to mean estimated value of the sampling collection. Subtype 1b was dominant in Republic Sakha (Yakutia).Conclusion: an increase in percent of HIVinfected people suffering from chronic hepatitis C has been noted during a 5-year period. Should be noted that the value varies in different constituent entities ofthe FEFD. Molecular-genetic analysis showed general prevalence of HCV 3a subtype while 1b HCV subtype is dominant in the Russian Federation in patients suffering from HCV-monoinfection as well as in those infected with HIV and HCV.

Prevalence of Naturally Occurring Resistance Associated Substitutions in NS3/4AProtease Inhibitors in Iranian HCV/HIV Infected Patients.

Hepatitis C virus (HCV) acts in the host as a complicated mixture of related variants with the potency to genetically escape host immune responses. Direct acting antivirals (DAAs) have been approved for HCV treatment with shorter duration, better cure rates and lower side effects. However, naturally occurring resistance associated substitutions (RASs) create some obstacles to this antiviral therapy success. In this study, we aimed at the determination of the naturally occurring NS3/4A RASs in HCV/human immunodeficiency virus (HIV)infected patients. A total of 120 DAA-naïve HCV-HIV co-infected patients were included. HCV NS3/4Agenome region was amplified with PCR and mutation analysis was performed by Sanger sequencing technique. The amino acid sequence diversity of the region was analyzed using geno2pheno HCV. Phylogenetic analysis showed that 73 cases were infected by 3a and 47 subjects by subtype1a. The overall RASs among studied subjects were observed in 6 (5%) individuals from 120 studied cases who were infected with HCV 1a. V36M/L, Q80L, S122G/L, R155T/G, A156S, D168Y/N and S174A/N/T mutations were detected in this study. Although the prevalence of RASs was totally low in this study, the presence of several cases of double and triple mutants among this population suggests prior evaluation of protease inhibitors related mutations before initiation of standard treatment and also an investigation on a large population could be of high value.

Retraction Note: Small interfering RNA targeted to stem-loop II of the 5\u2032 untranslated region effectively inhibits expression of six HCV genotypes

The antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance. Four different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains. These in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

High prevalence and genotype distribution of hepatitis C virus in people living with HIV in Kathmandu, Nepal

Background Hepatitis C virus (HCV) co-infection is still a significant cause of morbidity and mortality among HIV-positive individuals in many resource-limited countries. As prevalence rates of co-infection using the serological diagnosis of HCV infection might be imprecise, estimates of prevalence using polymerase chain reaction (PCR) confirmed diagnosis is needed to guide HCV treatment efforts among HIV-positive individuals in resource-limited countries. Methods We conducted this community-based cross-sectional study among 280 HIV-positive individuals recruited through the networks of five non-government organizations working with HIV-positive individuals in Kathmandu, Nepal. We collected blood samples from each participant and tested all the anti-HCV positive samples for HCV-RNA and genotypes. We calculated the prevalence of HCV/HIV co-infection and examined factors associated with it using multivariable logistic regression analysis. We also calculated the proportion of infection by different HCV genotypes and investigated HCV seroconversion. Results The prevalence of HCV/HIV co-infection was 29.6% (95% CI 24.25–34.95). History of a lifetime injecting drug use was associated with a higher likelihood of HCV/HIV co-infection (p < .001). Of the 81 individuals whose serum samples were available for genotype assessment, 55.7% tested positive for genotype 3A, 36.7% for genotype 1A and the remaining samples' genotype was undetermined (7.6%). Of the 100 anti-HCV positive samples, 17 (17.0%) tested negative for HCV RNA. Conclusions High prevalence of HCV/HIV co-infection, distribution of prevalent HCV genotype 1A and 3A and HCV seroconversion rate have important implications for the public health system in guiding HCV treatment and control efforts among HIV-positive individuals.

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

Background & AimsInfection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker.MethodsThe anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights.ResultsAvFc was capable of neutralizing cell culture–derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed.ConclusionsThese results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Impact of IL28 Genotypes and Modeling the Interactions of HCV Core Protein on Treatment of Hepatitis C

Mutations in the core CVR region of hepatitis C virus (HCV) and polymorphisms ofinterleukin 28B (IL28B) are associated with progressiontowardliver disease and in response to therapy. In addition, interactions of the core protein with some cell interactors can be related to HCV liver damage. This study aimed to evaluate the effect of core mutations as well as IL28B polymorphism on clinical features, sustained virological response (SVR) in 1a and 3a HCV genotypes amongst Iranian HCV infected patients, and the impact of mutations on core protein properties, antigenic properties, and interactions with HCV inhibitors, using several bioinformatics tools. Seventy-nine Iranian patients infected with HCV genotypes 1a and 3a and diagnosed with chronic active hepatitis were examined. Plasma viral RNA was used to amplify and sequence the HCV Core gene; also, HCV viral load, molecular genotyping, and the liver enzymes were determined for all samples. The sequencing results were analyzed by several reliable bioinformatics tools to determine the physicochemical properties, B cell epitopes, post-modification changes, and secondary/tertiary structures; and evaluate the interactions with 4 drugs by docking method. There were some substitutions in core CVR related to ALT and AST enzymes that can lead to HCV advanced liver disease. The most prevalent mutation for 3a genotypes was a substitution in aa 162 (I to V) while we did not find any mutation in 1a responder group. Polymorphism of the rs8099917 showed that the majority of patients had TG heterozygous and carried CT genotype at the rs12979860. Analysis indicated several phosphorylation sits for core protein as well as two important disulfide bonds. Immunogenic prediction showed that core protein can strongly induce the immune system. Interaction analysis, using the docking method revealed two potential interactors (Vitronectin and SETD2). Generally, mutations in all core CVR regions in all patients showed a relationship between such substitutions and higher liver enzymes that can result in advanced liver disease progression in HCV infected patients. Furthermore, immunoinformatics analysis determined the possible immunodominant regions to be considered in HCV vaccine designs. Furthermore, no association between SVR and IL28B polymorphism was shown. In silico analysis determined modification sites, structures, B-cell epitopes of core protein and interactions with several interactors can lead to persistentHCV infection in the cell and the progress of liver diseases.

Hepatitis C virus genotype/subtype distribution and evolution among Chineseblood donors: Revealing recent viral expansion.

Hepatitis C virus (HCV) genotype (GT) distribution in China shows significant geographical and demographic difference. As a routinely tested virus in Chinese blood bank systems, rare molecular epidemiology research in blood donors is reported. Our purpose is to investigate the HCV GT/subtypes distribution, phylogenetic analysis and population genetics in Chinese blood donors. Anti-HCV screen positive samples and donor demographics were collected. HCV Core and E1 gene fragments were amplified by RT-PCR, followed by sequencing and phylogenetic analysis to determine HCV GTs/subtypes using MEGA 7.0. The population genetics were performed using Arlequin v3.0 and Beast v1.10.4. SPSS Statistics 17.0 software was used to analyze the correlation between HCV GTs/subtypes distribution and demographic characteristics. 419 and 293 samples based on Core and E1 gene respectively were successfully amplified. HCV la, lb, 2a, 3a, 3b, 6a, 6e and 6n were found, and the corresponding proportions were 0.66% (3/455), 58.68% (267/455), 17.80% (81/455) and 5.05% (23/455), 3.52% (16/455), 12.31% (56/455), 0.88% (4/455) and 0.66% (3/455). Samples from Guangxi showed the most abundant genetic diversity with 8 subtypes were found. The number of haplotypes in HCV-1b is higher than 2a and 6a. The negative Tajima's D and Fu's Fs values of HCV-1b, 2a and 6a suggested the population expansion of those HCV subtypes. The distribution of HCV GT showed significant statistical difference by age and ethnicity. Conclusion: An abundance of HCV genetic diversity was found in Chinese blood donors with mainly 1b and then 2a subtype. There were significant geographical and demographic differences in HCV GTs/subtypes among Chinese blood donors. HCV subtype 1b has stronger viability and HCV subtype 6a has experienced significant expansion.

Resistance-associated substitutions (RASs) to HCV direct-acting antivirals (DAAs) at baseline of treatment in thalassemia patients: a referral center study.

Patients with thalassemia major are at high risk of hepatitis C through blood transfusion from donors infected by hepatitis C virus (HCV). The use of direct-acting antiviral (DAA) therapy against such HCV infections has increased in different populations. However, resistant viral variants can affect treatment outcomes, and therefore improved surveillance strategies are needed. Accordingly, we aimed to evaluate resistance-associated substitutions (RASs) to HCV DAAs at the baseline of treatment in thalassemia patients in a referral center. Out of 89 thalassemia patients who suffered from HCV infection and were referred to our center between 2016 and 2017, 43 underwent further analysis of the HCV nonstructural proteins NS5A and NS5B using polymerase chain reaction (PCR) sequencing methods. Unique primers were designed using bioinformatics software for separate detection of HCV subtypes 1a, 3a, and 1b. Detection of RASs was performed based on previously published literature. Statistical analysis was carried out using SPSS version 19. The participants, 60.4% (26/43) of whom were male, had a mean age ± standard deviation (SD) of 33.0 ± 5.0years. HCV subtype 1a was found in 27 cases, 3a in 13, and 1b in three. In HCV subtype 1a there were 163 mutations in NS5A and 212 mutations in NS5B. The frequency of RASs was 20.9% (8 RASs in 9 patients), including M28V and H58P in subtype 1a, L28M, R30Q, C316N, and C316S in subtype 1b, and S24F in subtype 3a. Statistically, the subtype 1b and a higher mutation rate in NS5A were associated with RASs (p-value < 0.05). The emergence of natural RASs to HCV DAAs serves as a warning of the risk of drug resistance in response to the broad usage of antivirals. However, relapses in these DAA-treated HCV-infected thalassemia patients are rarely reported. Our findings indicate that the prevalence of RASs prevalence at baseline was 20.9% in these patients, and this calls for extrapolation to a larger population study, as highlighted in other studies, with larger sample sizes, high-throughput methods, and follow-up in order to fully evaluate treatment outcomes in RASs-detected individuals. Optimized therapeutic strategies, particularly in complex, difficult-to-cure patients, can effectively prevent DAA treatment failure as a result of selection for RASs.

Association of the Sialylation of Antibodies Specific to the HCV E2 Envelope Glycoprotein with Hepatic Fibrosis Progression and Antiviral Therapy Efficacy.

The E2 envelope glycoprotein of the hepatitis C virus (HCV) is a major target of broadly neutralizing antibodies that are closely related to a spontaneous cure of HCV infection. There is still no data about the diversity of E2-specific antibodies (Abs) glycosylation. The aim of this study was to analyze the level and sialylation of E2 IgG Abs, the relation of the respective changes to hepatic fibrosis (F) progression and their possible association with the efficacy of interferon-α-2a plus ribavirin (IFN-RBV) antiviral therapy. One hundred three HCV infected treatment-naive patients were examined using ELISA with E2 recombinant protein as antigen and sialic acid-specific Sambucus nigra agglutinin. The efficacy of the IFN-RBV treatment of patients with HCV dominant 1b and 3a genotypes (GT) was evaluated. A significant decrease of E2 Abs sialylation in the late stages of fibrosis was found irrespective of HCV genotype. On this basis, the F4 stage of fibrosis can be discriminated from its F0 or F1-3 stage by an about 75-79% accuracy. HCV infection of 1b genotype is associated with the production of lower sialylated E2 Abs, a higher frequency of F4 stage fibrosis, and a worse response to antiviral therapy. The increased SNA reactivity of E2 Abs was observed in patients with a sustained virological response (SVR). The proportion of SVR responders was significantly higher among patients with 3a genotype. However, for both dominant HCV genotypes (3a and 1b), an increased sialylation of E2 IgG was associated with a higher rate of patients with sustained virological response to antiviral therapy. Thus, the association of alterations of anti-E2 IgG Abs sialylation with hepatic fibrosis stage, HCV genotype, and the efficacy of antiviral therapy enables using these changes as novel noninvasive predictive biomarkers. The clinical potential of these findings is discussed.

Mixing particles from various HCV genotypes increases the HBV-HCV vaccine ability to elicit broadly cross-neutralizing antibodies.

The development of a safe, effective and affordable prophylactic vaccine against hepatitis C virus (HCV) remains a medical priority. Hepatitis B-C subviral envelope particles, which could be produced by industrial procedures adapted from those established for the hepatitis B virus vaccine, appear promising for use for this purpose. The prototype HBV-HCV bivalent vaccine-bearing genotype 1a HCV envelopes can induce neutralizing antibodies against this genotype, but is less effective against other genotypes. We show here, in a small animal model, that the use of a set of vaccine particles harbouring envelopes from different HCV genotypes in various association strategies can induce broad neutralizing protection or an optimized protection against a particular genotype prevalent in a given region, such as genotype 4 in Egypt. This vaccine could help to control the hepatitis C epidemic worldwide.

Development of a Method for Screening and Genotyping of HCV 1a, 1b, 2, 3, 4, and 6 Genotypes.

The World Health Organization and the World Health Assembly recommended eradicating hepatitis as a public threat by 2030. The accurate genotyping of hepatitis C virus (HCV) is crucial to achieving this goal because it is vital for the selection of anti-HCV therapy required for complete cure of HCV infection. We report the development of a method for accurate genotyping of HCV 1a, 1b, 2, 3, 4, and 6 genotypes. The merits of the developed method for HCV genotyping include (i) requirement of a single polymerase chain reaction (PCR) primer set, (ii) room-temperature detection in 30 min after the PCR, (iii) no need of highly trained professionals, (iv) highly accurate HCV genotyping results afforded by highly specific DNA–DNA hybridization, and (v) probe sequences that can be used on other platforms.