A shift in the Hepatitis C virus genotype dominance in blood donor samples from Thailand

Hepatitis C virus (HCV) can be classified into six major genotypes. The HCV genotypes variability accounts for its geographical distribution, its responses to treatments and the clinical outcomes. The aim of this study was to determine the distribution of HCV genotypes among volunteer blood donors in Thailand. Samples from 135 anti-HCV positive blood donors were analyzed. HCV RNA and genotyping was carried out using nested polymerase chain reaction (PCR) and genotype-specific primer PCR for a portion of the core region. HCV RNA was detected in 109 samples (80.7%). Genotype analysis demonstrated four different genotypes. The most common was genotype 3a (36.7%), followed by genotype 6 (29.4%), 1a (19.3%), 1b (6.4%) and mixed infection (1.8%). Seven samples were untyped (6.4%) in the present study. In several previous reports, the prevalence found in Thailand was HCV genotypes 3, 1 and 6. The present results show an increasing importance of the genotype 6 in HCV infections. This study has also described for the first time in Thailand mixed infections of HCV genotypes.     

Prevalence of hepatitis C virus (HCV) genotypes among positive UAE patients

A molecular study was conducted to investigate the prevalence of Hepatitis C virus genotypes in HCV infected population of UAE. 67 HCV seropositive samples were collected from different health care centres. Quantitative analysis of these samples using PCR resulted in 67 positive samples. The PCR positive samples were subjected to genotyping using the method described by Simmonds et al. (J Gen Virol 74: 2391–2399, 1993). HCV genotype 4 was the predominant genotype (46.2%) followed by genotype 3a (23.8%) and 1a (15%). The predominant genotype among the female patients was genotype 4 (65.6%), while genotype 3a was the predominant among the male patients (42.8%). The predominance of HCV genotype 4 in our population confirms the predominance of HCV genotype 4 in UAE and most of the Arab countries in the Middle East. Implications of genotyping for clinical outcome of HCV infection, response to treatment as well as for vaccine development are discussed.     

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background

Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region.

Methodology/Principal Findings

Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a.

Conclusions/Significance

The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.     

Genotypic Distribution of Hepatitis C Virus in Thailand and Southeast Asia

The majority of hepatitis C virus (HCV) infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV) is estimated at 150 million individuals, or 3% of the world’s population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5’ untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%), followed by 1a (19.9%), 1b (12.6%), 3b (9.7%) and 2a (0.5%). While genotype 1 was prevalent throughout Thailand (27–36%), genotype 3 was more common in the south. Genotype 6 (20.9%) constituted subtype 6f (7.8%), 6n (7.7%), 6i (3.4%), 6j and 6m (0.7% each), 6c (0.3%), 6v and 6xa (0.2% each) and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively). Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.     

A comparative evaluation between real time Roche COBas TAQMAN 48 HCV and bDNA Bayer Versant HCV 3.0

The HCV virus is a common human pathogen made of a single stranded RNA genome with 9600nt. This work compared two different commercial methods used for HCV viral load, the bDNA Bayer Versant HCV 3.0 and the RealTime Roche COBAS TaqMan 48 HCV. We compared the reproducibility and linearity of the two methods. Seventy-five plasma samples with genotypes 1 to 4, which represent the population (45% genotype 1; 24% genotype 2; 13% genotype 3; 18% genotype 4) were directly processed with the Versanto method based upon signal amplification; the same samples were first extracted (COBAS Ampliprep - TNAI) and then amplified using RealTime PCR (COBAS TaqMan 48). The results obtained indicate the same performance for both methods if they have genotype 1, but in samples with genotypes 2, 3 and 4 the RealTime PCR Roche method gave an underestimation in respect to the Bayer bDNA assay.     

Hepatitis C virus genotypes in patients with chronic hepatitis C infection in southern Iran from 2016 to 2019

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The treatment of HCV infection has become more complicated due to various genotypes and subtypes of HCV. The treatment of HCV has made significant advances with direct-acting antivirals. However, for the choice of medicine or the combination of drugs for hepatitis C, it is imperative to detect and discriminate the crucial HCV genotypes. The main objective of this study was to determine the pattern of circulating HCV genotypes in southern Iran, from 2016 until 2019. The other aim of the study was to determine possible associations of patients’ risk factors with HCV genotypes. A total of 803 serum samples were collected in 4 years (2016–2019) from patients with HCV antibody positive results. A total of 728 serum samples were HCV-RNA positive. The prevalence of HCV genotypes was detected using the genotype-specific RT-PCR test for serum samples obtained from 615 patients. The HCV genotype 1 (G1) was the most prevalent (48.8%) genotype in the area, with G1a, G1b, and mixed G1a/b representing 38.4%, 10.1%, and 0.3%, respectively. Genotype 3a was the next most prevalent (47.2%). Mixed genotypes 1a/3a were detected in 22 (3.6%) and finally G4 was found in 3 (0.5%) patients. The other HCV genotypes were not detected in any patient. Genotype 1 (1a and 1b alone, 1a/1b and 1a/3a coinfections) is the most prevalent HCV genotype in southern Iran. HCV G1 shows a significantly higher rate in people under 40 years old.     

Prospective study of hepatitis C virus infection in hemodialysis patients by monthly analysis of HCV RNA and antibodies

The aims of this study were to (i) evaluate the prevalence and the incidence of hepatitis C virus (HCV) infection in hemodialysis patients in two different centers in S?o Paulo (Brazil), (ii) determine the time required to detect HCV infection among these patients by serology or PCR, (iii) establish the importance of alanine aminotransferase determination as a marker of HCV infection, and (iv) identify the HCV genotypes in this population. Serum samples were collected monthly for 1 year from 281 patients admitted to hospital for hemodialysis. Out of 281 patients, 41 patients (14.6%) were HCV positive; six patients seroconverted during this study (incidence = 3.1/1000 person-month). In 1.8% (5/281) of cases, RNA was detected before the appearance of antibodies (up to 5 months), and in 1.1% (3/281) of cases, RNA was the unique marker of HCV infection. The genotypes found were 1a, 1b, 3a, and 4a. The presence of genotype 4a is noteworthy, since it is a rare genotype in Brazil. These data pointed out the high prevalence and incidence of HCV infection at hemodialysis centers in Brazil and showed that routine PCR is fundamental for improving the detection of HCV carriers among patients undergoing hemodialysis.     

吉林省延边地区丙型肝炎病毒基因型分布

为了解延边地区不同丙型肝炎病毒(HCV)基因型的分布特点,应用逆转录-聚合酶链反应(RT-PCR)技术,检测了48例本地区抗HCV阳性血清,结果有29例出现HCVRNA阳性.再利用限制性片段长度多态性(RFLP)技术,对29例RT-PCR阳性标本进行HCV基因分型.结果延边地区主要流行HCVⅡ型,其次是HCVⅢ型,也存在HCVⅡ/Ⅲ混合型,且HCVⅢ型的感染率较高.     

502例丙型肝炎患者病毒基因分型及与性别和年龄的关系研究

目的:探讨502例丙型肝炎患者病毒基因分型的情况,分析其与性别和年龄的关系,为丙型肝炎的防治提供参考。方法:收集贵州地区502例丙型肝炎患者的血液标本,采用PCR-荧光探针法检测丙型肝炎病毒基因型,并对检测数据进行统计学分析。结果:共检出4种基因型及5种基因亚型,主要以1b型、3b型为主,其次是6a、3a型,2a型较少。不同性别丙型肝炎病毒(HCV)基因型分布比较,差异有统计学意义(x²=11.029,P=0.026),男、女性患者均以1b型、3b型为主要基因型,但男性感染者明显多于女性。各个年龄组HCV基因型分布比较,差异有统计学意义(x²=102.946,P=0.000),40-59岁组感染者最多,以1b型为主,其次是20-39岁组,以3b型为主,≥60岁组感染人数较少,以1b型为主。结论:贵州地区HCV呈现多基因型分布,1b、3b型为主要流行基因型,6a、3a型次之,其余基因型较少,不同性别、年龄的HCV基因型分布存在差异性。     

High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC₅₀ value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.     

Investigating the origin and spread of hepatitis C virus genotype 5a

Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.     

HCV基因型的差异性流行与进化

丙型肝炎病毒(Hepatitis C virus,HCV)是导致慢性肝炎的主要病原体之一,全球感染人数大约为1.7亿。HCV基因组具有高度变异特性,利用现代遗传分类方法,可将HCV分为6个基因型和80多个基因亚型。不同HCV基因型、亚型的分布与流行具有明显地域特性:1型、2型呈全球流行态势,3型主要流行于亚洲、北美及欧洲部分地区,4型主要流行于中非、中东和欧洲地区,5型主要发现于非洲和欧洲部分国家,6型则主要在东南亚和北美地区流行。我国流行的HCV有1、2、3和6四种基因型,北方仍以1b和2a型为主要流行基因型,近年来3型和6型在华南、西南地区快速传播。据推断,云南将可能成为我国HCV流行与传播的重要源头,引起目前HCV基因型/亚型分布的较大变化,并呈现多样化的传播方式。通过溯祖理论和进化分子钟等分析方法,了解HCV不同基因型差异性流行与进化,对研究HCV的分子流行病学特征,对应性制定丙型肝炎的预防控制策略具有重要意义。     

HCV genotype analysis in HCV-HIV-co-infected Puerto Ricans who are injecting drug users: undetermined and mixed infections.

Direct percutaneous exposure is the main route of HCV transmission. In Puerto Rico half of people infected with HIV use illicit drugs. The effects of HCV in the course of HIV infection and vice versa have been extensively studied, but remain highly controversial. This may be due to HCV genetic heterogeneity. Therefore, a complex classification into genotypes has emerged that prompted us to determined how this impacts a population of intravenous drug users (IDUs) co-infected with HIV-1. Using Inno-LiPa II technique, we analyzed samples from 171 HCV-HIV-1-co-infected IDUs and 375 from a general HCV population of unknown HIV or source of infection status. Similar HCV genotype distribution was detected in these populations. HCV genotype 1a was the most frequently in IDUs-co-infected with HIV-1, followed by 1b and 3a. Twenty mixed infections and 5 undetermined genotypes were reported. A reduced HCV viral load was observed in HIV-1 positives with wasting syndrome. Individuals with a high HIV-1 viral load presented a low HCV viral load. There were no correlation between HCV genotypes and AIDS-related event. Patients with genotype 1b showed a higher HCV viral load. Males presented higher HCV viral load than females. Females were predominantly affected by genotype 1a, and men by 1a and 1b. Neither the HCV viral load nor the frequency of genotypes were influenced by the antiretroviral modality. The importance of continuous genotype monitoring is stressed.     

Hepatitis C Virus Genotype Diversity among Intravenous Drug Users in Yunnan Province,Southwestern China

Background

Recently, high proportions (15.6%–98.7%) of intravenous drug users (IDUs) in China were found to be positive for hepatitis C virus (HCV). Yunnan Province is located in southwestern China and borders one of the world''s most important opium-producing regions, thus it is an important drug trafficking route to other regions of China.

Methodology/Principal Findings

Here, we assessed 100 HCV-positive plasma samples from IDUs who were enrolled through the Kunming Center for Disease Control and Prevention in 2012. HCV C/E1 fragments were PCR-amplified and sequenced. We identified eight HCV subtypes (1a, 1b, 3a, 3b, 6a, 6n, 6u and 6v), of which genotype 6 was most predominant (frequency, 47%) followed by genotypes 3 (41%) and 1 (12%). HCV subtypes 6n (30%) and 3b (29%) were most common and were identified in 59% of the IDUs. We compared HCV genotypes among IDUs in Yunnan Province with those from other regions and found that the distribution patterns of HCV genotypes in Yunnan Province were similar to those in southern China, but different from those in eastern China. However, the distribution patterns of HCV subtypes varied among Yunnan Province and southern China, despite the shared similar genotypes. A comparison of the current data with those previously reported showed that the frequency of HCV genotype 6 increased from 25% to 47% within 5 years, especially subtypes 6a (5% to 15%) and 6n (11.2% to 30%). In contrast, the frequencies of subtypes 3b and 1b decreased by almost 50% within 5 years.

Conclusion/Significance

Our results provided further information to support the assertion that drug trafficking routes influence HCV transmission patterns among IDUs in Yunnan Province. The frequency of HCV genotypes and subtypes changed rapidly among IDUs in Yunnan Province and subtypes 6a and 6n may have originated in Vietnam and Myanmar, respectively.     

Liver Fibrosis,Host Genetic and Hepatitis C Virus Related Parameters as Predictive Factors of Response to Therapy against Hepatitis C Virus in HIV/HCV Coinfected Patients

Objective

To establish the role of liver fibrosis as a predictive tool of response to pegylated interferon alpha (Peg-IFN) and ribavirin (RBV) treatment in human immunodeficiency (HIV)/hepatitis C virus (HCV) coinfected patients, in addition to recognized predictive factors (HCV load, HCV genotype, IL-28B polymorphism).

Patients and Methods

A sample of 267 HIV/HCV coinfected patients was treated with Peg-IFN and RBV. Predictive factors of rapid (RVR) and sustained (SVR) virological response were analyzed. Independent variables were age, sex, IL28B, −238 TNF-α and −592 IL-10 polymorphisms, HCV genotype, HCV-RNA levels, significant fibrosis or cirrhosis and CD4+ T cell count.

Results

Patients infected by HCV genotype 1 (n = 187) showed RVR and SVR in 12% and 39% of cases, respectively. The parameters associated with RVR were IL28B genotype CC and plasma HCV-RNA levels <600000 IU/ml. Advanced liver fibrosis was negatively associated with SVR in patients without RVR. A SVR was obtained in 42% of subjects with HCV genotype 4, and the independent factors associated with SVR were IL28B genotype CC and an HCV-RNA <600000 IU/ml. A SVR was obtained in 66% of patients with HCV genotypes 2/3; in this case, the independent parameter associated with SVR was the absence of significant liver fibrosis. TNF-α and IL-10 polymorphisms were not associated with SVR, although a significantly higher percentage of −238 TNF-α genotype GG was detected in patients with significant liver fibrosis.

Conclusions

In HIV/HCV coinfected patients with HCV genotypes 1 or 4, RVR, mainly influenced by genotype IL28B and HCV-RNA levels, reliably predicted SVR after 4 weeks of therapy with Peg-IFN plus RBV. In patients infected by HCV genotype 3, an elevated relapse rate compromised the influence of RVR on SVR. Relapses were related to the presence of advanced liver fibrosis. Liver cirrhosis was associated with a −238 TNF-α polymorphism in these patients.     

Test of IL28B Polymorphisms in Chronic Hepatitis C Patients Treated with PegIFN and Ribavirin Depends on HCV Genotypes: Results from a Meta-Analysis

Background

Many studies have been published on the association between single nucleotide polymorphisms (SNP) near the IL28B gene and response to the combined treatments of pegylated-interferon (PegIFN) and ribavirin (RBV) in chronic HCV-infected patients, but without identical conclusions. The aim of this study was to assess impact of the IL28B polymorphisms on the effect of HCV standard treatment using meta-analysis based method.

Methods

Association studies between polymorphisms of rs12979860 or rs8099917 and response to PegIFN/RBV treatment in chronic HCV patients were retrieved from PubMed. Data of qualified studies on sustained virological response (SVR) in different genotypes were extracted and analyzed using meta-analysis method in Stata 10 software.

Results

Thirty-four papers, containing 46 independent studies, were included in the analysis. In the HCV G1/4 patients without treatment history, individuals carrying rs12979860 CC genotype were more likely to achieve SVR (OR 3.97, 95%CI 3.29–4.80) compared to those carrying CT/TT genotypes. Similar results were observed in the HCV G1/4 patients with unsuccessful or unknown treatment history (OR 3.76, 95%CI 2.67–5.28) or in the patients co-infected with human immunodeficiency virus (OR 5.20, 95%CI 3.04–8.90). However, associations could not be observed in HCV G2/3 patients. For rs8099917, similar results were obtained for genotype TT compared to genotypes TG/GG, indicating that TT genotype was significantly associated with better treatment response in patients infected with genotype 1 or 4 HCV, but not genotype 2 or 3 HCV.

Conclusion

Polymorphisms of rs12979860 and rs8099917 near IL28B only associate with the treatment response to PegIFN/RBV in patients infected with HCV genotype 1 or 4 but not with genotype 2 or 3, irrespective of the previous treatment history or HIV co-infected status. Therefore, identification of IL28B genotypes is necessary only in patients infected with relatively difficult-to-treat genotype 1 or 4 HCV.     

Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease

Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC₅₀ values) below 10 µM with comparable direct binding affinities (K_D values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC₅₀ values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.     

Molecular epidemiology of the hepatitis C virus in Brazil

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The genome of this virus consists of approximately 10,000 bp and codes for 10 mature polypeptides. Genome sequence comparison has revealed the existence of six major genotypes and a large number of subtypes. The genotypes can be distinguished by whole genome or genome fragment sequencing, genotype specific amplification of a genomic region or PCR amplification, followed by hybridization or restriction digestion, among other methods. There is a markedly heterogeneous geographical distribution of the HCV genotypes in the world. Different genotypes have been linked to distinct clinical outcomes and to differences in the susceptibility of the virus to interferon treatment. Several studies have been conducted to determine the distribution of HCV genotypes among different groups of individuals in Brazil. Most of these studies indicate a higher prevalence of genotype 1, followed by genotypes 3 and 2. Differences in genotypes can affect serological detection as well as the clinical outcome of the disease and sensibility to interferon treatment. Further studies need to be conducted to determine the degree of differentiation of circulating HCV genotypes in different patient groups in Brazil.