Molecular characterization of genotype 2 and 4 hepatitis C virus isolates in French blood donors

The subtype distribution of 142 genotype 2 and 97 genotype 4 hepatitis C virus (HCV) isolates from the sera of 1,319 volunteer blood donors in France was determined by gene sequencing and by phylogenetic analysis of the NS5B region and E1 envelope. Findings underlined a wide range of subtypes in both genotypes, that is, 20 in HCV-2 and 11 in HCV-4. Eighteen of these 31 subtypes had not been defined previously. Some subtypes, that is, 2a, 2b, 2c, 2i, 2k, 4a, and 4d, showed numerous strains while subtypes in donors from West Africa or Central Africa showed an endemic profile with only a few strains. A Bayesian coalescence approach was used to estimate the demographic history of each HCV subtype. The estimated mean dates of the most recent common ancestors (MRCA) were 1,889 (confidence interval (CI), 1,842-1,930) for HCV-2a, 1,886 (CI, 1,843-1,921) for HCV-2b, 1,791 (CI, 1,699-1,848) for HCV-2c, 1,846 (CI, 1,803-1,878) for HCV-2i, 1,911 (CI, 1,879-1,937) for HCV-4a, and 1,957 (CI, 1,943-1,967) for HCV-4d. The period of spread for subtype 2b, 2c, and 2i was between 1900 and 1960 whereas rapid exponential spread for subtype 2a, 4a, and 4d occurred in the 1960s. The inferred histories of population growth indicated that transmission rates differed according to HCV subtype. These results may help to predict the future burden of HCV in France.     

Morocco underwent a drift of circulating hepatitis C virus subtypes in recent decades

Hepatitis C virus (HCV) isolates circulating in Morocco are poorly documented. To determine the subgenotype distribution of HCV in chronically infected patients, serum samples from 185 anti-HCV-positive patients were analyzed. Determination of the HCV genotype and subtype was performed by sequencing the 5'UTR, NS5B and core regions. According to the NS5B phylogeny, the HCV strains primarily belonged to subtypes 1b (75.2%), 2i (19.1%) and 2k (2.8%). Using a Bayesian approach, the mean date of appearance of the most recent common ancestor was estimated to be 1910 for HCV-1b and 1854 for HCV-2i. Although it is currently the most frequent genotype in Morocco and the dominant form in hepatocellular carcinoma, it thus appears that HCV-1b was introduced into the population subsequently to HCV-2i.     

High prevalence of hepatitis C virus subtypes 4c and 4d in Malaga (Spain): phylogenetic and epidemiological analyses

Hepatitis C virus (HCV) is a major aetiological agent of chronic hepatitis and it may lead to the development of liver cirrhosis and hepatocellular carcinoma. HCV has been classified into six clades as a result of high genetic variability. A commercial procedure to genotype HCV in 678 patients from Carlos Haya Regional University Hospital, Malaga was used to study the distribution of HCV genotypes in Malaga, southern Spain. A high prevalence of HCV-4 (10.2%) was found. This genotype is found more commonly in Egypt, Central Africa and the Middle East. The distribution of the different subtypes in the 69 patients with HCV-4 was as follows: 4.3% subtype 4e, 7.2% subtype 4a, 11.5% not subtypable, and 76.8% subtype 4c/4d. Of the 53 4c/4d patients, 69% were intravenous drug users and 31% non-intravenous drug users. In order to characterise further the HCV-4c/4d patients, sequences of the non-structural 5B gene (393 bp) were obtained from 36 HCV-4c/4d-infected untreated patients. Phylogenetic tree topologies distinguished clearly the two subtypes: 11 patients were infected by subtype 4c and 25 by 4d. This phylogenetic analysis, reinforced by the epidemiological characteristics, suggests the extension of the HCV-4c and -4d subtypes in the area of Malaga among both intravenous drug users and non-intravenous drug users.     

Genetic diversity of HCV genotype 2 strains in south western France

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. The genetic heterogeneity of HCV and its spread among infected patients can be examined accurately by nucleotide sequencing. The diversity of HCV genotype 2 strains (HCV-2) was studied in a large cohort of patients in the Midi Pyrénées area of southern France. Phylogenetic analysis was performed on 344 NS5B sequences from patients infected with HCV-2. These included 145 strains whose E2 region was also analyzed, and epidemiological data were collected for the corresponding patients. HCV-2 accounts for 11.3% of HCV infections in this area. Phylogenetic analysis of NS5B sequences revealed eight subtypes, while that of the E2 region provided congruent results for 100% of strains. The most frequent subtypes were 2i (24.7%), 2k (22.4%) 2c (17.4%), and 2a (10.8%). The mean age of HCV-2-infected patients was 55.5 years. Epidemiological data showed that blood transfusion is the major route of infection, but it was not associated with any particular subtype. By contrast, intravenous drug users were infected predominantly with genotype 2a. HCV-2a-infected patients were younger than patients infected with other subtypes (48 vs. 56.5 years, P < 0.01). This study shows substantial genetic diversity of HCV-2 subtypes in the south of France and the spread of 2a strains via intravenous drug users.     

Hepatitis C virus (HCV) subtype prevalence in Chiang Mai, Thailand, and identification of novel subtypes of HCV major type 6.

Subtype analysis of hepatitis C viruses (HCVs) obtained from patients with chronic liver disease in Chiang Mai, Thailand, was performed. Of 46 HCV isolates, 13 (28%) were shown to belong to HCV subtype 3a (HCV-3a), 10 (22%) to belong to HCV-1a, 7 (15%) to belong to HCV-1b, 1 (2%) to belong to HCV-3b, and 1 (2%) to belong to a variant group, as determined from partial nucleotide sequences of the NS5B region of the viral genome. Analysis of 5' untranslated region sequences identified five other isolates (11%) of HCV type 1 and two other isolates (4%) of type 3. Detailed phylogenetic positions for the variant described above and those previously obtained from blood donors and drug addicts in Chiang Mai were determined by a six-parameter neighbor-joining method on the basis of core, E1, and NS5B region sequences. The results revealed that those sequence variants represent novel subtypes of HCV type 6. The HCV type 6 isolates appear to be antigenically different from isolates of HCV types 1 and 2, as determined by a serotyping method that utilizes recombinant peptides corresponding to a portion of the NS4 protein. The significance of subtype analysis around this area is discussed.     

Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences

Nucleotide sequence analysis of the NS5B region was performed to identify genotypes of 8,479 hepatitis C virus (HCV) RNA-positive patient samples collected in the Canadian province of Quebec. Genotypes could be determined for 97.3% of patients. Genotypes 1 to 6 were found in 59.4, 9.0, 25.7, 3.6, 0.6, and 1.8% of patients, respectively. Two isolates did not classify within the six genotypes. The subtype 1 distribution was 76.7% 1a, 22.6% 1b, and 0.7% others, while the subtype 2 distribution was 31.8% 2a, 47.6% 2b, 10.9% 2c, 4.1% 2i, and 5.6% others. Subtype 3a accounted for 99.1% of genotype 3 strains, while all genotype 5 samples were of subtype 5a. The subtype 4 distribution was 39.2% 4a, 15.4% 4k, 11.6% 4d, 10.2% 4r, and 23.6% others. The subtype 6 distribution was 40.4% 6e, 20.5% 6a, and 39.1% others. The 5' untranslated region (5'UTR) sequences of subtype 6e were indistinguishable from those of genotype 1. All samples that did not classify within the established subtypes were also sequenced in C/E1 and 5'UTR. C/E1 phylogenetic reconstructions were analogous to those of NS5B. The sequences identified in this study allowed the provisional assignments of subtypes 1j, 1k, 2m, 2r, 3i, 4q, 6q, 6r, and 6s. Sixty-four (0.8%) isolates classifying within genotypes 1 to 6 could not be assigned to one of the recognized subtypes. Our results show that genotyping of HCV by nucleotide sequence analysis of NS5B is efficient, allows the accurate discrimination of subtypes, and is an effective tool for studying the molecular epidemiology of HCV.     

Hepatitis C virus recombinants are rare even among intravenous drug users

Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT‐PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti‐NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type‐specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type‐ and subtype‐specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU. J. Med. Virol. 82:232–238, 2010. © 2009 Wiley‐Liss, Inc.     

Hepatitis C virus sequences from different patients confirm the existence and transmissibility of subtype 2q, a rare subtype circulating in the metropolitan area of Barcelona, Spain

The hepatitis C virus (HCV) has been classified into six genotypes and more than 70 subtypes with distinct geographical and epidemiological distributions. While 18 genotype 2 subtypes have been proposed, only 5 have had their complete sequence determined. The aim of this study was to characterize HCV isolates from three patients from the Barcelona metropolitan area of Spain for whom commercial genotyping methods provided discordant results. Full-length genome sequencing was carried out for 2 of the 3 patients; for the third patient only partial NS5B sequences could be obtained. The generated sequences were subjected to phylogenetic, recombination, and identity analyses. Sequences covering most of the HCV genome (9398 and 9566 nt in length) were obtained and showed a 90.3% identity to each other at the nucleotide level, while both sequences differed by 17.5-22.6% from the other fully sequenced genotype 2 subtypes. No evidence of recombination was found. The NS5B phylogenetic tree showed that sequences from the three patients cluster together with the only representative sequence of the provisionally designed 2q subtype, which also corresponds to a patient from Barcelona. Phylogenetic analysis of the full coding sequence showed that subtype 2q was more closely related to subtype 2k. The results obtained in this study suggest that subtype 2q now meets the requirements for confirmed designation status according to consensus criteria for HCV classification and nomenclature, and its epidemiological value is ensured as it has spread among several patients in the Barcelona metropolitan area.     

Differential prevalence of hepatitis C virus subtypes in healthy blood donors, patients on maintenance hemodialysis, and patients with hepatocellular carcinoma in Surabaya, Indonesia.

Determination of the prevalence of liver disease caused by hepatitis C virus (HCV) of various genotypes helps provide an understanding of the virulences of these genotypes. Differences in the prevalences of these genotypes are known to exist in the various geographical regions of the world. Hence, we performed seroepidemiological and molecular epidemiological analyses of HCV in Surabaya, Indonesia. The prevalences of anti-HCV antibodies were 2.3, 76.3 and 64.7% in healthy blood donors, patients on maintenance hemodialysis, and patients with hepatocellular carcinoma (HCC), respectively. HCV-2a was the most common (52%) among the HCV clones obtained from blood donors; this was followed by HCV-1b (15%), HCV-1a (7%), and HCV-1d (7%), a unique Indonesian subtype. The high prevalence of HCV-2a in blood donors was further supported by serotyping analysis that could discriminate HCV type 2 (HCV-2a and -2b) from HCV type 1 (HCV-1a, -1b, and -1d). HCV-1a, -1b, and -1d were strongly associated with elevated serum alanine aminotransferase (ALT) levels in blood donors, suggesting a possibly more pathogenic feature of those subtypes than HCV-2a. In patients on maintenance hemodialysis, HCV-1a and -1b (each 31%) were among the most common subtypes, and in contrast to the case with blood donors, HCV-1a, -1b, and -1d were found in those with normal ALT as well as those with elevated ALT levels. Impaired immune responses of hemodialyzed patients might be responsible for the apparently decreased hepatocytic injury caused by infection with HCV type 1. In patients with HCC, HCV-1b (57%) was the most common; this was followed by HCV-1d (19%) and HCV-2a (5%). Subtype prevalence was not different between HCC patients with advanced liver cirrhosis and those without advanced cirrhosis.     

High-Resolution Hepatitis C Virus Subtyping Using NS5B Deep Sequencing and Phylogeny,an Alternative to Current Methods

Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.     

HCV在中国流行多样性和独特生长史的分析

目的 探讨中国丙型肝炎患者中HCV的基因型分布类型,并进行HCV在中国流行病学历史分析,进而研究HCV在中国的分子流行病学和进化动力学特征.方法 从423例丙型肝炎患者的血清中提取HCV RNA并进行cDNA反转录,扩增E1和NS5B两个基因区的核苷酸序列,排除不合格及不适合分析的病毒株序列,再对合格序列进行系统发育分析（phylogenetic analysis）.运用BEAST软件中的Bayesian MCMC （Markov Chain Monte Carlo）算法来进行Coalescence分析,通过重构BSPs（Bayesian skyline plots）图回溯HCV的流行病学历史.结果 HCV分离株的基因分型如下：共包括6个基因型,12个基因亚型（1b：65.9％,6a：17.1％,2a：7.4％,3a：3.6％,3b：3.3％,6e：0.76％,1a,1c,2b,2f,4d以及5a共占0.25％）,以及2个新基因型6变异体.所产生的5个BSP曲线图均凸显1993年至2000年这一时间段,中国的HCV感染数量呈现指数增长,随后则陡然下降.结论 证实了HUV在中国多样性的流行现状,反映了HCV不断变化的基因型流行模式;1993年至2000年期间由于“单采血浆”事件的影响,HCV在中国的感染数量经历了一段“指数”增长期.     

Molecular characterization of hepatitis C virus genotype 4 sequences in HIV-coinfected patients from Argentina

The prevalence of hepatitis C virus genotype 4 (HCV-4) is increasing in different parts of the World but in Latin America the data are still scarce. We aimed to characterize HCV-4 isolates from 383 HIV-coinfected patients in Argentina. Sequence analyses were based on the non-structural 5B region of HCV. Results from 18 patients indicated a genetic heterogeneity that involved three genotype 4 subtypes. Sequences were ascribed to subtype 4d (67%), 4a (22%), and 4m (11%). In spite of different sources of transmission were defined among patients, no statistical association was found with the genotype 4 subtype. The scenario is also compatible with multiple importation of the epidemic and there is no evidence for transmission-specific clusters or network-like transmission of HCV-4. This HCV-4 does not represent a recent introduction in Argentina, it circulates in all transmission groups and its presence is increasing among HIV-infected patients.     

Molecular epidemiology of hepatitis C virus among injection drug users in Iran: a slight change in prevalence of HCV genotypes over time

Injecting drug users (IDUs) are the main at-risk population for hepatitis C virus (HCV) transmission. We studied HCV infection, risk factors, and genotype distribution in relation to the year of first injection among Iranian IDUs. Of a total of 126 specimens positive for HCV antibody, 93 (74?%) had detectible HCV RNA, and the NS5B gene was sequenced for 83, with genotype 3a (n?=?48, 58?%) being predominant, followed by 1a (n?=?35, 42?%). Tattooing was an independent predictor for HCV infection. No significant difference was found between HCV genotypes and IDU characteristics. Although there was no change in the distribution of prevalent genotypes before and after 1997, a slight variation in the prevalence was observed (p?=?0.71). The difference in the prevalence of subtypes 1a and 3a (9.1?% in the period 1984-1996 and 18.2?% in the period 1997-2009) during 25?years was 9.1?%. These findings indicate a high prevalence of HCV infection among Iranian IDUs and highlights HCV-3a as the most prevalent subtype for the past 25?years. Harm-reduction strategies appear to be the most important measures to reduce the transmission of HCV in Iran.     

Hepatitis C virus genotype distribution in China: predominance of closely related subtype 1b isolates and existence of new genotype 6 variants

To determine hepatitis C virus (HCV) genotype distribution in China, a total of 148 HCV RNA positive serum samples were collected from nine geographic areas and subjected to RT-PCR followed by direct DNA sequencing and phylogenetic analysis of the core, E1, and NS5B regions. HCV was genotyped in 139 (93.9%) samples. Among them subtype 1b was the most predominant [66% (92/139)] followed by 2a [14% (19/139)]. Of 92 subtype 1b isolates, 35 (38%) and 30 (33%) formed two clusters, designated groups A and B. Group A was prevalent throughout China, while group B was predominant in the central and southern regions. In three cities in the Pearl River Delta, subtype 6a replaced 2a as the second most predominant subtype, and in Kunming (southwest) multiple HCV genotypes/subtypes were present. New variants of HCV genotype 6 were discovered in three samples from Kunming and one in Guangzhou in the Pearl River Delta.     

Génotypage du virus de l’hépatite C : comparaison du test Abbott RealTime HCV Genotype II au séquençage de la région NS5B

Purpose of the study

Hepatitis C virus genotyping is needed for treatment decision and monitoring. The results of a genotyping assay based on real-time PCR and TaqMan chemistry were compared with the results of NS5B region sequencing.

Materials and methods

One hundred and two sera (genotypes 1–6) were tested. Amplification and detection of viral RNA were performed with the Abbott RealTime HCV Genotype II assay targeting 5′non-coded region (5′NC) for the identification of genotypes 1 to 6 and NS5B, for 1a and 1b subtypes detection. Sequencing of 5′NC fragment was used to resolve discrepant results.

Results

No indeterminate results were obtained. Concordance with NS5B sequencing was 93% (95 on 102), 96% at the genotype level (98 on 102) and 93% for genotype 1 subtyping (40 on 43). Discordant genotyping results were a 2f subtype identified as 5, a 6a typed as 1, a 3a identified as a 1-3 co-infection and a 4r identified as a 1-4 co-infection. Discordant subtyping results were 2 1b subtypes only typed as 1 and a 1e identified as 1a.

Conclusion

Abbott RealTime HCV Genotype II assay is a rapid, automated and simple to interpret method for HCV genotyping. It allows the detection of possible mixed infections which might have a negative impact on therapeutic response. However, the discrepant results found in this small series underline the need for assay optimization.     

Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+ response in patients with chronic hepatitis C

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.     

Distribution and heterogeneity of hepatitis C genotypes in hepatitis patients in Cameroon

Hepatitis C virus infects humans world-wide. The virus genome varies greatly and it has several genotypes. HCV infection is highly prevalent in Central Africa and Cameroon. Initial studies on the genetic variability of HCV showed infection with HCV genotypes 1, 2, and 4. We have now sequenced the NS5b and E2 regions of 156 HCV isolates collected from patients presenting for diagnosis in Yaounde and used the data to describe the distribution of HCV genotypes and subtypes in patients with hepatitis in Cameroon. Genotype 1 was more frequent than Genotypes 4 and 2. Genotypes 1 and 4 were highly heterogeneous, containing many subtypes described previously (1b, 1c, 1e, 1h, 1l, 4f, 4t, 4p, 4k) and unsubtyped groups. There was a systematic phylogenetic concordance between NS5b and E2 sequence clustering. The Genotype 2 sequences did not vary. Neither subject age nor gender influenced HCV distribution. HCV Genotypes 1 and 4 are very heterogeneous in Cameroon, perhaps due to ancient infections. The homogeneity of HCV Genotype 2 indicates its more recent introduction from western Africa.