Assessment of HCV genotypes in Yunnan Province of Southwest China

Recently, we reported that the frequency of hepatitis C virus (HCV) genotypes and subtypes has rapidly changed among intravenous drug users (IDUs) in Yunnan Province over the last 5 years; this is especially true for subtype 6a which has increased in frequency from 5 to 15%. Here, we assessed 120 HCV-positive plasma samples from the general population (GP). HCV NS5B fragments were amplified and sequenced by PCR. We identified four HCV genotypes (1, 2, 3 and 6) and seven HCV subtypes (1b, 2a, 3a, 3b, 6a, 6n, and 6k) in this population. Genotype 3 was predominant, with a distribution frequency of 0.484, followed by genotype 1 (0.283), genotype 6 (0.133) and genotype 2 (0.100). HCV subtypes 3b (frequency 0.292) and 1b (frequency 0.283) were the most common subtypes. A comparison of the current data with previous results reported for IDUs showed that the distribution frequencies of genotypes 1, 2 and 6 were significantly different between patients in the GP and IDUs (P < 0.05). Among the HCV subtypes, the distribution frequencies of 1b, 2a, 6a, and 6n were significantly different between patients in the GP and IDU groups (P < 0.05). Moreover, Phylogenetic analyses showed that HCV subtype 6a strains isolated from IDUs and the GP were intermixed and not separately clustered. HCV subtype 6a was predominant not only among IDUs but also among those in the GP in the Guangdong Province and Vietnam. However, HCV subtype 6a was predominant only among IDUs and not among those in the GP in the Yunnan and Guangxi Provinces. Our results indicate that the HCV subtype 6a could rapidly spread across China.     

Subtyping genotype 2 hepatitis C viruses from Tunisia: identification of two putative new subtypes

HCV variants were classified into six genotypes (1–6) subdivided into several subtypes with different geographic distribution worldwide. Previous studies conducted in Tunisia showed that genotype 1 counts for more than 80 % of circulating HCV genotypes and most of the isolates belong to subtype 1b. Genotype 2 comes in the second position, however, few sequences have been analyzed and published. In the present study, 89 isolates from Tunisian patients, typed as genotype 2 by the InnoLIPA commercial probe hybridization test, were sequenced in the NS5B and Core/E1 regions. All the isolates, clustered with the genotype 2 reference sequences, in the NS5B and in the Core/E1 region and the phylogenetic analyses in the two genomic regions were perfectly concordant: subtype 2c was the most frequent (58 out of 89, 65.1 %) and few isolates belonged to subtypes 2k(n = 10), 2i(n = 5), and 2b(n = 1). Fifteen isolates did not match with any of the reference sequences representing the genotype 2 subtypes, identified up-to-date. They divided into 2 separate clusters with high bootstrap values in both genomic regions. This study shows perfect concordance between the NS5B and the Core/E1 region suggesting that any of the two regions can be used for genotyping and that intergenotypic and intragenotypic recombinants are not very frequent, at least for HCV isolates from genotype 2. The present study also shows a predominance of subtype 2c among genotype 2 HCV isolates circulating in Tunisia, the co-circulation of minor subtypes (2k, 2i, and 2b) and proposes the possible existence of two other new subtypes.     

Determining hepatitis C genotype by analyzing the sequence of the NS5b region

Assays to determine the hepatitis C virus (HCV) genotype have recently become useful for clinical decision making and may be suitable for epidemiological investigations, such as identifying HCV outbreaks in a given population. Molecular assays are the most common diagnostic tools for HCV genotyping. This study compares two genome typing assays, one, the Trugene 5'NC genotyping kit, uses the sequence of the 5' non-coding (5'NC) region and the other, a non-commercial assay, uses the non-structural 5b (NS5b) region. Serum samples from 203 chronically HCV-infected patients were tested. The 5'NC and the NS5b assays were both very effective in identifying the genotype (99 and 98.5%) and the results with the two methods were always concordant for the genotype. The NS5b analysis permitted the identification of the subtype in all samples, whereas the 5'NC region assay did not in 33% of samples. The NS5b analysis showed that one patient had a mixed infection with HCV subtypes 1a and 2c, while the 5'NC assay did not. It is concluded that phylogenetic analysis using both the 5'NC and the NS5b regions are reliable and convenient methods for HCV typing in clinical practice. But analysis of the NS5b region may be more useful for tracing the source of an HCV infection.     

慢性丙型肝炎患者HCV基因型调查

目的 研究丙型肝炎病毒（ＨＣＶ）基因型的流行病学特点。方法 采用ＩＮＮＯ－ＬＩＰＡ反向线形探针杂交法，对来自我国南方（上海，武汉，徐州）、北方（北京、天津）和东北（大连、沈阳）７市的１０７例慢性丙型肝炎患者进行了ＨＣＶ基因型调查。结果（１）地域性，ＨＣＶ１ｂ是主要的基因型，其检出率达８３．１７％，其中东北２市（７２．２２％）低于北方２市（８７．６５％，Ｐ〈０．０１）；２型的检出率较低，仅占６．８６     

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.     

Diversity of hepatitis B and C viruses in Chile

Although there is a low prevalence rate (around 1% of the population) of infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) in Chile, little is known about the diversity and molecular characteristics of the circulating viruses. In the present study, 40 HBV and 57 HCV samples from Santiago City, Chile, were examined. The phylogenetic analysis of HBV samples showed the autochthonous genotype F as the most represented genotype in the study (67.5%), while genotypes A, B, C, and D were less frequent (7.5%, 5%, 7.5%, and 12.5%, respectively). The frequency of circulation of HBV genotypes observed is in accordance with the genetic background of the Chilean population. Most of the HCV samples tested belonged to subtype 1b (82%). The coalescent analysis conducted for both the NS5A and NS5B regions of the HCV strains showed similar population growth rates, with a most recent common ancestor estimated to date between 1893 and 1901. This result may indicate that genotype 1b strains circulating in Chile have epidemiological features similar to those described for HCV genotype 1b in Brazil and the United States. However, the most recent common ancestor for Chile is older than that reported recently for genotype 1b in Argentina. J. Med. Virol. 81:1887–1894, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis C virus subtyping based on sequencing of the C/E1 and NS5B genomic regions in comparison to a commercially available line probe assay

Hepatitis C virus (HCV) genotype determination is required in clinical practice to establish the dose and duration of antiviral treatment. Although subtype identification does not impact on current therapy this is changing with new specific inhibitors of HCV enzymes and functions which are becoming available worldwide. These new drugs may yield different antiviral responses and resistance profiles. Accurate classification of HCV genotype and subtype is therefore crucial. An “in‐house” method was developed for improving HCV subtyping and the results were compared with a second‐generation line probe assay (LiPA) used extensively in Portugal. Phylogenetic analysis was undertaken of the C/E1 and NS5B genomic regions of HCV isolated from 72 prisoners with chronic HCV infection and from reference samples. Although LiPA is considered to be a good method for genotyping, HCV was subtyped in only 47.2% of cases compared with 95.8% of cases by the “in‐house” method. Molecular data for both C/E1 and NS5B regions were obtained in 88.9% of the samples. Two out of 23 cases of subtype 1a were misclassified as subtype 1b by LiPA. A putative recombinant like RF1_2k/1b, two potential inter‐genotypic recombinants 1b/4a and 3a/4a, and also a potential intra‐genotypic recombinant 2q/2k in C/E1 and 2k/2a in NS5B were also identified. The “in‐house” method enabled HCV to be subtyped accurately with the detection, in some cases, of recombinant viruses or dual HCV infections. Near full‐length genomic analysis to characterize these potential recombinant viruses is planned. J. Med. Virol. 85:815–822, 2013. © 2013 Wiley Periodicals, Inc.     

Changing molecular epidemiology of hepatitis C virus infection in Northeast Italy

To assess HCV genotype distribution and its determinants, 318 consecutive HCV RNA positive patients were examined. Subtype 1b infection was the most prevalent (35.5%), followed by subtype 1a (22%), 3a (21.4%) and 2 genotype (21.3%). Subtypes 1a, 1b and 3a had a comparable prevalence (30-35%) in the 0-15-, 16-30- and 31-45-year age groups. In subjects older than 45 years, genotype 2 prevalence increased, whereas subtype 1a and 3a infections decreased markedly. In this age group types 1b and 2 accounted for a prevalence of more than 90% in a comparable proportion. Genotype prevalence rates according to different risk factors were different statistically (P < 0.001): subtype 1a and 3a infections were predominant in injection drug users (42.9% and 37.7%, respectively), whereas community acquired infections and infections in patients with a history of transfusion were caused mainly by subtype 1b (38.5% and 66.6%, respectively). Logistic regression showed that age and injection drug use are independent determinants of genotype distribution.     

Hepatitis C plasma viral load is associated with HCV genotype but not with HIV coinfection

The influence of human immunodeficiency virus (HIV) coinfection and hepatitis C virus (HCV) genotype distribution on HCV viral load and alanine amino transferase (ALT) levels in chronically infected patients remains unclear. In the present study, serum samples from a group of haemophiliac patients were investigated retrospectively. HCV geno- and subtyping was carried out using the Inno line probe assay (Inno LIPA, Innogenetics, Zwijnaarde, Belgium) in 87 patients positive by HCV RT PCR. Of these patients, 31 (35.6%) were HIV coinfected. HCVRNA was quantified with the HCV Monitor kit (Roche, Basel, Switzerland) in 43 patients (22 HIV-negatives, 21 HIV-positives). The most prevalent genotypes were 1 (n = 52) and 3a (n = 16) followed by genotype 2 (n = 9) and 4 (n = 3). Mixed infections were detected in 7 patients. Of genotype 1 positive samples, 24 and 23 were classified as subtype a and b, respectively. Five samples could not be subtyped. Although higher mean values of ALT were observed in genotype 1 infected patients, there was no statistically significant association between HCV genotype or subtype and liver enzymes (P > 0.05). On the other hand, statistically significant higher HCV RNA titres were observed in haemophiliacs infected with HCV genotype 1 in comparison to those infected with other genotypes (P < 0.01). No relationship was found between the presence of HIV coinfection and viral load of HCV RNA. There was no evidence that HCV infection had a more severe outcome in HIV-positive patients who had been infected with HIV and HCV more than ten years ago, even in those with very low CD4+ cell counts. No clear association between high ALT levels and large amounts of viral RNA was observed. In conclusion, a large viral load is associated with HCV genotype 1 infection; HIV coinfection has no clear effect on the intensity of HCV replication. An ongoing prospective study will evaluate the respective role of viral load, genotype, HIV coinfection and ALT level in the response to interferon therapy. © 1996 Wiley-Liss, Inc.     

Hepatitis C virus genotyping: interrogation of the 5' untranslated region cannot accurately distinguish genotypes 1a and 1b

Although the 5' untranslated region (5' UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5' UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5' UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position -99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position -99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5' UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.     

High genetic diversity including potential new subtypes of hepatitis C virus genotype 6 in Lao People's Democratic Republic

Sera from 105 anti-HCV-positive first-time blood donors collected in 2004, 2005 and 2008 in different provinces in Laos were investigated by PCR. Forty-five samples were positive for HCV (42.86%); two belonged to subtype 1b (2/45, 4.4%) and all others to genotype 6 (43/45, 95.6%), including subtypes 6b, 6h, 6k, 6l, 6n and 6q. Three groups of sequences were not clearly attributable to any genotype 6 subtype, two of which may be regarded as candidates for new subtypes of genotype 6. Two samples were mixed infected with different subtypes or clusters of genotype 6 viruses.     

The effects of widespread methadone treatment on the molecular epidemiology of hepatitis C virus infection among injection drug users in Hong Kong

The distribution of HCV genotypes among injection drug users in Hong Kong was assessed in context of methadone treatment availability. Three time periods were defined by the year of initiating injection-on or before 1980, 1981-1994, and 1995-2006-with methadone becoming widely available since the second period. Of the 273 HCV RNA-positive cases, the most prevalent subtype was HCV 6a (52.4%), followed by HCV 1b (38.5%). The new variants of HCV subtypes 6e and 6h were detected. Both subtypes 1b and 6a were prevalent among older injectors, while subtype 3a was more common in young injectors and those initiating injection recently during the third time period. Age (P < 0.05) and recent injection frequency (P < 0.01) were independently associated with HCV 6a infection. Subtype 1b was predominant in the first period, whereas 6a was more common in the second and third. Subtype 1b sequences appeared to have originated at two positions on the phylogenetic tree, while 6a showed a more disperse distribution suggestive of multiple introductions. Phylogenetic analysis on the NS5B region did not reveal specific clustering of any subtype/genotype. Overall, there was no suggestion of outbreaks of HCV. The extensive use of methadone may have protected Hong Kong from the emergence of HCV clusters among injection drug users.     

Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5'-untranslated region (5'UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5'UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5'UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5'UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.     

The unique HCV genotype distribution and the discovery of a novel subtype 6u among IDUs co-infected with HIV-1 in Yunnan, China

The Yunnan province is the epicenter of HIV-1 epidemics in China and a center for drug trafficking to the other parts of the world. In six prefectures of this province, a total of 132 IDUs were recruited to determine the sero-prevalence of HCV and HIV-1 and the positive rates were 93.94% and 68.18%, respectively (P<0.001). Co-infection with HCV and HIV-1 was found among 89 IDUs, of whom several HCV fragments were amplified and sequenced. Sequences of the HCV 5'NCR-C and NS5B region were determined from 82 IDUs. Phylogenetic analyses showed consistent genotyping among 80 IDUs. Among them HCV genotypes 1a, 1b, 3a, 3b, 6a, 6n, and a tentatively assigned novel 6u subtype were found in 1 (1.25%), 16 (20%), 19 (23.75%), 24 (30%), 4 (5%), 9 (11.25%) and 7 (8.75%) individuals, respectively. In two IDUs, genotyping results were discordant, suggesting mixed HCV infections or recombination. The proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province. Genotype 3 and 6 strains were more frequent in the southern prefectures. The novel subtype 6u strains were only detected in Dehong which borders Myanmar. Our findings showed a unique pattern of HCV genotype distribution, which is similar to that in the southeastern Asian countries but distinct from that among the general population in China. Routes of drug trafficking and the resulting high prevalence of HIV-1 infection may have contributed to this pattern of HCV genotype distribution.     

PREVALENCE OF HEPATITIS C VIRUS (HCV) COINFECTION IN HIV INFECTED INDIVIDUALS IN SOUTH INDIA AND CHARACTERIZATION OF HCV GENOTYPES

Purpose: To determine anti-HCV antibodies and genomic subtype of HCV in 1487 confirmed human immunodeficiency virus (HIV) positive samples. Methods: A total of 1487 confirmed HIV-positive samples were tested for anti-HCV antibodies by using a third generation ELISA kit (Ortho 3.0) and by RT PCR for HCV. HIV and HCV coinfected samples were selected for HCV genotyping by RFLP and subtyping with NS5-type specific primers. Results: A total of 1487 HIV-infected serum samples were screened for HCV infection, of which, a 1443 (97.04%) were negative and 45 (3.02%) were coinfected. HIV–HCV coinfection was predominant in the age group 41–50 years (51.1%). HCV genotyping and subtyping was done for the 45 HCV RNA-positive specimens of which genotype 1 was observed in 31 (68.8%) and genotype 3 was observed in 14 (31.1%) subjects. Further subtyping analysis showed the genotype 1b in 23 (51.1%), 1a in eight (17.7%), 3a in 10 (22.2%) and 3b in four (8.8%) subjects. Conclusion: HIV and HCV seroprevalence is higher in South India, and the most prevalent genotype in coinfection was genotype 1b.     

Genetic characterization of hepatitis C virus strains in Estonia: fluctuations in the predominating subtype with time

During the last decade, there has been a dramatic increase in intravenous drug use in young adults in Estonia with an increased incidence of both hepatitis B and C as a consequence. Since genetic data are limited regarding hepatitis C virus (HCV) strains in Estonia, the aim of the study was to characterize HCV strains in different risk groups to determine their relatedness to strains from other geographical regions. Three hundred fifty-three anti-HCV positive sera collected during 1994-2004 from hospitalized patients, blood donors and health care workers were used as source of HCV RNA. Two hundred nine (59%) of the sera were positive for HCV RNA by PCR directed to the 5'-UTR region. For 174 strains the HCV subtype was determined by analyses of the NS5B and/or the 5'UTR-core regions. 1b (71%) was the most common subtype followed by 3a (24%), 2c (2%), 1a (1%), and 2a (1%). The 1b and 3a strains were similar to strains from other regions of the former USSR. Within genotype 1b there were several HCV lineages. However, for 3a there seemed to be two separate introductions into Estonia. There was a relative shift from subtype 1b to 3a in 1999-2000 with a further replacement of 3a with 1b in intravenous drug users in 2001 and onwards (P < 0.05). However, both subtypes were found to co-circulate in the community independent of risk factors. One patient was infected with the 2k/1b recombinant presumed to originate from St. Petersburg being the first isolate of this recombinant recovered outside Russia.     

Evaluation of a new-generation line-probe assay that detects 5' untranslated and core regions to genotype and subtype hepatitis C virus

The VERSANT HCV Genotype 2.0 Assay (LiPA 2.0; Innogenetics, Ghent, Belgium; distributed by Siemens Medical Solutions Diagnostics, Tarrytown, NY) is a new-generation line-probe assay that simultaneously detects sequences in the 5' untranslated (5'UTR) and core regions to genotype and subtype hepatitis C virus (HCV). We tested 60 specimens of known genotype and subtype and 2 specimens with mixed infections with the LiPA 2.0 assay. After arbitration based on genotype and subtype determined by sequencing, there was concordance in 58 of 60 specimens (specificity, 96.7%). Computer-assisted typing yielded comparable results, but much more rapidly. Of 67 clinical specimens, 64 readily yielded genotype and subtype; 3 indeterminate specimens were typed by sequencing and were uncommon types not in the database. The newgeneration line-probe assay that detects the 5'UTR and core regions to genotype and subtype HCV is applicable to more than 95% of specimens. Interpretation is facilitated by computer-assisted analysis.     

Molecular epidemiology of hepatitis C infection in cyprus: Evidence of polyphyletic infection

The genetic diversity of the hepatitis C virus (HCV) in Cyprus is investigated for the first time in this study. Nucleotide sequence analysis of the CORE‐E1 and NS5B regions of the HCV genome was performed on blood plasma samples obtained from 77 HCV patients in Cyprus, collected during 2005–2008. The amplified products were sequenced and compared to reference HCV strains of known genotype and subtype in order to classify the isolates found in this study. Genotype could be determined for all strains, and subtype for all but four isolates. Phylogenetic analysis revealed that 51 patients were genotype 1, of which 38 were subtype 1b, 9 were 1a, and 1 was unclassified, one patient was genotype 2c, 13 were genotype 3a, nine were genotype 4, of which six were subtype 4a, and three were of unclassified subtype, one was genotype 5a, two patients seem to carry a possible 2k/1b recombinant strain, and no genotype 6 strains were found. This study demonstrated a genetic heterogeneity of HCV infection in Cyprus, with five of the six known HCV genotypes on the island, including unclassified isolates in genotypes 1 and 4, and also the apparent introduction of the 2k/1b recombinant strain in intravenous drug users. J. Med. Virol. 81:238–248, 2009. © 2008 Wiley‐Liss, Inc.