Identification of a Naturally Occurring Recombinant Genotype 2/6 Hepatitis C Virus

Hepatitis C virus (HCV), an important causative agent of acute and chronic hepatitis, is an enveloped plus-strand RNA virus that belongs to the family Flaviviridae (33). Its genome, approximately 9.4 kb in length, encodes both structural (Core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4a/b, and NS5a/b) proteins in a single open reading frame (6). Short conserved untranslated regions (UTRs) located at the 5′ and 3′ ends of the genome are required for viral replication (9, 10). An internal ribosomal entry site in the 5′UTR is involved in protein translation (46). Because of its heterogeneity, HCV is classified into six major genotypes and a large number of subtypes (36-38). Different genotypes display up to 30% sequence diversity, whereas subtypes vary by more than 20% (37, 38). The variability is distributed unequally across the genome, with regions such as E1 and E2 displaying most sequence diversity, whereas 5′UTR and CORE sequences are more conserved (39). Several methods were developed to determine the HCV genotype, such as serological genotyping, reverse transcription-PCR (RT-PCR) amplification with genotype specific primers, restriction fragment length polymorphism analysis, reverse hybridization assay, and sequence analysis (28). Of these methods, sequence analysis of phylogenetically informative regions is more reliable for genotype and subtype identification. Genotyping of HCV is important for prediction of the responses to and determining the duration of antiviral therapy (23, 28). Moreover, assessment of the distribution of genotypes in different parts of the world may help to understand the epidemiology and evolution of HCV. The geographic distribution of HCV relates to different epidemic histories and routes of transmission. Some genotypes, such as 1a/b, 2, and 3a, are widely distributed in Western countries, the United States, and Japan as a result of transmission via blood transfusion and contaminated needles between intravenous drug users (2). These strains typically have limited sequence diversity, resulting from the recent introduction of a few strains from areas of endemicity (36). On the other hand, a more complex viral heterogeneity is observed in parts of Africa and Southeast Asia. In Western Africa, HCV infection is caused predominantly by genotype 2, whereas genotype 1 and 4 are most prevalent in Central Africa (4, 24). In both areas, a notable divergence of subtypes, especially broad genotype 2 variety in Ghana, was reported (5). Similar observations were made in Asia, where genotype 1, 3, and 6 dominate (33), and a large variety of genotype 6 was demonstrated in Vietnam, Thailand, Myanmar, China, and Hong Kong (8, 25, 34). This large sequence divergence points to a long-term presence of HCV infection among local populations through a variety of routes, including vertical, sexual, and household contact transmission (35, 41). The sequence diversity of HCV is supposedly caused by a high mutation rate of the RNA-dependent RNA-polymerase during replication (41). Interestingly, recombination, commonly seen among RNA viruses, including other members of the family Flaviviridae (14, 20, 47, 51), is not thought to play a major role (39, 40, 49). The fact that recombinant forms of HCV have been observed in nature, such as the St. Petersburg strains containing sequences of both genotype 2k and 1b (18) and a possible 1a/1b recombinant virus in Peru (7), would suggest that all requirements for genetic exchanges could be met. However, only a few chimeric HCV genomes were described, which may be due to the detection limits of currently used methods that are not suited for discovering recombinant strains. It is therefore possible that the true frequency of HCV recombination is underestimated (7, 18, 35), especially in areas of endemicity with a high prevalence of different HCV genotypes. In the present study, we analyzed sera from 58 HCV RNA-positive blood donors from Ho Chi Minh City, Vietnam, in order to survey the molecular heterogeneity of HCV in this area. The CORE and NS5B genes of each sample were sequenced and phylogenetic analysis was performed. Two samples gave discordant results and were analyzed by full-length genome sequencing. Our results revealed one mixed genotype infection, whereas the other sample contained an intergenotypic recombinant virus.