Recombinant HCV type 4 Nonstructural Protein 5 [GST]

The high prevalence of hepatitis C virus (HCV) markers in alcoholic liver cirrhosis (AL-LC) and hepatocellular carcinoma (HCC) suggests a close aetiopathogenic relationship between alcoholic liver disease (ALD) and HCV infection. In the present study, HCV markers in ALD were measured by the highly sensitive methods, and the changes of sequential HCV markers after abstinence in ALD patients were analysed in order to elucidate the effect of alcohol on HCV. Antibodies to HCV-related antigen were determined using the first or second generation test kit. HCV-RNA genomes encoding the NS-5 region were detected using the RT-PCR method. In the HCV-NS5 negative serum, HCV genomes of the 5'-noncoding region were detected using the two-stage PCR method. Titres of HCV-RNA were measured by multiple cyclic PCR and cDNA dot blotting. Typing of HCV genomes was carried out on the PCR product from the NS-5 region by slot blot hybridization using type-specific cDNA probes, or by restriction fragment length polymorphisms analysis. In alcoholic fibrosis and alcoholic hepatitis, the prevalence of HCV markers was low, suggesting that the main aetiological factor is alcohol but not HCV in these types of ALD. HCV markers were positive in the half of the patients with AL-LC, and in more than 80% of patients with AL-CH and AL-HCC, indicating that HCV infection closely relates to these types of ALD. The ratio of the K1 type to the K2 type of HCV genomes was 4:1 in all types of NANB liver disease. On the other hand, the prevalence of the KI type was significantly higher than that of the K2 type in AL-HCC, suggesting a possibility that alcohol misuse may modify the development of HCC caused by the K1 type of HCV. Disappearance of HCV-NS5 from blood was observed in four out of eight patients who were sequentially determined HCV-NS5 following abstinence. In one patient, the titres of HCV-RNA changed in parallel with drinking or abstinence, suggesting that alcohol misuse enhances the replication of HCV. These results indicate that the HCV-marker positive ALD patients had a high risk of developing HCC.

Variants of hepatitis C virus (HCV) have been classified by nucleotide sequence comparisons in different regions of the genome. Many investigators have defined the ranges of sequence similarity values or evolutionary distances corresponding to divisions of HCV into types, subtypes and isolates. Using these criteria, novel variants of HCV from Vietnam, Thailand and Indonesia have been classified as types 7, 8, 9, 10 and 11, many of which can be further subdivided into between two to four subtypes. In this study, this distance-based method of virus classification was compared with phylogenetic analysis and statistical measures to establish the confidence of the groupings. Using bootstrap resampling of phylogenetic trees in several subgenomic regions (core, E1, NS5) and with complete genomic sequences, we found that one set of novel HCV variants ('types 7, 8, 9 and 11') consistently grouped together into a single clade that also contained type 6a, while 'type 10a' grouped with type 3. In contrast, no robust higher-order groupings were observed between any of the other five previously described HCV genotypes (types 1-5). In each subgenomic region, the distribution of pairwise distances between members of the type 6 clade were consistently bi-modal and therefore provided no justification for classification of these variants into the three proposed categories (type, subtype, isolate). Based on these results, we propose that a more useful classification would regard all these variants as subtypes of type 6 or type 3, even though the level of sequence diversity within the clade was greater than observed for other genotypes. Classification by phylogenetic relatedness rules out simple sequence similarity measurements as a method for assigning HCV genotypes, but provides a more appropriate description of the evolutionary and epidemiological history of a virus.