Entirely sequencing the hepatitis C virus (HCV) genome has been a critical object of study but often difficult to attain for many reasons. However, this is a prerequisite for completely understanding of HCV molecular virology, epidemiology, evolution, and pathogenesis. Particularly, this is a major determinant associated with the outcome of -interferon therapy. When the recent HCV nomenclature was updated, only six subtypes of genotype 6 had their entire genome sequences clarified, while the other 11 subtypes remained to be fully described. The first purpose of this study was to entirely sequence isolates representing these 11 subtypes and two other variants that may qualify for two new subtypes. Six reference sequences representing subtypes of genotype 6 were retrieved from Genbank and co-analyzed with the obtained 13 full genomes. It showed pair-wise nucleotide similarities of 71.9% to 82.7% over the entire genome length. Notably, the nucleotide similarities among these four pairs of sequences were higher than the 75%-80% range that was previously defined to classify different HCV subtypes. To investigate further, four prototype isolates were also entirely sequenced. Consistently, analyses of the resulted full genome sequences gave a same range of nucleotide similarities, providing new information for HCV subtype classification. Phylogenetic analysis demonstrated that these 13 isolates were all classified into HCV genotype 6. Further analysis in conjunction with partial sequences from the Los Alamos HCV database led to the identification of many other closely related HCV isolates that were exclusively derived from south-eastern Asia or immigrants from that region. Common source of HCV infection was therefore strongly suggested. The second purpose of this study was to validate the hypothesis of interspousal HCV transmission. Three other HCV complete genomes from two infected Thai blood donors and their spouses were further entirely sequenced. The similarities firstly provided the complete genome information to support the interspousal HCV transmission. Two other HCV genotype 6 variants co-infection with HIV-1 from IDUs were also entirely sequenced. Analysis of the two sequences suggests that there is a trend for subtype 6e to change from a previous endemic pattern to a current epidemic pattern in southern China. This was likely promoted by network transmission of HCV among injection drug users. Conclusively, in this study a total of 21 new HCV variants were entirely sequenced, each from a 100 μl of serum sample remained. The strategy can be simply described as DNA walking over “bridges” and “islands” using conventional PCR with degenerate primers combined with strain specific primers. While this was efficient in HCV genomic study, the strategy would be also applicable to other studies of molecular epidemiology in which the viral sequence information is critical but the sample volume is small. Furthermore, These genomes represent a full panel of 17 subtypes within the oldest, the most diverse, the endemic, and possibly the zoonotic genotype 6. The utility of this panel of complete sequences for accurate detection and classification of infection, and for estimating the origin and evolution of this genotype of HCV, and for finding new variants, and for predicting the future epidemic trend and health burden, and for improving HCV diagnostic, treatment, and vaccination, is very important.
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