Determination of Hepatitis C Virus Genotypes by Direct Sequencing Analysis of 5' UTR of HCV Genome

High degree of sequence variations are present throughout in the coding regions of Hepatitis C virus (HCV) genome. However, a high degree of sequence conservation within the 5'untranslated region (UTR) has made this region a target of choice for most of detection assays based on nucleic acid amplification. The current study was designed to determine the HCV genotypes in samples of HCV chronically infected patients in Pakistan. Specific primers targeting 5'UTR region were designed, which were then used for both amplification and sequencing of all isolates. All HCV isolates were sequenced and genotyped based upon phylogenetically informative regions within the 5'UTR of HCV genome. Out of 15 samples, 9 (60 %) samples 5'UTR of HCV genome was successfully sequenced. However, only 6 (66.7 %) samples were assigned genotypes based upon sequence comparison with reference sequences of database. Results of this study revealed that five isolates that were assigned genotype 3a, showed 97 to 99 % sequence conservation to isolates of genotype 3a previously reported from Pakistan as well as from rest of world, grouped together and coincided with their positions in the phylogenetic tree. Moreover, one isolate that was assigned genotype 4a, showed 97 % sequence conservation to isolates of genotype 4a previously reported from rest of world, coincided with its position in the phylogenetic tree. Our findings suggest that direct sequencing analysis of the 5'UTR of HCV genome is a sensitive and efficient approach of HCV genotyping; it may be adopted as a routine diagnostic procedure for HCV genotyping in clinical settings.