Genotyping of GB virus C by restriction pattern analysis of the 5' untranslated region

GB virus C (GBV-C) is a virus that has been proposed as a member of the Flaviviridae family, distantly related to hepatitis C virus (HCV). The virus is able to infect humans parenterally and perinatally, although its true pathogenicity remains unknown. The 5' terminal region of GBV-C is the most highly conserved region of the virus genome. Comparison of 5' untranslated region (5' UTR) sequences from GBV-C infected individuals shows that variation is limited to particular sites that are often covariant and associated with different virus genotypes. Extensive sequence analysis of the GBV-C genome provides evidence for the existence of at least five major genotypes, some of which can be further divided into subtypes. For genotyping by restriction fragment length polymorphism (RFLP), it is essential to identify genomic positions that not only reflect genotype differences, but that also harbor restriction sites that allow recognition of these differences. Restriction site analysis of type-specific sequence motifs predicted that endonucleases BsmFI, HaeII, HinfI, and ScrFI could be used for the identification all known genotypes (types 1-5) with 99.6% accuracy. The method was applied to serum samples from 46 chronic GBV-C carriers of heterogeneous geographical and ethnic origin, comparing observed cleavage patterns of GBV-C variants amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) of the 5' UTR with the RFLP predicted from sequences deposited in GenBank database. cDNA sequencing and subsequent alignment of the 46 GBV-C isolates confirmed RFLP profiles predicted theoretically. The observed geographical distribution of genotypes is also in agreement with previous reports. This method may be useful for rapid and reliable characterization of GBV-C isolates when either epidemiological or transmission studies are carried out.