The hepatitis C virus 5’ untranslated region gene amplified by rapid amplification of cDNA ends and its secondary structure

Objectives: To obtain very end full-length cDNA ofhepatitis C virus (HCV) 5’ untranslated region(5’UTR) and analyze its primary and secondarystructure.Methods: A patient infected genotype 2a HCV wasidentified by reverse transcription-nested polymerasechain reaction (RT-PCR) and restriction fragmentlength polymorphism (RFLP). Total RNA isolatedfrom the serum was used as template, and the cDNAof the 5’ untranslated region was amplified using rap-id amplification of cDNA ends (RACE). The frag-ments were recombinated by A-T clone strategy, andthe recombinants were confirmed by RFLP andPCR, and sequenced subsequently. Secondary struc-tures were analysed by RNAdraw.Results: Very end full-length cDNA of genotype 2aHCV 5’ UTR was obtained by RACE. In five clonesobtained, three contained full-length 5’UTR cDNA;A21G, G170A, T222C, T247C, C339T substitutionswere found as compared to HC-J6. Homological re-sults of HCV-1, HC-J6, HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions didnot alter secondary structure. Two of 5 clones weredeletions of 53bp and 135bp at the 5’terminal ofHCV 5’UTR, respectively.Conclusions: RACE can be used to obtain the full-length cDNA of 2a genotype HCV 5’UTR. Genes de-leted at the 5’ terminal of HCV circulate in hepatitisC patients.