Antigenic Variations of Core Protein among Different Genotypes of Hepatitis C Virus

Abstract

Purpose: Antibodies against core antigen are reliable markers of virus replication, since their presence is found to be closely associated with the presence of specific mRNA. Hence the aim of the present study was to evaluate antigenic variations of 120 amino acids of core gene among 1a, 1b, 3a and 3b genotypes. Methods: Serum samples of 120 patients who were anti HCV positive by 3rd EIA, 50 subjects were negative for all hepatitis markers. Viral RNA extraction was carried out by QIAamp viral RNA kit followed by RT-PCR using core primes. The 120 amino acids of core gene were Cloned in BamHI and Eco RI sites of PET21 expression system (Novogen). Gene was transformed in BL21 DE3 cells. Gene expression was induced by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG). The bacteria were harvested after 6 h after IPTG activation. Cell lysates were subsequently prepared for protein purification. The purified 100 ng of core antigens were coated to a microtiter plates and tested with the patient's samples. Results: The amino acid sequences of the recombinant protein used from different genotypes in the ELISA were compared with each other. The sequence of genotype 1a differing from that of genotype 3b by 13 of 120 amino acids (11% divergence), from that of genotype 3a by 9 amino acids (7.5% divergence) and 1b by 6 amino acids (5% divergence). All the four recombinant proteins were tested with 120 anti HCV positive samples and it was observed that all four proteins did not pick up any negatives (n = 50) showing 100% specificity, and had > 97% sensitivity. The protein 1a was the most sensitive and had a sensitivity of 99%. There was a close correlation observed between reactivity to the core protein of type 1a, 1b, 3a & 3b, with correlation coefficients of 1.289, 1.176, 1.201 and 1.154 respectively. Conclusions: i) The structural antibody would appear much earlier and more persistently than antibodies against the non structural region and hence can be used as a good diagnostic marker. ii) It was observed that by using just one structural recombinant antigen, a significant correlation between our EIA and commercial 3rd EIA was noted. iii) Although slight amino acid difference was observed in the proteins among different genotypes, there was no significant difference was observed in the specificity and sensitivity of the test result.