CHARACTERIZATION OF THE GENOMIC SEQUENCE OF TYPE-V (OR 3A) HEPATITIS-C VIRUS ISOLATES AND PCR PRIMERS FOR SPECIFIC DETECTION

We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5' untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1 a, lb, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3' end of 5' UTR, C, El and 5' end of E2/NS1) and 8276 to 9394 (3' end of NS5 and 3' UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96 %, compared to 73 to 74 %, 73 %, 70 % or 69 to 70 % against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported 'type V (3a)' isolates was 88 to 100 % ; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3' UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5'CGTAAAACTTCT GAACGGTC, sense and no. 339: 5'GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.