Full-length Sequence of the Genome of Hepatitis C Virus Type 3a: Comparative Study With Different Genotypes

Abstract

Hepatitis C virus (HCV) type K3a (type 3a), which represents a minor genotype in Europe, the U.S.A. and Asia, appears to be significantly distributed throughout Australia and Brazil. We amplified the HCV-K3a/650 genome by reverse transcription polymerase chain reaction in ten overlapping fragments and determined the nucleotide sequences. The total sequence was 9454 bases in length and contained an open reading frame of 3021 amino acids, which is 10 or 11 amino acids longer than in HCV type 1 and 12 amino acids shorter than the sequence of type 2. These differences were due to the different lengths of both the putative envelope protein E2 and the NS5A regions, whose nucleotide lengths differ between types 1 and 2 also. Phylogenetic analysis of the putative core region and a portion of NS5B encoding the Gly-Asp-Asp motif indicated that HCVK3a closely matched the corresponding type 3a group. The deletion and addition of amino acids in both E2 and NS5A may be associated with their pathobiological features.

Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response

“…Numbering of codons and amino acid positions are according to the HCV-3a prototype NZL1. 26 Within the putative ISDR (codon 2215-2254) the HCV-3a prototype sequences NZL1 and HCV-K3a 25,26 were identical to the consensus sequence of the 33 isolates (treatment schedules indicated in Fig. 5).…”

“…The consensus sequence obtained by sequence alignment of all 33 isolates of the study and the HCV-3a prototype sequence NZL1 26 are identical between codons 638 and 704. In comparison with HCV-3a prototype HCV-K3a, 25 only one conservative amino acid mutation (I660L) was found ( Fig. 2A).…”

“…Sequencing of the (ϩ) strand and the (Ϫ) strand was performed by an automat (Applied Biosystems 310 DNA Sequencer). The deduced amino acid sequences of the E2 and NS5A region were compared with the E2 and NS5A sequences identified in the prototype isolates for HCV-1a (HCV-1 27 ), HCV-1b (HCV-J 19 ), HCV-3a (NZL1 and HCV-K3a 25,26 ) and the consensus sequence of all HCV-3a isolates (cons) investigated in the present study. Sequences of the E2 and NS5A region of HCV-1b-and HCV-3a-infected patients were submitted to GenBank.…”

“…After an initial denaturation step at 95°C for 2 minutes, 35 cycles of 95°C for 30 seconds, 55°C for 50 seconds, and 72°C for 2 minutes were performed in a PE9700 thermocycler (Perkin Elmer Cetus). In the second round of the nested PCR, 35 cycles using internal sense primer 3a5A20s (5Ј-TTCTTCACTGAAGTGGATGG-3Ј; nucleotide position 6718-6737 according to HCV-K3a 25 ) and the internal antisense primer 3a5A20a (5Ј-GTTGGTGGTACATAATCCGG-3Ј; nucleotide position 7267-7286) were performed as described for the first round of the PCR. The resulting amplification product was analyzed on a 1.8% agarose gel stained with ethidium bromide.…”

“…In HCV isolates of all HCV-3a-infected patients the NS5A region between codon 2144 and 2300 according to HCV-3a prototype NZL1 26 was amplified after reverse transcription by nested PCR. The first round of PCR was performed using external sense primer 3a5A6s (5Ј-GCCCAACACA-CAACTACACGCGCGC-3Ј; nucleotide position 6581-6605 according to HCV-K3a 25 ) and antisense primer 3a5A3a (5Ј-CTTTTCCTC-CGAGGAGG-3Ј; nucleotide position 7333-7349). After an initial denaturation step at 95°C for 2 minutes, 35 cycles of 95°C for 30 seconds, 55°C for 50 seconds, and 72°C for 2 minutes were performed in a PE9700 thermocycler (Perkin Elmer Cetus).…”