Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

Abstract

The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype.

Figure 1

Polyacrylamide gel electrophoresis of the reaction products of semi-nested PCR and DNA Markers. The first round of amplification products (1), the second round of amplification products (2), and the negative control (NC) are shown.

Figure 2

(a) Phylogenetic tree of the full-length nucleotide sequences of the eight HBV DNA standard genotypes (HBV GENOTYPE A-H). (b) Phylogenetic tree of the nucleotide sequences of the fragments of the eight HBV DNA standard genotype polymerase genes (hbv A-H).

Figure 3

Phylogenetic tree of the nucleotide sequences of the fragments of the semi-nested PCR products and HBV DNA standard genotype polymerase genes. Patients with chronic hepatitis B (P-), asymptomatic carriers of HBV (C-), and the fragments of the standard genotype polymerase genes (hbv A-H) are shown. Only genotypes B, C, and D were detected in the 50 HBV DNA-positive cases.