Identification and characterization of genotype A and D recombinant hepatitis B virus from Indian chronic HBV isolates

Abstract

Aim: To confirm the presence of recombination, full-length hepatitis B virus (HBV) from chronic patients was sequenced and analyzed.

Methods: Full-length HBV genomes from 12 patients were amplified and sequenced in an automated sequencer. Phylogenetic analysis was carried out on full-length, Core and preS2/Surface regions using MEGA software. SimPlot Boot Scanning and amino acid sequence analysis were performed for confirmation of recombination.

Results: Eight patients were infected with genotype D strain; one patient with genotype A and three patients had genotype A and D recombination; two of them had cirrhosis and one had hepatocellular carcinoma. Phylogenetic analysis of core and preS2/surface regions separately showed evidence of genotype A and D recombination. The breakpoints of recombination were found to be at the start of preS2 and at the end of surface coding regions.

Conclusion: We identified and characterized recombinant A and D genotype HBV in hepatitis B surface antigen (HBsAg)-positive patients.

Figure 1

Phylogenetic tree revealed the distribution of genotypes. A: Neighbor joining (NJ) phylogenetic tree was constructed based on the complete genome sequences of 26 HBV reference strains from DDBJ/EMBL/Genbank and 12 Indian HBV isolates in the present study. The bootstrap values obtained from 1000 replicas are shown at the nodes of the main branches. HBV strains from this study are marked as Ran as suffix followed by the isolate number. Reference sequences are denoted by their accession numbers. The capital letter A to H denotes the HBV genotype; B: Phylogenetic analysis done on the PreS2 and surface regions showing three additional (103,105,113) isolates to be grouped in genotype A branch. Phylogenetic trees were constructed by NJ Kimura two-parametric method. Bootstrap values are shown at the nodes of the main branch and at the bottom distance is given; C: Phylogenetic analysis using the Core region. Phylogenetic tree was constructed by NJ Kimura 2-parametric method Bootstrap values are shown at the nodes of the main branch and at the bottom distance given.

Figure 2

SimPlot analysis demonstrating the recombination in two isolates 103 and105, which were subjected to bootscan analysis over the entire genome using SimPlot program (Lole et al[18]).

Figure 3

Recombinant sequence similarities with genotype A in PreS2 and Surface region. A: Surface region alignment with genotype A and D amino acids, marked in red shows all three recombinant sequences having similarity with genotype A, whereas yellow marked amino acids shows at least one recombinant sequence similar to genotype A, over all 18 positions; B: Shows same in the preS2 region; C: Shows all the amino acids of MHR of genotype D in black, upward arrow indicates the change of amino acid; D: Shows “a” determinant region and corresponding alignment with 12 isolates.