Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala, India

Abstract

Aim: To molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.

Methods: Sera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05.

Results: Irrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D.

Conclusion: Kerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.

Keywords: Chronic hepatitis; Cirrhosis; Genotype; Hepatocellular carcinoma; India; Phylogenetic analysis.

Figure 1

Prevalence of genotypes in different geographical areas of India compiled from previous reports. Northern[22,23,28,41,42]; Western[18,43]; Eastern[8,24,32,43-46]; Southern[6,47]; Nicobar Islands[48]. No hepatitis B virus genotyping data was available for Kerala before this study.

Figure 2

Phylogenetic relationships among complete preS1/pre S2/S sequences (nt 2854-835 numbering according to GenBank accession AY233274). A: Subgenotype A1 from hepatitis B virus (HBV) positive patients from Kerala (marked in bold) compared with sequences obtained from GenBank established using the neighbor joining method; B: Genotype D isolates from HBV positive patients from Kerala (marked in bold) compared with HBV isolates obtained from GenBank established using the neighbor joining method. Bootstrap statistical analysis was performed using 1000 replicates. Each sequence obtained from GenBank is designated by its accession number and its country of origin. The characteristic amino acids in the preS1 and polymerase spacer regions are indicated next to the sequences or relevant clades.

Figure 3

Comparison of the distribution of mutations in the basic core promoter/precore region (1742-1901 from the EcoR1 site) in genotypes A (62 isolates) and D (23 isolates). Graphs showing the percentage of mutant residues relative to the reference motif found at the 15 loci of interest (1753, 1762, 1764, 1766, 1773, 1809-1812, 1814, 1858, 1862, 1888, 1896 and 1899). The study sequence files were submitted to the Mutation Reporter Tool[49] to produce the graphs. The reference motifs used were TAGCTGCACACGGGG (genotype A) and TAGCTGCACATGGGG (genotype D) for comparison. This is also shown by the letter preceding each locus on the X-axis. To facilitate direct comparisons between the graphs, conserved loci were not suppressed and the Y-axis was scaled to 100% by selecting the appropriate controls on the input page of the Mutation Reporter Tool. Nucleotides: A (green), C (dark blue), G (black), T (red). ^aSignificantly associated with the respective genotype.