Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.