Genotype D of hepatitis B virus and its subgenotypes: An update

Aim The aim of the present study was to systematically and comparatively analyze the subgenotypes of genotype D of hepatitis B virus. Methods In total, 304 complete genomes of all genotype D subgenotypes were downloaded from the public databases. The sequences were analyzed using nucleotide divergence calculations, phylogenetic analysis and bioinformatics to detect amino acids signature motifs for each subgenotype and to define their geographical distribution. Results Intragroup divergence ranged from 0.8 +/- 0.5 (%standard deviation) for subgenotype D6 to 3.0 +/- 0.3 for D8. Inter-subgenotype divergence mostly ranged 47.5%. Phylogenetic analysis of genotype D showed separation into six distinct clusters (subgenotypes D1, D2, D3/D6, D4, D5 and D7/D8) with good bootstrap support. The mean intergroup divergence between D3 and D6 was the lowest and fell below the threshold of 4%, which is required to define a subgenotype, suggesting that subgenotypes D3 and D6 belong to one subgenotype. D8 is a genotype D/E recombinant, which clusters with D7. A number of signature amino acids were found in all four open reading frames that could differentiate the subgenotypes, which also showed distinct geographical distribution. Conclusion There are six and not eight subgenotypes of D, D1D6, which can be differentiated by distinct clustering with high bootstrap support and signature amino acids. Subgenotypes D3 and D6 should be reclassified as a single subgenotype D3 and it would be more correct to classify D8 as a genotype D/E recombinant rather than a subgenotype.