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Hepatitis B virus genotype C (HBV/C) has the largest number of subgenotypes (C1-C16) that vary with geography and isolates. HBV/C prevails in Southeast Asia (C1, C5-C16), East Asia (C2), Oceania (C3), and Australia (C4). Suitable reference strains for different subgenotypes could greatly facilitate research into HBV/C, but unfortunately they are scarce. We retrieved 974 HBV/C full-length sequences from the GenBank database and subgenotyped them by phylogenetic analysis. Reference sequences of each subgenotype from different locations were established with the most frequent nucleotide present at each position of the isolates that belonged to the same subgenotype. The reference sequences of subgenotypes C1, C2, C5, and C6 have been constructed and deposited in GenBank (KM0-KM3). The homology between the reference sequences and almost all the isolates belonging to the corresponding subgenotype was higher than 96%. Similarly, bootstrap values in phylogenetic trees supported clustering of reference strains with isolates belonging to the same subgenotypes. Moreover, both homology and phylogeny analyses showed that reference sequences had significant heterogeneity with isolates from other genotypes and subgenotypes. Sequence analysis further revealed that the mutation rate in the basal core promoter (BCP) region was extremely high in HBV/C2, relatively high in HBV/C1, but lower in HBV/C5 and HBV/C6. Mutations in the pre-core (Pre-C) region were common in HBV/C but the mutation rate was lower than in the BCP. HBV/ C5 has the oldest ancestral age, followed by C6, which is much more ancient than C1 and C2. This study successfully established references for HBV/C subgenotypes.

Establishment of reference sequences of hepatitis B virus genotype C subgenotypes