Complete genome sequence and phylogenetic relatedness of hepatitis B virus isolates in Papua, Indonesia

Abstract

Each hepatitis B virus (HBV) genotype and subgenotype is associated with a particular geographic distribution, ethnicity, and anthropological history. Our previous study showed the novel HBV subgenotypes C6 (HBV/C6) and D6 (HBV/D6), based on the S gene sequences of isolates in Papua, Indonesia. The present study investigated the complete genome sequence of 22 strains from Papua and subjected them to molecular evolutionary analysis. A phylogenetic analysis revealed that 9 out of 22 strains were classified as HBV/C6, 3 strains as HBV/D6, and 9 strains as HBV/B3. A particular strain positioned between HBV/B3 and HBV/B5 remained unclassifiable into any known subgenotypes. This strain showed high homology with HBV/C5 from the Philippines in the core region and was thought to have undergone genetic recombination with HBV/C5. Further studies are needed to determine whether this strain belongs to a new subgenotype of HBV/B. Based on the amino acid alignment, HBV/C6 has subgenotype specific variations (G18V and V47M) in the S region. HBV/C6 strains were more closely related in terms of evolutionary distance to strains from the east Asia and Pacific regions than those found in southeast Asia. HBV/D6 strains were most closely related to strains from the Western countries (HBV/D3) rather than those from Asia and Papua New Guinea. In conclusion, we have confirmed by complete sequence analysis that two novel HBV subgenotypes, HBV/C6 and HBV/D6, are prevalent in Papua, Indonesia.

FIG. 1.

Phylogenetic trees of HBV strains isolated from 22 blood donors in Papua along with 52 reference strains. (A) Complete genome; (B) pre-C/C gene; (C) S gene. The number in the tree indicates the bootstrap reliability. The lengths of the horizontal bars indicate the number of nucleotide substitutions per site. *, HBV/C5 including P18 in this study cluster in the same branch as HBV/B due to the genetic recombination with HBV/B over the pre-C/C gene. Isolates from the database are indicated with accession numbers, and relevant country names are added to each HBV/B, HBV/C, and HBV/D strain. The nucleotide sequence accession numbers used as references in the phylogenetic trees are as follows: for HBV/B, EF473976, EF473977, M54923, AB033554, D00331, AB219426 through AB219429, AB241116, AB241117, DQ463791, DQ463792, DQ463787 through DQ463794, AY033073, AB073835, D00329, D23678, AF121243, and AF121251; for HBV/C, D23684, M38636, X75656, X75665, AB111946, AB112472, AB241109, AB241113, AB048704, and AB048705; for HBV/D, DQ315779, DQ315780, AB048701, AB048703, AB033559, AY161157, AF151735, AB090269, AB078032, AB078033, AJ132335, and AY902776; for HBV/A, AB014370; for HBV/E, EX75657; for HBV/F, FX75658; for HBV/G, GAF160501; and for HBV/H, HAY090457.

FIG. 1.

Phylogenetic trees of HBV strains isolated from 22 blood donors in Papua along with 52 reference strains. (A) Complete genome; (B) pre-C/C gene; (C) S gene. The number in the tree indicates the bootstrap reliability. The lengths of the horizontal bars indicate the number of nucleotide substitutions per site. *, HBV/C5 including P18 in this study cluster in the same branch as HBV/B due to the genetic recombination with HBV/B over the pre-C/C gene. Isolates from the database are indicated with accession numbers, and relevant country names are added to each HBV/B, HBV/C, and HBV/D strain. The nucleotide sequence accession numbers used as references in the phylogenetic trees are as follows: for HBV/B, EF473976, EF473977, M54923, AB033554, D00331, AB219426 through AB219429, AB241116, AB241117, DQ463791, DQ463792, DQ463787 through DQ463794, AY033073, AB073835, D00329, D23678, AF121243, and AF121251; for HBV/C, D23684, M38636, X75656, X75665, AB111946, AB112472, AB241109, AB241113, AB048704, and AB048705; for HBV/D, DQ315779, DQ315780, AB048701, AB048703, AB033559, AY161157, AF151735, AB090269, AB078032, AB078033, AJ132335, and AY902776; for HBV/A, AB014370; for HBV/E, EX75657; for HBV/F, FX75658; for HBV/G, GAF160501; and for HBV/H, HAY090457.

FIG. 2.

(A) Location of the recombination event in the viral genome of the HBV/B P18 strain determined by the SimPlot program and bootscanning analysis. The sequence of the P18 strain was compared with three representative HBV isolates, HBV/B (GenBank accession no. D23678), HBV/C (accession no. AB241109), and outgroup HBV/F (accession no. X75658) over the full genome with a 200-bp window size, a 20-bp step size, 100 bootstrap replicates, gap stripping, and neighbor-joining analysis. (B) Genetic distances of the P18 strain from HBV/B (GenBank accession no. D23678) and HBV/C (accession no. AB241109) over the complete genome, with a window size of 200 bp and a step size of 20 bp. The dotted vertical lines show the breakpoints of recombination.