Identification of four novel subgenotypes (C13–C16) and two inter-genotypic recombinants (C12/G and C13/B3) of hepatitis B virus in Papua Province, Indonesia☆
Highlights
► In this study, we identified and characterized four novel HBV subgenotypes (C13−C16) in Papua, Indonesia. ► Two inter-genotypic recombinants (C12/G and C13/B3), displaying recombination breakpoints in the 5′-terminus of the P gene, were also found. ► The distribution of indigenous subgenotypes C11−C16 was associated with particular language speakers in Papua.
Introduction
Hepatitis B virus (HBV) infection remains a major health problem causing significant morbidity and mortality worldwide despite the availability of vaccines and antiviral treatments (Lavanchy, 2004). Currently, at least 350 million individuals in the world are infected chronically with HBV, some of whom will develop severe liver diseases, including cirrhosis and hepatocellular carcinoma. The prevalence of HBV infection is generally high in Asia and Africa, where most infections are acquired at birth or during childhood (Lee, 1997). HBV is a DNA virus in the family Hepadnaviridae, and has a genome composed of approximately 3200 nucleotides (nt) that contains four open reading frames for the P, preC/C, preS1/preS2/S, and X genes (Tiollais et al., 1985).
Compared to most DNA viruses, HBV has a high mutation rate of approximately 2 × 10−5 nucleotide substitutions per site per year (Okamoto et al., 1987a), although this is lower than the rate of most RNA viruses. Up to now, eight well-established genotypes (A−H) have been identified, which differ by >8% over the entire genome (Arauz-Ruiz et al., 2002, Norder et al., 1994, Okamoto et al., 1988, Stuyver et al., 2000). With the exception of genotypes E, G, and H, the genotypes are divided into subgenotypes, i.e., A1–A6 in HBV of genotype A (HBV/A), B1–B9 in HBV/B, C1–C12 in HBV/C, D1–D7 in HBV/D, and F1–F4 in HBV/F, with distinct geographical clustering (Cavinta et al., 2009a, Cavinta et al., 2009b, Kramvis et al., 2008, Lusida et al., 2008, Meldal et al., 2009, Mulyanto et al., 2009, Mulyanto et al., 2010, Mulyanto et al., 2011, Norder et al., 2004, Nurainy et al., 2008, Pourkarim et al., 2010, Sakamoto et al., 2006, Utsumi et al., 2009, Wang et al., 2007). Recently, a potential new genotype (tentatively designated genotype I) was proposed for a complex recombinant of genotypes A, C, and G mainly found in Laos and Vietnam (Olinger et al., 2008, Tran et al., 2008). In addition, genotype J was proposed for an HBV strain isolated from a hepatitis patient living in Okinawa, Japan, which was positioned phylogenetically in between the human and ape genotypes (Tatematsu et al., 2009). Infection with multiple distinct HBV genotypes is common, and recombination between HBV genotypes has also been observed (Bekondi et al., 2007, Bollyky et al., 1996, Cui et al., 2002, Kato et al., 2002, Kurbanov et al., 2005, Morozov et al., 2000, Osiowy et al., 2008, Suwannakarn et al., 2005)
Indonesia has a moderate to high endemicity of HBV infection, where HBV/B is predominant (66%), followed by HBV/C (26%), HBV/D (7%), and HBV/A (0.8%) (Mulyanto et al., 2009). As one of the largest archipelagos in the world, Indonesia consists of more than 17,000 islands and is inhabited by hundreds of ethnic groups using more than 442 local languages (Sugono, 2008). Up to now, at least 20 subgenotypes within genotypes A−D have been found in Indonesia (Lusida et al., 2008, Mulyanto et al., 2009, Mulyanto et al., 2010, Mulyanto et al., 2011, Nurainy et al., 2008, Thedja et al., 2011). Subgenotype B3, presumably indigenous to Indonesia, is distributed widely all over Indonesia, whereas subgenotypes C6 and D6 are found predominantly in a restricted area, the Papua province of Indonesia (Lusida et al., 2008, Mulyanto et al., 2009, Mulyanto et al., 2011). Our previous study revealed the presence of novel HBV subgenotypes of C11 and C12 among inhabitants in Papua. However, to date, there have been only limited studies about the prevalence of HBV genotypes/subgenotypes and the genetic characteristics of HBV in relation to the different ethnic groups and language populations in Papua. Therefore, the present study was conducted to further identify and phylogenetically characterize the various HBV isolates circulating in an a previously unexamined area, Nabire, which is situated on the north coast of Papua in the center of the great Cendrawasih Bay, and is surrounded by mountains and dense forest.
Section snippets
Serum samples
Serum samples were collected from 515 indigenous inhabitants (244 males and 271 females; mean age 26.6 ± 9.6 years; age range 15−76 years) in Nabire, including senior high school students (n = 143) and outpatients without liver disease (n = 372) who visited Bumiwonorejo Public Health Center or Santo Rafael Clinic in Nabire, suffering from malaria, common cold, gastroenteritis, or tuberculosis, between April and November 2010. These inhabitants rarely move outside the town or to other islands in Papua.
Detection of HBsAg and HBV DNA and determination of the HBV genotype/subgenotype based on the 396-nt and 1.6-kb partial sequences
Among the 515 subjects studied, 53 (28 males and 25 females; mean age 26.2 ± 8.7 years; age range 16−46 years) had detectable HBsAg, thus indicating the positive rate of HBsAg to be 10.3%. When serum samples from the 53 HBsAg-positive subjects were tested for the presence of HBV DNA by PCR with primers targeting the 396-nt S gene sequence, 51 samples were found to be positive. Based on the pair-wise and phylogenetic analyses of the 396-nt sequence, 29 samples (57%) were classified into genotype C,
Discussion
The present study revealed the presence of at least 11 subgenotypes within HBV genotypes B, C, and D in Nabire, Papua, including four novel subgenotypes within genotype C (tentatively designated C13–C16) as well as C6, C12, and D6, which may be indigenous to Papua. In addition, two inter-genotypic recombinant (C12/G and C13/B3) HBV strains were found in the studied population.
The genetic variability within genotypes has been extensively investigated, and all genotypes, except for E, G, H, and
Acknowledgements
The authors are grateful to Drs. Soewignjo Soemohardjo and Makoto Mayumi for their advice and encouragement during this study.
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The nucleotide sequence data reported in this study have been assigned DDBJ/EMBL/GenBank accession numbers AB644280–AB644287 for the 8 full-length sequences, AB644288–AB644325 for the 38 1.6-kb partial sequences, and AB644326–AB644331 for the 5 396-nt S gene sequences.