Ultradeep Sequencing for Detection of Quasispecies Variants in the Major Hydrophilic Region of Hepatitis B Virus in Indonesian Patients

Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to <20%; and minor, 1% to <5%. The rates of MHR variation that were present in the major and intermediate viral population were significantly greater in patients with advanced liver disease than those in chronic patients. The most frequent MHR variants related to immune evasion in the major and intermediate populations were P120Q/T, T123A, P127T, Q129H/R, M133L/T, and G145R. The major population of MHR variants causing impaired of HBsAg secretion (e.g., G119R, Q129R, T140I, and G145R) was detected only in advanced liver disease patients. This is the first study to use ultradeep sequencing for the detection of MHR variants of HBV quasispecies in Indonesian patients. We found that a greater number of MHR variations was related to disease severity and reduced likelihood of HBsAg titer.     

Serologic and Molecular Characteristics of Hepatitis B Virus among School Children in East Java,Indonesia

Universal childhood hepatitis B vaccination was introduced in Indonesia in 1997; by 2008, coverage was estimated to be 78%. This study aimed to investigate the serologic status and virologic characteristics of hepatitis B virus (HBV) among the children in East Java. A total of 229 healthy children born during 1994–1999 were enrolled in this study. Overall, 3.1% were positive for hepatitis B surface antigen (HBsAg) and 23.6% were positive for antibody to HBsAg (anti-HBs). HBV DNA was detected in 5 of 222 HBsAg-negative carriers, which were suggested to be cases of occult HBV infection. A single amino substitution (T126I) in the S region was frequently found. HBV infection remains endemic, and the prevalence of anti-HBs remains insufficient among children in East Java, Indonesia.     

Prevalence of hepatitis E virus among swine and humans in two different ethnic communities in Indonesia

The purpose of this study was to investigate the prevalence of hepatitis E virus (HEV) infection in swine and humans in different environments in Java and Bali, Indonesia. The prevalence of anti-HEV antibodies in people over 20?years old living in communities in Bali was significantly higher than that in Java. While 68.8% and 90.0% of swine in Bali were anti-HEV positive at 1 and 2?months of age, respectively, swine in Java were at significantly lower risk of HEV infection by the age of 2?months. Our present data suggest that substantial differences in swine-breeding conditions and human living environments affect the rate of HEV infection in humans and swine.     

Complete Genome Sequence and Phylogenetic Relatedness of Hepatitis B Virus Isolates in Papua,Indonesia

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.Hepatitis B virus (HBV) is an etiological agent of chronic liver disease, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and this poses major health problems worldwide, especially in Asian Pacific countries (7, 10).HBV strains that infect humans show genetic and antigenic heterogeneity, and eight genotypes, designated A to H, have been identified so far by molecular evolutionary analysis (19). The HBV genotypes have distinct geographical distributions, which are associated with anthropological history (4, 13, 20, 31). Furthermore, previous studies have demonstrated the presence of several subgenotypes within the widely spread genotypes. HBV genotype B (HBV/B) is classified into six subgenotypes, B1 to B6, B1 dominating in Japan, B2 in China and Vietnam, B3 in Indonesia, B4 in Vietnam, B5 in the Philippines, and B6 in the Arctic (3, 16, 23, 24, 26, 27). As for HBV/C, C1 is common in southeast Asia, C2 in east Asia, C3 in Oceania, C4 in Aborigines, and C5 in the Philippines (15, 26). HBV/D has a worldwide distribution, with its highest prevalence in the Mediterranean region, and is classified as D1 to D5 (1, 15, 17). Our previous study revealed novel subgenotypes (HBV/C6 and HBV/D6) based on the S gene sequence of HBV isolates in Papua, Indonesia, where HBV infection is endemic (9).HBV genotyping with the S gene sequence is, in general, consistent with the genotyping of the full genomic sequence, and therefore, HBV genotypes can be assigned based upon S gene sequences (11, 16, 19). Subgenotype classification, however, may not be applicable to some HBV strains on the basis of the S region sequence alone (9, 14, 15). Accordingly, complete genome sequences are more reliable for the analysis of genotype and subgenotype classification for HBV (14). The data on the complete genome sequences of the HBV strains found in Papua are scant. The present study aimed to evaluate the HBV genotypes and subgenotypes present among the Papuan population using complete genome sequences. In addition, the phylogenetic relatedness of HBV strains isolated from Papua was assessed.     

Viral load in Indonesian patients with chronic liver disease and in blood donors infected with different subtypes of hepatitis C virus

The viral load of different hepatitis C virus (HCV) subtypes, including the globally distributed HCV-1b and the unique Indonesian subtype HCV-1c, was analyzed using serum samples obtained from Indonesian blood donors and patients with chronic liver disease. The mean viral load of HCV-1c was comparable with that of HCV-1b, suggesting that HCV-1c is as pathogenic as HCV-1b. On the other hand, the mean viral load of HCV-2a was lower than that of HCV-1b or HCV-1c, with this result being consistent with previous observations. Interestingly, some HCV-2a strains were associated with a high viral load that was almost equivalent to that of HCV-1b and HCV-1c. This result implies the possibility that there exists a minor fraction of HCV-2a strains that cause higher levels of viremia compared with the majority of ordinary HCV-2a strains.