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.     

Seroprevalence of anti-HBs antibodies and the need for booster vaccination in children under 5 years of age born to HBsAg-negative mothers

Objective: To determine the proportion of HBV surface antigen(anti-HBs) antibody positive children under five years of age born to HBs Ag-negative mothers and to analyze the possible related factors following implementation of a hepatitis B vaccination program for infants in Indonesia 22 years ago.Methods: Blood samples were taken from children under five years of age born to HBsAg-negative mothers who have completed primary vaccination series. Anti-HBs antibodies were determined by using rapid test. Data of age, gender, nutritional status, vaccination timing or vaccination compliance, and booster vaccination were collected from vaccination card.Results: Ninety children were enrolled, consisting of 47 females and 43 males with a mean age of 2.3 years. Twenty two(24.4%)children received booster vaccine between 18 and 24 months and 55(61.1%) were anti-HBs positive. Among factors of age, gender,nutritional status, compliance to vaccination and booster vaccine,only administration of booster vaccine was significantly associated with anti-HBs status(OR 5.45, 95% CI 1.45, 20.52). Children who received booster vaccine at age of 18-24 months were 5.45 times more likely to be anti-HBs positive than that of children who did not receive booster vaccine.Conclusions: Booster vaccine rate is low among children under 5 years of age but is associated with anti-HBs positivity. Booster vaccination may be required to improve anti-HBs seropositivity.     

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.     

Analysis of genetic and serology of hepatitis A virus infection during and after outbreak in two junior high schools in Surabaya,Indonesia

Outbreaks of hepatitis A have occurred in some cities in Indonesia. In Surabaya, the capital city of East Java province, Indonesia, hepatitis A outbreaks have been reported since2013, with a marked increase in the number of cases in 2015. The aim of the present study was to analyze the genetic and serology of acute symptomatic cases (early infection) during a hepatitis A outbreak and asymptomatic cases after the outbreak in two junior high schools in Surabaya in 2015 to 2016. Students with acute symptomatic hepatitis A during the outbreak and other students who were asymptomatic 3 to 4 months after the outbreak were enrolled. Asymptomatic students had no symptoms from the outbreak until they were enrolled. Sera were collected to identify anti-hepatitis A virus (HAV) IgM (by enzyme-linked immunosorbent assay) and HAV genetic variations/genotypes (using polymerase chain reaction [PCR]-sequencing and phylogenetic analysis). A total of 33 (97.1%) out of 34 sera of students with acute symptoms were positive for anti-HAV IgM and 18% of them were positive by PCR, identified as HAV subgenotype IA. No prominent amino acid variations were observed from reported HAV sequences from Indonesia. Among 38 sera of asymptomatic students, most (55.3%) were positive for anti-HAV IgM, while none were positive by PCR. In conclusion, HAV-IA was the only subgenotype identified in acute symptomatic cases during the outbreak. The percentage of HAV-specific IgM-positive cases was very high among acute symptomatic students, but that was also high among asymptomatic students, which might contribute as the important source of infection during the outbreak.     

Interleukin-28B Polymorphisms and Response of Chronic Hepatitis C Patients from Indonesia to Pegylated Interferon/Ribavirin Treatment

This study demonstrated that Indonesian patients with chronic hepatitis C (mostly ethnic Java people) mostly were infected with hepatitis C virus (HCV) genotype 1; however, they carried mainly the major genotypes of interleukin 28B (IL-28B) single nucleotide polymorphisms (SNPs) (rs12979860 CC, rs11881222 TT, rs8103142 AA, and rs8099917 TT), and they mostly achieved sustained virological responses to pegylated interferon/ribavirin treatment.     

Current hepatitis B virus infection situation in Indonesia and its genetic diversity

Indonesia has a moderate to high endemicity of hepatitis B virus(HBV) infection. The risk for chronic HBV infection is highest among those infected during infancy. Since 1997, hepatitis B(Hep B) vaccination of newborns has been fully integrated into the National Immunization Program. Al though HBV infection has been reduced by the universal newborn Hep B immunization program, it continues to occur in Indonesia. The low birth dose coverage and the presence of vaccine escape mutants might contribute to this endemicity among children. Although limited information is available for an analysis of occult HBV infection(OBI), several variations and substitutions in the pre-S/S region have been detected in Indonesian HBV strains. Additionally, persistent infection and disease progression of chronic hepatitis B are related to not only viral factors but also the host genome. Indonesia is one of the most ethnically heterogeneous nations, with Javanese and Sundanese as the two highest ethnic groups. This multi-ethnicity makes genomic research in Indonesia difficult. In this article, we focused on and reviewed the following aspects: the current hepatitis B immunization program and its efficacy, OBI, HBV infection among high-risk patients, such as hemodialysis patients, and research regarding the host genome in Indonesia.     

Another novel subgenotype of hepatitis B virus genotype C from papuans of Highland origin

Hepatitis B virus (HBV) genotypes and subtypes have been identified worldwide. As HBV genotypes/subtypes, the HBV subgenotypes seem to be associated with their geographical distribution and ethnic origin. A previous study showed the novel HBV subgenotype C6 based on the complete genome sequences of isolates in Papua, Indonesia. In the present study, further characterization of HBV in Jayapura (capital of Papua Province), particularly from native people of Papua originating from the highland (highland Papuans) and those from the lowland (lowland Papuans) were examined. Of 32 HBV isolates from both highland and lowland Papuan blood donors with HBsAg positive, part of the S gene and the core gene sequences were analyzed. Analyses of some isolates from highland Papuans were confirmed by the complete genome sequences. Most HBV isolates were classified into genotype C (78.1%), followed by genotype B (18.8%), and genotype D (3.1%). The subtype adr was predominant (71.9%), followed by adw2 (25.1%), and ayw2 (3.1%). As with previous findings, phylogenetic analyses revealed that most HBV isolates from Papuans, C/adr, belonged to subgenotype C6. Interestingly, some C/adr isolates from highland Papuans formed a distinct cluster from all reported subgenotypes of HBV/C, and they differed from HBV/C1-C10 by 4.2-7.2% over the complete genome. SimPlot analysis showed no evidence of recombination with HBV/C1-C10. The isolated life and closed social systems of highland Papuans, even though some have been moving to Jayapura, likely contribute to the formation of this unique cluster of infection with a novel subgenotype of HBV, named C11.