Maria Inge Lusida

Novel Subgenotypes of Hepatitis B Virus Genotypes C and D in Papua, Indonesia

ABSTRACT Eight genotypes (A to H) and nine subtypes (adw2, adw4, ayw1, ayw2, ayw3, ayw4, adrq+, adrq−, and ayr) of hepatitis B virus (HBV) have been identified worldwide. They appear to be associated with geographical distribution, virological characteristics, and possibly clinical outcomes. We performed sequence analysis of part of the S gene and the entire precore/core gene of HBV isolates obtained from HBsAg-positive blood donors in Papua Province, Indonesia. Phylogenetic analysis of the S gene sequences revealed that 23 (85.2%) of the 27 HBV isolates tested belonged to genotype C (HBV/C) and 2 (7.4%) each to HBV/B and HBV/D. Interestingly, 19 (82.6%) of the 23 isolates of HBV/C clustered in a branch that was distinct from the previously reported subgenotypes C1 to C5 (HBV/C1 to HBV/C5). Similarly, two isolates of HBV/D clustered in a branch distinct from the reported subgenotypes HBV/D1 to HBV/D5. Phylogenetic analysis of the entire precore/core gene confirmed the consistent presence of the distinct branches in HBV/C and HBV/D. We therefore propose novel subgenotypes designated HBV/C6 and HBV/D6. The majority of HBV/C6 isolates in Papua had alanine at positions 159 and 177 (A159/A177) in the HBsAg. A159/A177 is different from the determinants for adrq+ (A159/V177), found throughout Asia, and adrq− (V159/A177), found in New Caledonia and Polynesia, possibly representing a unique antigenic group (provisionally referred to as adrq indeterminate). In conclusion, we have identified two novel HBV subgenotypes, HBV/C6 and HBV/D6, the first of which is the most prevalent subgenotype of HBV in Papua, Indonesia.

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.

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti‐HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti‐HCV activity with 50%‐inhibitory concentrations (IC50) of 20.0 and 8.0 μg/mL, respectively. Through bioactivity‐guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti‐HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4 μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti‐HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti‐HCV activity, their IC50 being 2.5 and 6.2 μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti‐HCV activity, with an IC50 of 3.7 μg/mL. Time‐of‐addition analysis revealed that all Glycyrrhiza‐derived anti‐HCV compounds tested in this study act at the post‐entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia.

Four subtypes (adw, adr, ayw, and ayr) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg‐positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co‐infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co‐infection than in those without HCV co‐infection. We also found that prevalence of hepatitis D virus (HDV) co‐infection was <0.5% among HBV carriers in Surabaya.

Correlation between Mutations in the Interferon Sensitivity-Determining Region of NS5A Protein and Viral Load of Hepatitis C Virus Subtypes 1b, 1c, and 2a

ABSTRACT In the present study, we analyzed the possible relationship between interferon (IFN) sensitivity-determining region (ISDR) sequence variation of various hepatitis C virus (HCV) subtypes and serum HCV titers in Indonesian patients without IFN treatment. The viremia titers (mean ± standard deviation) of HCV subtype 1b (HCV-1b) isolates with low (three or fewer) and high (four or more) numbers of ISDR mutations were 5.4 ± 0.6 and 4.2 ± 0.9 log10RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Similarly, the viremia titers of HCV-1c isolates with low and high numbers of ISDR mutations were 5.3 ± 0.6 and <3.0 ± 0.0 log10 RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Also, the virus titers of HCV-2a isolates with low and high numbers of ISDR mutations were 4.3 ± 0.7 and 3.5 ± 0.4 log10 RNA copies/ml, respectively, with the difference between the two groups being statistically significant (P < 0.01). Thus, our results demonstrated that virus load in Indonesian patients infected with HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of mutations in the ISDR sequence, implying the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia.

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.

Antiviral activities of Indonesian medicinal plants in the East Java region against hepatitis C virus

BackgroundHepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities.MethodsEthanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b).ResultsFour of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes.ConclusionsEthanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.

Hepatitis B and C virus infection among hemodialysis patients in yogyakarta, Indonesia: Prevalence and molecular evidence for nosocomial transmission

Hemodialysis patients are at an increased risk of acquiring hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. However, the prevalence of hepatitis viral infection and its genotype distribution among hemodialysis patients in Indonesia are unclear. In order to investigate these issues and the possibility of nosocomial transmission, 161 hemodialysis patients and 35 staff members at one of the hemodialysis unit in Yogyakarta, Indonesia, were tested for serological and virological markers of both viruses. HBV surface antigen (HBsAg) was detected in 18 patients (11.2%) and in two staff members (5.7%). Anti‐HCV was detected in 130 patients (80.7%) but not in any staff members. Occult HBV and HCV infection were detected in 21 (14.7%) and 4 (12.9%) patients, respectively. The overall prevalence rates of HBV and HCV infection among patients were 24.2% and 83.2%, respectively. HCV infection was independently associated with hemodialysis duration and the number of blood transfusions. Phylogenetic analysis revealed that 23 of 39 tested HBV strains (59%) were genotype B, 11 (28.2%) were genotype C, and 5 (12.8%) were genotype A. HCV genotype 1a was dominant (95%) among 100 tested HCV strains. Nosocomial transmission was suspected because the genotype distribution differed from that of the general population in Indonesia, and because the viral genomes of several strains were identical. These findings suggest that HBV and HCV infection is common among hemodialysis patients in Yogyakarta, and probably occurs through nosocomial infection. Implementation of strict infection‐control programs is necessary in hemodialysis units in Indonesia. J. Med. Virol. 85:1348–1361, 2013.

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. J. Med. Virol. 83:225–234, 2011.

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

ABSTRACT 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.