Isolation, Identification and Molecular Characterization of Hepatitis B Virus in Javan Gibbon in Indonesia

Abstract

Hepatitis B virus (HBV) was reported not only able to infect human being, but also non-human primates, especially the great apes group such as orangutans, gorillas, chimpanzees, as well as the lesser apes from the family of Hylobatidae. Among other species within the genus, Hylobates moloch (owa jawa or silvery/ javan gibbons), which is considered as one of Indonesia’s endemic endangered species, has been reported to harbor HBV of their own strain. Analyses of HBV isolated from infected gibbons were done in this study to reveal their relatedness to the published data. Plasma samples from nine javan gibbons were obtained as part of diagnostic purpose. Those animals were previously tested positive for the presence of hepatisis B-surface antigen (HBsAg) by enzyme-linked immunosorbent assay (ELISA). DNA samples were extracted from these nine plasma samples to be used as templates for amplification of the Pre-S1 region by polymerase chain reactions (PCR) technique using semi degenerate primers that could amplify several strains of HBVs. The PCR products of 457 - 459 base pairs were then subject to restriction fragment length polymorphism (RFLP) analyses using Bst2UI enzyme to evaluate the pattern of cleaved fragments. The PCR products were sequenced for further homology analyses and to create phylogenetic tree of the sequenced data obtained from the study in comparison with published data. Two plasma samples were used as templates for whole region amplification using sets of 4 primer. The PCR products of 3192 base pairs were sequenced for further homology analyses and to create phylogenetic tree. Phylogenetic tree analyses for Pre-S1 nucleotide region and whole region showed that the javan gibbon isolates formed their own clusters, separate from the other nonhuman primate isolates.