Molecular characterization and surface antigen mapping of gibbon hepatitis B virus

Abstract

Hepatitis B virus (HBV) infection represents a global health burden. There are approximately 350 million chronic carriers worldwide off which 25-30% eventually proceed towards chronic liver disease or liver cancer, HBV is also found in several species of nonhuman primates like chimpanzee, gibbon, orangutan, gorilla and woolly monkey. Since illegal captive gibbons in Thailand were housed in Krabok Koo wildlife Breeding Center, Cha-Cheng-sao, HBV infection screening were established to prevent viral spread among animals. Seroprevalence studies of 101 captive gibbons revealed that 38.61% (39/101) of the animals were positive for at least one marker of HBV infection and 19 gibbons were chronic carriers as defined by the presence of HBV DNA and HBsAg, in the absence of antibodies to the S protein. Alanine-aminotransferase levels of the carrier animals (68.75±48.12 U/I) were significantly higher compared to healthy control animals (33.04±15.91 U/I, p˂0.05). Evidence of vertical and horizontal transmission in captive gibbons was obtained by testing HBsAg, HBV-DNA in sera and saliva specimens. The 98.61% and 99.45% sequence similarity of the PreS1 gene-the most divergent part of HBV genome-isolated from sera of two HBV carrier mothers and their newborn babies further proved the fact that vertical transmission occurred. HBV horizontal spread by saliva contamination was suggested due to the fact that HBV-DNA was detected in saliva of HBV carriers. Although the similarity of gibbon and human viral particles was confirmed by EM study, molecular characterization of gibbon HBV revealed untypable genotype using RFLP profiles of human viruses. Precore promoter 1762/1764 point mutation was determined in four animals while some of the chronic animals were found to be anti-HBc negative (4/19). Phylogenetic tree analysis of PreS1 and HBV whole genome revealed clustering of the gibbon viruses separate from Hepadnaviruses of the other hosts. Compared to human hepatitis viruses, surface antigen mapping of gibbon HBV indicated similar binding sites of human HBsAg specific antibodies, as detected by ELISA assay. Particles of gibbon HBV was caught by immune anti-HBs from vaccinated patients measured by inhibition binding assay. Gibbon HBV surface protein was cloned and expressed in CHO mammalian cells for testing the attachment between viruses and host cells. Because of low amounts of viral particles produced, binding of gibbon viruses to human host cells could not be confirmed thus far. DMSO treated HepG2 cells used as target cells for infection by human and gibbon HBV positive serum revealed the presence of the HBV replicative form (cccDNA), 14 days after infection. In conclusion, HBV seroprevalence and viral spreading routes in captive gibbons were investigated. Although genotyping and phylogenetic analysis of gibbon viruses clearly revealed a separate group compared to human HBV, with respect to virion morphology and S antigen conformation structure these viruses are quite similar. Additionally, anti-S antibodies from vaccines specifically bound to gibbon HBV and replication of gibbon viruses in human hepatocyte cells was demonstrated. However, specific binding of gibbon viruses to human cells could not be confirmed thus far. Thus, the possibility of HBV cross transmission between gibbon and human should be taken into account. HBV immunization was suggested for animal caretakers and non-immune gibbons to prevent viral transmission.