1 Viral Hepatitis Research Unit, Department of Paediatrics, Chulalongkorn University and Hospital, Bangkok 10330, Thailand
4 Department of Microbiology, Faculty of Medicine, Chulalongkorn University and Hospital, Bangkok 10330, Thailand
2 Inter-Department of Medical Microbiology, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, Thailand
3 Institute of Virology, Erasmus University Rotterdam, The Netherlands
5 Faculty of Veterinary Science, Mahidol University, Nakornpathom, Thailand
Correspondence
Yong Poovorawan
Yong.P{at}chula.ac.th
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this paper are AF27449596, AF274499, AF275378, AF47748294, AY07773536 and AF52930809.
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Introduction |
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Phylogenetically, HBV isolated from gibbons and chimpanzees share an early lineage, indicating that these viruses were indigenous to their respective hosts (Norder et al., 1996). On the other hand, infection of chimpanzees with human and gibbon HBV can be accomplished (Gallagher et al., 1991
). Experimental transmission of human HBV to gibbons by exposure to human saliva containing HBV has been reported also (Bancroft et al., 1977
; Scott et al., 1980
). Replication of human HBV in the respective animals supported the close relation of these hosts and may indicate natural HBV cross-transmission. On the other hand, no evidence has been obtained thus far for HBV transmission from gibbon or chimpanzee to human. HBV is present at levels as high as 1x1013 virions ml-1 in the blood of HBV e antigen (HBeAg)-positive patients but virus particles have also been found in other body fluids, including saliva/nasopharyngeal fluids, semen, cervical secretions and leukocytes (Alter et al., 1977
; Davison et al., 1987
). The possibility of human HBV transmission through contact with saliva from HBV chronic carriers has been obtained both in humans as well as in gibbons (Bancroft et al., 1977
; MacQuarrie et al., 1974
; Scott et al., 1980
; Stornello, 1991
).
In order to analyse possible routes of gibbon HBV transmission, we determined the presence of HBV in captive gibbons in Thailand by serological testing and HBV DNA detection in chronic carriers. Sequencing and RFLP analysis of the viruses permits molecular characterization of gibbon HBV and possible routes of transmission between gibbons, including vertical transmission. To investigate horizontal transmission to humans, animal caretakers, some of whom are HBV carriers, were analysed for the presence of gibbon HBV. The unique deletion at the preS1 gene present in gibbon HBV permits the accurate identification of a zoonotic event.
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Methods |
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Animal caretakers at the Krabok Koo Centre (n=34) were screened for the HBV surface antigen (HBsAg) during the prevaccination screening programme.
Sample collection.
Gibbon blood was obtained by venepuncture during a brief period of anaesthesia by ketamine, part of the routine health-care programme. Sera were separated from clotted blood within 12 h by centrifuging at 1500 r.p.m. for 10 min and kept at -70 °C. Human blood was separated using the same procedure. Saliva samples were collected from 30 gibbons using the OraSure collection system (OraSure Technologie) following the manufacturer's protocol. Samples were kept at -70 °C until further testing.
HBV detection and liver function test analysis.
All sera were analysed for HBsAg, antibodies to HBsAg (anti-HBs) and anti-HBc (antibodies to the HBV core antigen HBcAg) by enzyme immunoassay methodology (EIA) using a commercially available kit (DiaSorin), according to the manufacturer's instructions. HBeAg, anti-HBc and anti-HBs antibodies of some samples were reconfirmed by EIA kits obtained from Abbott Laboratories.
Gibbon HBV DNA was detected as described previously (Theamboonlers et al., 1999). Briefly, DNA was extracted from 200 µl of serum with proteinase K in lysis buffer followed by phenol/chloroform extraction and ethanol precipitation. The S gene was amplified by nested PCR using two sets of primers: primer F1 (5'-GGAGCGGGAGCATTCGGGCCA-3', nt 30223042) and R6 (5'-GGCGAGAAAGTGAAAGCCTG-3', nt 11031084) were used for first-round amplification and primers F2 (5'-CATCCTCAGGCCATGCAGTGGA-3', nt 31923214) and R5 (5'-AGCCCAAAAGACCCAGAAATTC-3', nt 1015995) were used for second-round amplification. The amplification reaction required 30 cycles comprising initiation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, concluded by a final extension step at 72 °C for 10 min. The 1038 bp PCR product was detected by electrophoresis on a 1.5 % agarose gel with ethidium bromide and visualized under UV light.
The preS1/preS2 gene was detected using PCR primers P1 (5'-TCACCATATTCTTGGGAACAAGA-3', nt 28172839) and P2 (5'-TTCCTGAACTGGAGCCACCA-3', nt 8061). The 478 bp PCR product was used as template for RFLP analysis and DNA sequencing. The X gene was amplified using primers Xo1 (5'-CTCTGCCGATCCATACTGC-3', nt 12561274) and PC1 (5'-GGAAAGAAGTCAGAAGGC-3', nt 19741957). Amplified products were used for precore mutant detection of gibbon HBV.
Sera of gibbon HBV carriers and HBsAg-negative animals were analysed for alanine aminotransferase (ALT) at the Central Laboratory of the Chulalongkorn University and Hospital (Bangkok, Thailand) using an automated analyser (Hitachi 912). Data were expressed as mean±SD. The normal range of ALT is 038 U l-1. Student's t-test was used to test statistical differences between groups.
Identification of gibbon HBV genotypes by RFLP.
Two restriction enzymes, AvaII and DpnII (New England Biolabs), were used for digestion of the preS1/preS2 PCR products. The RFLP patterns obtained were compared with the restriction endonuclease analysis reference profiles for human HBV genotype classification as described previously (Lindh et al., 1998). Samples with different RFLP patterns were classified by direct sequencing.
The 720 bp PCR product of the X gene was digested with Sau3A I (New England Biolabs) for precore promoter mutant gene detection (nt 1762 and 1764), as reported previously (Takahashi et al., 1995).
Gibbon HBV sequencing and phylogenetic analysis.
HBV genes amplified by PCR were sequenced using the primer pairs P1 and P2, F2 and R5, and Xo1 and PC1 for the preS1/preS2, S and core region, respectively. Sequencing reactions were performed using the commercially available PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems). The sequence products were electrophoresed on a Perkin Elmer 310 sequencer (PE Biosystems). Each sample was sequenced in both directions and nucleotide sequences were edited and assembled using SEQMAN (LASERGENE program package, DNASTAR) and submitted to GenBank.
PreS1, S and core genes of two HBV carrier families were aligned using BIOEDIT, the sequence alignment editor (version 5.0.9), while other gibbon HBV strains were used as a control. For phylogenetic analysis, S nucleotide sequences of 19 HBV carrier gibbons and two animal caretakers were multiply aligned using the CLUSTAL X program. Genetic distances were calculated using the Kimura two-parameter method within DNADIST of PHYLIP, version 3.6 (J. Felsenstein, Department of Genetics, University of Washington, USA) and the result was illustrated graphically as a neighbour-joining tree. Bootstrap values representing 1000 replicates (100 multiple data sets) were determined using the SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs from the PHYLIP package. The TREEVIEW program (version 1.5) was run for unrooted phylogenetic construction.
To analyse the relationship between HBV isolates from animals in cage C, the best phylogenetic tree of the complete S gene was generated with the maximum-likelihood algorithms within the DNAML program using 1000 replicates of bootstrap values. The transition/transversion ratio was 2·0.
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Results |
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Molecular characterization of gibbon HBV
HBV DNA isolated from 19 positive animals was used as target for DNA sequencing and RFLP analysis. The absence of the 33 bp after the preS1 start codon of gibbon HBV was confirmed. Grouping of gibbon HBV was performed by phylogenetic tree analysis; S phylogram analysis, including human genotype AG, orang-utan, chimpanzee and woolly monkey, revealed the separate clustering of these gibbon HBVs (Fig. 1). Most animals from the three different cages cluster into the three separate groups. Animals in the closed cage, such as area C, were infected with a closely related strain of HBV since these viruses shared the root of the phylogenetic tree. HBV from two chronically infected mothers (Jieb and Ni) was more closely related to their infants. The R6 family showed a higher internal edge value at the node of the phylogram than the C15 family.
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A total of 34 animal caretakers of the Krabok Koo Centre were screened during part of a prevaccination programme and we found that 5·9 % (2 of 34) were positive for HBsAg and HBV DNA. Subsequently, gibbon and human HBV were identified by RFLP using AvaII/DpnII digestion of preS1/preS2 PCR products, as indicated in Fig. 4(b, c), respectively. Shown are representative digestion patterns observed in samples G1G4 and G6, gibbon virus strains spreading in the Krabok Koo Centre; the G5 strain is similar to G3. Two workers, H1 and H2, found to be HBs DNA positive might have been exposed to gibbon viruses. Patterns of restriction enzyme digestion of gibbon HBV (G1G6), however, showed different profiles as compared to human HBV (H1H2). The 33 bp deletion in the preS1 region caused the different RFLP patterns, which cannot be grouped to any human virus genotype, including genotype D viruses, which are reported to have a preS1 deletion in the same region. These results were confirmed by sequencing and phylogenetic analysis of the S gene (Fig. 1
).
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Discussion |
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Serological analysis of HBV infection in the gibbons kept at the Krabok Koo Centre showed that approximately 40 % of the animals were infected with HBV; 19 animals were HBV DNA-positive carriers and could be a source of virus spread in the gibbon population. Similar figures have been reported recently in captive gibbons housed in the Centre for Gibbon Studies in California, USA, by Lanford et al. (2000). On the other hand, Grethe et al. (2000)
reported even higher frequencies in animals housed at different zoos. Similar to the first report on gibbon HBV (Mimms et al., 1993
), four of the chronically infected gibbons were negative for anti-HBc, even when tested repeatedly with different EIA tests. The specific immune response against core may be undetectable due to the lack of specific anti-gibbon IgG reagents.
Precore promoter mutation AGGTGA at nt 1762 and 1764, linked previously to a relatively moderate downregulation of the synthesis of HBeAg (Buckwold et al., 1996
; Moriyama et al., 1996
) but also observed in the HBeAg-positive stage (Takahashi et al., 1995
), has been associated with more severe liver damage (Lindh et al., 1999
) and increased responses to interferon (Kanai et al., 1996
). In gibbon HBV carriers, this mutation was detected in four HBeAg-positive animals, including Pok C2, Midnight R27, Nin L14 (all anti-core positive) and Caesar L10 (anti-core negative). Unfortunately, core gene sequencing and ALT status in these gibbons could not be analysed in this study due to the limitation of samples.
Molecular characterization by RFLP and phylogenetic analyses confirmed the separate clustering of gibbon HBV compared to human and nonhuman primate HBV. The three different clusters within the gibbon viruses observed could relate to genomic variants as described by Grethe et al. (2000). Interestingly, animals in the C and L cage areas shared the same branches of each group, suggesting the possibility of brother-and-sister relationships of these animals or horizontal transmission by infectious viruses in saliva during fights or feeding at an early age.
The recent finding that chimpanzees as well as orang-utans can be infected with HBV in the wild makes any proposed spread of HBV from the New World extremely unlikely (Hu et al., 2000; MacDonald et al., 2000
; Takahashi et al., 2000
; Warren et al., 1999
). In contrast, all data support the idea that these viruses are indigenous to the different nonhuman primate populations. The geographical distribution of human genotype C viruses, the orang-utan HBV and gibbon HBV, all found in Southeast Asia, could indicate that spread through cross-species transmission in Southeast Asia may have occurred (Simmonds, 2001
). However, gibbon and orang-utan HBV are more closely related to each other than to the human genotypes. Thus, more sequence data on different isolates are needed as well as possible evidence for transmission of nonhuman primate HBV to humans.
HBV is transmitted by sexual contact and parental exposure, although it is thought that mother-to-child prenatal transmission is responsible for high rates endemically in several regions of the world; 2550 % of the HBV chronic carriers result through vertical transmission or horizontal transmission by nosocomial exposure in early childhood (Mast et al., 1999; Mahoney, 1999
; Wang & Zhu, 2000
). In gibbons, vertical transmission was supported by 99.5 % identity of the S gene of HBV isolated from mother and baby gibbons. Hence, base sequence changing may have occurred through mutations in time; for example, Baby R6, younger than Tao at the date of blood sampling, showed higher sequence similarity of the preS1 region (Fig. 2
). Unexpectedly, HBV isolated from Nongchai (C16 cage) showed a high percentage of sequence similarity to Ni and Tao (cage C15), possibly due to horizontal transmission. However, HBsAg isolated from Ni and Tao were more closely related, as determined by maximum-likelihood phylogram analysis. Noticeably, all animals that share cages with gibbon carriers were infected with HBV and anti-core antibodies were detected (Table 2
). These data suggest that sexual contact or horizontal HBV transmission of family members could be a source of virus spread in this gibbon population.
Similar to observations made in human HBV (Noppornpanth et al., 2000), HBsAg could be detected in the saliva of all HBV carriers. Until now, few cases have been reported on HBV infection from saliva by a human bite (MacQuarrie et al., 1974
; Stornello, 1991
) but the high prevalence of HBV among dentist personnel and family members suggests that HBV might spread by saliva (Heathcote et al., 1974
). However, no report of HBV transmission from captive animals to humans currently exists. In the present study, HBV DNA was detected in the saliva of six gibbons, representing circulating HBV infectious particles in body fluids (Ljunggren et al., 1993
). HBV-positive human saliva administered intradermally, but not orally, to gibbons caused HBV transmission (Scott et al., 1980
). Thus, HBV DNA detected in gibbon saliva may indicate a potential risk for horizontal transmission, especially if the animals are injured after fighting or through accidental injury of animal caretakers. Two workers of the Kabok Koo Centre were found to be positive for HBsAg and HBV DNA, which corresponds to seroprevalence of HBV in the general adult Thai population (Tandon & Tandon, 1997
). RFLP and phylogenetic analyses of the S gene from both isolates indicated that they were infected by human HBV.
The high prevalence of HBV infection in captive gibbons of the Krabok Koo Centre shows that HBV is an important infectious agent in captive gibbons. Since chronic carrier gibbons are the main source of HBV in the Centre, separating them and vaccination of newborn babies should be done to reduce the number of infected animals. Although transmission of HBV between nonhuman primates and humans, similar to zoonotic human immunodeficiency virus transmission (Hahn et al., 2000), could not be confirmed, these studies could provide more insight in the molecular evolution and transmission routes of HBV and facilitate our understanding of the origin of HBV and its pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 22 April 2002;
accepted 17 September 2002.