Each of 4 experimentally naive, domestically raised baboons was injected with serum from 1 of 4 wild-caught HBV DNA–positive baboons. Serum samples from the experimentally naive baboons were HBV DNA negative before injection, and baseline levels of aspartate aminotransferase, alanine aminotransferase, and IgG were also determined. The baboons were transiently positive for several of these markers and were intermittently positive for HBV DNA in serum. Results for a representative sample (baboon 2) infected with serum from baboon 9732 are shown in Figure 5. DNA extracted from liver tissue obtained at necropsy from baboon 2 was used to amplify and sequence a portion of the surface gene (nt 409–1101). The sequence of virus extracted from liver tissue of baboon 2 six months after injection was identical to the HBV sequence of this region found in original baboon 9732.
Discussion
Detection of HBV DNA in serum samples of 2 Chacma baboons before injection with human HBV (16) suggested that baboons might be chronically infected with HBV. Our objective was to determine the prevalence of HBV in wild-caught Chacma baboons and to characterize the virus isolated from these animals. The overall prevalence (21.7%) of HBV in the baboons is similar to the HBV prevalence in other nonhuman primates in areas to which HBV is highly endemic, including sub-Saharan Africa (25).
The baboons were serologically negative, and the 1 baboon examined histologically had mild focal lobular hepatitis, and HBcAg was detected in liver tissue by immunohistochemical staining. Detection of low levels of HBV DNA in baboon liver tissue and in serum in the absence of HBsAg and at relatively low levels of HBV DNA classifies this infection as occult (26). Lack of any serologic markers of HBV infection further distinguishes this infection as a seronegative occult HBV infection (26). This finding is analogous to a secondary occult infection in the woodchuck (27). Furthermore, HBV DNA was detected in juvenile and adult baboons, suggesting lifelong persistence of the virus, which was successfully transmitted to experimentally naive baboons. Detection of HBV DNA alone does not necessarily correspond to an HBV infection (28). Thus, detection of cccDNA and viral RNA in liver tissue of baboon 9732 showed that HBV was replicating. The low levels of viral nucleic acids detected in baboon liver are a further characteristic of occult infections (27). This study demonstrates a naturally occurring occult HBV infection in a nonhuman primate.
Phylogenetic analysis of the baboon HBV genome showed that it belonged to genotype A, clustering with subgenotype A2. This finding is an unexpected result because subgenotype A1 predominates in South Africa (24). However, in the basal core promoter/precore region, the baboon HBV had distinct characteristics of subgenotype A1. Four additional mutations in the polymerase, surface, X, and core regions of the baboon HBV strain differentiated the baboon isolate from most subgenotype A2 isolates. These mutations are not known to cause any major functional or conformational changes.
Paradoxical identification of subgenotype A2 in the baboon when subgenotype A1 predominates in Africa might indicate that subgenotype A2 is an older strain that previously circulated in Africa, which has been replaced by other strains, including subgenotype A1 and genotypes D and E (29). An analogous trend might have occurred in the Mediterranean region where genotype D now predominates over genotype A (30). Similarly, a change in the prevalent HBV genotype in central and western Africa has been postulated to have occurred over the past 200 years, and genotype E, originally restricted to the west coast of Africa, now spreads over a large crescent stretching from The Gambia, through Nigeria and the Democratic Republic of the Congo into Namibia and Mozambique (31). There is a paucity of sequencing data for subgenotype A2 from Africa. Only 4 complete subgenotype A2 genomes from South Africa have been deposited in GenBank. Shorter subgenomic sequences of subgenotype A2 from South Africa (32), Tunisia (33), and Kenya (34) have also been deposited. More extensive molecular epidemiologic studies in Africa might uncover a higher circulation of subgenotype A2 in more remote regions.
Another explanation for the paradoxical finding of subgenotype A2 in the baboon could be that in Africa, as shown in India, subgenotype A2 isolates are confined to peripheral blood leukocytes (PBLs). In India, subgenotype A1 and genotype D circulate, but actively replicating HBV subgenotype A2 isolates have been detected in PBLs (35). These subgenotype A2 isolates were confined to PBLs and were not detected in serum. Differential immune pressures are believed to compartmentalize HBV to different parts of the body in which different strains evolve independently (35). Further study will be needed to determine whether subgenotype A2 is compartmentalized to PBLs in Africa.
In sub-Saharan Africa, most human HBV carriers are negative for HBV e antigen, and in these persons, spread of HBV is mainly horizontal (36). In baboons, their natural habits indicate that horizontal baboon-to-baboon transmission of HBV is highly probable. Baboons are social animals, and bonds are strengthened by daily grooming with several related and unrelated partners, including offspring (37). Young baboons, like their human counterparts, spend much of the day playing together. The games can become quite physical, often leading to mock fights. Physical fights among male and female adult baboons are rare. Alpha males tend to be aggressive, and displays of dominance and chases occur daily.
The well-documented interactions between humans and baboons make cross-species transmission of this virus extremely plausible (37). Baboons are the most widely distributed nonhuman primates in Africa and are found in virtually all parts of sub-Saharan Africa. They often appear in ancient Egyptian mythology and art, depicted as captives brought from southern Africa or as pets on leashes (37). In southern Africa, baboons have been kept as pets and trained to work as oxcart drivers, railway laborers, and as goat herders on farms (37). However, more common are reports of conflict between humans and baboons. In the rural areas, baboons raid orchards, destroy irrigation pipes, and kill sheep and goats. Because baboons can become aggressive when challenged, they are often killed by farmers for being pests. Slaughter of baboons for bush meat could be another source of exposure to the virus (38).
From the results of the present study, it is impossible to determine whether baboons were infected with HBV by humans, as has been hypothesized (39), or whether humans were infected by baboons. However, as noted by Michael Lai, a virus expert, “When we expose ourselves to exotic animals, there is always a risk of being exposed to something unknown…When we perturb the existing peace between human beings and nature, we are opening a Pandora’s box, which may contain surprises” (40).
Dr Dickens is a researcher in the Department of Internal Medicine, University of the Witwatersrand, Johannesburg, South Africa. Her main research interests include molecular virology and cancer.
Acknowledgments
We thank C. Brechot for providing the HBV DNA probe and Allan Paterson for performing serologic and immunohistochemical analysis.
M.C.K. was supported by a grant (NRF2K421) from the National Research Foundation, South Africa. A.K. was supported by a grant (00/52) from the Polio Research Foundation, South Africa. C.D. was supported by bursaries from the National Research Foundation and the Polio Research Foundation. R.H.P. was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.