Members of the family Hepadnaviridae have been detected in both avian and
mammalian species. They have a very limited host range, and among the nonhuman
primates, have been found to occur naturally in chimpanzees, gorillas,
gibbons, orang-utans and woolly monkeys. The human hepatitis B virus (HBV)
has been shown to infect chimpanzees, Barbary macaques and tree shrews.
During the course of a previous study, to determine the susceptibility of baboons
(Papio ursinus orientalis) to HBV infection, HBV DNA was detected in the
serum of 2 baboons prior to their inoculation with HBV-positive human serum,
raising the possibility that baboons are naturally infected with a hepadnavirus.
Therefore the aim of this study was to determine the prevalence of HBV in wildcaught
baboons and to molecularly and functionally characterise the virus isolated
from these baboons.
DNA was extracted from the sera of wild-caught baboons and four separate
regions of the HBV genome amplified by nested polymerase chain reaction
(PCR). Samples were only considered to be positive for HBV if at least three of
these regions amplified. DNA was extracted from the liver tissue of one of the
HBV DNA-positive baboons using a proteinase K digestion followed by a phenolchloroform
extraction and ethanol precipitation. From this extract, the complete
HBV genome was amplified by nested PCR of eight overlapping subgenomic
fragments, and sequenced. This sequence was analysed phylogenetically using
both the PHYLIP and Simmonic software packages. A selective real time PCR
using SYBR®-green detection was used to detect covalently closed circular (ccc)
DNA. RNA was extracted from the baboon liver tissue using a guanidinium-acidphenol
extraction method, reverse transcribed and portions of the HBV genome
amplified by nested PCR. Transmissibility of the virus was tested by injecting
four experimentally naïve baboons individually with serum from four HBV DNApositive
baboons and followed for 26 weeks.
HBV was detected in the serum of 5/69 (7,2%) wild-caught baboons by Southern
hybridization and in 11/49 (22,4%) adult and 4/20 (20,0%) juvenile wild caught baboons. This gave an overall prevalence of 21,7% in the baboon population
surveyed. Serologically, the baboon sera were negative for all markers of HBV
infection and alanine aminotransferse (ALT) levels were normal. In the one
baboon liver tissue available, HBcAg was detected by immunohistochemical
staining in some of the hepatocyte nuclei, but HBsAg was not detected.
Phylogenetic analysis of the complete genome of the HBV isolate found it to
cluster with subgenotype A2, a surprising result considering that subgenotype A1
predominates in South Africa. However, unlike other subgenotype A2 isolates,
the basic core promoter had the G1809T / C1812T double mutation characteristic
of subgenotypes A1 and A3 and the precore region had the G1888A mutation
unique to subgenotype A1. These mutations in the basic core promoter and precore
regions have previously been shown to reduce the expression of the precore
and core proteins. Four additional mutations in the polymerase, surface, X and
core open reading frames (ORFs) further differentiated the baboon HBV strain
form the majority of previously sequenced subgenotype A2 isolates.
cccDNA was detected at low levels in the baboon liver tissue. Regions of the
precore/core and surface ORFs were amplified off reverse transcribed cDNA.
These results demonstrate HBV replication in the baboon liver. Transmission of
the virus was shown by the detection of HBV DNA in the sera of the four
inoculated baboons at various times throughout the 26 week follow-up period.
These baboons also showed transient seroconversion for HBsAg and HBeAg
during this period with intermittent fluctuations in ALT levels. Moreover, using
DNA extracted from liver tissue obtained at necropsy from one of the injected
baboons, the sequence of the HBV surface gene amplified was found to be
identical to the sequence of the isolate from inoculum.
The finding of subgenotype A2 in the baboon is paradoxical because
subgenotypes A1 and A3 as well as genotypes D and E predominate in Africa.
The possibility exists that subgenotype A2 is an older strain that has been
overtaken by these other strains. There is however a scarcity of subgenotype A2
sequencing data from Africa and a higher circulation of this subgenotype could be
uncovered with more extensive molecular epidemiological studies in more remote areas. Alternatively, a recent discovery of alternative compartmentalization of
subgenotype A2 infections in the peripheral blood lymphocyte population of
individuals from India, where subgenotype A1 also predominates, could explain
the lack of detection of this subgenotype in Africa.
Occult hepatitis B infection is defined as the presence of HBV DNA in the liver
(with detectable or undetectable HBV DNA in the serum) of individuals testing
negative for HBsAg by currently available assays. The detection of HBV DNA in
the baboon liver and serum in the absence of serological markers therefore
classifies this infection as occult. To our knowledge, this is the first study to
demonstrate a naturally occurring occult HBV infection in non-human primates.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/10848 |
| Date | 23 November 2011 |
| Creators | Dickens, Caroline |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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