|dc.description.abstract||This study details the search for, and characterisation of, retroviruses in a marsupial, the common brush-tailed possum (Trichosurus vulpecula). Initial efforts were directed at detecting exogenous retroviruses in possums, but the majority of the work described in this thesis involved the isolation and characterisation of a possum endogenous retrovirus.
Endogenous retroviruses were detected in possum genomic DNA by PCR amplification using degenerate primers derived from the retroviral pol gene. Cloning, sequencing, and analysis of these PCR products revealed the presence of several families of endogenous retroviruses in the possum genome.
Reverse transcriptase activity was detected in the blood of all possums tested using the product enhanced reverse transcriptase (PERT) assay. RT-PCR was performed on RNA isolated from possum blood plasma using the pol-derived degenerate primers. Cloning and sequencing of the products indicated that a homogeneous retroviral RNA species was present in the blood of possums, and that this RNA was related to, but not identical to, the endogenous possum retroviruses already detected.
A 3'-RACE approach was used to amplify the majority of the 3' end of the possum retroviral RNA. The subsequent discovery that this RNA species was derived from an endogenous retrovirus in the possum genome allowed amplification of the remainder of the genome by a combination of PCR, single primer PCR, and RT-PCR. The sequences were assembled into a contiguous sequence, the TvERV-K1 contig. In addition, a near-full-length TvERV-K1-related fragment, was amplified from possum genomic DNA, cloned, and sequenced. It was named pTvERV-K2. The TvERV-K elements are the first full-length marsupial retrovirus sequences to be reported.
Analysis of the sequences of the TvERV-K1 contig and pTvERV-K2 revealed most of the regulatory regions required for replication of a retroviral genome, as well as uninterrupted, or minimally interrupted, open reading frames (ORFs) for the gag, pro, and pol genes. Only a short region of sequence with homology to the Env proteins of other retroviruses was detected. All of the TvERV-K proteins displayed highest homology to those of the simian type D retroviruses. Likewise, phylogenetic analysis using the deduced amino acid sequences of the Pro and Pol proteins, placed the possum endogenous retrovirus with the exogenous and endogenous type D retroviruses of Old World and New World monkeys. Thus, a cross-species transmission event - from marsupials to primates, from primates to marsupials, or from an unidentified source to both marsupials and primates - appears to have occurred.
There are 15-20 copies of the TvERV-K element in the possum genome, as determined by Southern hybridisation. However, integration sites appeared to be variable between possums, suggesting recent (or ongoing) retrotranspositional activity. Both PCR and Southern hybridisation analyses suggest that TvERV-K elements shorter than those from which the TvERV-K1 contig and pTvERV-K2 were derived are present in the possum genome.
The implications of these findings are discussed.||