From the data presented, it is clear that a provirus (KoRV) related to the simian type C retroviruses is present in the DNA of koalas. The observation that retrovirus-like particles were produced by both tumor cells in vivo and cultured PBMCs in vitro suggests that proviral transcription and virus assembly occur. This is supported by the detection of retroviral transcripts homologous to proviral sequences in the blood of koalas and the detection of viral RNA and RT activity in serum and in PBMC culture supernatants.
Provirus was detected in all koalas and in all tissues by PCR, and virus-like particles were demonstrated by TEM in 98% of mitogen-stimulated PBMC cultures. Southern hybridization of genomic DNA demonstrated similar intensity and banding patterns across a range of tissues in koalas with and without lymphoid neoplasia. These data suggest that KoRV is an ERV, although exogenous forms may also exist. Full-length provirus sequences were detected and contained all of the basic genetic elements common to type C retroviruses (44
), namely, flanking LTRs; gag
, and env
genes; a tRNA primer binding site; a polypurine tract; a CAAT box; a TATA box; a Cys-His box; and a polyadenylation signal. The sequences showed the most similarity to the GALV-related simian retroviruses and also showed ORFs and spliced env
transcripts consistent with the type C mammalian group of retroviruses.
While full-length provirus is present in koalas, there is also strong evidence for the presence of truncated proviruses. Undersized PCR products derived from primers that span the complete proviral genome were detected in all animals, although the molecular size ranges of these products differed between animals. These amplimers hybridized to labelled KoRV pro-pol
, and partial sequencing confirmed their KoRV derivation, suggesting that they represent truncated proviruses. Tissue-, animal-, or population-specific truncated KoRV variants may have arisen in the koala genome either via recombination excision events or novel superinfection or reinsertion events. Considering the marked variation between koalas in the lengths of apparently truncated proviruses detected by PCR, it appears that either KoRV is not stable, in contrast to most other endogenous viruses (3
), or PCR detection is confounded by the presence of related exogenous virus.
Detection of some viral transcripts containing gag
sequences and failure to detect viral transcripts containing pro-pol
sequences by RT-PCR of serum and cell-free culture supernatants from some koalas may be due to transcription and expression of truncated proviruses and assembly of virions containing defective genomic RNA. In humans, there are numerous examples of expression of defective or truncated endogenous proviruses in both normal and transformed tissues (1
). Furthermore, the expression of the retroviral Gag protein alone may be sufficient to cause assembly and budding of virus-like particles from the cell (58
). Clearly, in the koala genome, there are multiple copies of proviruses, including both full-length and truncated proviruses, at least some of which are expressed both in vivo and in vitro.
The inability to detect strong RT activity in the serum or PBMC culture supernatants of some koalas may be explained by a number of possibilities. First, inhibition of RT activity by tissue-derived specific inhibitors has been demonstrated with some human ERVs (59
). A second possibility is that virions may not contain detectable RT because of a defective or absent pro-pol
gene. This is consistent with the observed expression of defective or truncated proviruses and has been previously demonstrated for HERV-K, which encodes a functional enzyme with weak activity (57
). A third possibility is that assay conditions may not have been optimal for the detection of KoRV RT activity.
The DNA sequence data from gag
, and env
unambiguously support a grouping of KoRV and GALV, to the exclusion of PERV and the MLV group. A similar grouping was also obtained from an independent phylogenetic study that compared pol
fragments derived from a wide range of host taxa (37
). This is an intriguing grouping, because gibbons (Hylobates
spp.) and koalas are taxonomically distant, making virus acquisition from a common ancestor unlikely and because gibbons and koalas are from remote biogeographic regions, making direct natural transmission improbable. A preliminary investigation of small numbers of animals failed to detect KoRV-like retroviruses in other marsupials, including an eastern grey kangaroo (Macropus giganteus
) and common wombats (Vombatus ursinus
) and southern hairy-nosed wombats (Lasiorhinus latifrons
) (data not shown). Wombats are the closest living relatives of koalas, although still relatively distantly related. Other studies have also failed to demonstrate KoRV-like viruses in marsupials other than koalas (23
). This suggests that endogenization of KoRV in koalas postdates the divergence of koalas from other marsupials.
There are two possible explanations for the close relationship between KoRV and GALV. Either KoRV and GALV share a more recent common ancestor than all other ERVs sequenced to date, and therefore the grouping reflects the true evolutionary history of these sequences, or the grouping is an artifact of sequence analysis. The potential causes of spurious phylogenetic signal include recombination between strains, base composition bias, sequence saturation, and molecular convergence. Each of these artifacts can be discounted on the basis of detailed DNA sequence analysis, suggesting that the KoRV-GALV grouping represents a true biological relationship.
Recombination between virus lineages can cause unusual phylogenetic groupings, because different regions of the genome have different phylogenetic histories. However, there is no evidence that recombination is causing the KoRV-GALV grouping, because all viral genes gave the same phylogenetic signal. There is little evidence of saturation (true signal obscured by multiple hits) in the DNA sequences (Fig. ). Nor is there any evidence for base composition bias pulling KoRV and GALV together, because the base composition of these sequences does not differ noticeably from the base compositions of the Friend MLV, MMLV, and PERV sequences. The KoRV-GALV grouping is unlikely to be caused by molecular convergence, which would require independent acquisition of many identical substitutions in both KoRV and GALV lineages and would expect to result in a strong conflicting signal in the data set. However, spectral analysis shows little conflicting signal (Fig. ). Strong support for the KoRV-GALV grouping from both the 1st and 2nd codon positions, which predominantly cause nonsynonymous changes, and 3rd codon positions, which are mostly “silent,” provides further evidence against molecular convergence, since a convergent signal would be expected only in the nonsynonymous changes.
Some ERV phylogenies closely match the phylogeny of their hosts, indicating that the viruses are evolving as an endogenous element of the host genome, rarely infecting other hosts. Examples include the MLV-related retroviruses in reptiles and amphibians (23
) and some ERVs of great apes (27
). Because the phylogeny does not match the order of branching of the mammalian tree (37
) (Fig. ), KoRV and GALV could only be considered to be strictly host tracking if the KoRV-GALV ancestor represents a novel insertion into the ancestral mammalian genome independently of the other mammalian ERVs sequenced to date. The similarity of KoRV and GALV to FeLV, the MLVs, and PERV would then be due to the similarity of the free-living viruses that inserted into the mammalian genome multiple times at least 130 million years ago, at the time of metatherian and eutherian mammal divergence (26
). This could be confirmed by identifying the host genome insertion points for these viruses: if insertion is random, then independent acquisitions of ERVs will occur at different loci (27
). However, the depth of molecular divergence in the KoRV-GALV clade is less than half that observed in the MLV clade, so the host-tracking hypothesis would require that the rate of molecular evolution in the KoRV-GALV lineage was dramatically slower than that of the MLV genomes. Given that GALV is exogenous, it seems highly unlikely that its rate of molecular evolution would have decreased to such an extent. Indeed, it would be expected that the rate of molecular evolution in an actively infectious virus would be greater than that of an endogenous virus (49
). A host-tracking explanation for the KoRV-GALV grouping is therefore inconsistent with the sequence data.
There seems to be no reason to cast doubt on the phylogenetic relationship between KoRV and GALV to the exclusion of other retroviruses sequenced to date. There are two possible explanations for the relationship: inheritance from the genome of the last common ancestor of gibbons and koalas or cross-species transmission. The former explanation is unlikely, given the DNA sequence data and the preliminary assessment that KoRV-like viruses are absent in other marsupials. It seems most likely that the KoRV-GALV grouping is the result of a relatively recent cross-species transmission of a KoRV-like virus into gibbon and/or koala populations. Because gibbons and koalas are from distinct biogeographical regions with very different faunas, direct cross-infection in the wild is improbable. However, natural transfer via an intermediate host, for example, a mobile species such as rodents, bats, or birds or an arthropod vector, cannot currently be ruled out. A scenario suggested by Martin and coworkers (37
) is that transmission was dependent upon an intermediate host, possibly a species of Asian rodent in which ERVs related to the primate type C viruses have been detected (6
KoRV has been isolated from both captive and wild koalas, but to our knowledge, GALV has been detected only in captive gibbons. This observation suggests that iatrogenic infection of captive gibbons with a KoRV-like virus derived either from live captive koalas, from tissue or fomites, is also a possible mechanism of recent cross-species transfer. If this observation holds true, it would imply that GALV infection in gibbons is a recent artifact of captivity. Alternatively species mixing and opportunities for either direct or indirect cross-infection between koalas and gibbons may have been promoted by indigenous trade between southeast Asia and mainland Australia in precolonial times. This mechanism has been proposed as a transmission method of an Australian marsupial arthropod parasite, Heterodoxus spiniger
, to Asian dogs (16
Whether iatrogenic or natural transmission has occurred, pinpointing the date of such a host jump through “molecular clock” dating may help to resolve the path of cross-species infection. However, dating virus divergence times from molecular data is made difficult by lack of a suitable calibration rate. There are no currently known divergence dates that can be used to calibrate the rate of change in KoRV or GALV. Given that substitution rates can vary enormously for different virus lineages (49
) and between genes within a lineage (32
), it is prudent to be wary of assuming a molecular clock to date virus divergences. However, the relatively low degree of divergence between the env
genes of KoRV and the various GALV strains supports a recent divergence. In view of the apparently widespread endogenous infection of koalas and the suggestion that the exogenous GALV viruses are relatively recently acquired by simians (33
), the possibility remains that the GALV group originated in koalas and spread to gibbons by a yet to be discovered mechanism. The parallel finding that macropodid herpesviruses paradoxically cluster with the simian herpesviruses (36
) also supports the possibility of an occult epidemiological link between Australian marsupials and old-world primates.
Lymphoid malignancy is a relatively common disease of both free-living and captive koalas (11
), and its clinical similarity to retrovirus-associated disease in other mammals has been noted (7
). The ability of GALV and SSV to cause neoplasia in their host species has been demonstrated in both natural (28
) and experimental infections (12
). In view of the relatively high prevalence of hematopoietic neoplasia and related diseases in koalas (4
) and anecdotal evidence for lymphoid neoplasia epizootics in captive koala colonies, it is reasonable to suspect a pathogenic role for KoRV. However, KoRV was detected in all diseased and healthy koalas tested and appears to be endogenous. Although this lack of clinical correlation does not rule out a pathogenic role for the virus, it implies that other factors are necessary for disease to occur. Potential cofactors include superinfection with KoRV exogenous pathogenic variants, recombination with other retroviruses, interactions with host haplotypes, interactions with somatic mutations, or coinfection with other viruses, such as herpesviruses.
Serious consideration should be given to the zoonotic potential of the GALV-KoRV group. Early experimental work on GALV and more recent investigation of GALV as a vector for gene therapy have demonstrated multiple host and tissue tropisms (17
). The broad in vitro host range of GALV, its apparently well-conserved receptor (42
), and the evidence presented for taxonomic cross-transmission in vivo suggest that the members of the GALV-KoRV group have the potential for future host jumping. Furthermore, simian type C-related virus sequences and cross-reactive proteins have been detected in humans (14
), and a novel strain of GALV (GALVX) has been isolated from a human cell line (5
). Continuing studies are aimed at further defining the epizootiology of the KoRV-GALV group and at clarifying the pathogenic and zoonotic potential of this virus.