EPIDEMIOLOGY
Our understanding of the epidemiology of herpesvirus infections in Australian marsupials has been informed largely through sero-epidemiological studies and through more recent studies that have used PCR techniques to detect herpesvirus DNA.
Serological techniques detect both latent and lytic herpesvirus infections and do not discriminate between these different stages of infection. Serological studies are currently restricted to the detection of serum antibodies against the macropo- did alphaherpesviruses that have been successfully isolated and can be readily propagated in cell culture. Some serological cross-reactivity between these viruses in serum-virus neutralisation assays has been demonstrated and it is likely that these assays are also able to detect antibodies to other related alphaherpesviruses that have not yet been characterised (Vaz et al. 2013). In contrast, PCR-based epidemiological studies use primers that target a conserved region of the herpesvirus genome and thus can detect all viruses within the Herpesviridae. These PCR-based studies are generally used to detect lytic infections, although they can be used to detect latent virus; for example, virus within latently infectedTable 23.1. Typical characteristics of viruses in each of the three Herpesviridae Subfamilies (source: Pellett and Roizman 2013)
| Subfamily | Host range | Sites of latency | Replication |
| Alphaherpesvirinae | Variable | Sensory ganglia | Short reproductive cycle; rapid spread in cell culture; efficient destruction of infected cells |
| Betaherpesvirinae | Restricted | Secretory glands, lymphoreticular cells, kidneys, other tissues | Long reproductive cycle; slow progression of infection in cell culture; infected cells frequently become enlarged (cytomegalia) |
Gammaherpesvirinae Restricted Lymphoid cells In vitro replication in lymphoblastoid cells; lytic
infection of specific types of epithelioid and fibroblastic cells; viruses usually specific for either T or B lymphocytes
lymphocytes that may be present in blood or tissues (Stalder et al.
2015). Because of the latency characteristics of herpesviruses, animals that test positive for either latent or lytic herpesvirus infections should be assumed to have lifelong infections.An early serological study demonstrated a high prevalence of antibodies to macropodid herpesvirus 1 (MaHV- 1) in marsupials and monotremes from different locations in Australia. The prevalence was higher in managed animals (41%) compared with free-ranging animals (23%). Most of the animals examined were different species of macropods, but non-macropods and the two monotreme species were also examined. Of these, bandicoots (Isoodon spp.), the short-beaked echidna (Tachyglossus aculeatus), Antechinus spp. and Dasycercus spp. were positive for the presence of neutralising antibodies to MaHV-1 (Webber and Whalley 1978), although cross-reactivity with other related but uncharacterised herpesviruses was also a possibility. A follow-up study showed that the prevalence of neutralising antibodies was particularly high (69%) in free-ranging eastern (Macropus giganteus) and western (M. fuliginosus) grey kangaroos in Vic. and increased with age, reaching around 90% prevalence of neutralising antibodies among older animals. These findings are consistent with these animals being the natural host species of the virus to which antibodies were detected (this could be MaHV-1, or a closely related virus) and a source of infection for other marsupial species (Kerr et al. 1981). A more recent serological survey of mostly free-ranging marsupials in Vic., spanning five different Families, has confirmed these earlier studies and demonstrated a high overall prevalence (40%) of neutralising antibodies to MaHV-1 and/or MaHV-2. In eastern grey kangaroos the prevalence of neutralising antibodies was even higher (92%). This same study identified a high prevalence (67%) of neutralising antibodies in free-ranging bare-nosed wombats (Vombatus ursinus) (Stalder et al. 2015). Virus neutralising antibodies to MaHV-1 and MaHV-2 have also been identified in managed Lumholtz’s tree-kangaroos (Dendrolagus lumholtzi) and quokkas (Setonix brachyurus) during clinical investigations into sudden death of animals held within wildlife parks (Callinan and Kefford 1981; Shima et al.
2020). This raises concern for inter-specific transmission of alphaherpesviruses from other macropod species in managed environments and highlights the need for improved quarantine and other biosecurity measures. The biosecurity risk of introducing alphaherpesviruses into naive free-living populations should also be considered in planning managed carerelease programs (Shima et al. 2020).The largest study to utilise PCR-based techniques to detect herpesvirus DNA in Australian marsupials examined 397 animals (309 free-ranging animals and 88 animals in managed care) across eight different Families (Stalder et al. 2015). The overall prevalence of infection was 25%, with a high prevalence detected in eastern grey kangaroos (25%), koalas (Phascolarctos cinereus) (33%), Tasmanian devils (Sarcophilus harrisii) (34%) and barenosed wombats (45%). Overall, infection was significantly higher in males, in animals with a lower body condition score and in animals sampled during the summer months. The sex difference could potentially be explained by behavioural characteristics of males (fighting, promiscuous mating, greater dispersion distances) allowing more opportunities for infection. The lower body condition could potentially be from herpesvirus infection causing disease, but is more likely to be associated with an increased risk of new or reactivated infections in animals suffering ill thrift from other causes, including concurrent infections. The higher prevalence in summer months could be from increased activity, such as breeding or fighting in spring and summer, dispersal of adolescents from the previous breeding year and displacement from habitat because of extreme weather events or other natural disasters (e.g. fire and drought), which are more common in summer. In koalas, a strong association was observed between the detection of herpesvirus DNA and detection of Chlamydia pecorum DNA. The mechanism behind this association is unknown, but could involve concomitant transmission of the pathogens or immune suppression caused by infection with one pathogen facilitating infection with the second pathogen (Stalder et al.
2015). A recent case report identified a novel alphaherpesvirus, tentatively denoted Phascolarctid alphaherpesvirus 3 (PhaHV-3), in a zoo-housed female adult koala with concurrent cryptococcosis. The authors postulated that the alphaherpesvirus infection was likely opportunistic given that the only individual affected within the enclosure was co-infected with Cryptoccocus gattii and immunologically compromised because of advanced age and lactation (Bowater et al. 2022).Sequence analysis of the DNA products amplified by PCR showed that both alphaherpesviruses and gammaherpesviruses were present in the marsupials examined. Alphaherpesviruses were detected in bare-nosed wombats and eastern grey kangaroos. The only alphaherpesvirus detected in eastern grey kangaroos was MaHV-4, which has serological cross-reactivity with MaHV-2 (Vaz et al. 2013). Based on these results it is possible that MaH V-4 infection is contributing to the high prevalence of neutralising antibodies to MaHV-2 detected in this species. Gammaherpesviruses were detected more frequently and were identified in eastern grey kangaroos, koalas, eastern bettongs (Bettongia gaimardi), swamp wallabies (Wallabia bicolor), Tasmanian devils, bare-nosed wombats and in a southern brown bandicoot (I. obesulus) (Stalder et al. 2015). A gammaherpesvirus was also detected in brushtailed bettongs (B. penicillata) (Skogvold et al. 2017). Each different species of herpesvirus was only identified in a single host species. Multiple individual animals were found to be infected with more than one species of herpesvirus, including koalas, eastern grey kangaroos and bare-nosed wombats (Stalder et al. 2015). In general, the results from these epidemiological studies are consistent with what is known about herpesvirus biology and epidemiology more broadly. In particular, the identification of many herpesviruses that are host species-specific, as well as multiple different herpesviruses being present within a single host species, including within individual animals, is consistent with findings from studies of other (nonmarsupial) animals (Pellett and Roizman 2013).
Further studies of herpesviruses in free-ranging and managed marsupials have recently also identified novel gammaherpesviruses in the yellow-bellied glider (Petau- rus australis), the critically endangered Leadbeaters possum (Gymnobelideus leadbeateri) (Douch et al. 2022), among quokka populations on Rottnest Is. and the WA mainland (Martinez-Perez et al. 2021), and in healthy managed and free-ranging Lumholtz’s tree-kangaroos (Shima et al. 2020).
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