Characterization of the putative wobbly possum disease virus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Virology at Massey University, Manawatū, New Zealand
The objective of this PhD was to characterise a marsupial arterivirus, termed wobbly possum
disease virus (WPDV), and to confirm aetiological involvement of the virus in the development
of a neurological disease of the Australian brushtail possum (Trichosurus vulpecula), termed
wobbly possum disease (WPD). An in vitro culture system supporting the growth of the virus,
comprising primary possum macrophages was developed. Purified virus stock was prepared
using iodixanol density gradient ultracentrifugation of infected cell culture lysates and the in
vitro growth kinetics of WPDV in primary possum macrophages was investigated using a
previously described WPDV-specific RT-qPCR. The steepest increase in the levels of
intracellular viral RNA was observed between six and 12 hours post infection, followed by a
gradual release of cell-free viral RNA between nine and 24 hours post infection. Maximum
levels of intracellular and extracellular viral RNA levels occurred at 24 and 48 hours post
Aetiological involvement of the virus in the development of WPD was supported by induction
of disease in healthy wild-caught possums following infection with the purified virus. The
pathogenesis of viral infection was explored by characterisation of histological lesions and
quantification of WPDV RNA in various tissues from experimentally infected possums.
Mononuclear inflammatory cell infiltrates of variable size were consistently observed in the
liver, kidney, salivary gland and brain. The highest viral RNA levels were found in lymphoid,
splenic and liver tissues, suggesting virus tropism for cells of the immune origin, most likely of
the monocyte-macrophage system. High levels of viral RNA in tissues and sera from possums
euthanased nearly four weeks post-infection indicates that immune response was ineffective in
clearing the virus in that time-frame.
To investigate the presence of the virus in wild possum populations in New Zealand, an indirect
ELISA using Escherichia coli-expressed recombinant viral nucleocapsid (rN) protein as antigen
was developed. Pre and post-infection sera from experimentally challenged possums was used
for ELISA development. These sera were also characterised using Western-blot against rN
antigen. A serological survey of archival possum serum samples that had been collected
between the years 2000 and 2016 and from five different regions of New Zealand was also
performed using indirect ELISA. Bayesian estimates of parameters for a model of the ELISA
data were used to establish ELISA cut-offs for WPDV antibody positive and negative samples.
Applying these cut-offs, 50/230 (22%) archival samples were seropositive by indirect ELISA.
Altogether, our data suggest that WPDV has been circulating in wild possum populations in
New Zealand. Five out of 14 (36%) of pre-infection sera from the challenge study were also
seropositive by Western-blot. Development of WPD in these possums following challenge
suggests that pre-existing immunity was insufficient for protection against the development of
disease. As such, further exploration of virus, host and environmental factors that govern
development of disease is required.