Wobbly possum disease (WPD) is a fatal neurological disease of the Australian brushtail possum (Trichosurus vulpecula) that was first identified in a colony of research possums, and later in free-living possums in New Zealand. Recently, a novel nidovirus has been proposed to be aetiologically involved in the development of WPD based on PCR data. In the present report, we describe development of the cell culture system to support the growth of the novel virus and the in-vivo infection of adult possums with the cell culture isolate of the virus.
The virus was grown in primary liver macrophages and purified using density gradient ultracentrifugation. Possums caught from the wild (n=16) were individually housed. Following acclimatization period of up to 4 weeks, each possum in group 1 (n=4) received 1 mL (2 x 107 TCID50) of a purified cell culture isolate, each possum in group 2 (n=4) received 1.8 mL (1 x 106 TCID50) of a cell lysate from inoculated cultures, and each possum in group 3 (n=4) received 1 mL of a previously used standard inoculum that had been prepared from tissues of WPD-affected possums. Control possums (n=4) received either a gradient material prepared from uninoculated cultures (n=2) or nothing (n=2). All inoculations were performed via intraperitoneal injection. All but one possums in groups 1-3 developed clinical signs of WPD approximately 2 weeks post inoculations and were euthanized. Decreased appetite was observed in 12/12, weight loss in 11/12, and severe neurological signs in 11/12 of infected possums. One possum died prior to the development of neurological signs, and 3/5 joeys died. Control possums did not demonstrate any clinical signs of WPD. WPD-specific PCR detected high levels of viral RNA in tissues from all possums that received infectious inocula, but not from control possums, confirming the aetiological involvement of the novel nidovirus in development of WPD.