Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease

The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9 × 108/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12 h post-infection. Maximum levels of cell-associated viral RNA were detected at 24 h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9 h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.