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 6.9×10(8)/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 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h 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.

Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

Ribavirin efficiently suppresses porcine nidovirus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are porcine nidoviruses that represent emerging viral pathogens causing heavy economic impacts on the swine industry. Although ribavirin is a well-known antiviral drug against a broad range of both DNA and RNA viruses in vitro, its inhibitory effect and mechanism of action on porcine nidovirus replication remains to be elucidated. Therefore, the present study was conducted to determine whether ribavirin suppresses porcine nidovirus infection. Our results demonstrated that ribavirin treatment dose-dependently inhibited the replication of both nidoviruses. The antiviral activity of ribavirin on porcine nidovirus replication was found to be primarily exerted at early times post-infection. Treatment with ribavirin resulted in marked reduction of viral genomic and subgenomic RNA synthesis, viral protein expression, and progeny virus production in a dose-dependent manner. Investigations into the mechanism of action of ribavirin against PRRSV and PEDV revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular GTP pool by inhibiting IMP dehydrogenase may be essential for ribavirin activity. Further sequencing analysis showed that the mutation frequency in ribavirin-treated cells was similar to that in untreated cells, indicating that ribavirin did not induce error-prone replication. Taken together, our data indicate that ribavirin might not only be a good therapeutic agent against porcine nidovirus, but also a potential candidate to be evaluated against other human and animal coronaviruses.