Metagenomic discovery and co-infection of diverse wobbly possum disease viruses and a novel hepacivirus in Australian brushtail possums.

Background: Australian brushtail possums (Trichosurus vulpecula) are an introduced pest species in New Zealand, but native to Australia where they are protected for biodiversity conservation. Wobbly possum disease (WPD) is a fatal neurological disease of Australian brushtail possums described in New Zealand populations that has been associated with infection by the arterivirus (Arteriviridae) wobbly possum disease virus (WPDV-NZ). Clinically, WPD-infected possums present with chronic meningoencephalitis, choroiditis and multifocal neurological symptoms including ataxia, incoordination, and abnormal gait.

Methods: We conducted a retrospective investigation to characterise WPD in native Australian brushtail possums, and used a bulk meta-transcriptomic approach (i.e. total RNA-sequencing) to investigate its potential viral aetiology. PCR assays were developed for case diagnosis and full genome recovery in the face of extensive genetic variation.

Results: We identified genetically distinct lineages of arteriviruses from archival tissues of WPD-infected possums in Australia, termed wobbly possum disease virus AU1 and AU2. Phylogenetically, WPDV-AU1 and WPDV-AU2 shared only ~ 70% nucleotide similarity to each other and the WPDV-NZ strain, suggestive of a relatively ancient divergence. Notably, we also identified a novel and divergent hepacivirus (Flaviviridae) - the first in a marsupial - in both WPD-infected and uninfected possums, indicative of virus co-infection.

Conclusions: We have identified marsupial-specific lineages of arteriviruses in mainland Australia that are genetically distinct from that in New Zealand, in some cases co-infecting animals with a novel hepacivirus. Our study provides new insight into the hidden genetic diversity of arteriviruses, the capacity for virus co-infection, and highlights the utility of meta-transcriptomics for disease investigation in a One Health context.