Discovery and rescue of porcine bastroviruses associated with polioencephalomyelitis in domestic pigs

Bastroviruses (BastV) are non-enveloped single-stranded positive-sense RNA viruses that have been discovered recently in feces samples of different animals and humans. The non-structural proteins of these viruses show similarities to those of hepevirids (<i>Hepeviridae</i>), and the structural proteins exhibit similarities to those of astrovirids (<i>Astroviridae</i>). BastVs have been found in fecal samples of mammals, amphibians, and invertebrates, but the association of infection and clinical disease manifestations has not been established. Here, we report the identification of porcine bastroviruses (PoBastV) in central nervous system (CNS) tissue samples of domestic pigs that presented fatal neurological disease in two unrelated disease outbreak scenarios in Australia and Switzerland. Viral metatranscriptomics of formalin-fixed paraffin-embedded (FFPE) CNS tissues identified genomic sequences of two genetically closely related PoBastV strains (PoBastV AUS/2015 and CHE/2022). Genomic RNA of both strains was readily detected by <i>in situ</i> RNA hybridization in neurons and glial cells of CNS tissues presenting histopathological lesions, thus supporting a plausible causal relationship between neurotropism and disease. We generated a molecular cDNA clone of PoBastV CHE/2022 and rescued infectious virus by reverse genetics in swine kidney cells (SK6) and further virus passage in intestinal porcine enterocytes (IPEC-J2). We used transmission electron microscopy to demonstrate PoBastV CHE/2022 virions in infected IPEC-J2 cells. These findings pave the way toward PoBastV as one of the etiologies of neurological disease outbreaks. Additionally, they allow studies further elucidating the molecular biology and pathogenesis of emerging BastV infections.IMPORTANCEBastroviruses (BastV) have been discovered recently in feces samples of different animals and humans. To date, BastV infections have not been associated with clinical diseases. Here, we report the identification of porcine BastV (PoBastV) in central nervous system tissue samples of domestic pigs that presented fatal neurological disease in two unrelated disease outbreak scenarios in Australia and Switzerland. This finding supports the hypothesis that PoBastV infections may cause clinical disease. We further rescued infectious PoBastV <i>in vitro</i> using the genome sequence data of one neuroinvasive PoBastV strain. With these tools, we can now start deciphering the molecular biology of BastV replication and the interaction of the virus with the host, which will lay the ground for future prophylactic and therapeutic strategies.