Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

Lagoviruses, family Caliciviridae, are some of the most virulent vertebrate viruses known. They infect leporids, i.e., rabbits (Oryctolagus spp.), hares (Lepus spp.) and cottontails (Sylvilagus spp.) and rapidly kill over 95% of susceptible animals. Pathogenic lagoviruses are hepatotropic and induce a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 hours of infection. However, the pathophysiological mechanisms governing this extreme phenotype and other aspects of the fundamental biology of these viruses are poorly understood due to a lack of cell culture systems. Here, we report on a robust and reliable ex vivo model for the cultivation of hepatotropic lagoviruses. We show that three rabbit haemorrhagic disease virus (RHDV) variants, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids. Viral replication was demonstrated by a (i) increase in viral RNA levels of greater than one log10 over a 23-hour period, (ii) detection of viral structural and non-structural proteins, and (iii) detection of double-stranded RNA viral replication intermediates. Furthermore, we generated hepatobiliary organoids from a feral cat (Felis domesticus), a wild mouse (Mus musculus) and a European brown hare (Lepus europaeus) and showed that monolayer cultures derived from the cat and mouse organoids were not permissive for lagovirus infection, while those derived from the hare organoids only supported replication of RHDV2, recapitulating the species tropism that has been observed with these viruses. Our organoid culture system will facilitate future studies into the molecular biology of lagoviruses, which will have considerable import for the conservation of endangered leporid species in Europe and North America and the biocontrol of overabundant rabbit populations in Australia and New Zealand.