Identification of novel hepaciviruses and Sylvilagus-associated viruses via metatranscriptomics in North American lagomorphs

Cottontails (Sylvilagus spp.) and jackrabbits (Lepus spp.) within the Leporidae family are native to North America and are found in a wide range of habitats, including deserts, forests, and grasslands. Although there is a growing body of research describing the arrival of the highly virulent rabbit haemorrhagic disease virus 2 (RHDV2, GI.2) on this continent, and its impact on native lagomorphs, information about the natural virome and microbiome of healthy and deceased American lagomorphs is relatively limited. In this study, we used a meta-transcriptomics approach to conduct whole pathogen profiling on healthy and deceased animals in the USA. We analysed 48 matched liver and lung sample pools from apparently healthy cottontails and jackrabbits in Texas and an additional 48 liver samples from deceased animals from nine other US states. This approach enabled the discovery of three distinct new viruses and revealed additional new insights into the lung and liver microbiomes of North American lagomorphs. Of the three new viruses, a tetnovirus and a novel picorna-like virus were likely of insect origin and therefore considered environmental contaminants. Of particular interest was a new species of hepacivirus, with around 50% sequence identity to a known hepacivirus from a xeric four-striped grass rat (Rhabdomys pumilio). Phylogenetic analysis from 41 individual hepacivirus genomes recovered from our lagomorph samples revealed two distinct clades, corresponding with different cottontail species. No hepaciviruses were detected in any of the jackrabbit samples. This is the first description of a hepacivirus in lagomorphs. Our findings extend the Hepacivirus genus, provide new insights into its evolution, and describe the first baseline on microbial diversity in North American lagomorphs, an important step towards understanding the role of potential pathogens for population management and conservation.