Virus-virus interactions and host ecology are associated with RNA virome structure in wild birds

Little is known about the factors that shape the ecology of RNA viruses in nature. Wild birds are an important case in point, as other than influenza A virus, avian samples are rarely tested for viruses, especially in the absence of overt disease. Using bulk RNA-sequencing ("meta-transcriptomics"), we revealed the viral diversity present in Australian wild birds through the lens of the ecological factors that may determine virome structure and abundance. A meta-transcriptomic analysis of four Anseriformes (waterfowl) and Charadriiformes (shorebird) species sampled in temperate and arid Australia revealed the presence of 27 RNA virus genomes, 18 of which represent newly described species. The viruses identified included a previously described gammacoronavirus and influenza A viruses. Additionally, we identified novel virus species from the families Astroviridae, Caliciviridae, Reoviridae, Rhabdoviridae, Picobirnaviridae and Picornaviridae. We noted differences in virome structure that reflected underlying differences in location and influenza A infection status. Red-necked Avocets (Recurvirostra novaehollandiae) from Australia's arid interior possessed the greatest viral diversity and abundance, markedly higher than individuals sampled in temperate Australia. In Ruddy Turnstones (Arenaria interpres) and dabbling ducks (Anas spp.), viral abundance and diversity were higher and more similar in hosts that were positive for influenza A infection compared to those that were negative for this virus, despite samples being collected on the same day and from the same location. This study highlights the extent and diversity of RNA viruses in wild birds and lays the foundation for understanding the factors that determine virome structure in wild populations.