Non-invasive surveys of mammalian viruses using environmental DNA

Niccolo Alfano; Anisha Dayaram; Jan Axtner; Kyriakos Tsangaras; Marie-Louise Kampmann; Azlan Mohamed; Seth Timothy Wong; M. Thomas P. Gilbert; Andreas Wilting; Alex Daivd Greenwood

Tiger leeches (Haemadipsa picta) and brown leeches (Haemadipsa zeylanica) were collected in Malaysian Borneo and processed as pools (bulk samples) consisting of 1 to 77 individual leeches separated by leech species and sampling location. Viruses were identified in 40 of the 68 leech pools analysed (59%) ( Fig. 2 ; Suppl. Tab. 1). In 18 of these (45%), two to three viruses were identified. Sequence data from six vertebrate-infecting viral families were detected, including the Anelloviridae, Circoviridae, Coronaviridae, Parvoviridae, Retroviridae and Rhabdoviridae. The most common viral group detected was Rhabdoviridae which was found in 37% of samples (25 samples out of 68), followed by Coronaviridae which was identified in 24% of samples (16 samples Fig. 2 ). The other contigs clustered phylogenetically, suggesting they represent two new species of a rhabdovirus related to lyssaviruses (Suppl. Fig. 2 ). Although in most cases one contig per sample was observed, in five samples (L4, L12, L23, L58, L68) two different viruses were found. Several viral regions for Rhabdoviridae were represented in the baits. However, most of the oligonucleotides were specific for the L gene which encodes the RNA-dependent RNA polymerase. All the recovered contigs mapped to the L gene (Suppl. Fig. 3A -C). The viral contig sequences were confirmed by PCR and Sanger sequencing for L55 and L58 (Suppl. Fig. 3D ).