Detection and Genetic Characterization of Viruses Present in Free-Ranging Snow Leopards Using Next-Generation Sequencing

Abstract

Snow leopards inhabit the cold, arid environments of the high mountains of South and Central Asia. These living conditions likely affect the abundance and composition of microbes with the capacity to infect these animals. It is important to investigate the microbes that snow leopards are exposed to detect infectious disease threats and define a baseline for future changes that may impact the health of this endangered felid. In this work, next-generation sequencing is used to investigate the fecal (and in a few cases serum) virome of seven snow leopards from the Tost Mountains of Mongolia. The viral species to which the greatest number of sequences reads showed high similarity was rotavirus. Excluding one animal with overall very few sequence reads, four of six animals (67%) displayed evidence of rotavirus infection. A serum sample of a male and a rectal swab of a female snow leopard produced sequence reads identical or closely similar to felid herpesvirus 1, providing the first evidence that this virus infects snow leopards. In addition, the rectal swab from the same female also displayed sequence reads most similar to feline papillomavirus 2, which is the first evidence for this virus infecting snow leopards. The rectal swabs from all animals also showed evidence for the presence of small circular DNA viruses, predominantly Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses and in one case feline anellovirus. Several of the viruses implicated in the present study could affect the health of snow leopards. In animals which are under environmental stress, for example, young dispersing individuals and lactating females, health issues may be exacerbated by latent virus infections.

Keywords: Mongolia; Panthera unica; free-ranging; next-generating sequencing; rectal swabs; snow leopard; virome.

Figure 1

Neighbor-joining phylogenetic tree for a 1,127-nt-long fragment of the rotavirus VP3 gene obtained from the rectal swab of animal M10 together with selected sequences from GenBank (NCBI accessions are given within parentheses). These were selected to include the representative from different hosts with the highest similarity, except that all available isolates from felids were included if not identical. The phylogenetic trees were constructed with the CLC genomics workbench using the neighbor-joining method with the Jukes–Cantor distance measure with 1,000 bootstrap calculations. Only branches with at least 60% bootstrap support are shown. The NCBI accession of the novel snow leopard VP3 sequence is shown in red.

Figure 2

Neighbor-joining phylogenetic tree for a 771-nt-long fragment of the rotavirus NSP1 gene obtained from the rectal swab of animal M1 together with selected sequences from GenBank (NCBI accessions are given within parentheses). These were selected to include the representative from different hosts with the highest similarity, except that all available isolates from felids were included if not identical. The phylogenetic trees were constructed with the CLC genomics workbench using the neighbor-joining method with the Jukes–Cantor distance measure with 1,000 bootstrap calculations. Only branches with at least 60% bootstrap support are shown. The NCBI accession of the novel snow leopard NSP1 sequence is shown in red.

Figure 3

Neighbor-joining phylogenetic tree for a 435-nt-long fragment of the papillomavirus E1 gene obtained from the rectal swab of animal F9 together with selected sequences from GenBank (NCBI accessions are given within parentheses). These were selected to include viral sequences obtained from related host species or for showing high similarity. The phylogenetic trees were constructed with the CLC genomics workbench using the neighbor-joining method with the Jukes–Cantor distance measure with 1,000 bootstrap calculations. Only branches with at least 60% bootstrap support are shown. The NCBI accession of the snow leopard papillomavirus sequence is shown in red.