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.

Screening of Eurasian Tundra Reindeer for Viral Sequences by Next-Generation Sequencing

“…As found in many other studies of fecal microbiome [37,41], many reads from small circular DNA viruses were observed in the present study, with most sequences belonging to the families Circoviridae (e.g., CRESS virus), Genomoviridae (e.g., Alces alces faeces assoc. genomovirus) and Smacoviridae (e.g., ovine faeces assoc.…”

“…Then 250 µL was extracted from the pool with a magnetic bead-based kit (Viral NA Extraction Kit, Diasorin, Ireland) in an Arrow extraction robot (NorDiag, Oslo, Norway). Extracted RNA in the eluted total NA was converted to cDNA using random hexamers or the FR20RV-6N primer [ 37 ] with the SuperScript IV first-strand synthesis kit (Invitrogen). Double-stranded DNA was obtained by incubation of cDNA with Klenow Fragment DNA polymerase (New England Biolabs, Ipswich, MA, USA) at 37 °C for 1 h. The Klenow enzyme was then inactivated at 75 °C for 10 min.…”

“…Double-stranded DNA was obtained by incubation of cDNA with Klenow Fragment DNA polymerase (New England Biolabs, Ipswich, MA, USA) at 37 • C for 1 h. The Klenow enzyme was then inactivated at 75 • C for 10 min. When the tagged primer was used for cDNA synthesis, random amplification of the tagged cDNA was performed using the FR20RV primer [37] under the following conditions: 10 min at 95 • C, followed by 40 cycles of 30 s at 95 • C, 30 s at 58 • C, and 90 s at 72 • C. The reaction was ended with an extra elongation step at 72 • C for 10 min. The PCR reaction contained 1x PCR buffer, 2.5 mM MgCl2, 2.5 mM dNTPs, 0.4 mM primer, and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA).…”

Increasing risks for emerging infectious diseases within a rapidly changing High Asia

“…The only study on viral diseases in free-ranging snow leopards to date found that four of six sampled snow leopards had been infected by rotavirus. The study also reported infections of felid herpesvirus 1 and feline papillomavirus 2 (Johansson et al 2020b). Snow leopards from the same study area also carried antibodies against feline corona virus (Snow Leopard Trust, unpublished data).…”

Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review)

“…Quantitative real-time PCR (qPCR) [37], next-generation sequencing (NGS) [38,39], enzyme-linked immunosorbent assays (ELISA) [40] are currently the most widely used "gold standard" methods, which are applied for the detection and identification of viral DNA in clinical practice [41][42][43]. Therefore, during the recent epidemic of COVID-19 [44,45], it is especially relevant to develop a new method or to adapt/optimize other specific, sensitive, rapid, inexpensive, accurate, already applicable techniques for early detection of a nucleic acid of interest in specific and environmentally friendly methods.…”