Viral Pathogen Screening in Fishing Cats

The fresh tissue samples including heart, lung, liver, spleen, and kidney of all fishing cats plus additional intestinal and mesenteric lymph node tissues of fishing cat no. 3, were subjected to viral nucleic acid extraction by individually homogenizing them with 1% (v/v) phosphate-buffered saline (PBS). Subsequently, the total viral nucleic acid was extracted using a commercial viral nucleic acid extraction II kit (Geneaid, Taipei, Taiwan) following the manufacturer’s suggestion. The extracted nucleic acids were then qualified and quantified by their A₂₆₀/A₂₈₀ absorbance ratio using the spectrophotometry method (NanoDrop, Thermo Scientific™, Waltham, MA, USA). All extracted nucleic acids were further subjected to viral molecular investigation using polymerase chain reaction (PCR) analysis with several pan-virologic-family PCR testing panels including the detection of the herpesvirus [23 (link)], paramyxovirus [24 (link)], pneumovirus [25 (link)], calicivirus [26 (link)], influenza virus [27 (link)], bocavirus [26 (link), 28 (link)], parvovirus [29 (link)] and coronavirus [30 (link), 31 (link)]. The PCR protocols, reagents, cycling conditions and positive/negative controls used in the reactions are described in the S1 File. PCR detection specific of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of feline was used as an internal control as described previously [32 (link)]. Subsequently, the positive PCR amplicons were visualized using a QIAxcel capillary electrophoresis platform (Qiagen, Hilden, Germany) as previously described [33 (link)]. The positive amplicons of each PCR assay were purified and subjected to bidirectional Sanger sequencing (Macrogen Inc, Seoul, South Korea). Due to moderate autolysis of the tissues, it was not possible to perform bacteriological investigation on these fishing cats.