Virus screening, isolation, and transmission electron microscopy observations
Viruses known to be associated with diarrheal diseases in pangolins, including CPV, canine coronavirus (CCoV), canine distemper virus (CDV), and dog circovirus (DogCV), were screened based on PCR analyses using previously described primers (Deng et al., 2018; Hsu et al., 2016). For the purposes of virus isolation, samples of pangolin tissues were homogenized, diluted with phosphate-buffered saline, and purified by passing through a 0.22-µm sterile filter. These filtered samples were subsequently added to monolayers of F81 cells (ATCC-CL-0081) maintained in minimum essential media supplemented with 10% fetal bovine serum at an amount equal to 10% of the cell culture medium volume. The cells were thereafter inspected daily for cytopathic effects. Cells showing evidence of infection were collected, purified by ultracentrifugation, and following negative staining with phosphotungstic acid, subjected to transmission electron microscopy (Hitachi).