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).