Virus Isolation and Plaque Purification

PK15 cells (American Type Culture Collection [ATCC] CCL-33) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Bovogen, Australia) at 37°C in a 5% CO₂ incubator. Growth medium was removed from confluent monolayer cells; the cells were washed twice with DMEM and inoculated with a mixture of the supernatants of the positive tissue samples and DMEM containing 20 μg/ml trypsin (GIBCO, 1:250) at a ratio of 1:1. After adsorption for 60 min at 37°C, the cells were washed with DMEM, and maintenance medium consisting of DMEM supplemented with 10 μg/ml trypsin was added. The inoculated cell cultures were observed for CPE for 3–5 days, harvested, and blindly passaged for five times. The viruses in a CPE positive sample was cloned by repeating plaque purify three times and designated as HQ2016.