Viral isolation and purification
The sample supernatant filtered by 0.22 μm filter (Millipore) for viral
isolation. Marc-145 cells (stored in our lab) were cultured in 10 cm
dishes up to confluency at 37oC in a 5%
CO2 incubator. 1 mL of filtered supernatant per dish was
added into monolayer Marc-145 cells and incubated for an hour. Viral
solution was discards and washed out with PBS twice. The cells were
continued to culture with 2% fetal bovine serum in DMEM and monitored
cytopathic effect (CPE) daily. Once CPE were observed, the
virus-containing supernatant was collected, subjected to three
freeze-thaw cycles at -70oC and room temperature, and
then used to inoculate Marc-145 cells for viral passages.
To purify the virus, single plaques were picked and amplified with
Marc-145 cells for three generations. And then virus was identified by
RT-PCR using GETV C gene primers. The purified GETV were conducted the
viral titer assessment at each time point, and the
TCID50 value was calculated according to the Reed-Muench
method.