Virus release assay
MDCK cells were divided into 2 treated groups including approximately
106 cells per group, and the group without cells was
as control, then the 3 groups were treated with 1,000
TCID50 H9N2 virus in ice bath for 1 h. The first group
and the control group were incubated at 37°C for 30 min; and the second
group was incubated in ice. The supernatant was collected after
centrifugation, then viral total RNA was extracted from the supernatant
using the TIANamp Virus RNA kit (Tiangen, Beijing, China). Reverse
transcription into cDNA was performed by HiScript® II Reverse
Transcriptase (Vazyme Biotech Co., Ltd., Nanjing, China) (U12 A/G:
AGC/AAAAGCAGG). The copies of M gene were determined by quantitative
real-time reverse transcriptase PCR (qRT-PCR) according to the
instructions provided in the ChamQ SYBR qPCR Master Mix (Vazyme Biotech
Co., Ltd., Nanjing, China)(Spackman et al., 2002). Each group had 3
replications. The data were analyzed using absolute quantitative method.
Data of the relative viral infection was (the copies of M gene in the
control group – the copies of M gene in ice group) / (the copies of M
gene in the control group), and the data of the relative viral release
was (the copies of M gene in the control group – the copies of M gene
in ice group) / (the copies of M gene in the 37 °C group – the copies
of M gene in ice group).