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).