Isolation and Characterization of Human Norovirus GII.4

The sequence of HuNoV GII.4 used here (Gene bank: OL721917) was isolated from positive stool samples of diarrhea patients [8 (link)]. Briefly, QIAamp RNA Blood Mini Kit (Qiagen, 52304, Hilden, Germany) was used to isolate the HuNoV genome from the positive stool sample, and then moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, M170B, Dane County, WI, USA) was used to synthesize the HuNoV cDNA. The progeny viruses were obtained by transfecting HEK293T cells with the plasmid, which encoded the full-length cDNA of HuNoV. Virus stocks were aliquoted and stored at −80 °C until use, and the genome copy was determined. Human colon epithelial cell lines Caco2 cells and HEK293T cells were purchased from ATCC and cultured in Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), purchased from Thermo Scientific, Sydney, Australia, and 100 U/mL penicillin/streptomycin (Genom, Hangzhou, China) at 37 °C with 5% CO2 in an incubator. Antibodies against NLRP3 (19771-1-AP), caspase1 (22915-1-AP), N-GSDMD (20770-1-AP), and β-tubulin (10068-1-AP) were purchased from Proteintech, Wuhan, China. Besides, HRP-conjugated goat anti-rabbit IgG (AS063) or goat anti-mouse IgG (AS003) were also purchased from Abclonal, Wuhan, China.