Cell Culture and Virus Isolation Protocol

IPI-2I cells (porcine intestinal epithelial cells), BHK-21 cells, and HeLa cells were obtained from the China Center for Type Culture Collection (Wuhan, China). LLC-PK1 cells were acquired from the American Type Culture Collection (ATCC CL-101; Manassas, VA, USA). IPI-2I, BHK-21, and LLC-PK1 cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Invitrogen, USA). HeLa cells were grown in RPMI 1640 medium with 10% fetal bovine serum. These cells were maintained in 5% CO₂ at 37 °C. PDCoV strain CHN-HN-2014 (GenBank accession no. KT336560)^{10 (link)} and TGEV strain WH1 (GenBank accession no. HQ462571) were isolated in 2014 and 2010, respectively, in China. Mouse monoclonal antibodies against TGEV M, TGEV N or PDCoV S, PDCoV N were created in-house. APN antibody was purchased from ABclonal (China). An anti-Flag rabbit polyclonal antibody (MBL, Japan), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Santa Cruz, USA), and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Santa Cruz, USA) were used for indirect IFA. Horseradish peroxidase-conjugated goat anti-mouse antibody (Beyotime, China) and horseradish peroxidase-conjugated goat anti-rabbit antibody (Beyotime, China) were applied in western blots.