PDCoV Isolation and Propagation Protocol

HEK-293T cells were cultured and maintained in RPMI-1640 (HyClone, UT), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (PAN-biotech, Bavaria, Germany) at 37 °C in a humidified 5% CO₂ incubator. The PDCoV strain CHN-HN-2014 (GenBank accession number KT336560) used in this study was isolated from a suckling piglet with acute diarrhea in China in 2014 [26 (link)]. LLC-PK1 cells, a porcine kidney cell line purchased from the ATCC, were cultured at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated FBS and were used to amplify PDCoV. SEV was acquired from the Centre of Virus Resource and Information at the Wuhan Institute of Virology. Recombinant VSV-GFP was generously provided by Prof. Zhigao Bu at Harbin Veterinary Research Institute, China. Poly(I) and poly(C)-agaroses were bought from Sigma-Aldrich (MO, USA). Mouse monoclonal antibodies against Flag, HA, Myc and β-actin were purchased from Medical & Biological Laboratories (Nagoya, Japan). The PDCoV-N-protein-specific monoclonal antibody was produced in our laboratory as described previously [27 (link)]. Rabbit polyclonal antibodies directed against Flag were bought from ABclone (Wuhan, China). Alexa Fluor 488-conjugated donkey anti-mouse and 594-conjugated donkey anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (CA, United States).