Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs

Lee, Seung Heon; Yang, Dong-Kun; Kim, Ha-Hyun; Cho, In-Soo

A Korean PEDV strain, QIAP1401, isolated in our laboratory was propagated on African green monkey kidney (Vero) cells (ATCC CCL-81, Manassas, VA, USA), as described previously [17] , and was used after 70 passages (QIAP1401-p70). In brief, for viral infection, confluent monolayers of Vero cells were washed with phosphate-buffered saline (PBS) and inoculated with the PEDV isolate at a 0.1 multiplicity of infection in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 1 µg/mL crystallized trypsin (Sigma, St Louis, MO, USA). After incubation for 1 hour at 37°C in an atmosphere of 5% CO2, the culture medium was replaced to remove extracellular virions. The PEDV-infected cells were maintained at 37°C in 5% CO2 and monitored daily for a cytopathic effect (CPE) using a microscope. Upon observation of a CPE, the culture flask was subjected to three rounds of freezing and thawing, and clarified viral fluid was collected by centrifugation at 1,000×g for 10 minutes. Serial passaging of the virus was routinely conducted in six-well plates using the sequential limit dilution culture method. After each passage, an aliquot of the virus was stored at -70°C until use. PEDV QIAP-1401-p11, which was passaged 11 times after isolation, was used as the challenge strain.