Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs

Liu, Xinsheng; Zhao, Donghong; Zhou, Peng; Zhang, Yongguang; Wang, Yonglu

Virus. HEK293A cells were seeded in a 6 cm dish (Corning, USA), placed in a 37°C incubator with 5% CO 2 until a 70-80% confluent cell monolayer formed, and then transfected with 1.5 μg of the transfer bacmid pDC316-S-EGFP and 6 μg of pBHGlox(delta)E1,3 using the Lipofectamine ™ 2000 transfection reagent (Invitrogen, USA). After incubation for 6-10 hours at 37°C with 5% CO 2 , the fresh growth medium (containing DMEM and 10% heatinactivated FBS) was added, and the cell culture continued at 37°C and 5% CO 2 , with daily observation for cytopathic effects (CPEs). When more than 90% of cells showed CPEs, the cell cultures were harvested, frozen, and thawed three times and centrifuged for the next passage of the adenovirus-PEDV-S recombinant virus (rAd-PEDV-S). After three passages, the third passage (P3) rAd-PEDV-S was purified by cesium chloride gradient centrifugation, and the titers of the recombinant adenoviruses were tested by fluorescence dilution as follows: 1 × 10 4 HEK293A cells were seeded into 96-well plates and then maintained at 37°C in a humidified 5% CO 2 incubator for 24 hours. e harvested recombinant adenoviruses were 10-fold serially diluted in DMEM (Invitrogen, USA) (from 10 − 1 to 10 − 11 ), mixed well, and vortexed. After confluence, the monolayer cells were washed with PBS (Gibco ™ , USA). en, 100 μl of 10-fold serially diluted virus suspensions was inoculated in two replicates per dilution. After 24 hours of incubation, 100 μl of DMEM (Invitrogen, USA) with 10% heat-inactivated FBS (Gibco ™ , USA) was added. Cells were monitored for 72 hours, and the viral titers were determined using a fluorescence microscope. Furthermore, the expression level of the PEDV S gene in rAd-PEDV-S was measured by real-time fluorescence quantitative RT-PCR according to the method used in the previous study [29] .