PRRSV protein counteracts the anti-PRRSV effects of pCH25H. (A) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged pCH25H and HA-tagged PRRSV E protein. The cells were harvested for quantitation of 25HC by ELISA at 48 h posttransfection. (B) PAMs were infected with different doses of PRRSV. The cells were harvested for quantitation of 25HC by ELISA at 48 h postinfection. (C) PK-15CD163 cells were cotransfected with expression vectors encoding FLAG-tagged pCH25H or FLAG-tagged hCH25H and HA-tagged PRRSV E protein. At 48 h posttransfection, cells were infected with PRRSV (MOI of 1.0); 24 h later, they were harvested for a plaque assay. All experiments were performed in triplicate, and data shown are representative of three independent experiments. Standard errors of the mean are indicated by error bars. *, P < 0.05; **, P < 0.01. N.S., not significant.