Evaluating DiNap's Antiviral Effects on PRRSV

MARC-145 cells and PAMs were prepared in 6-well cell culture plates (BD, Falcon) 24 to 48 h before the start of the experiment. Following this, the cell monolayers were pretreated for 2 h before infection with growth medium containing DiNap at five different concentrations (0, 0.01, 0.02, 0.04, and 0.06 mM). After the pretreatment incubation, the cell monolayers were washed with growth medium, inoculated with VR2332 at a multiplicity of infection (MOI) of 0.01 and incubated for 1 h. After the final 1-h incubation, the viral inoculum was discarded, and the cell monolayers were replenished with growth medium containing the same concentrations of DiNap as a treatment and incubated for 4 more days under the same culture conditions. The cell culture supernatants were collected every 24 h, centrifuged, and stored at −80 °C until analysis. The progeny virus titers were measured using a microtitration infectivity assay [52 (link)]; the virus titration assay was described briefly in a previous study [53 (link)]. The virus titers were calculated based on the cytopathic effect (CPE) and are expressed as TCID₅₀/mL [54 ].