Kinetics of viral internalization

EECs grown in 12-well plates (3 × 10⁵ cells/well) were infected with PPRV at a multiplicity of infection (MOI) of 2, and incubated at 37 °C. To separate the adsorption and internalization processes, the EECs were pretreated with PPRV at 4 °C for 1 h and then shifted to 37 °C. Proteinase K treatment significantly affected the number of virions attached to the cell surface, suggesting that proteinase K removes the viruses attached to cells [26]. At different time points, the cells were washed with phosphate-buffered saline (PBS) and treated with proteinase K (2 mg/mL) (Solarbio, China) for 45 min at 4 °C to remove the adsorbed but not internalized virus. The proteinase K was then inactivated with 2 mM phenylmethylsulfonyl fluoride in PBS with 5% bovine serum albumin (BSA), and the cells were washed with PBS–0.5% BSA with low-speed centrifugation. Finally, the cell pellet was resuspended in DMEM/F12 and serial tenfold dilutions of the cell suspension were plated. EEC monolayers were grown in 96-well plates containing DMEM with 2% FBS. Eight replicates were established for each dilution, and 100 μL of virus diluent was added to each well. The cells were incubated at 37 °C under 5% CO₂ for about 5–7 days, and the numbers of wells with or without CPE were counted. TCID₅₀ was calculated with the Reed–Muench method and used to calculate the infectivity of the viral stocks: infectivity (plaque-forming units/mL) = 0.69 × TCID₅₀. Each test was performed in triplicate. To determine the rate of virus internalization, a parallel set of cultures was processed under the same conditions, except that proteinase K was replaced with PBS.