*2.7. Analysis of Virus Attachment and Internalization*

MARC-145 cells or PAMs were pre-incubated with the 40 kDa linear PEI for 1 h at 37 ◦C or left untreated (control), followed by pre-chilling at 4 ◦C for 30 min prior to PRRSV inoculation. The PRRSV-JXA1 strain was used to inoculate the cells at 4 ◦C for 1 h at the indicated MOI. Similarly, pre-chilled MARC-145 cells or PAMs were co-incubated with a mixture of PRRSV-JXA1 virus and the 40 kDa linear PEI at 4 ◦C for 1 h or PRRSV-JXA1 virus only. Subsequently, the cells with the indicated treatment were washed three times with cold PBS to remove unbound virions before being harvested for RT-qPCR to evaluate viral RNA level from the virions bound.

For the virion internalization assay, PRRSV-JXA1 at the indicated MOI was used to inoculate MARC-145 cells or PAMs at 4 ◦C for 1 h to allow the sufficient attachment of virions to the cell surface without triggering endocytosis as previously instructed [32]. After removing unbound viral particles by washing cells with cold PBS for three times, fresh medium containing the 40 kDa linear PEI was used to treat MARC-145 or PAMs followed by shifting cells to 37 ◦C to trigger endocytosis, which allows the internalization of bound PRRSV virions on the cell surface. One hour later, the cells were washed with cold PBS again and further incubated with 50 μg proteinase K (Sigma-Aldrich) for 45 min at 4 ◦C to remove non-internalized virions located on the cell surface. After the inactivation of proteinase K by a protease inhibitor cocktail (Roche, Basel, Switzerland), the cells were harvested for RT-qPCR analysis of the viral RNA of internalized PRRSV virions.
