*2.2. Cell Viability and Cytotoxicity*

Cell viability and cytotoxicity were evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA) by following the manufacturer's instructions. Briefly, MARC-145 cells or PAMs were seeded into 24-well plates at a density of 5 <sup>×</sup> 104 cells/well or 4 <sup>×</sup> 105 cells/well and cultured for 24 h. Then, fresh medium containing the PEI at the indicated concentration was added to the cells. After incubation for another 24 h, the cells were lysed by using 1× passive cell lysis buffer (Promega). The cell lysate was transferred into a 96-well black polystyrene microplate (Corning Inc, Corning, NY, USA). CellTiter-Glo® reagent was added and mixed with the cell lysate. After incubation for 10 min at room temperature (RT), luminescence signal was determined with VICTORX™ X5 Multilabel Reader (Perkin-Elmer Life and Analytical Sciences, Wellesley, MA, USA).

### *2.3. PRRSV Inhibition Assay*

MARC-145 cells were seeded into 24-well plates at a density of 5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well and cultured for 24 h. The cells were inoculated with a mixture of PRRSV (0.1 or 1 MOI) in plain DMEM and linear PEI or branched PEI (with indicated concentration). The cells were then incubated at 37 ◦C for 5 h. The mixture containing PEIs and virions was discarded and the cells were rinsed with phosphate-buffered saline (PBS), pH7.2, followed by culturing for another 24 h before further experiments. Subsequently, cells were rinsed with PBS twice and prepared for immunofluorescence assay (IFA), or harvested for Western blot and qPCR analyses. Cell culture supernatant containing progeny virus were further titrated to evaluate the infectious virions as well.

To evaluate the antiviral activity of PEIs in PAMs, cells were seeded into 24-well plates at a density of 5 <sup>×</sup> 10<sup>5</sup> cells/well and incubated for 6 h before further experiments. A fresh RPMI 1640 medium containing PRRSV (0.01 MOI) and PEI was added to the cells. After 1 h inoculation, PAMs were rinsed with PBS to remove the virus and PEI mixture unbosund and cultured for another 24 h before further analysis with qPCR, Western blot, and virus titration.
