*3.4. The PEI-Linear Blocks PRRSV Replication in PAMs*

Since PAMs are the primary target cells for PRRSV infection in vivo, we also evaluated the antiviral activity of PEI-linear in PAMs. According to the cell viability assay results (Figure 1), PEI-linear at a concentration of 6 μg/mL was used in PAMs. As our preliminary results demonstrated the attachment and infection efficiency of PRRSV to PAMs is nearly as high as 1000-fold in comparison to MARC-145 cells [33]. Therefore, 106 PAMs were inoculated with the PRRSV-2 strains: SD16, JXA1, GD-HD, and NADC30-like, at 0.01 MOI due to the high susceptibility of PAMs to PRRSV. Our results demonstrated that PEI inhibited the replication of the heterogeneous PRRSV isolates in PAMs (Figure 4A). Meanwhile, the RT-qPCR results confirmed the PEI inhibition of PRRSV in PAMs as well (Figure 4B). Furthermore, the titration results showed that PEI-linear inhibited all the PRRSV isolates tested (Figure 4C). Interestingly, it appears that the NADC30-like isolate replicated similarly in

PAMs as the other isolates (Figure 4C). Taken together, these data demonstrated that PEI-linear inhibits PRRSV infection in PAMs as well.

**Figure 4.** PEI-linear inhibits the replication of PRRSV-2 isolates in PAMs. (**A**). PAMs were inoculated with the virus and PEI mixture containing the indicated PRRSV strains (SD16, JXA1, GD-HD, NADC30-Like, 0.01 MOI) and PEI-linear 6 μg/mL for 1 h. The cells were then washed with PBS buffer and incubated for another 24 h. PRRSV N level was determined with Western blot. (**B**). PRRSV RNA levels in PAMs inoculated with the virus and PEI mixture containing the indicated PRRSV strains (0.01 MOI) and 6 μg/mL linear PEI. (**C**) Progeny PRRSV virions in cell culture supernatant of PAMs infected with different PRRSV strains at an MOI of 0.01 in the presence of linear PEI (6 μg/mL) for 24 h. All experiments were repeated at least three times, and the data were presented as the mean ± SD, which is further subjected to Student's *t*-test. Significant differences between indicated groups were marked by "\*" (*P* < 0.05) and "\*\*" (*P* < 0.01).
