*3.3. The PEI-linear Demonstrates Broad inhibition of Heterogeneous PRRSV-2 Isolates*

As a chemical reagent, the inhibitory effect of PEI-linear on PRRSV is expected to be broad spectrum. We next tested whether this PEI could inhibit other *PRRSV-2* isolates in MARC-145 cells. Our results showed that the replication of other *PRRSV-2* isolates (JXA1, GD-HD, and VR-2332) was significantly inhibited by PEI in a dose-dependent manner (Figure 3A–C). Moreover, the progeny virus from cell culture supernatants were titrated. Based on our data, it appears that PEI-linear demonstrated the strongest inhibition to PRRSV-2 VR-2332 for at least two Log10 reductions of infectious viral particles (Figure 3D). Notably, the NADC30-like isolate replicated poorly in MARC-145 with a viral titer below 10<sup>5</sup> TCID50/mL (Figure 3D). The inhibitory effect of PEI against different PRRSV strains was not uniform as the titers of certain viral isolates such as JXA1 were higher than the others. Taken together, these data showed that PEI-linear could effectively inhibit several PRRSV-2 isolates tested in MARC-145 cells.

**Figure 3.** PEI demonstrates broad inhibition of heterogeneous *PRRSV-2* isolates in MARC-145 cells. MARC-145 cells were incubated with a mixture containing 1 MOI of PRRSV JXA1 (**A**), GD-HD (**B**) and VR-2332 (**C**) mixed with the 40 kDa linear PEI at the indicated concentrations for 1 h. Then the cells were washed and cultured for an additional 24 h. Replication of PRRSV was determined in Western blot with Mab-6D10 against N protein. (**D**) PRRSV yields in MARC-145 cells infected with the mixture of different PRRSV virus strains at 0.1 MOI and PEI-linear (6 μg/mL). The mixture was removed and replaced by a fresh medium. The cells were incubated for 24 h before the cell culture supernatant was harvested for titration. 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).
