*3.1. EGCG Inhibits the Infectivity of PCV2*

To assess the effect of EGCG on the infectivity of PCV2, the cytotoxicity on PK15 cells of the compounds was first examined using the CCK8 assay. The results showed that EGCG at a concentration of 100 μM did not generate significant cytotoxicity to PK-15 cells with a duration time of 24 h, 72 h, and 120 h, and EGCG at a concentration of 200 μM could produce cytotoxicity to cells, which limited the maximum concentration of EGCG at 100 μM (Figure 1b). The infectivity of PCV2 was significantly inhibited at the 100 μM EGCG treatment during the whole infective process, the virus titer of PCV2 infected cells with EGCG treatment showed a significant decrease at various time points compared to that without EGCG treatment (Figure 1c). Inhibition of PCV2 infectivity was also manifested by reduced level of viral capsid protein expression measured by immunoblotting (Figure 1d), and the reduction of viral genome copy number detected by qPCR (Figure 1e) at 72 hpi with the 100 μM EGCG treatment. The inhibitory effect of PCV2 infection was still detectable for the 10 μM EGCG treatment (Figure 1d,e). Furthermore, the cells infected with PCV2 at MOI = 1.0 were detected with IFA, which showed that the quantity of the PCV2 infected cells with the 100 μM EGCG treatment was obviously lower than that without EGCG treatment (Figure 1f). The original catechin EC displayed no considerable anti-infective effect on PCV2 under similar treatment conditions (Figure 1c–f). In addition, the EC50 of the antiviral effect of EGCG on PCV2 was further determined to be 37.79 ± 1.64 μM, which was consistent with its inhibitory concentration of 100 μM. Accordingly, EGCG could exert a prominent antiviral effect against the PCV2 infection.
