*3.2. EGCG Exerts the Antiviral E*ff*ect via Directly Targeting Virions*

The antiviral effect of EGCG might be exerted by targeting PCV2 virions, such as the effect on certain viral nucleic acids and proteins, or targeting host cells [23]. To assess this issue, the time of addition assay was preformed, the cells were pretreated with EGCG and then inoculated with PCV2, or the PCV2 was premixed with EGCG before seeding to cells. Surprisingly, the inhibitory effect could not be detected with immunoblotting in the pre-treated cells group (Figure 2a), which was consistent with results of viral nucleic acid and titer determination (Figure 2b,c). Meanwhile, the prominent antiviral effect could be displayed in the pre-treated virions group with high EGCG concentration (100 μM), but non-sensitive to the low EGCG concentration (10 μM) pretreatment (Figure 2a–c), suggesting the compound might interact with virions. To rule out the possibility that the EGCG might also act on some cellular factors activated only in the presence of virions or some intracellular signaling pathways induced by the virus stimulation, we performed the dilution assay. Briefly, virus samples were pretreated at a high EGCG concentration (100 μM), and thereafter diluted 10-fold before being inoculated on the cells, ensuring the cells were treated with compound at 10-fold lower concentrations (10 μM) than the viral particles; in such a way, the inhibitory phenomenon would disappear if the compound really targeted host cells only in the presence of virions. However, the antiviral effect could

still be detected with immunoblotting, qPCR and virus titer determination (Figure 2d–f). Considering that the pretreatment of cells with 10 μM EGCG could not trigger antiviral activity (Figure 2a–c), this inhibition was attributed to the effect of 100 μM EGCG on virions, further supporting that EGCG performed an antiviral effect via directly targeting virions.

**Figure 1.** EGCG exhibits an anti-viral effect on PCV2 infection. (**a**) Molecular structure of EGCG and EC. (**b**) Cytotoxicity assay of soluble EGCG and EC. PK-15 cells were added with a concentration of 0, 10, 50, 100 and 200 μM EGCG or EC and cultured for 24 h, 72 h and 120 h, respectively. Cell vitality was detected by CCK8 assay and calculated as (A450compound / A450mock) × 100%. (**c**) One-step growth curve of PCV2 with EGCG treatment. PK-15 cells were inoculated with PCV2 at MOI = 1.0 and cultured with 100 μM EGCG or EC treatment. TCID50 at various time points were determined by IFA and calculated according to the Reed-Muench method. (**d**–**f**) EGCG inhibited the infectivity of PCV2. PK-15 cells were inoculated with PCV2 at MOI=1.0 with EGCG or EC addition. (**d**) The expression inhibition of PCV2 capsid protein was detected by immunoblotting at 72 hpi. (**e**) PCV2 genome copies were measured by real-time PCR. (**f**) Indirect IFA detection of PCV2 infected cells. (**g**) Dose response curve and EC50 of the EGCG's antiviral effect on PCV2. The mean ± SD of three independent experiments was compared using Student's *t*-test (n.s., not significant; \* *p* < 0.05; \*\* *p* < 0.01).

**Figure 2.** EGCG inhibits the PCV2 infectivity by directly targeting virions. (**a**–**c**) Cells were pre-exposed to 10 μM or 100 μM EGCG for 60 min at 37 ◦C, then infected with PCV2 at MOI = 1.0 for 1 h, alternatively, virus was pre-mixed with EGCG under the same condition before being added to the cells, then the cells were cultured without EGCG. At 72 hpi PCV2 infectivity was assessed by viral protein expression (**a**), viral genome copies (**b**), and virus titers (**c**). (**d**–**f**) Virions were pretreated with 100 μM EGCG and thereafter diluted 10-fold prior to infecting cells, which were cultured without EGCG and tested with immunoblotting (**d**), qPCR (**e**), and virus titers determination (**f**). Three independent experiments were performed and the results were compared using Student's *t*-test (n.s., not significant; \*\* *p* < 0.01).
