*3.6. Replacement of Key Amino Acids in Capsid Weakens the Antiviral E*ff*ect of EGCG*

To further confirm the role of the key amino acids in capsid towards the antiviral activity of EGCG, we tried to use virus rescue strategy to generate mutant viruses and evaluate the regulation in EGCG's antiviral activity. The circularized PCV2 genome with R51A, G54A, D70A, R73A, N75A, and D78A mutations were transfected into PK-15 cells, respectively. Also, the genome with the A135A mutation was designated as a positive control. Transfected cells were serially passaged and cultured to harvest the rescued virus particles, which were then inoculated into PK-15 cells to test the infectivity. The results of IFA showed that PCV2 (G54A), PCV2 (D70A), PCV2 (N75A) and PCV2 (D78A) could be detectable at 72 hpi, similar to PCV2 (WT) and PCV2 (A135A) (Figure 7a). The results of immunoblotting also confirmed the capsid expression of these mutant viruses at 72 hpi (Figure 7b). One-step growth curves of PCV2 (G54A), PCV2 (D70A), PCV2 (N75A), and PCV2 (D78A) also displayed a similar replication ability to wild-type PCV2 (A135A) (Figure 7c). Surprisingly, PCV2 (G51A) and PCV2 (G73A) could not be rescued and obtained with the same operation (Figure 7a–c), which might be due to the replacement of these key arginine residues related to HS binding ability would cause the impairment of virus binding to cell surface, which eventually lead to the inability of virions to complete the infection process [18].

We next performed the infectivity assay of mutant viruses with 100 μM EGCG treatment during the whole infective process. The titers of mutant viruses were detected at 72 hpi and the results showed that EGCG could inhibit the infectivity of PCV2 (G54A) and PCV2 (N75A) (Figure 7f,h), similar to its antiviral effect on PCV2 (WT) and PCV2 (A135A) (Figure 7d,e). In contrast, the antiviral effect of EGCG could not be detected during the infection of PCV2 (D70A) and PCV2 (D78A) (Figure 7g,i), These findings are consistent with the results of affinity determinations (Figure 6c,f,g), indicating that ASP70 and ASP78 of capsid are essential for binding of PCV virions to EGCG.

**Figure 7.** Regulation of EGCG antiviral activity by replacement of key amino acids in capsid. (**a**,**b**) The infectivity of rescued PCV2 mutants. The rescued PCV2 mutant virus was inoculated into PK-15 cells at

MOI = 1.0; at 72 hpi the cell samples were detected by IFA (**a**), and the capsid expression was measured by immunoblotting (**b**). (**c**) One-step growth curve of PCV2 mutants. PK-15 cells were inoculated with mutant viruses at MOI = 1.0, TCID50 at various time points were determined by IFA and calculated according to the Reed-Muench method. (**d**–**i**) Antiviral effect of EGCG on PCV2 mutants. PK-15 cells were infected by mutant viruses at MOI = 1.0 with 100 μM EGCG treatment during the whole infective process and the titers were detected by IFA at 72 hpi. Mean ± SD of three independent experiments were statistically analyzed using Student's *t*-test (\* *p* < 0.05; \*\* *p* < 0.01).
