*2.6. Infectivity Assay*

PCV2 samples were pretreated with chemical compound at 37 ◦C for 10 min, then inoculated into near-confluent PK-15 cell monolayers at a MOI = 1.0 at 37 ◦C. Virus inoculum was removed after 1 h and the infected cells were maintained in medium containing the compound. Virus infectivity was assessed by titer determination at various time points. Meanwhile, at 72 hpi the virions in the infected cells were detected by IFA with mAb 5E11 FITC-conjugated goat anti-mouse IgG; expression level of the PCV2 capsid protein was measured by western immunoblotting using 5E11 antibody and HRP- goat anti-mouse IgG; copy numbers of the viral genome were determined with quantitative real-time PCR (qRT-PCR) with forward primer (TACTGCTGTGAGTACCTTGTTGGA) and reverse primer (TCTGCATTTTCCCGCTCACT) targeted to the 359~483 bp sequence (124 bps) of PCV2 *Rep* gene, and the probe was designed as 'FAM-AGTCTGGTGACCGTTGTTGCAGAGCAGCAC-BHQ'. The pMD18-*Rep* plasmid was serially diluted 10-fold to 103~107 to generate the standard curve and the copy numbers of samples were quantified using absolute quantification method [35].
