*2.10. Heparin Column Chromatography*

The purified recombinant PCV2 dcapsid were bound to a 5-mL HiTrap™ Heparin-Sepharose HP Column (17040701, GE Healthcare, Los Angeles, CA, USA) in loading buffer (10 mM sodium phosphate, 0.3 M NaCl, pH 7.4) at a flow rate of 1 mL per minute. The column was washed with 10 mL loading buffer to remove excess molecules, and thereafter eluted with soluble heparin, EGCG or EC competitor in loading buffer. The remaining bound protein was ultimately eluted with 3 M NaCl in loading buffer because the electrostatic interactions between Heparin-Sepharose and heparin-binding proteins could be disrupted by high salt concentrations. The original and eluted fractions were diluted to equal volumes, and analyzed using Western immunoblotting with the mAb 5E11 and HRP- goat anti-mouse IgG. The intensities of the immunoreactive protein bands were measured using Image J software (Version 1.52 g, ImageJ, Bethesda, MD, USA).

#### *2.11. Microscale Thermophoresis Assay*

The purified dcapsid proteins were labelled with the Cy5 fluorophore based on the chemical reaction between the 108cysteine of capsid and Cy5-maleimide (PA25031, GE Healthcare Life Sciences). The labelled molecules were separated from the unreacted and excess dyes with a PD Mini-Trap G-25 desalting column, and mixed at 10–50 nM concentration with 2 fold serially dilutions of EGCG, EC or heparin in reaction buffer (150 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 0.05% Tween-20, pH 7.4). The binding reactions were fixed into equal volume and incubated at room temperature for 15 min. The mixtures were then enclosed in standard pretreated glass capillaries and analyzed with MST machine (Monolith NT.115, NanoTemper Technologies, Munich, Bavaria, Germany), and the Kd values of each reaction were computed by NanoTemper Analysis software (Version 2.1, NanoTemper Technologies, Munich, Bavaria, Germany).

#### *2.12. Construction of PCV2 Infectious Clone and Virus Rescue*

The whole genome of PCV2 strain HZ0201 was cloned and inserted into pMD18-T vector (Takara, Tokyo, Japan). Site-specific mutagenesis was performed using PCR to construct the mutants. The linear genomes of the WT and mutants were extracted, digested by *Eco* RI restriction enzyme (NEB), cyclized

by T4 DNA ligase (Takara), and transfected into PK15 cells for virus rescue with the jetPRIME® in vitro transfection reagent (Polyplus-transfection, New York, NY, USA). The transfected cells were maintained for 72 h, then continuously passaged and cultured. The rescued virus samples were harvested and inoculated to PK15 cells and the infectivity was evaluated with immunoblotting in an indirect immunofluorescence assay. The virus titers were calculated according to the Reed-Muench method.
