Fig 5. Synthetic DVN peptides significantly inhibit RHDV internalization.

(A) RK-13 cells were pretreated with DVN or control peptides at a concentration of 100 μg/mL for 6 h. Then, the attachment and internalization assays were performed with mRHDV-infected cells (MOI = 1) and the cells harvested for RNA analysis. The percentage of RHDV VP60 copies was calculated as a ratio to the value obtained from untreated RK-13 cells. The control peptide was employed as a negative control. (B) Kinetics of mRHDV internalization into RK-13 cells. Internalization of mRHDV (MOI = 1) or mRHDV-FITC into RK-13 cells was assessed by mRHDV incubation for 10, 30, and 60 min at 37°C. The amount of internalized virus was determined by qRT-PCR and expressed as VP60 RNA copies per 100,000 of GADPH. (C) RK-13 cells were pretreated with the DVN and control peptides at concentrations of 5, 10, 20, 40, 80, and 100 μg/mL for 6 h, respectively. mRHDV-FITC-infected cells (MOI = 1) were then used for internalization assays and harvested for flow cytometry analyses. The control peptide was employed as a negative control. The data are representative of the means and standard deviations (error bars) of three samples per group (**p < 0.01). All experiments were repeated at least three times with consistent results. (D) RK-13 cells were pretreated with the DVN peptides (100 μg) or anti-NCL mAb (50 μg) for 6 h. Then, the cells were used for the internalization assay with mRHDV-FITC (MOI = 1) and subsequent immunofluorescence analysis. The RHDV virion is green and cell nucleus in blue. Mouse IgG and control peptides were employed as negative controls.