Fig.3. Effect of hesperetin on BPXV attachment, entry and budding
(A) Attachment assay. Vero cells, in triplicates, were treated with hesperetin or DMSO for 1 h, followed by BPXV infection at an MOI of 5 for 1 h at 4°C. Thereafter, the cells were washed with PBS to remove the unattached virus, and cell lysates were prepared by the rapid freeze-thaw cycles. The viral titers in cell lysates were quantified by plaque assay. (B) Entry assay. Pre-chilled confluent monolayers of Vero cells were infected with BPXV at 5 MOI for 1 hr at 4°C to permit attachment, followed by washing with PBS and addition of serum-free DMEM containing hesperetin or vehicle control, and incubation at 37°C for 1 h (to allow virus entry). At 1hpi, cells were washed with PBS and supplemented with serum free DMEM without drug. The infectious virus particles released in the supernatant at 48 hpi were titrated by plaque assay. (C) Virus release assay. Vero cells, in triplicates, were infected with BPXV at MOI of 5 for 1 h, followed by washing with PBS and the addition of fresh medium without any inhibitor. The cells were incubated at 37°C. At 36 hpi, when BPXV presumably starts releasing from the infected cells, hesperetin or DMSO were applied. The supernatants were harvested at the indicated time points (0.5 h and 4 h) following the addition of hesperetin and quantified by plaque assay. Values are representative of three independent experiments. Error bars indicate SD. Pair-wise statistical comparisons were performed using the Student’s t test. ns = non-significant difference.
Fig. 4. Effect of hesperetin on BPXV mRNA, DNA and protein synthesis
Confluent monolayers of Vero cells, in triplicates. were infected with BPXV at an MOI of 5 for 1 h, followed by washing with PBS and addition of fresh DMEM. Hesperetin or DMSO were added at 3 hpi. Cells were scraped at 24 hpi for quantitation of viral DNA, RNA and proteins (A) mRNA synthesis: Total RNA was isolated from the cells, and cDNA was prepared using oligo (dT) primers. The levels of BPXV C18L gene in hesperetin-treated and DMSO-treated cells were quantified using qRT-PCR. (B) Genome synthesis : DNA was isolated from hesperetin-treated or DMSO-treated cells and the BPXVC18L gene was quantified by qRT-PCR. (C) Protein synthesis: Cell lysates were prepared in RIPA buffer and subjected to western blot analysis by using anti-BPXV hyper-immune serum (upper panel) or β-actin (housekeeping control) antibody (lower panel). Values are representative of three independent separate experiments. Error bars indicate SD. Pair-wise statistical comparisons were performed using the Student’s t-test (** = P<0.01, *** = P<0.001).