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).