2.1 Virus detection, identification, isolation, and growth
kinetics
Vesicular fluids and tissue samples were collected from a pig farm in
Guangxi Province in the southwest of China. Total RNA was extracted from
each sample using TRIzol reagent in accordance with the manufacturer’s
instructions (Sangon, China). Next, we synthesized cDNA from the total
RNA using random primers (primer 9) and M-MLV reverse transcriptase
(Takara, China), as described by the manufacturer’s guidelines. We also
designed a range of specific primers to amplify different viruses (FMDV,
SVDV, VESV, VSV and SVV); these primers were designed by Primer Premier
5 (Table S1). Using baby hamster kidney (BHK-21) cell line, SVV was
isolated as described previously
(Saeng-Chuto et al., 2018b).
BHK-21 cells were seeded into
6-well plates and infected with SVV at a multiplicity of infection (MOI)
of 0.1. The cells were then cultured until 14 hours post-infection
(HPI). Cells were then fixed with 4% polyformaldehyde (Beyotime) for 20
min, permeabilized with 0.1% Triton X-100 for 7 min, and blocked with
5% skimmed milk (diluted in PBST) for 2 hours. The cells were then
incubated for 2 h with a primary mouse-derived anti-VP1 polyclonal
antibody (1:500; synthesized in-house). Next, the cells were washed
three times in PBST and then incubated with a FITC-labeled goat
anti-mouse IgG (H+L) secondary antibody (1:2000) for 30 min. Finally,
cells were washed five times in PBST and analyzed under a fluorescent
microscope.
In a second experiment, we seeded BHK-21 cells into 6-well plates and
infected the cells with SVV (0.1 MOI). Cell culture supernatants were
then harvested at 6, 12, 24, 36, 48, and 60 HPI. We then measured the
titer of virus in each sample of supernatant by titration. Results are
reported as 50% tissue culture infective doses per milliliter
(TCID50/ml), in accordance with the Reed-Muench method
(Chen et al., 2016). Data were analyzed by
GraphPad Prism (version 6.0) software (GraphPad Software Inc., La Jolla,
CA).