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).