Cell reagents and virus isolation
Baby hamster kidney 21
(BHK-21)
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified incubator in a
5% CO2 atmosphere. The cells were used for the
proliferation of SVV recovered from tissue samples of infected piglets
on a farm in Heilongjiang Province. SVV viral RNA was extracted from
fecal samples and was converted to cDNA by using reverse transcriptase
HiScript®Q RT SuperMix (+gDNA wiper) and synthetic cDNA primers (Vazyme,
Nanjing, China) (J. Zhang et al., 2019). cDNA was used as a follow-up
template for PCR analysis of SVV-specific primers. The purified PCR
products were sequenced (GenScript, Nanjing, China). The SVV-1/2 primer
was used to amplify the VP1 of SVV. Other vesicular disease viruses,
such as foot-and-mouth disease virus (FMDV, serotypes Asia 1, O, and A),
Vesicular stomatitis virus (VSV), swine vesicular disease virus
(SVDV),
and Vesicular exanthema of swine virus (VESV) were amplified using
RT-PCR. The virus isolation method was used with BHK-21 cells as
previously reported(Leme et al., 2015). The cells were incubated for 2
days until a cytopathic effect (CPE) was observed(Yang et al., 2018)
(Figure 1A). Cells displaying CPE were tested for SVV by RT-PCR as
described above. The isolated strain was designated as SVV-CH-09-2018.
SVV-CH-09-2018 was added to BHK-21
cells at a multiplicity of infection of 0.5 and 1 for one-step growth
curves (Fernandes et al., 2018) (Figure 1B)