3.1 Isolation and purification of the SVV strain
RT-PCR was not designated to detect Teschovirus A, Sapelovirus A, Enterovirus G, FMD, VS, and SVD virus in the vesicular outbreak clinical samples. The expected 542-bp product of SSV was detected in all vesicular fluid swabs from the pigs. After two sequential passages in BHK-21 cells, SVV was successfully isolated from the vesicular fluid samples. A cytopathic effect was evident 2 days following the addition of SVV to BHK-21 cell cultures. The changes were not observed in the NC (Figure 1A). RT-PCR detected only SSV. FMD, VS, SVD, Teschovirus A, Sapelovirus A, and Enterovirus G were not detected in the infected BHK-21 cells. A one-step growth curve of SVV-CH-09-2018 in BHK-21 cells was performed at a multiplicity of infection of 0.1 and 0.5. The infected cells were collected at 0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h post-infection (hpi) for determination of TCID50. The virus began to quickly replicate at 4 hpi, with the highest titers at 32 hpi. The maximum viral titer was 2.59×109 at 32 hpi (Figure 1B). At 24 hpi BHK-21 cells were examined by electron microscopy. The virions observed were round with a diameter of approximately 30 nm (Figure 1A).
3.2SVV-CH-09-2018 sequence and evolutionary analysis
An evolutionary tree was drawn and analyzed using MEGA7.0 and OMICSTUDIO evolutionary tree software. A genome schema map (Figure 2) was constructed based on prototype SVV-CH-09-2018 isolated by our lab. The general structure of the genome included leader protein, 5’ and 3’ UTRs, P1 region proteins (capsid proteins), P2 region proteins (nonstructural proteins), and P3 region proteins (nonstructural proteins). The evolutionary tree revealed that SVV-CH-09-2018 had the typical L-4 genome layout of picornaviruses. A previous phylogenetic analysis showed that SVV strains could also be divided into four branches. The SVV-CH-09-2018 strain belonged to clade III; it shared the highest homology with the American Senecavirus A strain HB-CH-2016 (GenBank, KX377924.1; 99.66%) and the Senecavirus A strain CH-01-2015 isolated in China (GenBank, KT321458.1; 99.67%).
The evolutionary tree revealed the presence of Seneca genes from isolates from the United States, China, Canada, Vietnam, Colombia, and Brazil, indicating the global distribution of the virus. The relationship of various strains isolated from different countries is indicated in Figure 2A with different colors used to denote different strains. The spread of global SVV genomes was evident, with three major evolutionary clusters: United States, China, and Canada-like strain clusters. To date, more than half of the SVVs in China was similar with SVV from the US, and a part of the SVVs in China were from the US and Canada strains. To shed more light on how the Seneca virus is spreading globally, we compiled an SSV chronology of landmark incidents. The chronology was combined with the evolutionary tree to facilitate the analysis of the evolution and prevalence of the Seneca molecular epidemiology of SVV. As shown in Figure 2 and Table 2, the strains from different locations or dates, were separated, which implies that the geographic distribution and infectious host may contribute to the codon usage pattern in the evolution of SVA. The geographic distribution and host are the two main mutational-pressures of natural selection. The origin of the strain that was the focus of the present study, was not clear. The collective data favored the view that the pathogenicity of SSV has increased since 2015, with increased morbidity and mortality rates associated with the infections. The strain that we have examined has been continuously evolving in China for some time. Further investigations into the evolution and prevalence of SVV, including identification of the pathogenesis and molecular epidemiology, are urgently needed.