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.