Discussion
Since SADS-CoV was first reported in pigs in early February 2017 in
Guangdong, China (P. Zhou et al., 2018),
this novel swine enteric CoV has been widely detected in areas of
southern China, including Guangdong and Fujian
(K. Li et al., 2018;
L. Zhou, Li, et al., 2019), results in
significant economic losses to the pig industry. Vaccines are essential
for the prevention and control of porcine enteric CoVs. Serological
methods are helpful to evaluate the immune effect of vaccines and
monitor the prevalence of viruses. Currently there are no suitable
serological method to assess the infection of SADV-CoV and effectiveness
of vaccines. In the present study, an indirect ELISA method using a
recombinant S protein from SADS-CoV was developed and utilized this
ELISA to investigate the prevalence rates of SADS-CoV in the pig farms
of China.
The basic principle of ELISA is based on the specific reaction between
antigen and antibody. Therefore, the key to establish a specific ELISA
method for detecting virus antibodies is to select the appropriate
coating antigen. S protein of CoVs has higher antigenicity and persist
longer in vivo (Knuchel et al.,
1992). In addition, the S protein from SADS-CoV has low homology
(22.4%-23.9%) with the S proteins from TGEV, PEDV and PDCoV,
indicating that there may be no cross-reaction of antibodies against
SADS-CoV S protein. These informations prompted us to select the
expression and purification of S protein from SADS-CoV as the coating
antigen. Prokaryotic and eukaryotic are two common protein express
systems (Verma, Boleti, & George, 1998).
The prokaryotic expression mainly depends on the expression system ofEscherichia coli (E. coli ), which is characterized by high
expression of exogenous proteins but no capable of glycosylating
proteins (Verma et al., 1998). On the
contrary, the eukaryotic expression systems like baculovirus to
express exogenous proteins can retain the functional activity of viral
proteins (Verma et al., 1998). Our
previous study found that the PEDV S protein expressed by E. coliimmunized mice could not induce the body to produce neutralizing
antibodies (data not shown), while the expression of Infectious
bronchitis virus (IBV) S1 protein by baculovirus could provide a
protective immune response in chickens
(Yin et al., 2016), indicating that loss
of glycosylation of S protein expressed by E. coli might affect
its immunogenicity. We also confirmed the presence of glycosylation of S
protein from SADS-CoV by using O -Glycosidase &α2-3,6,8,9
Neuraminidase (data not shown). Therefore, the S protein from SADS-CoV
was expressed by baculovirus -HEK 293F cells expression system in
this study, and finally obtained the S protein with high purity and good
immunogenicity that met the coating antigen requirements of establishing
ELISA (Fig 2). We further successfully established a specific indirect
ELISA method for the detection of SADS-CoV antibodies by ELISA reaction
conditions optimizing, threshold determination, repeatability and
specificity analysis. IFA is another common serological method, which is
also widely used to identify viruses (Xu
et al., 2018). To evaluate the practicality of the S-iELISA, we used
IPI-FX, a stable porcine ileum epithelial cell line
(X. Wang et al., 2019), to perform IFA to
test the same samples tested by S-iELISA. We did not use Vero cells for
IFA due to some pig virus vaccines like PEDV vaccine were made with Vero
cells (Won et al., 2019), and antibodies
against Vero cells might exist in the pig serum, leading to false
positive results. Our results showed that the overall coincidence rate
between IFA and S-iELISA was 97.3% when IFA as standard evaluation
method, indicating that S-iELISA can accurately detect whether the body
is infected with SADS-CoV. It is known that the S protein of CoVs plays
an important role in virus attachment and entry, and induction of
neutralizing antibodies in vivo(Wen et al., 2018). The titer of
neutralizing antibodies often indicates the protection rate
(Q. Li et al., 2018), but the detection of
neutralizing antibody is time-consuming. Combining virus neutralization
results with 7 different OD450nm values sera, we found
that OD450nm values obtained by S-iELISA were positively
correlated with SADS-CoV neutralization. This will help to assess the
protective effect of the vaccines, but more samples need to be tested to
reinforce this conclusion.
Pathogens are detected mainly by nucleic acids and specific antibodies
(Thachil, Gerber, Xiao, Huang, &
Opriessnig, 2015; Xu et al., 2018). A
positive test for enterovirus nucleic acid in diarrhea samples usually
indicated that the virus has infected the body, but sometimes some hosts
infected with the viruses are in a non-detoxification state or the
target organs are not easy to collect, which required serological tests
compensate for nucleic acids test at this time. In addition, serological
tests can also track whether a healthy body has been infected with
pathogen, which could help choose the right time to immunize vaccines.
Although only Guangdong and Fujian reported SADS-CoV infection so far
(K. Li et al., 2018;
L. Zhou, Li, et al., 2019), we used
S-iELSA to test the pig sera from eleven different provinces in China
and confirmed that up to 81.7% of the sera were positive, indicating
that these places had existed SADS-CoV infection and certain measures
should be taken to prevent the SADS-CoV outbreak. In addition, PEDV,
TGEV, PDCoV were also tested by ELISA showed that these viruses all had
different degrees of infection in China, and there were co-infections of
multiple viruses, which there is a risk of outbreak, indicating that the
importance of virus surveillance. Together, all these results confirmed
that indirect ELISA for SADS-CoV
was established in this study is
specific, sensitive, repeatable and can be applied for detection the
SADS-CoV infection in swine industry. However, there are still several
important questions remain for us to do. For instance, how to improve
the expression of S protein in baculovirus -HEK 293F cells
expression system? Whether the specificity of detection can be improved
by selecting part of S protein as coating antigen? Elucidation of these
questions will help us to develop a specific ELISA method to detect
SADS-CoV infection. In addition, IgA induced by mucosal immunity play an
key role against enteroviruses (Wen et
al., 2018), indicating that further development of an ELISA method
based on S protein to detect IgA will help to more accurate evaluate of
the protection of vaccines.
In conclusion, an indirect ELISA method with strong specificity, high
sensitivity and good repeatability was established in the present study
by using a recombinant S protein from SADS-CoV, which might facilitate
the development of a reliable tool for the large-scale detection of
SADS-CoV in pig farm and evaluate the effectiveness of vaccines.