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.