A Customized Novel Blocking ELISA for Detection of Bat-Origin Swine Acute Diarrhea Syndrome Coronavirus Infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered emerging alphacoronavirus. SADS-CoV shares over 90% genome sequence identity with bat alphacoronavirus HKU2. SADS-CoV was associated with severe diarrhea and high mortality rates in piglets. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. Here, monoclonal antibody (MAb) 6E8 against SADS-CoV N protein accurately recognized SADS-CoV infection. Then, MAb 6E8 was utilized as a blocking antibody to develop blocking ELISA (bELISA). We customized the rN coating antigen with concentration 0.25 µg/mL. According to receiver operator characteristic curve analysis, the cutoff value of the bELISA was determined as 38.19% when the max Youden index was 0.955, and specificity was 100%, and sensitivity was 95.5%. Specificity testing showed that there was no cross-reactivity with other serum positive swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine rotavirus (PoRV), and porcine sapelovirus (PSV). In conclusion, we customized a novel and high-quality blocking ELISA for detection of SADS-CoV infection, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection. IMPORTANCE SADS-CoV was reported to be of high potential for dissemination among various of host species. Accurate serological diagnosis of SADS-CoV infection is key in managing the emerging SADS-CoV. However, thus far there have been no effective antibody-based diagnostic tests for diagnose of SADS-CoV exposure. We customed a novel and high-quality bELISA assay for detection of SADS-CoV N protein antibodies, and the current bELISA will be linked to a clinical and epidemiological assessment of SADS-CoV infection.

Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1 ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. KEY POINTS: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread.

Comprehensive Subcellular Localization of Swine Acute Diarrhea Syndrome Coronavirus Proteins.

Bats are reservoirs for diverse coronaviruses, including swine acute diarrhea syndrome coronavirus (SADS-CoV). SADS-CoV was first identified in diarrheal piglets in 2017. As a novel alphacoronavirus, SADS-CoV shares ~95% identity with bat alphacoronavirus HKU2. SADS-CoV has been reported to have broad cell tropism and inherent potential to cross host species barriers for dissemination. Thus far, no effective antiviral drugs or vaccines are available to treat infections with SADS-CoV. Therefore, knowledge of the protein-coding gene set and a subcellular localization map of SADS-CoV proteins are fundamental first steps in this endeavor. Here, all SADS-CoV genes were cloned separately into Flag-tagged plasmids, and the subcellular localizations of viral proteins, with the exception of nsp11, were detected using confocal microscopy techniques. As a result, nsp1, nsp3-N, nsp4, nsp5, nsp7, nsp8, nsp9, nsp10, nsp14, and nsp15 were localized in the cytoplasm and nuclear spaces, and these viral proteins may perform specific functions in the nucleus. All structural and accessory proteins were mainly localized in the cytoplasm. NS7a and membrane protein M colocalized with the Golgi compartment, and they may regulate the assembly of SADS-CoV virions. Maturation of SADS-CoV may occur in the late endosomes, during which envelope protein E is involved in the assembly and release of the virus. In summary, the present study demonstrates for the first time the location of all the viral proteins of SADS-CoV. These fundamental studies of SADS-CoV will promote studies of basic virology of SADS-CoV and support preventive strategies for animals with infection of SADS-CoV. IMPORTANCE SADS-CoV is the first documented spillover of a bat coronavirus that causes severe diseases in domestic animals. Our study is an in-depth annotation of the newly discovered swine coronavirus SADS-CoV genome and viral protein expression. Systematic subcellular localization of SADS-CoV proteins can have dramatic significance in revealing viral protein biological functions in the subcellular locations. Furthermore, our study promote understanding the fundamental science behind the novel swine coronavirus to pave the way for treatments and cures.

TET2 is required for Type I IFN-mediated inhibition of bat-origin swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered bat-origin coronavirus with fatal pathogenicity for neonatal piglets. There is no vaccine to prevent SADS-CoV infection or clinically approved drugs targeting SADS-CoV. Therefore, unraveling cellular factors that regulate SADS-CoV for cell entry is critical to understanding the viral transmission mechanism and provides a potential therapeutic target for SADS-CoV cure. Here, we showed that Type I interferon (IFN-I) pretreatment potently blocks SADS-CoV entry into cells using lentiviral pseudo-virions as targets whose entry is driven by the SADS-CoV Spike glycoprotein. IFN-I-mediated inhibition of SADS-CoV entry and replication was dramatically impaired in the absence of TET2. These results suggest TET2 is found to serve as a checkpoint of IFN-I-meditated inhibition on the cell entry of SADS-CoV, and our discovery might constitute a novel treatment option to combat against SADS-CoV.