Development and validation of real-time RT-PCR assays for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV) and investigation of its presence in US swine

There are currently six coronaviruses that can infect pigs and these include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (pHEV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). Five of them (PEDV, PDCoV, TGEV, PRCV and pHEV) are endemic in U.S. swine. SADS-CoV has only been reported in China; its presence in the U.S. remained unknown. However, transboundary spread of infectious diseases from countries to countries via known or unknown routes including the potential spread of virus via feed and feed ingredients have occurred in recent years. Therefore, it is critical to conduct ongoing surveillance to monitor the potential emergence of SADS-CoV in U.S. pigs. In addition, PEDV, PDCoV, TGEV and SADS-CoV are all enteric pathogens and cause indistinguishable clinical signs, making differential diagnosis crucial. This study included three objectives. Objective 1: develop and validate a SADS-CoV singleplex reverse transcription real-time PCR (RT-rtPCR). Objective 2: develop and validate a PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex RT-rtPCR for simultaneous detection and differentiation of the four viruses (XIPC serves as an internal positive control for the PCR). Objective 3: investigate if SADS-CoV is present in the U.S. and determine the detection frequency of these swine enteric coronaviruses in U.S. swine.

Four singleplex SADS-CoV RT-rtPCR assays were evaluated and one SADS-CoV PCR was chosen for the 5-plex assay development. Some previously published singleplex PDCoV PCRs cross-reacted with sparrow deltacoronaviruses. In this study, we evaluated three singleplex PDCoV PCRs and selected one PDCoV PCR that did not cross-react with sparrow deltacoronaviruses for developing the PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR assay.

The 5-plex PCR includes primers and probe for an exogenous internal positive control (XIPC) in addition to including primers and probes for PEDV, PDCoV, TGEV, and SADS-CoV. The internal positive control provides an additional quality assurance approach to ensure the accuracy of the PCR results. The 5-plex PCR was optimized and validated thoroughly for its analytical specificity, analytical sensitivity, and diagnostic performance. The 5-plex PCR was proved to have excellent specificity, sensitivity, and diagnostic performances.

Subsequently, the 5-plex PCR was used to determine if SADS-CoV was present in U.S. swine. Based on testing 288 clinical samples archived during 2019-2020 from diarrheic pigs in the U.S. negative for PEDV, PDCoV, TGEV, rotavirus A, B, C and several thousand clinical samples submitted to the ISU VDL during 2019-2021, there was no evidence of SADS-CoV presence in U.S. swine. Among these tested samples, 1,028 samples were found positive for at least one of PEDV, PDCoV and TGEV and the following detection frequency data were obtained. These included (1) 71.2% PEDV positive only, (2) 18.1% PDCoV positive only, (3) 0% TGEV positive only, (4) 10.1% PEDV and PDCoV positive, (5) 0.19% PEDV and TGEV positive, (6) 0% PDCoV and TGEV positive, and (7) 0.39% PEDV, PDCoV, and TGEV positive.

In summary, a specific and sensitive PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex RT-rtPCR assay was developed and thoroughly validated. This 5-plex PCR can simultaneously detect and differentiate PEDV, PDCoV, TGEV and SADS-CoV in one PCR reaction and it is an invaluable tool to determine the presence and frequency of these swine enteric coronaviruses in either single infection or co-infections. Although there is no evidence of SADS-CoV presence in the U.S. now, the availability of the PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR will enable us to conduct ongoing surveillance and thereby we are better prepared to respond to introduction.

In addition to infecting pigs, PDCoV has been reported to cross species to infect calves, chicken embryos and chicken, turkey, and humans. SADS-CoV has also been reported for its broad cross-species tropism of infecting cell lines of various host species and primary human cells. The PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR developed in this study should be able to not only for testing swine samples but also for testing samples from other host species if any of these CoVs are suspected in such samples.

For more information, please contact Dr. Jianqiang Zhang at Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University. E-mail: [email protected]

Key Findings:
• SADS-CoV singelplex real-time RT-PCR assays were developed and validated.
• PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and thoroughly validated. It has excellent sensitivity, specificity, and diagnostic performance.
• There is no evidence of SADS-CoV presence in U.S. swine at this point.
• The PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR is an invaluable tool to conduct ongoing surveillance to determine the presence and frequency of these swine enteric coronaviruses in either single infection or co-infections.
• Some swine coronaviruses such as PDCoV and SADS-CoV could potentially infect other host species. The PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR should be able to not only for testing swine samples but also for testing samples from other host species if any of these CoVs are suspected in such samples.