Development of Multiplex Real-Time PCR- and Antibody Reagents for the Detection of Swine Acute Diarrhea Syndrome Coronavirus (SADS-CoV)
Principal Investigator: Diego Diel
DESCRIPTION (provided by applicant):
A new swine coronavirus - related to the bat coronavirus HKU2 - named swine acute diarrhea syndrome coronavirus (SADS-CoV) was recently associated with severe outbreaks of diarrhea with high mortality rates in pigs in China. Given the impact that the closely related porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) had after their introduction in the US, the emergence of SADS-CoV in Asia raises significant concerns to the pork industry in the US. Should SADS-CoV follow the same path that PEDV and PDCoV and be introduced into the US, the industry must be prepared to rapidly implement adequate control strategies to mitigate the impact of the disease to pork producers. One of the first tools to combat emerging infectious disease agents is a diagnostic assay capable of rapidly detecting such pathogens. The overall goal of the proposed project is to develop rapid diagnostic tools that will allow the timely detection of SADS-CoV nucleic acid and/or antigens in clinical samples. The objectives of the proposed study are: Objective 1: To develop and evaluate a real-time multiplex PCR for SADs-CoV, PEDV and PDCoV. This objective is directly aligned with the top research priority listed in the SHIC SADS-CoV diagnostics RFP and will lead to a diagnostic tool that allows rapid detection of SADS-CoV and its differentiation from other endemic SECs including PEDV and PDCoV. This assay would provide an invaluable tool to veterinarians and producers and could be immediately used by diagnostic laboratories for the detection of the endemic SECs PEDV and PDCoV and for surveillance for the exotic SADS-CoV. Objective 2. To develop antibody reagents for SADs-CoV. We propose to develop SADS-CoV-specific antibodies (polyclonal and monoclonal) for use in serological- (fluorescent focus neutralization [FFN] and ELISAs) or antigen detection assays (immunohistochemistry [IHC] and immunofluorescence [IFA]). This objective is directly aligned with the second and third research priorities listed in the SHIC SADS-CoV diagnostics RFP and will provide invaluable tools for the detection of SADS-CoV antibodies or for its direct detection in infected tissues or cell culture (for verification of virus isolation). Successful completion of the proposed study will result in a diagnostic tool set to detect SADS-CoV in clinical samples. Availability of a multiplex real-time PCR for SADS-CoV, PEDV and PDCoV will allow precise and rapid diagnosis of specific SEC associated with outbreaks of enteric disease in pigs. Additionally, given that, the SADS-CoV assay will replace TGEV in a pre-optimized and commercially available PEDV/PDCoV/TGEV assay (assay name, Tetracore Inc.), the PCR developed here will be readily available for use by the D-labs in the country and will allow monitoring for SADS-CoV while performing routine diagnostic testing for the endemic PEDV and PDCoV. Antibodies developed in objective 2 will allow the development of serological assays as well as antigen detection assays for SADS-CoV sero-surveillance or yet for the direct detection of the virus.