Development and validation of molecular diagnostic assays for emerging swine and human coronaviruses

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Zhu, Jinhui
Major Professor
Zhang, Jianqiang
Wang, Chong
Gauger, Phillip C
Main, Rodger G
Committee Member
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Veterinary Diagnostic and Production Animal Medicine
Coronaviruses (CoVs) are a group of highly diverse, enveloped, positive-sense and single-stranded RNA viruses in the order Nidovirales and the family Coronaviridae. Coronaviruses have a wide range of host species and can infect humans, mammal animals, and various birds. New coronaviruses and/or CoV variants have periodically emerged or re-emerged and have significant impacts on the health of humans and animals. Sensitive, specific, and rapid methods for detecting the emerging coronaviruses are critical for control the spread of these viruses. In pigs, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are important enteric pathogens. Some of them (e.g. a highly virulent PEDV variant, PDCoV and SADS-CoV) emerged or re-emerged relatively recently. Since these swine enteric CoVs cause similar clinical signs, lab-based differential diagnosis is necessary. Reverse transcription real-time PCR (RT-rtPCR) is a sensitive and specific assay and has been widely used for detection of many RNA viruses, including swine enteric coronaviruses. However, there are concerns with some previously described PCR assays that cross-reacted with other pathogens such as PRCV and sparrow deltacoronaviruses. Thus, in the first study, we developed and evaluated multiple singleplex RT-rtPCR assays for each swine enteric CoV and then selected one singleplex RT-rtPCR each for the detection of PEDV, PDCoV, TGEV, and SADS-CoV, together with a singleplex PCR for an exogenous internal positive control (XIPC) to develop the PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex RT-rtPCR. The 5-plex PCR had excellent analytical specificity, analytical sensitivity, repeatability, and diagnostic performance. After thorough validation, the 5-plex PCR was used to screen 1,807 clinical samples collected from U.S. swine during 2019–2021 to investigate whether SADS-CoV was present in the U.S. and also to determine the detection frequency of swine enteric CoVs in U.S. swine. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. The emergence of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused unprecedented impacts on the global public health and many other aspects. Meanwhile, many types of methods have been developed to detect SARS-CoV-2; this has greatly advanced the technologies in the diagnostic field. However, point-of-care (POC) testing, which is needed in some resource-limited areas and/or in nursing homes or long-term care facilities, is lacking. In the second study, we describe development and validation of a sample-in-result-out POCKIT Central PCR system for detecting SARS-CoV-2 in comparison with a commercial reference real-time RT-PCR assay (TaqPath COVID-19 Combo Kit). Both assays were able to detect various SARS-CoV-2 strains including some variants. The limit of detection for both assays were determined by testing serial dilutions of SARS-CoV-2 USA-WA1/2020 isolate. Subsequently, 183 clinical samples were tested by both assays and the diagnostic sensitivity, specificity and agreement were great. The compact sample-to-result POCKIT Central SARS-CoV-2 PCR system (combing nucleic acid extraction and PCR amplification into one instrument) is a simplified and efficient point-of-care tool for SARS-CoV-2 detection. 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. Although our data in the current study indicate that there was no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance and be better prepared to respond to any future introduction. The 5-plex PCR will be valuable for detecting and differentiating these CoVs not only in swine samples but also in other host species samples as some of these CoVs can cause cross-species infection. In addition, a novel, automated sample-to-result POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR assay that combined nucleic acid extraction and PCR reaction in one instrument was described in this thesis, which had comparable diagnostic accuracy to the reference real-time RT-PCR, especially for samples with Ct < 30. The compact POCKIT Central SARS-CoV-2 PCR system can be easily set up and implemented in some areas where high-throughput testing of a large number of samples is not needed. In addition, this platform can be readily adapted to detect other human and animal viruses.