Multiplex Reverse Transcription-PCR for Rapid Differential Detection of Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus, and Porcine Group a Rotavirus

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.

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One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719883834 ◽

2019 ◽

Vol 31 (6) ◽

pp. 909-912 ◽

Cited By ~ 1

Author(s):

Chunyan Jiang ◽

Haijian He ◽

Chaoying Zhang ◽

Xiaoju Zhang ◽

Jianfeng Han ◽

...

Keyword(s):

Reverse Transcription ◽

Porcine Epidemic Diarrhea Virus ◽

Detection Method ◽

Rt Pcr ◽

Diarrhea Virus ◽

Reverse Transcription Pcr ◽

Porcine Sapovirus ◽

One Step ◽

Porcine Epidemic Diarrhea ◽

Porcine Sapelovirus

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 μM for PEDV, 0.15 μM for PSV, and 0.2 μM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR–based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

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