Abstract
Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.
Highlights
Porcine epidemic diarrhea virus (PEDV), a member of swine enteric coronaviruses (CoVs), causes diarrhea, vomiting, anorexia, and dehydration in neonatal piglets [1]
We found that the growth kinetics of rPEDVNLuc was comparable to that of wild-type PEDV, and the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells
The reverse genetics system for the PEDV PC22A strain has been successfully developed based on the unidirectional assembly of a full-length genome cDNA from a set of consecutive shorter cDNA fragments [26]
Summary
Porcine epidemic diarrhea virus (PEDV), a member of swine enteric coronaviruses (CoVs), causes diarrhea, vomiting, anorexia, and dehydration in neonatal piglets [1]. Virulent PEDV variants (GII) have caused epidemic outbreaks, resulting in high morbidity and mortality rates in newborn piglets [8]. To prevent and control the outbreaks, several commercial vaccines, such as inactivated, live-attenuated, and S subunit vaccines, have been approved for use against PEDV in China, South Korea, and the USA [10,11,12]. These vaccines cannot provide adequate protective immunity against the prevalent PEDV strains, and large-scale outbreaks of PEDV still occur and frequently recur [13]. The development of antiviral agents is urgently required to prevent PEDV infection from compensating for the vaccine’s effect
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