Red recombination enables a wide variety of markerless manipulation of porcine epidemic diarrhea virus genome to generate recombinant virus

Shuonan Pan; Chunxiao Mou; Zhenhai Chen

doi:10.3389/fcimb.2023.1338740

Red recombination enables a wide variety of markerless manipulation of porcine epidemic diarrhea virus genome to generate recombinant virus

Front Cell Infect Microbiol. 2024 Jan 22:13:1338740. doi: 10.3389/fcimb.2023.1338740. eCollection 2023.

Authors

Shuonan Pan¹, Chunxiao Mou^{1

2

3}, Zhenhai Chen^{1

2

4}

Affiliations

¹ College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
² Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, China.
³ Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China.
⁴ Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, China.

Abstract

Porcine epidemic diarrhea virus (PEDV) is a member of the genera Alphacoronavirus that has been associated with acute watery diarrhea and vomiting in swine. Unfortunately, no effective vaccines and antiviral drugs for PEDV are currently available. Reverse genetics systems are crucial tools for these researches. Here, a PEDV full-length cDNA clone was constructed. Furtherly, three PEDV reporter virus plasmids containing red fluorescent protein (RFP), Nano luciferase (Nluc), or green fluorescence protein (GFP) were generated using Red recombination with the GS1783 E. coli strain. These reporter-expressing recombinant (r) PEDVs showed similar growth properties to the rPEDV, and the foreign genes were stable to culture up to P9 in Vero cells. Using the Nluc-expressing rPEDV, the replication of PEDV was easily quantified, and a platform for rapid anti-PEDV drug screening was constructed. Among the three drugs, Bergenin, Umifenovir hydrochloride (Arbidol), and Ganoderma lucidum triterpenoids (GLTs), we found that GLTs inhibited PEDV replication mainly after the stage of virus "Entry". Overall, this study will broaden insight into the method for manipulating the PEDV genome and provide a powerful tool for screening anti-PEDV agents.

Keywords: ORF3; RED recombination; antiviral; porcine epidemic diarrhea virus; reporter virus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chlorocebus aethiops
Coronavirus Infections* / veterinary
Diarrhea
Escherichia coli
Porcine epidemic diarrhea virus* / genetics
Recombination, Genetic
Swine
Swine Diseases*
Vero Cells

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province [CX (21) 2014], Guangxi Key Research and Development Plan (Grant No. AB21238003), the 111 Project D18007, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) to ZC. It was also supported by the Open Project Program of Jiangsu Key Laboratory of Zoonosis (No. R2107) to CM.