Abstract
Porcine epidemic diarrhea virus (PEDV), a swine enteric coronavirus causing acute diarrhea in piglets, is one of the major threatens to the pork industry globally. Reverse genetics is a valuable tool for the virological study and vaccine development for coronaviruses. Due to the large size and unstable problem in Escherichia coli of coronavirus genome, construction and manipulation of reverse genetics system for coronaviruses remain laborious and time-consuming. In this study, a reverse genetics system of the genotype II PEDV strain HM was generated using the transformation-associated recombination (TAR) technology in yeast within 1 week. The rescued virus (rPEDV) exhibited similar growth properties to the wild-type virus in vitro. With this PEDV infectious cDNA clone, CRISPR/Cas9 technology and homologous recombination were combined to generate a recombinant virus rPEDV-EGFP in which the ORF3 gene was swapped with an EGFP gene. The reporter virus displayed similar growth properties to the parental virus rPEDV and remained stable during serial passage in vitro. Of note, the strategies of construction and manipulation of PEDV infectious cDNA clone are extremely simple and efficient, which could be applied for other RNA viruses and DNA viruses.
Highlights
Porcine epidemic diarrhea (PED) is a swine enteric disease caused by the porcine epidemic diarrhea virus (PEDV)
We further conducted a phylogenetic analysis with this genome and 25 representative Porcine epidemic diarrhea virus (PEDV) genomes downloaded from the GenBank database using MEGAX (Kumar et al, 2018)
Based on the phylogenetic tree generated with the neighbor-joining method, PEDV strain HM was clustered with genotype II (GII) PEDV strains (Figure 1B) which are new PEDV variants and shared 98.2% sequence identity with AJ1102 strain which was isolated from a neonatal piglet with acute diarrhea in China in 2011 (Bi et al, 2012)
Summary
Porcine epidemic diarrhea (PED) is a swine enteric disease caused by the porcine epidemic diarrhea virus (PEDV). It was first reported in the United Kingdom and Belgium in the 1970s (Wood, 1977) and spread to European and Asian countries before 2010 (Song and Park, 2012). The complete genome of PEDV encodes at least seven open reading frames (ORFs), including ORF1a, ORF1b, and ORF2–6. Two polyproteins encoded by ORF1a and ORF1b were further cleaved by viral proteases into 16 non-structural proteins (nsps), nsp1-nsp. ORF3 was demonstrated to be non-essential for PEDV replication and can be replaced for the expression of reporter genes (Wang et al, 2012; Beall et al, 2016; Wongthida et al, 2017; Peng et al, 2020)
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