One-Step Assembly of a Porcine Epidemic Diarrhea Virus Infectious cDNA Clone by Homologous Recombination in Yeast: Rapid Manipulation of Viral Genome With CRISPR/Cas9 Gene-Editing Technology

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.

Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection

“…Then, the cell lines were exposed to a reporter virus (rPEDV-EGFP) engineered to express enhanced GFP (EGFP) during replication. This reporter virus replicated well in Vero CCL-81 cells [16]. We repeatedly detected some EGFP expression in HepG2, Hep3B217, and SNU387 cells as well as the Huh7 cells, whereas there was no GFP expressed in Li7, SNU182, and SNU761 cells (Figure 1C).…”

“…The PEDV-SD strain was isolated and stored in our laboratory. Recombinant rPEDV-EGFP was produced by transfecting infectious cDNA clones into BHK21 cells as previously described [16]. The viruses were titrated on the Vero CCL-81 cells by TCID 50 .…”

Reverse Genetics Systems for Emerging and Re-Emerging Swine Coronaviruses and Applications

“…Rapid assembly of the whole CoV genome in one time is the main advantage for yeast-based method. However, this method needs extract yeast plasmid from yeast culture; yeast plasmid extraction is more complicated, time-consuming, and expensive than bacterial plasmid extraction [42].…”

“…An advantage of using the yeast system for constructing a CoV reverse genetics system is that > 10 cDNA fragments can be ligated simultaneously such that the total fragment length exceeds 110 kb. In 2022, Zhou et al reported the construction of a PEDV reverse genetics system using the yeast system [42]. The sequences of CMV promoter, HDVr, and BGH termination signal sequence were cloned into pYES1L vector to obtain the vector pYES1L-CMV-HDVrbz-BGH.…”

Development of a highly sensitive Gaussia luciferase immunoprecipitation assay for the detection of antibodies against African swine fever virus

“…The gaussia luciferase assay was performed with coelenterazine h substrate (Maokang Biochem, Shanghai, China) as described previously ( Zhou et al., 2022 ). The substrate was prepared by diluting coelenterazine h stock to 20 μM with PBS supplemented with 5 mM NaCl, pH 7.2, and incubated at RT in the dark for 30 min.…”