Construction And Application Of A Porcine Epidemic Diarrhea Virus Reverse Genetics Via Yeast Transformation-Associated Recombination

Porcine epidemic diarrhea(PED)is a highly contagious swine disease that mainly causes clinical symptoms such as acute diarrhea,vomiting,and enteritis in piglets.The disease is one of the major threats to the global swine industry with high morbidity and mortality,especially in newborn piglets.A reverse genetics system is an important tool for virological research and vaccine development of coronaviruses.Due to the large size of coronavirus genomes and their instability in Escherichia coli,the construction and manipulation of coronaviruses’ infectious cDNA clones remain laborious and time-consuming.The purpose of this study is to establish a reverse genetics system based on yeast transformation-associated recombination and rapidly manipulate the viral genome through CRISPR/Cas9 gene-editing technology and homologous recombination in vitro.In addition,PEDV reporter viruses were generated and applied for the antiviral drug screening assay.Firstly,to overcome the issues in constructing a full-length cDNA clone of coronaviruses,we employed the transformation-associated(TAR)recombination technology to establish a reverse genetics system of genotype II PEDV strain HM in yeast within 1 week.The rescued virus(rPEDV)exhibited similar growth properties to the wild-type(WT)virus in vitro.With this infectious cDNA clone,CRISPR/Cas9 technology and homologous recombination were combined to generate a recombinant virus rPEDV-EGFP in which the ORF3 gene was replaced 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 this infectious cDNA clone are extremely simple and efficient,which could be applied to other RNA viruses and DNA viruses.Secondly,the reporter virus is of great value for research on antiviral drug screening.The low expression level of EGFP expressed by rPEDV-EGFP has hampered its application in antiviral drug screening.To improve EGFP expression by reporter virus,we provided an alternative strategy to construct a PEDV reporter virus expressing an EGFP.In this strategy,an EGFP gene followed by Thesea asigna virus 2A(T2A)was introduced to the 5’ terminus of the nucleocapsid(N)gene.Based on this design,during the infection of this reporter virus,EGFP and N will be encoded by a subgenomic RNA and separated via a noncanonical translational mechanism of ribosome skipping.The rescued rPEDV-EGFPN exhibited similar growth properties to WT virus and rPEDV-EGFP and remained stable for at least 10 passages in vitro.In comparison with rPEDV-EGFP,rPEDV-EGFPN expressed a significantly high level of EGFP,suggesting that EGFP could serve as an indicator of PEDV infection in vitro.Using the rPEDV-EGFPN,we developed an antiviral drug screening platform for PEDV and confirmed the inhibitory effects of GC376 and Melatonin on PEDV infection which has been reported previously.We demonstrate the antiviral activity of Allicin in PEDV-infected cell cultures.In summary,we established a reverse genetics system for the GⅡ PEDV variant using the TAR technology in yeast.Using the CRISPR/Cas9 gene-editing technology,we provided two strategies to construct PEDV reporter viruses.Furthermore,an antiviral drug screening assay against PEDV was established using the reporter virus,and the inhibitory effect of Allicin on PEDV was confirmed.