Rescue Of Porcine Epidemic Diarrhea Virus Carrying Different ORF3 Genes And Study Of Their Proliferation Characteristics On VERO Cells

Porcine epidemic diarrhea?PED?caused by porcine epidemic diarrhea virus?PEDV?is an acute intestinal infectious disease of pigs that is characterized by diarrhea,vomiting and dehydration.Its massive outbreak has caused huge losses to the pig industry since 2010.PEDV is an alphacoronavirus with a positive-sense,single stranded RNA,which encods 1A/1B non-structural proteins and S,E,M,N structural proteins.The orf3 located between the s and e genes encodes an accessory gene called ORF3,which is the only accessory gene found in PEDV.The function of ORF3 is one of the research hotspots of PEDV.The veterinary community generally believes that ORF3 is related to the virulence of PEDV.However,in the existing research,due to the limitations of research methods,little is known about its biological effects and mechanisms.The contents and results of this study are as follows:?1?Rescue and identification of three recombinant viruses.We constructed recombinant plasmids with different orf3 genes and without orf3 gene.Recombinant PEDVs with different orf3 genes and without orf3 gene were rescued by using reverse genetic operating system based on targeted RNA recombination.The viruses were purified by terminal dilution and the recombinant viruses were identified by RT-PCR and immunehistochemistry?IHC?.Successful rescue of recombinant PEDV strains with or without orf3 was verified by using RT-PCR and observing the cytopathic effect?CPE?.The results of IHC assay indicated that orf3 gene of recombinant PEDV was expressed in Vero cells.?2?Generation of virus growth curve.Vero cells were infected with the newly acquired recombinant PEDV strains as well as rDR13^att-ORF3^wt and PEDV DR13?attenuated?at a multiplicity of infection?MOI?of 0.1.The viral titers(TCID₅₀)at 8,16,24,32,40,48 hours post infection?hpi?were measured and corresponding virus growth curves were plotted with virus infection time as abscissa and TCID₅₀ as ordinate.SPSS analysis showed that the viral titers of recombinant PEDV strains with orf3 gene were significantly higher than that without orf3 gene?p<0.05?.?3?Determination of the Vero cell number at different times infected with five PEDV strains.1st method: Number of living cells was counted using cellometer auto T4.The Vero cells were infected with the recombinant PEDV strains as well as rDR13^att-ORF3^wt and PEDV DR13?attenuated?strain at a multiplicity of infection?MOI?of 0.1 and the intact cells at 25 and 36 hpi were counted.SPSS analysis showed that the numbers of intact cells left in the flasks infected with the recombinant PEDV strain with orf3 gene at both 25 and 36 hpi were significantly higher than that infected with the strain without orf3 gene?p<0.05?.2nd method: Number of living cells was measured by using Cell Counting Kit-8.The Vero cells were infected with the above mentioned viral strains at a multiplicity of infection?MOI?of 0.1 and the OD were measured by a microplate reader at 450 nm.The statistical analysis using SPSS showed that the OD₄₅₀ of Vero cells infected with the five viruses was not significantly different at 12 hpi.The OD₄₅₀ of Vero cells with the PEDV strain with orf3 gene at both 24 and 48 hpi were significantly higher than that without orf3 gene?p<0.05?.And at 36 hpi the result was similar,but the difference is extremely significant?p<0.01?.This result further confirmed the results of the cell counting by cellometer auto T4.?4?Determination of cell viability.The Vero cells were infected with the aboved mentioned strains at a multiplicity of infection?MOI?of 0.1 and the OD were measured by a microplate reader at 450 nm.Calculate the measured OD₄₅₀ as cell viability based on the cell viability calculation formula,The statistical analysis using SPSS showed that the OD₄₅₀ of Vero cells infected with the five viral strains at 12 hpi was not significantly different.The OD₄₅₀ of Vero cells with the PEDV strain with orf3 gene at both 24 and 48 hpi were significantly higher than that without orf3 gene?p<0.05?.And at 36 hpi the result was similar,but the difference is extremely significant?p<0.01?.Through the above experiments,we conclude that ORF3 protein promotes the proliferation of PEDV on Vero cells,the mechanism of which is the time delay of death of infected cells by the ORF3 protein.These results provided information on the function of PEDV orf3 gene and were helpful in understanding the replication mechanisms of PEDV.