Establishment Of PDCoV And PEAV Diagnostic Methods,genetic Evolution Analysis And The Mechanism Of N Protein Action

Porice deltacoronavirus(PDCoV)and porcine enteric alpha-coronavirus(PEAV)are two newly discovered porcine infectious coronaviruses,which have similar clinical symptoms,including diarrhea and vomiting.Pigs of all ages can be infected,and the mortality rate of young piglets is 100%,causing huge economic losses to the pig industry.This study aim to establish a rapid and accurate duplex RT-PCR method to distinguish PDCoV and PEAV,and tested PDCoV and PEAV in porcine diarrhea samples in some areas of Guangxi Province,and explored the genetic variants and the epidemic status of two viruses.To study the mechanism of PDCoV and PEAV N protein inhibiting the production of type I IFN.According to the PDCoV and PEAV N gene sequences,design specific primers,respectively.The positive plasmid as a template,and perform specificity and sensitivity tests after optimizing the duplex RT-PCR reaction conditions.From 2017 to 2018,62 intestinal and diarrhea samples collected from Guangxi were tested.The positive samples were amplified by RT-PCR and spliced to obtain the full length of the PDCoV genome.Using PDCoV and PEAV genomes as templates,construct the N gene eukaryotic expression plasmid,transfect HEK-293T cells,confirm whether PDCoV and PEAV N protein inhibit the production of IFN-?by q PCR,and use Western blot,Co-IP to verify its specific mechanism.The specific fragment sizes of PDCoV and PEAV were 690bp and 208bp,respectively.There were no amplification bands for PEDV,TGEV,PRV and PCV2.The lowest detection rates for PDCoV and PEAV were 5.13×10~2copies/?l and 4.73×10~1copies/?l.The positive rate of PDCoV in samples from Guangxi Province was 19.4%(12/62),and the positive rates of PEDV,TGEV and Po RV were 32.3%(20/62),4.8%(3/62),and 6.5%(4/62),respectively.PEAV was not detected.The 5 whole genome sequences analysis showed that there were 6-nt and 9-nt deletions in the non-structural protein 2(Nsp2)and non-structural protein 3(Nsp3),respectively,CHN-GX81-2018 strain had no gene deletion or insertion in S gene and 3'-UTR.The genetic evolution analysis of PDCoV showed that4 strains had 98.2%-98.4%nucleotide homology with CHN-AH-2004,which belonged to the Chinese lineage.The CHN-GX81-2018 strain has98.7%nucleotide homology with Vietnam/Binh21/2015 and belongs to the Vietnam/Laos/Thailand lineage.Both PDCoV and PEAV inhibited the production of IFN-?stimulated by Poly I:C.Further research found that PDCoV and PEAV N proteins inhibit the ubiquitination modification of RIG-I by interacting with RIG-I,at the same time inhibit the phosphorylation of IRF3 and p65(NF-?B subunit)in the downstream signaling pathway,thereby inhibiting the production of IFN-?.