| Porcine epidemic diarrhea(PED)is a severe acute,contagious intestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV).The disease is mainly characterized by severe enteritis such as diarrhea,vomiting and dehydration in infected pigs,and has a high infectivity and mortality.Since PEDV was first reported in the UK in 1971,PEDV has broken out many times and has spread to many countries and regions in the world,causing huge economic losses to the world pig industry.As for the mechanism of PEDV infection in vitro and vivo,many scholars at home and abroad have done a lot of research.According to relevant literature,some viral proteins can effectively inhibit or promote the replication and proliferation of PEDV in vitro and in vivo,and the influence of some viral non-structural proteins on the PEDV replication mechanism is related to the activation of NF-κB signaling pathway.NF-κB is an important intracellular nuclear transcription factor,involved in the inflammatory response,immune response and other processes.In order to understand the genetic variation of PEDV in Guangdong province from2018 to 2019,RT-PCR was used to detect the PEDV antigen in 74 samples collected in the experiment,and S1,M and ORF3 of some positive samples were sequenced,and the sequencing results were constructed with reference strains for evolutionary tree construction,homology analysis,and amino acid sequence alignment.The results showed that 38 samples were positive for PEDV by RT-PCR,with a positive rate of 51.35%.In PEDV S1 genetic evolution analysis,the detected strains are distributed in the G2 branch,and have higher homology with VN-VAP1113,about 96.0%-97.6%,with 3 amino acid deletions,5 amino acid insertions,and multiple amino acid mutations.In PEDV M genetic evolution analysis,the GDxh strain is distributed in the G1 branch,with DR13 homology was 98.3%.GDhz strain is distributed in the G2-1branch,with VN-VAP1113 homology was 98.3%.The other 12 detected strains are distributed in the G2-2 branch and have higher homology with OH851,which are 97.4%-99.1%,there are mainly 5 amino acid mutations.In PEDV ORF3 genetic evolution analysis,the GDxh strain is distributed in the G1 branch,with DR13 homology was 99.6%,there are 5 amino acid mutations and deletion of an amino acid fragment.Other strains in G2 branch with OH851 and VN-VAP1113 homology is higher,82.7%-92.0%,83.1%-87.6%,respectively,of which 8 strains has mainly 5 amino acid mutations,and other 5 strains has mainly 11 amino acid mutations,all without amino acid insertions or deletions.This study conducted a preliminary study on PEDV non-structural proteins nsp6,nsp7,nsp8,nsp9 and nsp10 by qPCR,TCID50 detection and Western blot.It was found that 12 h,24 h and 36 h after PEDV infection in Vero E6 cells,these PEDV non-structural proteins can promote PEDV replication and proliferation in cells.The double luciferase reporter gene test found that PEDV nsp9 significantly inhibited NF-κB signaling pathway in 293 T cells.Further studies have found that PEDV nsp9 can significantly inhibit the expression of CCL2,IFNα,IFNβ,and TNFα,and gradually reduce the expression of cytokines IL8,and PEDV nsp9 can inhibit the phosphorylation of P65 and IKBα.In this study,it was found that the PEDV epidemic strains in Guangdong province are mainly G1 and G2 strains,and there are multiple amino acid mutation sites in S1 and ORF3 gene segments,which are relatively unstable,and the M gene is relatively conservative.Studies have found that PEDV non-structural proteins nsp6,nsp7,nsp8,nsp9 and nsp10 can promote PEDV replication.PEDV nsp9 may inhibit the activation of NF-κB signaling pathway by inhibiting the phosphorylation of P65 and IKBα,and inhibit the expression of cytokines IL8,CCL2,IFNα,IFNβ,and TNFα,thereby promoting the intracellular replication process of PEDV.These results provide a theoretical basis for the prevention and control of PEDV epidemic in Guangdong province and further elucidation of the mechanism of PEDV replication in cell. |
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