Prevalence Analysis Of Porcine Epidemic Diarrhea Virus In Shanxi Province And Surrounding Areas And The Mechanism Of ORF3 Protein In The Immune Escape

Porcine Epidemic Diarrhea(PED)can cause infection in pigs of all ages,including fattening pigs,with mortality rates of up to 100% in piglets up to 7 days of age.With the internationalization of the pig trade,frequent pig transfers have led to increasing reported PED cases,causing huge economic losses to the global pig industry.The genome of the Porcine Epidemic Diarrhea Virus(PEDV)has mutated and recombined over a long period of genetic evolution,making the existing vaccine strains unable to provide effective immune protection and making the prevention and control of PED difficult.PEDV infection induces the host’s natural immune response,producing type Ⅰ interferon(IFN-Ⅰ)and other cytokines,which exert antiviral effects.At the same time,the virus has evolved different immune escape mechanisms to evade the host’s immune response as it co-evolved with the host.PEDV has been found to encode multiple interferon antagonists,including both non-structural and structural proteins.Of the 23 proteins encoded by PEDV,at least 10 were identified as antagonists of IFN-Ⅰ,and 11 were identified as antagonists of type Ⅲ interferon(IFN-Ⅲ).The mechanisms by which the nsp1,PLP2,nsp5,N,and M proteins inhibit IFN-Ⅰ production have been elucidated in detail.In contrast,the mechanisms by which the other encoded proteins inhibit IFN-Ⅰ production need to be further investigated.nsp1,nsp15,and N proteins have also been proposed to inhibit IFN-Ⅲ,but detailed mechanisms of action need to be elucidated.PEDV encodes the only accessory protein(ORF3),which has ion channel activity and plays an important role in influencing viral titers and viral pathogenicity.To further unravel the relationship between PEDV viral proteins and natural immunity,overexpression of ORF3 was investigated for its effect on interferon production and downstream signaling pathways and to probe the mechanism of action of ORF3 in escaping natural immunity.The main components include:1.screening PEDV-positive disease material using multiplex RT-PCR,HE,and IHC,isolating,purifying,and identifying virulent strains.In this study,a total of 47 clinical disease materials within 10 days of age in which dams were immunized against PEDV and piglets became sick or died were collected from a total of 13 pig farms in Henan,Hebei,and Shanxi provinces from November 2019 to April 2021.Multiple testing showed that the PEDV positivity rate was 42.6%.Single positive samples from seven pig farms were selected for Sanger sequencing.The M,N,and ORF3 gene sequences of the seven PEDV-positive samples were determined and combined with the reported sequences of 24 strains for homology comparison and evolutionary analysis.The results showed that the M sequence was highly homologous to the reference sequence,with a low variation rate,and could be used as one of the target genes for antigen detection.The N gene sequence was highly homologous and different from the existing strains and may have undergone different degrees of variation during genetic evolution.The ORF3 gene sequence was less homologous,and the E6 sample had a partial deletion of the gene sequence after multiple passages,thus making ORF3 evolutionarily Diversity.The test isolate is distantly related to the classical CV777,LZC,and the Korean strain DR13.TCID50 and histological analysis of the JZ-SX6 isolate showed a low virulence multi-site,multi-cellular transmission,all of which deserve our attention.2.Effect of JZ-SX6 isolate on early infection of host cells and the role of its encoded viral protein on promoter activation.The JZ-SX6 isolate was passaged on Vero cells and infected with PK-15 model cells at viral infection plural MOI 1 or MOI 0.1.The results showed that the isolate inhibited the expression of type I and type Ⅲ interferons in the early stages of infection,and the expression of the pro-inflammatory factors TNF-α and IL-1β was also inhibited.Using JZ-SX6 as a template,viral proteins S1,S2,M,N,E,and ORF3 were constructed.Dual luciferase reporter gene assays showed that S2,M,N,ORF3,and E all significantly reduced sus-IFN-β promoter activity,and S1,S2,M,N,E,and ORF3 all significantly reduced sus-IL-29 promoter activity.Thus ORF3 inhibits the activation of sus-IFN-β and sus-IL-29 promoters.3.Validation of the PEDV auxiliary protein ORF3Its secondary and tertiary structural features are predicted online as a monomeric protein consisting of several α-helices surrounding a thrice transmembrane structure.The pc DNA3.1-ORF3 and p EGFPN1-ORF3 overexpression vectors were constructed,and CCK-8 assays showed little effect on cell proliferation and no cytotoxicity.Indirect immunofluorescence showed that ORF3 protein was localized in the nucleus and cytoplasm of cells at the early stage of virus infection.4.The role and mechanism of ORF3 in immune escapeTo elucidate the mechanism of action of ORF3 in the inhibition of interferon,first overexpression of ORF3,with or without PEDV infection of host cells PK-15,and endogenous assays revealed that ORF3 significantly reduced TLR3,TLR7,IRF3,and IRF7 at m RNA expression levels.Stimulation with the agonists poly(I:C)or R837 showed by fluorescence quantification and immunoblotting that ORF3 decreased interferon production by down-regulating TLR3 and TLR7 expression and that IRF3 and IRF7 expression and corresponding phosphorylation levels were reduced.Infection of PEDV in heterologous cells 293 T,ORF3 inhibited the expression of TLR3,TLR4,and TLR7 and their downstream IRF3 and IRF7 genes in heterologous cells.Their type Ⅰ and type Ⅲ interferon gene expression levels were suppressed under poly(I:C)or R837 stimulation,ORF3 suppressed type Ⅰ and type Ⅲ by downregulating TLR3 and TLR7 interferon production by down-regulating TLR3 and TLR7.Secondly,the dual luciferase reporter gene,WB,and IFA assays suggested that ORF3 inhibited interferon production via the NF-κB pathway.The RLRs pathway plasmid was constructed and cotransfected with ORF3,respectively.The results showed that overexpression of ORF3 protein significantly affected the abundance of MAVS and IRF3 proteins,and Co-IP assay showed that there was an interaction between ORF3 protein and MAVS or IRF3 protein,thus suggesting that ORF3 inhibits IFN-β production through the IRF3 pathway.The results indicate that the continuous variation of PEDV genes shows evolutionary diversity and complexity and is one of the important factors in escaping traditional vaccine immunity.ORF3 affects the expression of interferon by inhibiting the TLRs signaling pathway,NF-κB pathway,and IRF3 pathway,thus evading natural immunity.