Research On The Regulation Of FcRn Expression By Infection Of Three Porcine Enteric Coronaviruses

Most pathogens microorganisms invade the body through the mucosal surface of the digestive tract or urogenital tract.The mucosal immune system has a vital role in immune response and protection.SIg A,Ig M andIgG in mucous secretion were mediated by the transfer of the polymeric immunoglobulin receptor(pIgR)and the newborn Fc receptor(FcRn).FcRn can also transport antigen antibody complexes,which plays a key role in protecting the organism from the invasion of pathogenic microorganisms.When the pathogen invades through the mucosa,the mucosal epithelium of FcRn expression could change,which subsequently affect the host’s anti-infection.Porcine epidemic diarrhea virus(PEDV),porcine delta coronavirus(PDCoV)and transmissible gastroenteritis virus(TGEV)could infect pigs of all ages,especially for suckling piglets,the fatality rate is extremely high.The cyclical epidemic worldwide has caused serious losses to the pig industry.In this study,it was isolated and identified one strain of PEDV and PDCoV,completed the whole genome sequencing,and explored its regulation of FcRn expression through animal infection experiments.Previous studies have found that TGEV infection up-regulates the expression of FcRn in IPEC-J2 cells,this study explored the specific molecular mechanism of TGEV-encoded protein and its secreted inflammatory factors to regulate the expression of FcRn.The main research results obtained are as follows:1.Isolation and identification of PEDV and its effects on the host’s natural immune responseThe PEDV positive samples were inoculated with Vero cells for virus isolation.Through cytopathic,IFA,Western blot and TEM observations,it was confirmed that the isolated virus was PEDV and was named JS-A strain.After sequencing the genome of the JS-A isolate and performing a phylogenetic tree analysis,it is shown that PEDV JS-A belongs to the G2 a subtype,is closely related to the prevalent PEDV in many countries,and different from many current vaccine strains.Animal regression tests showed that piglets that were orally infected with the virus continue to develop diarrhea watery stools,and weight loss.The small intestine of infected piglets was transparent,thin-walled and intestinal villi have severe atrophy and shedding.In addition,PEDV infected piglets early could significantly down-regulate the expression of TLRs/RIG-I,downstream mediator,NF-κB transcription factor and FcRn expressions in the intestinal mucosa,but these genes were completely opposite in the later stage of infection.It was indicated that PEDV inhibited natural immune response in early stage of infection,but it was activated in later stage of infection.2.Isolation and identification of PDCoV and its effects on the expression of immunoglobulin transport receptors in the intestinal mucosa of infected pigletsThe PDCoV positive samples were inoculated with LLC-PK1 cells for virus isolation.The strain CHN-JS-2017 was isolated and identified by cytopathology,IFA,transmission electron microscopy,and sequence analysis.After sequencing the genome of the CHN-JS-2017 isolate and performing a phylogenetic tree analysis,it is shown that whole genome of CHN-JS-2017 was 97.4%–99.6% identical to other PDCoV strains.Animal experiments confirmed that the isolated strain is highly pathogenic to 5-day-old piglets,and can cause piglets to have typical symptoms such as vomiting and watery diarrhea.The small intestine of infected piglets was transparent,thin-walled and intestinal villi have partly atrophy and shedding.CHN-JS-2017 infected five-day-old piglets could significantly down-regulate the expression of FcRn,pIgR,and NF-κB in the intestinal mucosa,and there was a positive correlation between two of the three.These results may help explain the immunological and pathological changes associated with PDCoV infection.3.The effect of TGEV-encoded protein on the regulation of FcRn expressionBoth TGEV and UV-TGEV could up-regulate the expression of FcRn,but the inactivated virus is lower than the regulation of live virus.It can be seen that TGEVencoded protein also participates in the up-regulated expression of FcRn.In order to investigate the effect of TGEV-encoded protein on FcRn expression,the TGEV-encoded protein eukaryotic expression plasmids were constructed.IPEC-J2 cells were cotransfected with NF-κB-Luc or FcRn,p RL-TK and different TGEV protein expression vectors and analyzed by the dual luciferase reporter system.We found that TGEV N protein can both significantly activate NF-κB and up-regulate the expression of FcRn.The si RNA experiment showed that after transfection of si TLR3,si TLR9,si My D88,si TRIF and si RIGI,TGEV could significantly inhibit the expression of FcRn by RT-qPCR and Western blot,while si TLR2,si TLR4,si TLR8 have no effect.The results showed that TGEV could upregulate the expression of FcRn through the TLR3/TLR9-My D88/TRIF and RIG-I pathways.4.The effect of cytokines secreted by TGEV on the regulation of FcRn expressionTGEV significantly increased the expression levels of IL-1β,IL-6,IL-8,TNF-α,TGF-β1 in IPEC-J2 cells using RT-qPCR and a porcine cytokine array,suggesting that TGEV infection can activate the natural immune response of IPEC-J2 cells.TGF-β1 up-regulates the expression of FcRn in a dose-dependent and time-dependent manner by RT-qPCR and Western blot.IPEC-J2 cells were pretreated with JNK inhibitor(SP600125),followed by TGF-β1 incubation,we found that TGF-β1 significantly down-regulated the expression of FcRn and c-JUN/JUN,indicating that TGF-β1 upregulated FcRn expression via the JNK/cJUN pathway.At the same time,we analyzed the presence of c-JUN transcriptional binding sites of FcRn promoter regions with online software.According to the transcriptional binding sites of FcRn promoter regions,we continuously truncated the FcRn promoter region,and finally constructed 9 FcRn promoter luciferase reporter plasmids with different lengths.Luciferase reporter assays revealed that the main binding site of c-JUN was located between-1215～-140 of the FcRn promoter.Finally,chromatin immunoprecipitation(Ch IP)was used to prove that this region has 3 c-JUN transcription factor binding sites.We used IPEC-J2 cells to construct the transwell model in vitro and found that TGF-β1 can promote the transport of IgG.