Investigation Of JE Infection In Breeding Boars In Guizhou Province And The Effect Of JEV On TLRs Signaling Pathway In Leydig Cells

Japanese encephalitis(JE)is an insect-borne,zoonotic infectious disease caused by Japanese encephalitis virus(JEV).harm.More than 2 billion people are currently at risk of becoming infected with Japanese encephalitis in an endemic or regional setting.Most animals infected with JEV develop mild or even no symptoms.Boars infected with JEV developed orchitis,which was mostly unilateral in the initial stage,with swelling of the testis and hot pain,loss of libido,decreased semen quality,loss of breeding ability and culling;pregnant sows infected with JEV suffered from miscarriage,stillbirth or mummified fetus and other symptoms,leading to reproductive disorders;after the fattening pigs are infected with JEV,they have persistent high fever and loss of appetite,causing huge economic losses to the pig industry.The TLR receptor(Toll-like receptor,TLRs)family is the first identified transmembrane host pattern recognition receptors,which can recognize a variety of pathogen-related molecules.In mammals,there are more than 10 TLRs members that recognize microorganisms.Studies have shown that TLR3,TLR7 and TLR8 signaling pathways play an important role in the activation of innate immunity in flavivirus infection,and JEV is a member of the flavivirus genus.This study will investigate the immune status and infection of Japanese Japanese encephalitis in boars in Guizhou Province,the effects of JEV-infected Leydig cells on TLR3,TLR7 and TLR8 signaling pathways,and the effects of JEV-infected cells on inflammatory factors after silencing TLRs signaling pathways.Three aspects of secretion effects were studied to explore the effects of JEV infection on Leydig cells on its TLR3,TLR7 and TLR8 signaling pathways and the secretion of inflammatory factors IL-1β,IL-6 and TNF-α.The relevant research contents of this experiment are as follows:1.Immune status of Japanese Japanese encephalitis and JEV infection in breeding boars in Guizhou ProvinceIn this study,134 blood samples of boars were collected from 26 large-scale pig farms in Guiyang,Liupanshui,Zunyi,Anshun,Tongren,Bijie,Qiandongnan,Qiannan and Qianxinan in Guizhou Province.There are 134 semen samples of boars and breeding boars,and these pig farms are regularly immunized with the attenuated Japanese encephalitis vaccine SA14-14-2 every year.ELISA detection method was used to detect the serological antibody of Japanese encephalitis in some pig farms in our province,and the immune antibody level of Japanese encephalitis in pigs was analyzed to clarify the immune status of Japanese boars in our province.The blood and semen samples were detected by RT-PCR,and the etiological investigation of boar Japanese encephalitis was carried out in some pig farms in our province,so as to understand the distribution area and prevalence of pig Japanese encephalitis in Guizhou province.The results showed that the positive rate of Japanese encephalitis antibody level in boars in Guizhou Province was 88.05%,which reached the national standard.The results of RT-PCR in the blood and semen of the breeding boar were negative,and there was no JEV infection.2.Effects of JEV GZ strain infection on Leydig cells on TLR3,TLR7 and TLR8 signaling pathwaysThe JEV GZ strain(full gene sequence Gen Bank No.KC915016)isolated and identified in our laboratory was used to infect Leydig cells,and the Leydig cell pathological effect(CPE)of JEV GZ strain infection was observed.Transcription level and protein expression changes of TLR8 signaling pathway-related m RNA.The results showed that after infection of Leydig cells by JEV GZ strain,TLR3 and TLR7 signaling pathways were activated,TLR3 and TLR7 and adaptor protein molecules TRIF and My D88 m RNA began to be up-regulated at the transcriptional level at 6 h,and the m RNA remained at a relatively low level at 12 h and 24 h.high transcription levels.The expression levels of TLR3,TLR7 and adaptor protein molecules TRIF and My D88 were detected by Western blotting at 6 h.The transcription level of TLR8 was inhibited at 6 h,and the transcription level of TLR8 was significantly inhibited at 12 h and 24 h.ELISA was used to detect the changes of inflammatory factors IL-1β and TNF-α at 6 h,12 h and 24 h after infection of Leydig cells by JEV GZ strain.The results showed that inflammatory factors IL-1β and TNF-α were not detected,but inflammatory factors were not detected.The expression of IL-6 gradually increased over time,indicating that the expression levels of inflammatory factors IL-1β and TNF-α did not change after JEV GZ strain infected Leydig cells.The experimental results show that: JEV GZ strain activates the TLR3 and TLR7 signaling pathways after infection of Leydig cells,and induces the up-regulation of the inflammatory factor IL-6.3.The effect of JEV GZ strain on the secretion of inflammatory factors in Leydig cells infected with Leydig cells after silencing the TLRs signaling pathway in Leydig cellsIn this study,three groups of TLR3-si RNA,TLR7-si RNA,TLR8-si RNA,TRIF-si RNA,My D88-si RNA gene silencing primers were designed to silence Leydig cells by si RNA gene silencing technology.Western blot was used to detect the protein silencing effect of TLR3,TLR7,TLR8 and the adaptor protein molecules TRIF and My D88,and a set of primers with the best silencing effect were used for gene silencing of TLR3,TLR7,TLR8,TRIF and My D88 in Leydig cells,and JEV was detected by ELISA.The effect of GZ strain-infected Leydig cells silencing related genes was detected 6 h after inflammatory factor secretion.The results showed that after gene silencing of TLR3,TLR7,TLR8,TRIF and My D88,respectively,the expression of inflammatory factor IL-6 was significantly higher in JEV GZ strains infecting Leydig cells for 6 h and JEV GZ strains infecting Leydig cells for 6 h compared with the control.both increased.Compared with Leydig cells infected with JEV GZ strain for 6 h,the expression of inflammatory factor IL-6 decreased after TLR3,TLR7,TLR8,TRIF and My D88 genes were silenced.The experimental results further showed that the infection of Leydig cells by JEV GZ strain induced the expression of inflammatory factor IL-6 through TLR3 and TLR7 signaling pathways.