| Flaviviruses are single-stranded RNA viruses transmitted by blood-sucking arthropods.These include Japanese encephalitis virus(JEV),Zika virus(ZIKV),dengue virus(DENV)and West Nile Virus(WNV)etc.Flavivirus has obvious neurotropism.For example,JEV and WNV infection can target the central nervous system of the host and lead to viral encephalitis.ZIKV,on the other hand,can infect a fetus from mother to child and lead to congenital Zika syndrome(CZS)due to brain infection.In addition,the reproductive system is a major target for flavivirus infection.For example,JEV infection can lead to orchitis in boars,and ZIKV infection can lead to orchitis in men and affect semen quality.Male reproductive system is not only related to reproduction,but also closely related to hormone secretion and immune regulation.However,the molecular mechanism of male reproductive system damage caused by flavivirus infection such as JEV and ZIKV,and the effects of orchitis caused by flavivirus infection on hormone secretion and immune regulation are still unknown.Therefore,this study focuses on the subject of JEV and ZIKV,in constructing the animal model of virus infection and the cell model,analytical JEV and ZIKV infection of testicular damage mechanisms,evaluation of testicular injury after the secretion of testosterone and the effects on the body,and to explore the supplement testosterone treatment of viral infection and immune regulation function,the main research content is as follows:1.In vivo model establishment of viral infection induced orchitisAdult boars with negative JEV antibody and antigen were infected with JEV P3 strain.Testicular tissue of boars was collected 7 days after infection for pathological examination.The results of HE staining showed that the testicular tissue of boars infected with JEV showed pathological changes such as lymphocyte infiltration and hemorrhage,abnormal arrangement of spermatogenic tubules,and a large number of spermatogenic cells were shed from spermatogenic tubules.Further immunohistochemical analysis showed that JEV mainly infected Sertoli cells and spermatogenic cells after invading testicular tissue.Fluorescence quantitative detection showed that JEV could up-regulate the expression of inflammatory factors such as IL-1β,IL-6,IL-8,RANTES and TNF-αafter infection with swine testis.It was found that testosterone levels in boars infected with JEV were significantly reduced by ELISA.In order to establish a model of ZIKV infection induced orchitis,this study infected 8-week-old male A129 mice with ZIKV H/PF/2013 virus strain.On the 8th day after infection,the mice showed neurological symptoms such as malaise and claudication,and the autopsy was performed on the mice.The appearance observation showed that after ZIKV infection,the male reproductive system of mice appeared obvious hyperemia,testis hyperemia atrophy;The results of HE staining showed that the testicular spermatogenic tubules in mice infected with ZIKV lost the original structure,the spermatogenic tubules were disordered,and spermatogenic cells were shed in the middle of the spermatogenic tubules.The results showed that ZIKV infection in A129 mice could significantly up-regulate inflammatory factors such as RANTES,TNF-αand IL-6 in testis.An ELISA assay found that testosterone levels in ZIKV infected mice were also significantly reduced.These results indicate that in vivo models of orchitis induced by JEV infection in swine and ZIKV infection in mice have been successfully constructed in this study.2.Study on the signal pathway of orchitis induced by JEVIn order to further elucidate the molecular mechanism and signaling pathway of flavivirus-induced orchitis,we studied porcine testicular cell line ST cells infected with JEV.After infection with 1 MOI JEV for 24 h,ST cells showed obvious pathological changes,and viral proteins could be detected by Western-blot and indirect immunofluorescence,indicating that JEV successfully infected ST cells.Fluorescence quantitative results showed that inflammatory factors such as IL-1β,IL-6,IL-8,RANTES and TNF-αwere significantly up-regulated.The expression of pattern-recognition receptors was quantified by fluorescence,and the results showed that RIG-I expression was significantly upregulated after JEV infection.On this basis,we used CRISPR/Cas9 to knock down RIG-I in ST cells,and found that the knockdown of RIG-I could significantly reduce the expression of inflammatory factors induced by JEV infection,suggesting that RIG-I plays an important role in orchitis induced by JEV infection.Further studies showed that p65,as a transcription factor,was phosphorylated and enriched in the nucleus after JEV infection.Inhibition of p65 activation with its inhibitor QNZ significantly inhibited the expression of inflammatory factors induced by JEV infection,suggesting that p65 is also involved in the testicular inflammatory response induced by JEV infection.In addition,WB results showed that knockdown RIG-I reduced phosphorylation of p65,suggesting that RIG-I regulates p65 activation.These studies suggest that JEV activates p65 by upregulation of pattern-recognition receptor RIG-I in porcine testicular cells,enabling it to enter the nucleus for its transcriptional function,and resulting in increased transcription levels of inflammatory factors and inflammatory responses.3.The effect of ZIKV infection on testosterone secretion in mice and the effect of testosterone supplement on the virulence of ZIKVAs mentioned above,both JEV and ZIKV infection can lead to a significant decrease in testosterone secretion,and testosterone,as an important male hormone,plays a regulatory role in body immunity.Therefore,we speculated that if in the case of virus infection,artificial testosterone supplement will have a therapeutic effect on flavivirus infection?To verify this hypothesis,a mouse model infected with ZIKV was used as a model for study.A129 mice aged 8 weeks were infected with ZIKV H/PF/2013 strain10~3p FU/m L by intraperitoneal injection of 100μL.On days 2 and 4 after ZIKV infection,mice in the testosterone protected group were intraperitoneally injected with 0.05mg of testosterone dissolved in 100μL of sesame oil,and mice in the ZIKV infected group were injected with the same dose of sesame oil.The results showed that testosterone supplementation improved the survival rate of mice.In order to study the specific mechanism of testosterone protection in mice,we infected ZIKV and injected testosterone with the same method.On day 8 after ZIKV infection,samples of mouse brain and spleen were collected for pathological section and flow cytometry.Pathological examination showed that testosterone supplementation reduced immune cell infiltration and lesions in the brain.Flow cytometry was used to measure the number of T cells in the spleen and brain,and we found that testosterone injection led to a decrease in the number of CD8~+T cells.Fluorescence quantitative results also showed that testosterone injection can reduce the immune response in the brain.These results suggest that testosterone protects ZIKV infected mice by inhibiting the immune response in the central nervous system and can be used as a candidate drug for treatment of ZIKV infection.In conclusion,this study established an animal model of orchitis induced by JEV and ZIKV,initially clarified the mechanism of inflammatory response in porcine testicular cells caused by JEV infection,revealed the therapeutic effect of testosterone on flavivirus infection,and provided a new idea for the prevention and treatment of subsequent flavivirus infection. |
