Study On The Antiviral Effect Of IFN-Beta On Porcine Hemagglutinating Encephalomyelitis Virus Infection In Macrophages And Mice

Poreine hemagglutinating encephalomyelitis is a kind of piglets disease causedby Poreine Hemagglutinating encephalomyelitis virus (PHEV), showed neuroloicalsymptomys or vomiting and failure as the clinical symptoms of acute and highlycontagious disease,mainly affects pigs from1to3weeks. the mortality rate was upto20-100%. Serologieal survey showed that PHEV infeection was distributed byworldwide, and has caused great economic losses to the Pig industry.Particularly,there is no effective vaccines and drugs to prevent and control this disease. It is soimportant to detect an efficient antiviral drug to prevent and treat the disease inclinical Veterinary areas currently.The early innate immune response against viruse’s infection is the first line ofdefense, but there are some ways for the virus escape antiviral immune response byinhibiting the expression of IFN-β.Not only does IFN-β prevent viral duplication bytaking part in the different processes of viral replication, such as adsorption,shelling,transcription, but also strengthen the phagocytosis fuction of the natural killercells to killing viruses in host cells.Therefore, IFN-β is one of the key factors to affectthe antiviral immune response.Currently, the use of genetic engineering techniques inrecombinant IFN-β has provide new idears for the prevention and treatment of viralinfection-related diseases.The study has found that paramyxovirus, hepatitis B virus,Japanese encephalitis virus and other viruses are able to escape antiviral immuneresponse by inhibiting IFN-β expression. This disease has a typical neurotropicproperties and primarily infected the central nervous system of the host. The virus ismainly transmitted to the central nervous system along the peripheral nervoussystem.Early in this laboratory, we found that PHEV inhibit the expression of IFN-βin dendritic or N2a cells. The immune escaption of IFN-β may be one of the factorsleading to immunosuppression in the infection of PHEV. We not clear that the hostantiviral immune mechanism before neurological damage with the infection of PHEV..Therefore, It is significantly important to establish a vivo model to studyimmune escape mechanisms and explore whether the IFN-β is involved inantiviral-PHEV immunity.First, The mon-olayer cells were infected with PHEV-67N in strain macrophages(J774A.1cell lines) to analyze proliferation characteristics by CPE observation,TCID50immunofluorescence assay (IFA) and other measuring methods.In order toexplore the role of Procine hemagglutinatin encephalomyelitis virus in macrophages(J774A.1) and the mechanism for celiular inflammatory responses between the hostand the virus,the level of inflammatory cytokine mRNA and protein expressionincluding the TNF-a, IL-6IL-1β and IFN-β mRNA after infected in different timeswere tested by ELISA, RT-PCR．The results showed that the amount of inflammatorycytokines IL-6, TNF-a, IL-1β expression are raised, and the amount of IFN-βexpression is down-regulated during infection of PHEV. We clear that J774A.1cellsby inhibiting IFN-β expression to evade the immune response, for in-depth study toevade the body’s immune response PHEV pathogenesis of the foundation. It is deplyin order to study the evade of the immune response and the pathogenesis of PHEV.Then, this study also extracted RNA from a mouse brain by the infection model ofPHEV and amplified RNA-specific gene of IFN-β by using the pGEX-4T-1prokaryotic expression vector with a GST tag. We successfully establishedpGEX-4T-1-IFN-β fusion protein prokaryotic expression system, To achieve a solublerecombinant protein expression of IFN-β in E. coli by using Western-blot andSDS-PAGE analysis to identify and successfully got the soluble histonepGEX-4T-1-IFN-β. This is in-depth study of pathogenic mechanisms and viralimmune escape phenomenon.Then in order to further explore whether IFN-β involvedof anti-viral immunity in cell models and animal models.In the cell infection model,when the6-well plates confluented monolayer774A.1macrophage, we could dividedthis study into two groups (the IFN-β experimental group and the control group).TheIFN-β experimental group were inoculated active IFN-β, after which the two groupswere inoculated PHEV-67N respectively.At last, the samples of two groups werecollected after viral inoculation at different times(12h,24h,32h,48h,60h).Inanimal infection models,10-week-old BALB/c mice were randomly divided into twogroups.The IFN-β experimental group were injected of active IFN-β in Lateralventricle of mice at first day, after which the two groups were inoculated intranasally with PHEV-67N respectively.At last,the samples of two groups were collected afterviral inoculation at different days.By using immunofluorescence, PCR and HE biopsy,etc. we have found that compared with the control group, the IFN-β experimentalgroup inhibited expression of PHEV and more significantly suppressed PHEVreplication in early infection. The results of HE sections showed that, in the earlystages of infection, the control group showed normal neuronophagia and accompaniedby a large number of blood vessels sets and satellite phenomenon, and the IFN-βexperimental group only have a small part of the neuronal degeneration andnecrosis.Because in vitro studies do not fully simulate the complexity of the immuneresponse in vivo, we also considered the effect of IFN-β in vivo anti-PHEV infectionin an animal model, The results prove that the recombinant IFN-β can effectivelyinhibit the replication of PHEV and evade the innate immune system surveillance.The results also suggest that we can use genetic engineering technology to produceactive IFN-β for the prevention and treatment of diseases of the PHEV.