Preliminary Study On The Changes Of Cytokines In The Brain Of IFNAR Knockout Mice During Rabies Virus Infection

Rabies virus(RV)is a nonsegmented negative-stranded RNA virus of the Rhabdoviridae family and induces a fatal neurological disease in humans and animals.Neurological invasiveness and neurological impairment are the main characteristics of typical rabies virus infection.Although significant advances have been made in rabies prevention and control,the disease remains a major threat to public health and continues to cause more than 59,000 deaths around the world each years.Type Ⅰ interferons(IFNα/β)are a group of signaling proteins made and released by host cells in response to the presence of viruses.In a typical scenario,a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.All type Ⅰ IFNs bind to a specific cell surface receptor complex known as the IFN-α/β receptor(IFNAR).By interacting with their specific receptors,IFN activates signal transducer genes and induces interferon-stimulated genes(ISG)expression finally.Previous studies have found that lower level of IFN-α and IFN-β can be caused by intracerebral injection of the vaccine strain RC-HL in normal mice.But fixed rabies virus strain CVS-24 can induce high levels of type Ⅰ interferon production.Further studies showed that the production of type I interferon was related to the Glycoprotein of rabies virus.In this study,we used gene knockout technique to constructed IFNAR gene knockout mice.Then we inject various rabies virus strains into the mice and collected the brain tissues on the 4th and 7th day after injection.The changes of cytokines in brain were detected by qPCR,ELISA and Western blot.As a result,It was found that the knockout of the IFNAR gene cause the morbidity and death of mice injected with rabies virus was earlier.Moreover,the vaccine strain RC-HL and the attenuated strain rRC-HL△G/R333Q,which did not kill normal mice,also caused 100%death of IFNAR-/-mice.The result showed that IFNAR knockout mice infected with rabies virus caused higher levels of IFN-α and IFN-β gene expression than normal mice.But lack of IFNAR receptor prevents type Ⅰ interferon from functioning properly.And some cytokines such as IL-6,IL-1β and TNF-α are produced earlier than normal mices.The gene expression level of other cytokines also increased in varying degrees,which was related to the virulence of the virus.The expression levels of IL-6,IL-12,IL-1β and TNF-α in IFNAR-/-mice infected with attenuated strains RC-HL and rRC-HL△G/R333Q were higher than those in normal mice on the 4th and 7th days.IFNAR-/-mice infected with Street Virus GX074 or G gene recombinant strains rRC-HL△G242-288 and rRC-HL△G expressed higher levels of IFN-γ,IL-6,IL-12,IL-1β and TNF-α at the near-death stage.In this study,we found that high levels of cytokine expression were positively correlated with the onset of symptoms and the early death time of mice.Moreover,high levels of cytokine expression did not prevent the replication and proliferation of the virus,but formed excessive cytokine storms to aggravate the clinical symptoms of mice.By detecting the mRNA and protein expression of the antiviral proteins MX1,OAS-1 and Viperin induced by type Ⅰ interferon,The results showed that the mRNA and protein expression of IFNAR-/-mice induced by fixed strain CVS-24 in OAS-1 and Viperin were similar to those in normal mice,and there were high levels of OAS-1 expression.rRC-HL,rRC-HL△G,rRC-HL△G242-288 and Mull strains induced higher levels of OAS-1 and Viperin in the brain of IFNAR-/-mice than in normal mice.The expression of MX1 mRNA in IFNAR-/-mice was significantly inhibited.Except the RCHL,the expression of MX1 protein in IFNAR-/-mice infected with rRC-HL,rRC-HL△G,Mu11,rRC-HL△G242-288 and rRC-HL△G/R333Q strains on the 7th day after infection was also significantly lower than that in normal mice.Rabies virus infection in mice can lead to a high level of cytokine expression,but the high level of cytokines did not allow mice to get the corresponding protection,and even the time of onset and death was advanced.The mechanism of these abnormal phenomena still needs to be further explored.It is of great significance to study the pathogenesis of rabies virus.