Study On The Immune Response Of Multi-cytokine To Tree Shrew Infected With Influenza Virus

Influenza is a severe acute respiratory syndrome that caused by influenza virus infection,it is the leading case among global epidemic infectious diseases.Influenza is characterized as widespread,outbreaking,acute and repid,and sometimes also can lead to deadly pneumonia.Influenza is one of the most sevre diseases threatening human health,it's urgent to estabilish the effective animal model to research the viral pathogenesis and infection mechanism.However,there are various defections and limitations in current laboratory animal models.It is crucial to develop new alternative animal models for influenza virus research.Tree shrew is regarded as the newly potential animal model that can be used as the replace for primate model due to its unique features of evolution and biology,and the similarities with human at metastasis,physiology and biochemistry and immunology.Now,tree shrew has been widely used in human diseases such as viral infection diseases,nervous system diseases and tumor.In this study,peripheral blood mononuclear cells?PBMC?of tree shrews were firstly isolated and cultured.Then,PBMC was infected with influenza virus in vitro,and the mRNA expression level of relevant cytokines was detected by The Real-time quantitative PCR.The detect results were used to evaluate the immune response of PBMC against influenza virus.The results showed that the 11 cytokines in tree shrew PBMCs post infection with influenza virus were found up-regulated at mRNA expression level.Except the expression levels of IL-4 and MCP-1 changed insignificantly?P>0.05?,the others such as IL-6,IL-10,IFN?,TNF-?increased 3-5folds?P<0.05?;IL-13,CCL-5,CXCL-9 and IP10 increased 8-18 folds?P<0.05?.Notably,the up-regulation of IL-8 expression was the most significant,which increased 800-folds?P<0.05?.The tree shrews were then inoculated intranasally with H9N2 influenza virus(at a concentration of 10⁶ TCID₅₀/250?l).At 48 hours and 96 hours post infection,the respiratory tract tissue?turbinates,trachea,and lung tissues?were obtained by dissecting the tree shrews and followed by partial homogenization.Respiratory virus titers were determined by cytopathic assay?CPE?.The expression levels of cytokines and chemokines were evaluated by the detection for mRNA.And histopathological HE staining was also evaluated.The results showed that influenza virus was detected in the lung,trachea and turbinate homogenate at day2 and day4 post infection.The virus titers rang from 10^2.5TCID50 to 10⁶TCID50/ml.Furthermore,the mRNA expression of cytokines and chemotaxis of the infected respiratory tissue,including IL-4,IL-6,IL-10,TNF-a,IL-8,IL-13,IP-10,RANTES,MIG,and IFN-beta were up-regulated at varying degrees.HE staining revealed the accumulation of inflammatory cells in the iliac bone and trachea mucosa from the infected tree shrew,as well as the widening of the space.And HE staining also revealed the presentation of a large number of red blood cells and obvious exudates in the lung tissue.These results indicating that influenza virus successfully infected the tree shrew and replicated in its respiratory tissue.Finally,in order to screen the available human ELISA kits that can be used for the detection of tree shrew cytokines effectively,the cross-reactivity of the human IL-8 and IP-10 cytokine antibodies and the tree shrew IL-8 and IP-10 cytokine antigens was detected.The results revealed that the concentration of IL-8 protein in infected tree shrews ranged from 212.46 pg/ml to 642.12 pg/ml,and the IL-8 protein concentrations in normal tree shrews were 73.37 pg/ml to 83.99 pg/ml respectively,which indicating that the protein level of IL-8 in the infected group was significantly higher than that of the normal group.The concentration of IP-10 protein in infected tree shrews range from 49.44 pg/ml to 93.20 pg/ml,whereas the IP-10 protein concentrations in normal tree shrews were 33.73 pg/ml to 79.43 pg/ml,which indicating that the expression level of in the infected group was not significant compared with the normal group.In summary,firstly,the mRNA expression of cytokines of the PBMCs isolated from the infected tree shrews increased varyly indicating the basical immune response against to the infection of influenza virus.Secondly,the detection of viral titles,cytokines and chemotaxis and histopathological changes for tree shrews inoculated intranasally with influenza virus,indicated that influenza virus can successfully infect tree shrews and replicate in their respiratory tissues.Finally,Human IL-8 antibody had immune cross-reaction with tree shrew IL-8 antigen,while human IP-10 antibody did not cross react with tree shrew IP-10 antigen.These results proved the feasibility of tree shrew served as the influenza animal model,but developing for the specific testing kit is required.