Study On Inflammatory Reaction Mechanism Of Enterovirus Molecular Epidemiology And Enterovirus 71 Infection

Enterovirus71(EV71) is one of the main pathogens causing severe hand, foot andmouth disease, with obvious neurotropic. EV71can cause aseptic meningitis, encephalitis,brainstem encephalitis, encephalomyelitis, spinal cord gray matter like syndrome and otherinflammatory diseases of the nervous system with high morbidity and mortality. EV71wasfirst isolated in1969in California from infants suffering from central nervous system disease.Then it spread widely in Australia, Sweden, Bulgaria, Hungary, Japan, Singapore, Malaysiaand China. In China, EV71has a large distribution of each provinces and autonomous regions.In1998, a largest outbreak caused by EV71occurred in Taiwan, with405children infectedwith severe symptom and78patients died. Based on the statistics of Chinese Health Ministrystatistics, in2012, the number of HFMD patients nationwide had reached2,168,737peopleand567cases died. The cause of death was mainly due to EV71infection-inducedinflammation in the central nervous system and pulmonary edema, myocarditis and othercomplications. However, the mechanism of EV71induced inflammation is not yet clear.There is still a lack of specific and effective antiviral drugs in terms of treatment, whilesuppression of inflammation and symptomatic treatment are the main treatment. In the clinicaltreatment of EV71infection, glucocorticoids are often used to suppress inflammation innervous system, but of which systemic immune suppression has side effects which can lead toserious adverse reactions such as viral spread further and secondary bacterial infection. Thus,in response to reduce EV71epidemic and mortality, fully aware of pathogenesis of EV71infection induced inflammation in nervous system is extremely important.First, we use EV71strains (AH08/06) to establish a mouse model of EV71infectioninduced inflammation in central nervous system (CNS). To determine the inflammation inCNS,1-day-old Kunming mouse was intraperitoneally injected with EV71virus (10-7TCID50).The features of the survival, body weight, pathology, viral distribution, cytokines andcomplement molecules of the model confirm to the typical symptoms of EV71infection. Themice infected EV71died at6days post infection and grow slowly from2days post infection.The average weight of infection group from3days to6days post infection was significantly lower than that of control group (P <0.05). Clinical symptoms of infected mice appear from3days post infection such as weight loss, tremors, stiff neck, convulsions, paralysis and death.Cases of skin rash do not appear.We use real-time quantitative PCR, tissue virus titer determination andimmunohistochemical techniques to observe the viral distribution in blood, brain, lung,intestine, muscle of hindlimb, liver, spleen, kidney of infected mice1-6days post infection.Virus was detected first in blood at1day post infection, indicating that the virus transmittedfrom abdominal cavity into the blood in some way. The virus was detected in the smallintestine, lung, skeletal muscle at2days post infection, indicating infection of these organsmay be caused by blood-borne. Virus was finally detected in brain at3days post infection.Viral titers in brain, lung, intestine, skeletal muscle increase over time, indicating that virusreplication occur. Persistent viremia was found during the whole survival course. Virus titersin skeletal tissues were significantly higher than other organs, indicating that skeletal muscleis the main target for EV71replication. Virus was not detected in liver and spleen.HE staining was used in various organs of mice infected with EV71for observation ofhistopathological change. EV71only infected brain stem and a fraction of cerebellum sectionin mouse brain. In agreement with the neurological symptoms, neuronal degeneration and loss,neuronophagia, and vessel congestion accompanying a mild neutrophilic infiltration wereconsistently observed in the spinal cords, particularly in the ventral horn areas, ofEV71-infected mice before death. Sleeve like infiltration appeared around the small bloodvessels, indicating that severe inflammation occurred in brain stem. Viral inclusions were notfound in CNS. EV71infection leads to severe necrotizing myositis with rupture of musclefiber and a large number of inflammatory cells. There are not significant pathological changesin intestine indicating that the tolerance of intestine is better than other organs. Edema ofcardiac muscle cells and focal myocardial contraction band necrosis are found in heart.Furthermore, the ratio of monocytes and lymphocytes are found increase in part of lumens.Inflammatory cell infiltration is found in pulmonary bronchial wall, and alveolar wallwidened with varying amounts of monocytes and lymphocytes. There are not significantpathological changes in liver, spleen and kidney.Several cytokines and chemokines such as G-CSF, IL-1a, IL-6, IP-10, KC, MCP-1,MIP-1b, RANTES, TNF-a, Eotaxin, GM-CSF, IFN-gamma, M-CSF, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-13, IL-15, IL-17, MIP-2, LIF, MIP-1alpha, MIG,IL-12p70, VEGF, IL-9were measured in serum EV71infected mice by Flow Cytometry.G-CSF, IL-1a, IL-6, IP-10, KC, MCP-1, MIP-1b, RANTES, IFN-γ, TNF-α have changedsignificantly in EV71infection.Serum biochemical index of alanine aminotransferase (ALT), aspartate aminotransferase(AST), creatinine (Cr) and blood urea nitrogen (BUN) are measured to observe the effect ofEV71infection on liver and kidney function. ALT, AST, BUN have changed significantly atthe late stage of infection, indicating the dysfunction of liver and kidney which may be one ofthe reasons for death of EV71infected mice.To further understand the molecular pathogenesis of EV71, we profiled cellular miRNAsof brains from Kunming mice infected with EV71(AH08/06) at3days and6days postinfection for comparison. Microarray analysis showed that398miRNAs in total were detectedin infected and control mouse brain. The two groups cause a group of common and distinctdifferentially expressed miRNAs. Characteristically, more differentially expressedmicroRNAs were aroused on6days post infection than on3days and more down-regulateddifferentially expressed microRNAs were provoked than the up-regulated. down-regulatedmicroRNAs. Among them, up-regulated miRNAs were not observed and only2microRNAswere differentially down-regulated on3dpi. On6dpi, the number of differentially expressedmicroRNAs dramatically increased to60; among them,22microRNAs were down-regulatedand38microRNAs were upregulated. Go analysis of the predicted targets showed that theactivity of transcription, transcription factor activity, sequence-specific DNA binding, proteinserine/threonine kinase activity, transcriptional inhibition of protein kinase activity mayinvolved in EV71infection. Pathway enrichment analysis of the predicted targets revealedthat axon guidance pathway, MAPK signaling pathway, ErbB signaling pathway, focaladhesion, neurotrophic factor signaling pathway, Wnt signaling pathway played an importantrole during EV71infection. To our knowledge, this is the first report of microRNA expressionprofiles of EV71in a mouse model, and our findings might offer novel therapy targets forEV71infection.In conclusion, we successfully established a model with CNS inflammation caused byEV71(AH08/06) infection in1-day-old mice. We use real-time quantitative PCR, tissue virustiter determination, immunohistochemical staining to describe the distribution of EV71in mouse model. We also use histopathology, cytokine, organ function index to illustrate theextent of inflammatory damage caused by EV71infection. Furthermore, we profile cellularmiRNAs of brains from Kunming mice infected with EV71at3days and6days postinfection for comparison. This study provides important data to investigation of pathogenesisof EV71infection induced CNS inflammation in mouse, and attempt to contribute thedevelopment of genetic vaccines and antiviral drugs.