Study On The Severe Pathogenesis Of Enterovirus71-associated Hand, Foot, And Mouth Disease

Enterovirus71(EV71) is a common pathogen that causes hand, foot, and mouth disease (HFMD) and severe neurological complications mainly in the Asia-Pacific region. HFMD associated with EV71infection in young children may cause severe complications, such as myocarditis, acute flaccid paralysis, aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema and even death. However, the mechanism of severe EV71infection remains unclear.Objectives The objective of this study was to investigate the mechanism of severe EV71infection through virus and host, respectively. By the establishment and comparative study of severe and mild mouse models of EV71infection, our research explores the severe pathogenesis from the aspects of experimental animal models. In order to explore the early biological factors associated with severe outcome, analysis of the differencial expressed proteins in sera of mild and severe patients was performed. Through the pathological comparison of fatal patients and mouse model, our study may improve the mechanistic understanding of the pathogenesis of the severe neurological diseases and complications. From the perspective of the immunoregulation of intestinal probiotics, we explore the anti-injury and anti-infective effects of Lactobacillus casei on EV71infection, which may provide basic information for further expounding the mucosal immunoregulation.Methods1. In vitro experimentations were carried out for comparison of replication and complete genome sequencing of nine EV71clinical isolates.2. Severe and mild mouse models of EV71infection were established to compare the pathological characteristics, viral replication in tissues, as well as the proinflammatory cytokine production in sera.3. To understand the pathogenesis of severe EV71-associated disease, we carried out pathological detections for locating EV71antigen and receptor (SCARB2) as well as the basic structural changes of blood-brain barrier (BBB) in human cases and mouse model. We conducted in situ hybridization for detecting EV71viral RNA in tissues of fatal patients, and meanwhile, we elucidated immunopathological mechanism in EV71infection via classification and distribution of inflammatory cells.4. We adopted clinical index statistics and iTRAQ analysis to determine biomarker candidates for a risk assessment for severe development in sera of patients with severe or mild HFMD.5. We investigated the anti-infective effects of Lactobacillus casei on severe EV71infection in mice, and the tissue lesions and virus distribution were detected, which may provide basic experimental data for further explaining the regulatory mechanism of Lactobacillus casei in EV71infection.Results1. Nine EV71isolates, which belong to subgenogroup C4, showed infectivity to SK-N-SH cells, suggesting strong neurotropism.2. Severe and mild EV71mouse models were firstly established, and the survival rate of severe model was71.43%, and60%of the surviving mice of severe model had sequelae of paralysis while the mice of mild model showed ruffled fur. Dynamic detection of serum cytokines and chemokines showed that IL-5(6hpi), IL-13(2dpi), IL-6, MCP-1, CCL5/RANTES (3dpi) were significantly up-regulated at the early stage of severe EV71infection, suggesting that these factors might herald a severe outcome.3. Microscopically, human fatal cases exhibited severe encephalomyelitis, pulmonary edema and necrotic enteritis. In human cases, EV71antigen and SCARB2were observed mainly in the neurons, microglia cells and inflammatory cells in the central nervous system (CNS), epithelial cells in the intestines. EV71RNA was mainly detected in inflammatory cells located in the CNS, intestines, lungs and tonsils. Morphologically, most of EV71RNA-positive inflammatory cells in CNS were macrophages/microglia and neutrophils infiltrated in perivascular cuffing, microglial nodule, neuronophagia and meninge. Moreover, the BBB in patients with EV71viral encephalomyelitis was severely disrupted. Comparatively, in our mouse model, we observed massive necrotic myositis, different degrees of viral diseases in CNS, lesions in anterior motor neurons of the spinal cord, extensive interstitial pneumonia. And the systemic necrotic myositis may contribute to the respiratory failure and other complications.4. We identified that high level of body temperature, blood sugar, neutrophilic granulocyte ratio, CK-MB and LDH, which might be highly related to the overactive immune response. Using iTRAQ-method and pathways analysis, we found that differentially expressed proteins in sera of severe and mild patients are mainly involved in the immune response and cell death related process.5. Administration of Lactobacillus casei could reduce the viral loads and pathological changes, ameliorate the focal lesions in the mucous membrane of the intestines. The mucosal immune may be up-regulated by Lactobacillus casei, and the virus loads in the intestines and lungs were then reduced, which improve survival rate of the EV71infection in mice. Our data suggest that Lactobacillus casei showed protective effect and anti-infection to EV71infection.Conclusions EV71strains commonly possess neurotropism to humans. The elevated cytokines and chemokines, including IL-5, IL-13, IL-6, MCP-1and CCL5/RANTES, may have potential value as severe prognostic markers in severe mouse model. Fatal patients of EV71infection are characterized as severe neurological lesions with intense inflammatory cells reaction. Massively infiltrated macrophages/microglia and neutrophils might be associated with EV71replication, transmission to CNS, neurological impairment, and neurological complications. EV71mouse model mainly exhibits necrotizing inflammation in skeletal muscle, which is the typical characteristics of severe mouse model. And changes in the CNS, especially in anterior motor neurons may have certain association with paralysis in limbs. Overactive immune response induced by EV71infection may lead to severe and fatal prognosis in HFMD patients. Lactobacillus casei may enhance the mucosal immune to play a role in the anti-infective effect against EV71infection, but the specific regulatory mechanism remains to be furthur study. Our study may provide valuable theoretical foundation and disease models for understanding neurologic pathogenesis of fatal EV71prognosis, and diagnostic and therapeutic values for EV71infection.