| 1. Background and objectiveEV71belongs to the human enterovirus, a species of the enterovirus genus within the family Picornaviridae. It is a common cause of hand, foot and mouth disease, aseptic meningitis, encephalitis, poliomyelitis-like paralysis and a lot of epidemics correlated with nerve system. EV71associated with neurological diseases was first reported in1974, the documented outbreak in California. After the first identification, the epidemic of EV71in different regions have been reported, it have been lead to repeatedly outbreak and epidemic in the world. Although the genome and the life cycle of EV71is similar to the picornavirus family members, but it induced pathological symptoms are significantly different. The mechanisms of EV71pathogenesis still remain unclear.There is no specific treatment and effective vaccine to prevent EV71infection. It has very important significance that clearly the pathogenesis of EV71. Studies have also detected EV71viral genomes and antigens by histopathology, immunohistochemistry, and reverse transcription polymerase chain reaction in the cells of CNS tissue, including neurons, neuronal processes and associated inflammatory cells. This indicate EV71can into CNS, however, it is not clearly how EV71infected CNS. As for poliovirus (PV), two possible routes by which the virus reaches the central nervous system (CNS) have been suggested:the virus either enters the CNS from the blood across the blood-brain barrier (BBB) or is transmitted to the CNS through peripheral nerves via retrograde axonal transport. Although the study confirmed after newborn mice taken orally EV71, it may be a cause of persistent viremia and blood cerebrospinal fluid barrier permeability increasely. But low levels of virus number in CNS indicated hematogenous pathway is not main way. Therefore, it has important significance for the prevention and treatment of EV71infected how EV71effects on the structure and function of BBB. The BBB constitutes the interface between the blood and the central nervous system (CNS). Under physiological conditions, the BBB maintains CNS homeostasis and selectively regulates intracellular and paracellular passage of ions, molecules and cells. So it is not clearly how EV71penetrate BBB. Virus infection process is the first step in virus by and located at the cell surface receptor binding and specificity of adsorbed on the surface of infected cells, then the cells by endocytosis into the cell or virus nucleic acids are released into the cell.Viruses use specific receptor to bind target cells. Then the virus partical by endocytosis into cells and the virus nucleic acids are released into cells. Therefore, we hypothesized the major components of BBB (endothelial cells) might be susceptible cell on EV71. EV71can lead to the structural changes and dysfunction of BBB. Although researchers have reported cynomolgus monkeys and young mice as animal models to represent CNS involved diseases by EV71infection. However, some differences still remain between EV71replication sites in these animal models and those in humans. Therefore, it is still room for find better model to unmask neural pathogenesis of EV71. In this study, EV71was isolated from the stool of a patient with poliomyelitis-like paralytic disease. HBMEC was infected by EV71. To explore whether EV71can infect and replicate in HBMEC, the morphological of HBMEC was observed, EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants were detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected.Viral invasion will modify the patterns of host cell protein expression, which may affect the normal physiological function of host cell and determines viral pathogenic progress and consequence. Therefore, studies on viral infections proteomics contributes to uncover the mechanism of interaction between EV71and HBMEC and viral molecular pathogenesis, found early biomarker of EV71infection, develop earlier diagnostic method, evaluate therapeutic effect and prognosis and so on. In this paper, techniques of viral infection proteomics, the progress of changes of HBMEC proteome induced by EV71were explored. The protein expression of culture supernatants and intracellular at Oh,1h,16h,24h after EV71infected HBMEC were analyzed by two-dimensional-electrophoresis and matrix assisted laser-desorption ionization time of flight mass spectrometry.2. Methods2.1. An EV71strain was isolated and identificated which come from severe hand-foot-mouth disease patients.2.2. HBMEC was infected by EV71, the CPE were observed; EV71antigen in HBMEC was observed by immunofluorescence; EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants was detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected. EV71related HBEMC and normal HBMEC were examined by2-D. Intensity changes of protein spots detected with statistic significance were identified by MALDI-TOF MS or MS/MS. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin in the development of EV71ralated HBMEC.3. Statistical analysisAll data in this study are presented as mean±SD. All these statistical analyses were carried out using prism6.0or SPSS13.0for window.The apotosis rate and EV71copies were evaluated by the ANOVA for the repeated measures, The normality of results was evaluated by the Kolmogorov-Smirnov test. Comparison between groups were performed with the independent samples t-test. P<0.05was considered statistically significant. 4. Results4.1. An EV71strain was isolated.4.2. According to the morphological observation, EV71can result in CPE of HBEMC, along with the prolonged of infection, the CPE became more severe. Diameter20-30nm virus particles was found in HBEMC by transmission electron microscope. EV71antigen was founded in HBMEC by using a polyclonal antibody. The copy number of EV71in HBMEC and RD cells were detected by using Real-Time PCR method, the copy number reached peak at third days, the replication effect in HBMEC was lower than in RD cells (P<0.01). EV71infection can lead to HBMEC cytoskeletal rearrangement by using rhodamine phalloidin staining. After HBEMC was infected by EV71at8h, the early apoptosis of HBMEC was detected with JC-1kit. Using the annexin V-FITC/PI kit, EV71can lead to the apoptosis of HBMEC, accompany with the prolong of infection time, mainly in cell necrosis (P<0.01).4.3. By two-dimensional gel electrophoresis and mass spectrometry identification,28proteins were found expressed differently in these groups.4.3. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin protein and demonstrated the expression of vimentin can lead to cytoskeletal alterations.5. Conclusion5.1. The morphological observation and immunofluorescence and transmission electron microscopy method confirmed HBMEC can be infected with EV71.EV71can replicated in HBMEC. This indicated HBMEC is susceptible cell of EV71, however, the replication efficiency is more lower than RD. BBB may exist specific receptors of EV71. EV71may be binding with endothelial cells of BBB and through endocytosis into cells, at last, lead to the morphological and functional changes of BBB and invasion into the brain.5.2. Using JC-1mitochondrial apoptosis detection kit has confirmed infection of EV71can induce the mitochondrial apoptosis in HBMEC, along with the prolonged duration of infection, cells mainly in necrosis. 5.3. HBMEC cell micro filaments structural rearrangements by EV71infected HBMEC. Microfilament cytoskeleton changes also played a supporting role for viral replication and activation.5.4. Proteomics research discovered28expression of protein, according to the functions are divided into9categories. Combining differential spectrum expression change of protein function and the infection of EV71attack and attack HBMEC protein expression variation analysis, some important molecular targets were found. |
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