Pathological Study On Fatal EV71Infection And Molecular Biology Study On EV71Virus

HFMD (Hand, foot and mouth disease, HFMD) is an infectious disease caused byenterovirus infections. In recent years enterovirus type71(Enterovirus71, EV71) wasfound to be the major epidemic pathogen. Children are common susceptible to EV71, andhave diverse clinical manifestations after infected with EV71. In addition to thecharacteristic HFMD, they often show serious complications of the central nervous systemdisease and pulmonary edema and/or pulmonary congestion and even death. It is unclearabout the pathogenesis of HFMD leaded by EV71, and lacks of effective treatment, soclinical disputes often occur. Therefor, it is important to identify the HFMD’s pathogen,analyse comprehensively the pathological changes with the EV71infection to investigatethe pathogenesis, subsequently research security and effective HFMD vaccine to preventand control disease outbreaks effectively. Accordingly, this thesis, which contains fivechapters, is focus on the pathologic changes infected with EV71and attempt to researchvaccine candidate.Chapter Ⅰ Introduction of the background of HFMD and EV71, including the prevalenceof HFMD, the virology characteristics of EV71, epidemiological studies, pathogenesis,diagnosis and treatment of EV71infection. The research on genetic engineering vaccineand the baculovirus expression system were also presented. Finally the content andsignificance of this work were summarized. Chapter Ⅱ From one fatal case of suspected with EV71infection, the pathology researchwas performed. With routine forensic pathology analyses, severe pathologic lesions in thecentral nervous system (CNS) were found. Under the microscopy, typical features of acuteviral encephalitis were identified, inclusding neure necrosis, multifocal areas of necrosis,widespread perivascular cuffing by mononuclear cells, extensive microglial proliferation,and many glial nodules. Pulmonary edema and congestion were significant, inflammationwas prominent. So acute viral encephalitis and viral pneumonia leading to acute respiratoryand circulatory failure were affirmed as the cause of death. On this basis, EV71wasconfirmed as the pathogen by reverse transcription polymerase chain reaction (RT-PCR)with the EV71universal detection primers published by national CDC. Further, theorganization relative viral load was investigated by using the nested PCR amplificationwith specific primers for EV71conserved region. And then anti-EV71polyclonal antibodyas the first antibody was used to analysis the virus antigen distribution byimmunohistochemical staining on paraffin sections. The results showed that the viral loadon brain tissue was relatively high, the brain stem and cervical spinal cord were the highest.Viral antigens were found in the cytoplasm of neurons, neuronal processes, andinflammatory cells, most often associated with glial nodules. The nerve cells in the CNSand inflammatory cells were the main site of virus infection and replication. The lung tissuewas obvious edema/congestion and inflammation, but the virus nucleic acid and antigenwere negative, not the virus infection and replication site.Chapter Ⅲ Virus isolation was performed from the highest viral load in brainstem, andthen virus culture and plaque purification were proceeded on RD cells in vitro. Virusinfectivity was affirmed by One-step growth curve and virus genotypes were identifiedaccording to VP1gene sequence. EV71genome complete sequence was cloned with fouroverlapping segments, the genome sequencing and analysis were finished then submitted toGenebank. Results showed EV71-xf live virus could be successfully isolated from brain stem, which showed the typical virus-specific cytopathic effect (CPE) on RD cells in vitro.There could find uniform size, shape consistent with a diameter of about20～30nm EV71virus particle in vitro by transmission electron microscopy. Four purified virus palques wereamplified and their infectivity showed no significant difference and all of them were the C4subtype. The complete genomic sequence of EV71-xf was7405bp, and the Genebankaccession number was JQ804832. So it suggested that EV71-xf virus was still the domesticepidemic strains C4, the whole geome sequence would help the follow-up study.Chapter Ⅳ The one-day-old Balb/c sucking mice were infected by EV71-xf virus withintracranial injection(IC) ways, then their performance were observed every day. Bloodsamples were adopted for detection of EV71specific IgG and neutealizing antibody fromorbital blood. Virus was isolated from the mice brain tissue and then infected freshone-day-old mice by IC ways again, and so forth to generate a mouse-adapted EV71strain.Results showed that one-day-old Balb/c sucking mice could be infected by EV71-xf withIC ways, but there was no obvious nervous system involved manifestation. The virusspecific antibody IgG showed higher in the two weeks after infection, but still wassub-neutealized antibody levels, and then declined. Secondary infection by IC couldstimulate mice to produce higher concentrations of IgG, which exhibited good specificityand cross protective effect against EV71on RD cells in vitro. Maternal antibodies hadstrong protective effect for newborn mice, but the sustaining time was very short, about4-week-age its protective effect disappeared. A mouse-adapted EV71strain EV71-xf/A/J/Qwere obtained by subjecting to three passages in the mouse brain tissue, which displayedincreased infectious in mice, but its virulence properties requires further study.Chapter Ⅴ EV71-xf polyprotein P1and3CD were cloned under pPh and pP10promotersof vector pFastBac-Dual, respectively. Doner palsmid pFastBac Dual EV71-xf-3CD-P1wasconstructed, the recombinant Bacmid was constructed by transpose and recombinantbaculovirus AcBac-EV71-xf-3CD-P1was obtained. Bacmid DNA was transfected into Sf9 cells, EV71virus like particles (VLPs) were assembled and preliminary purified by suroseand CSCl density gradient centrifugation. The expressed protein was detected by WesternBlot and VLPs were identified by Transmission electron microscopy (TEM). Furthermore,a stable expressed3CD cell line of Sf9-3CD was constructed to product VLPs after Zeocinselection and real-time PCR identification. The results showed that recombinantbaculovirus AcBac-EV71-xf-3CD-P1could express the precursor peotein P1andvirus-encoded protease3CD simultaneously, and P1could be digested into structuralprotein VP1by3CD. EV71virus like particles (VLPs) could be observed self-assembledunder the TEM. Through preliminary purification by sucrose and cesium chloride densitygradient centrifugation, VLPs was observed to have similar shape, size, and VP1epitope asnatural EV71virus particles by TEM and Western Blot. It suggested that the VLPs could bea candidate for vaccine produce. Sf9-3CD cell lines could stably express3CD protein anddigest P1to assemble VLPs, so it is a novel way could to be used to produce VLPs.In summary, the forensic pathology analysis of one fatal case of suspected HFMD wascompleted and enterovirus71was identified as the major pathogen. A highly activeEV71-xf stain was isolated from the brainstem tissue and purified by plaque purification onRD cells in vitro. Followed by completion of virus infection activity detection andwhole-genome sequencing of one EV71-xf strain, the preliminary study of the mice wereinfected with EV71-xf virus as well as genetically engineering vaccines were proceeded.This study provided useful data to analyze the mechanism of the fatal EV71infection, andalso attempted to contribute the foundation for genetic engineering vaccines for the EV71,made important contributions to the work on the prevention and control of HFMD.