Construction Of H₅N₁ HPAIVP Animal Model And Study On It's Pathogeny

Avian influenza (AI) is a severe avian contagious disease caused by avianinfluenza virus (AIV). In recent years, outbreak of AI caused by H5N1 subtypeAIV has brought a serious disaster to many poultry cultivation in manycountries.Even more it has brokendown the species barrier to contract humanbeings,which is serious threatening the health of pelple all over the world.Furthermore, human'mortality is very high. Respiratory tract is the targetorganization of H5N1 AIV. It was reported that severely progressive interstitialpneumonia are the most important symptom of people who were infected by H5N1AIV, which is the direct cause of death. At present, most of study on H5N1 AI arein etiology, especially in molecular biology research of virus virulence variation.Few reports were found about pathogenic mechanism about human infectioncaused by HPAIV. The purpose of this study is to set up a mice model of H5N1HPAIVP and detect the change of ICAM-1 and it's receptors and kinds ofcytokines in H₅N₁ HPAIVP mice in different time in order to reveal the roles ofthem in H₅N₁ HPAIVP employing immunology, molecular biology andimmunohisto-chemistry techneques, it can give some understandings aboutpathogenic mechanism of H₅N₁ HPAIVP and provide theory basis for medicinesused for prevention and treatment for H₅N₁ HPAIV.Specific study are asfollowing: Firstly, in the experiment of animal model building of H5N1 HPAIVP,65 Kunming mice were divided averagely to 13 groups randomly, experimentgroups consisted of 12 groups, the thirteen group is control group. At first, wedilute by 10 multiproportion chick embreyo allantoic fluid of H₅N₁ AI to 10-12which came from tiger and has been already isolated, identified and purified.Every experiment mice were inoculated by nasal cavity with 50 microlite10-1¹0-12 dilution of this virus post etherisation respectively, control mice weregiven 50 microlite nomal chick embreyo allantoic fluid by rhinovaccination. Toobserve mice two weeks continuely. Indexes of observation consisted of clinicalsymptom, change of body weigh, death number, pathological change of lung, lungindex, detecting of HI antibody, searching for and isolating virus antigen andpurifyingt it by electron microscope and RT-PCR methods. On the basis of H5N1HPAIVP animal model, experiment of detecting expression of ICAM-1 and it'sreceptors in lung and serum in H5N1 HPAIVP mice were carried out. Incontracting toxic experiment by means of different virus concentration,experiment groups were divided from 1 to 12 by different toxic virusconcentration, every experiment groups mouse was inoculated by nasal cavity 50microlite H5N1 AIV allantoic fluid which concentration is from 10-1to 10-12, everycontrolgroup mouse was inoculated by nasal cavitywith 50 microlite DMEM. Toobserve closely clinical symptomsof every group mice post infection, sacrifice themouse who was on the brink of death, dissect mouse and get lung sterilitasly. Atthe same time, lung tissue were fixed which were got acrossly in biggest diameterof right-down lung vertical plane, paraffin imbedding routinely and cutting sheetto prepare for immunohistochemistry staining. In conteracting toxic experimentby means of the same virus concentration, every experiment mouse wasinoculated by nasal cavity with 50 microlite H5N1 AIV allantoic fluid whichconcentration is 10-5, it is about 100-150LD50.Every control mouse wasinoculated by nasal cavity with 50 microliteDMEM. To sacrifice respectively 5experiment mice and control mice by eyeball bloodletting in following 6 days postconteracting toxic, get 0.5 millilitre peripheral blood from every mouse toprepare for detecting directly the expression content of CD11b, CD18 and CD54of experiment mice and group mice in different moment on the surface ofperipheral blood leucocyte employing whole blood immuoflurescence flowcytometry, dissect mouse and get lung sterilitasly, figure out lung index. At thesame time, a part of lung tissue were fixed which were got acrossly in biggestdiameter of right-down lung vertical plane, paraffin imbedding routinely andcutting sheet to prepare for immunohistochemistry staining. Meanwhile, to grindleft lung to get lung supernate fluid. At last we detect the expression contents ofICAM-1 and CD11b/CD18 in lung employing ELASA method. At the same time,significance and assaying of serum IL-1β, IL-6, IL-8 and TNF-α in HPAIVPneumonia mice were studied.Every experiment mouse was inoculated by nasalcavity 50 microlite H5N1 AIV allantoic fluid (100-150LD50) which was dilutedbyDMEM to 10-5 post etherisation respectively.Every control mouse wasinoculated with 50 microlite normal by nasal cavity. To sacrifice respectively 5experiment mice and control mice in continual 6 days post contracting toxic, toprepare serum by eyebal bloodletting and reserve in 20℃for ELASA experiment.Meanwhile, to grind lung by glass grinder in sterility room, then add to DMEMwhich volume is 5 times of lung tissue to make 1: 10 emulsion then shift in germfree tube and centrifuge 10 minutes in 2000rpm, centrifuge of it's supernatant 15minutes in 5000rpm to get supernatant to reserve in 4 ℃ to prepare for RT-PCR todetect mRNA expression of IL-1β,IL-6,IL-8 and TNF-α in lung. At last,H5N1HPAIVP were treated by ribavirin, observing and recording mice deathnumber, dissecting mouse to get lung sterilitasly, evaluating index of medicineanti-virus activity consist of: weigh change of mice, lung index,survival rate ofmice,virus titer in lung of mice,average survival days. Meanwhile, detectingexpression contents of ICAM-1 and it's receptors, as well as kinds of cytokines.Result indicates: Animal model H5N1 HPAIVP is built successfully, miceinoculated by nasal cavity with H5N1 inluenza virus were deprementia, clothinghair reverse erecting, sticky secretionin eyes, some mice appeared nervalsymptoms, such as parlysis in forward or hind limbs, circling blindly, et al.Compared with control groups mice, the weigh of dying mice in experimentgroups decreased or not increased;The most principle diseased region of ill anddead mice is lung. There were mottling hemorrhage, severe pneumonia occurredin mice and lung index (lung weigh /body weigh) heightened to 2³%. Alveolarseptum widened in pathological section, a lot of inflamming cells and protein-likeserofluid effusioning in alveolar space. AT the same time, H5N1 virus positiveparticles can be seen in immunohistochemistry staining of lung preparations,influenza virus-like particles can be found on the pavement epithelium cellssurface of lung alveolus and in the alveolar space in ultrastructure observing to thelung preparation. It was verificated that the virus isolated from lung of dead miceis H5N1 influenza virus by RT-PCR and specific HI test. This experiment showedthat lung is the target organization of H5N11 influenza virus, H5N1 AIV canreplicate and evoke infection in corpore of mice, even more, make mice fall ill orresult in death. H5N1 AIV have high pathogenicity and high fatality rate in mice, itcan stimulate survival mice to generate specific immunoreaction, HI antibody inserum increased obviously. H5N1 AIV can infect mice and cause the same clinicalsymptoms and pathological change as human, furthermore, onset of illnessfollows certain rules, the indexes which can be detected are clear. Above all, micecan be used for building good animal model of H5N1 HPAIVP, as a mammalmodel to study pathogenesis and treatment and immunity research of H5N1 AIV.The intercellular adhension molecular-1 (ICAM-1) and it's receptors in the lungsof HPAIV and control mice pneumonia were detected by usingimmunohistochemistry staining. The expressive levels of ICAM-1 and itsreceptors of CD11b and CD18 in bronchial alveolar epithelial cells,bronchopulmonary capillary endothelial cells, and infiltrativing lymphomonocyteand monocytes were significantly higher in HPAIV group than that in controlgroup 24 hours after infection. The peak level was detected 4 days after H5N1HPAIV infection. Among the different viral dosage,the highest expression of·9·ICAM-1 and its receptors were detected in 10-5 dilution group. The change wasconfirmed by the method of ELASA. To detect directly the expression content ofCD11b, CD18 and CD54 of H5N1 HPAIVP model mice and group mice indifferent moment on the surface of peripheral blood leucocyte employing wholeblood immuoflurescence flow cytometry, percentage of positive cell(PPC)andmean fluorescence intensity(MFI)of CD11b, CD18 and CD54 on the surface ofperipheral blood leucocyte in experiment group has increasing tendency 24 hoursafter infection, and it's higher remarkably than that in control group. The peaklevel was detected 4 or 5 days after infection. The result indicates that ICAM-1and its receptors have the high expression level in HPAIVP pneumonia. Perhapsthey play an important role in the pathopoiesis of HPAIV pneumonia mice. Tostudy the interrelation between HPAIV Pneumonia and cellular factorsinterleukin-1β (IL-1β) interleukin-6, (IL-6) interleukin-8 (IL-8) and tumornecrosis factor– a (TNF-α) and HPAIV pneumonia in mice by HPAIVP model.The expression content of serum and mRNA expression level of lung of IL-1β,IL-6, IL-8 and TNF-α were detected directly in experiment and control groupmice in different moment employing ELASA and RT-PCR methods. The levels ofIL-1β, IL-6 and TNF–αin model groups mice increased in different degree 24hours after infection, the level of IL-8 increased is the most light, the level ofIL-1βgot to the peak level quickly 24 hours post infection, the peak level of IL-6and TNF-α was arrived 2 days post infection, increasing extent of TNF-α is themost obvious, it's level is 3 times of control group. This result indicates thatcytokines IL-1β, IL-6, IL-8 and TNF-α might participate in the whole process ofH5N1 HPAIVP. Ribavirin was used to treat H5N1 avian influenza pneumonia mice,weigh decreasing of treatment group mice was amended in Ribavirin group mice,weigh of treating group mice didn't decrease evidently, during experimentalobserving period changing tendency of weigh is the same as that of normal controlgroup. Meanwhile, virus titer in lung and lung index of Ribavirin group were alsolower significantly than that of virus control group. Average survival days werelonger than that of virus control group. In short, Ribavirin can improve the weighdecreasing, delay course of disease, raise survival rate, improve pathologicalchanges in lung obviously, lung index and virus titer decreased. At the same time,expression contents of ICAM-1 and cytokines were lower than those of viruscontrol group in lung, which verified the roles of ICAM-1 and it's receptors andkinds of cytokines in the pathopoiesis of H5N1 HPAIVP.