Study On Pathology And Establishment Of Mice Model Infected By H5N1 Highly Pathogenic Avian Influenza Virus

Tiger is a kind of wild animal. In may 2002, some tiger in Shanxiprovince, Harbin and Tangshan city, an acute infectious disease wasoccured which characterized by clinical signs fever, anorexia and twitch.Isolation of virus, genetic test and HA/HI test, the pathogen was identifiedinfluenza virus type A, subtype H5N1, by means of named TIV. Themortality of TIV reached 40%, and caused great economic loss. Due tostrong pathogenicity of the virus and pose a serious threat to tiger, so thedisease received much attention. So far the pathogenic mechanism of TIV isnot yet understanded, so it is difficult to prevent and control. In our studywe established mice infected model of TIV, and have done thehistopathology research on H5N1 highly pathogenic avian influenza virus.It supplies the basis and direction for studying on the pathogenesismechanism and therapeutic agent of this disease, and also establishedfoundation for evolving reserve of therapeutic agent for infection highlypathogenic avian influenza virus.TIV HPAIV A/Tiger/Harbin/01/2003 strain was cultured in MDCKcell, and infected mice by inoculation nasal cavity, TCID50 of virus was10-7.36/0.05ml on MDCK cell. Results showed that the inoculated micedisplayed the clinical signs such as mental deterioration, behavioralretardation and mental symptom. Body weight of the mice decrease, andlung index reached 2-3%. There were bleeding and swelling in the lung ofdied mouse. TIV could be isolated from lung, brain, liver and kidney ofthem, MLD50 value was 10-7.25/0.05ml. The highest HI titer of the toleratedmurine Sera got to 1:2-8. The control mouse remained normal. The highestHI titer of the control murine sera could not reach 1:2. The results showedthat was high pathogenic to mice Which could be used as mammalianmodel to study TIV.After mice died of the infection of TIV HPAIVA/Tiger/Harbin/01/2003 strain, TIV was isolated from heart, liver, spleen,lung, kidney and brain with cytopathic effect in MDCK cell, TCID50 wascalculated by Reed-Muench method. At the same time, the virus wasidentified through RT-PCR, HA/HI and observed under electronmicroscope. Results showed that HPAIV could be propagate in lungs, livers,kidneys and brains of mice,and the virus titer was highest inlungs,respectively ranged from 4.5 ± 0.53logTCID50/0.5ml, 1.5 ±0.63logTCID50/0.5ml, 1.33 ± 0.12logTCID50/0.5ml and 1.67 ±0.37logTCID50/0.5ml. The specific nucleate straps of HPAIV wereexpanded in lungs, kidneys, livers and brains and the infected cell culturesas same as the theory value with 464bp by evaluation of RT-PCR. We candetect that the HA titer was 1:23 and the typical influenza virus-likeparticles were observed with the electron microscope in the infected cellculture and the supernate of lung emulsion.Through pathologic study, the results indicated that the damages oflungs from the infected dead mice were the most severe. Under themicroscope, sackful infiltrative of macrophage and manipulus protein-likeeffusion in the alveolar. Histopathology of lung showed widening ofalveolar space and effusions. It developed telangiectasis and congestion inthe small vessels inner lungs, and hyperemia in the bronchus. Byimmunohistochemistry, the positive staining particles of virus were foundon the epithelium cells of bronchus and alveolar and in the endochylema oflymphomonocyte. We could observe influenza virion which was short-axletype and spherical from pavement epithelium cells of alveoli pulmonis inmice by electron microscope ultramicrostructure observation. Some apartvirion had not isolated from alveolar epithelium. This study possesses veryimportant rational significance and clinical application value in the aspectof medical science research area.