Waterfowl Origin H5N1 Subtype Avian Influenza: Ultrastructural Pathogenicity,Developing Its Monoclonal Antibody Against M Protein And Preliminary Application

We have systemicly compared the influences on various organism cells of ducks, geese, chickens which artificially infected with the three isolates (ZM, SG and JC) were observed with transmission electron microscope (TEM) and scanning electron microscope (SEM) at first. Ultrathin sections of infected tissues and organs were observed with TEM, the results suggested that the three isolates could cause severe ultrastructural pathogenesis widely in many organs and tissues such as hepar, pancreas, lien, ren, pulmo, cerebral, cerebellum, cor, proventriculus, muscular and so on. AIV virions can be found in lien and proventriculus of infected duck inoculated ZM strain, in cerebellum and ren of infected goose inoculated SG strain, in cerebral and proventriculus of infected chicken inoculated JC strain in ultrathin sections . Apoptosis induced by avian influenza virus ZM strain infection in the tissue cells of cor and recta and SG strain infection in pancreas also can be found. The results of SEM suggested that all of the three isolates could cause obvious and severe ultrastructure pathogenesis in epithelial cells of digestive tract and respiratory tract ,such as desquamation of microvilli and cilia, many secretory granules ,creaking on the surface of epithelial cells could be observed. Avian influenza virions could be found on the surface of mucous membrane in ZM infected esophagus, trachea, caecum and proventriculus, in SG stain-infected esophagus, and in JC strain-infected esophagus, bursal and caecum. All of the results indicate that avian influenza viruses can cause the infected host organs with severe ultrastructural pathogenesis. The waterfowls are not the single role of natural hosts of avian influenza virus. Our result can be took as a piece of detailed datum and afford references and proofs to the correlative study on avian influenza in future.H5 subtype avian influenza virus ZM strain was isolated from muscovy and propagated in the allantotic cavity of 11 day-old embryonated SPF chicken eggs. The allantotic fluid infected ZM was purified, men the purified virus was prepared as antigen and immunized BALB/C mice by introperitoneal injection. By using the hybridoma technique, SP2/0 myelomacells and spleen cells of the immunized BALB/C mice were fused by PEG-1700, one monoclonal antibody named B2B11 was obtained by indirect-ELISA. This monoclonal antibody only reacted with a 41kD protein band in western-blot That is to say, this monoclonal antibody was the one against the M protein (M1 and M2) of H5 subtype avian influenza virus. The results of heat stability test, secretion stability test and specificity test suggested mat mis monoclonal antibody has wonderful stability and specificity. All of the properties make the obtained monoclonal antibody has a bright future in Mab-based diagnosticassay for fast detection of H5 subtype avian influenza virus.