Preparing The Monoclonal Antibodies Of CK/SH/F/98(H9N2)and Analyzing The Biological Characteristics And The Two Surface Gene Of CK/SD/C9/11(H9N2) Avian Influenza Virus

H9N2subtype avian influenza virus (ATV) is widely popular and spread in China, and has caused severe economic losses in our country. The first case of human infection with H9N2subtype AIV was reported in1999. Recently a new subtype H7N9AIV, which may result from gene rearrangement, has emerged in some province in China, and infected human, therefor AIV has an enormous threat to human health and life safety. In this study, the monoclonal antibodies (McAbs) of A/Chicken/Shanghai/F/98(H9N2) AIV were prepared, and identified by many methods; The transmission characteristics of A/Chicken/Shandong/C9/2011H9N2subtype AIV was studied with histopathology and immunohistochemistry; The HA gene and NA gene of A/Chicken/Shandong/C9/2011H9N2subtype AIV were sequenced and analyzed. The results in this study may contribute to rapid clinical diagnosis and new vaccine candidate against influenza virus.First, A/Chicken/Shanghai/F/98(H9N2) AIV was purified by differential gradient centrifugation, then immunized eight-week-old BABL/c mice. Using hybridoma technique, positive hybridoma clones were screened by indirect enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition test (HI), and limiting dilution method to get positive subclone. Two hybridoma cell lines of A/Chicken/Shanghai/F/98H9N2subtype AIV, named1C10and6D7strains, were obtained, and their HI titer of the cell cultures supernatant and the ascites were27-8,213-14, respectively. The ELISA titers of the ascites of1C10and6D7were up to5×104, which were specific to H9subtype AIV, but not reacted with H5subtype ATV, New-casttlet Disease vius,avian infectious bronchitis virus, and avian egg drop syndrome virus by HI and ELISA. Two McAbs could react with75kd HA protein of H9subtype AIV confirmed by Western blotting method. A/Chicken/Shanghai/F/98(H9N2) AIV antigen were detected with1C10McAbs by immunohistochemistry in the lung of four weeks of SPF chickens inoculated by F strain. A/Chicken/Shandong/C9/2011(H9N2) AIV, called C9, was isolated from Shandong. The HA titers of C9strain were210-11,50%egg infectious dose (EID50) and mean embryo lethal dose (ELD50) were101125/0.2mL, and109/0.2mL, respectively. In order to investigate the transmission and replication characteristics of C9strain.4-week-old SPF chickens were inoculated with107EID50allantoic fluids of C9strain through oral, intranasal and eye drops. The results showed that C9strain replicated mainly in the respiratory tract and was isolated from the digestive tract, but could transmit from inoculated chickens to uninoculated chickens by direct contact, and transmit by aerosol and by fecal contact in contact. The pathological changes of infected chickens appeared in the trachea and lungs, but did not found in the digestive system. Antigen of C9strain was detected in the trachea and lungs of SPF chickens by histochemistry with McAbs at3d and5d after inoculation. The results showed that C9strain can spread by direct contact, aerosol and fecal.Viral RNAs of C9were extracted and RT-PCR was performed to amplify the HA gene and NA gene segment with specific primer designed according to the HA gene and NA gene of H9N2subtype AIV from Genbank. Phylogenetic analysis showed the HA gene and NA gene of C9strain belonged to Y280-like, and had92.8%、93.6%of nucleotide homology and94.4%,95.3%of animo acid homology to the HA gene and NA gene of A/Duck/Hong kong/Y280/97(H9N2) respectively, a Y280-like strain. But the HA gene and NA gene of C9strain had88.4%,89.7%low nucleotide homology to A/HK/1073/99(H9N2), a human derived influenza virus. The deduced amino acid sequences of C9at the hemagglutinin cleavage site were R-S-S-R, indicated that C9strain is typical of lowly pathogenic AIV. A potential glycosylation site was missed at position218in the HA gene of C9AIV, The alanine at position190in HA gene of C9strain was mutated to alanine. The glutamine at position226in HA gene of C9strain was changed to leucine, where have defined the RBS of HA as a key component in influenza host range.3animo acids were deleted in the NA gene of C9strain at position63-65, it resulted in deletion of one glycosylation site, but the rest potential glycosylation site were highly conservative. The aspartic acid at position369of the hemadsorbtion (HB) site of the NA gene of C9strain was mutated to asparagine. Further studies are needed to ascertain the effect of HA and NA genes in the biological characteristics of H9viruses.