| Avian influenza, which was called avian flu (Avian Influenza, AI) for short, is the infection and / or disease syndrome caused by A-type avian influenza virus that was assigned to Orthomyxoviridae family. There have been a number of scholars have who had isolated the same subtype of avian flu virus from sick chickens of different area since H9N2 subtype avian influenza was first reported epidemic in China in 1994. H9N2 subtype avian influenza virus has become the main type in China nowadays analyzed by the existing report. It makes more and more serious damages to the poultry industry and it aroused people widespread interest in it especially for the links with public health aspects. In View of the HA gene plays a key role in the all aspects of virus adsorption, transmembrane processes and pathogenicity of the virus, to start the study of the HA gene mutation analysis of H9N2 subtype avian influenza virus and study of its immunosuppressive effects are both significant in aspects of virology and veterinary science.1 The isolation, identification and analysis of HA gene mutation of H9N2 subtype avian influenza virus.We collected 5 hundreds of pathological material of disease which suspected H9N2 subtype influenza virus infection from different commercial broilers farms in Taian, Weifang, Yantai and 12 other regions in different seasons during 2007 to 2010. Then the virus were isolated and identificated. And the HA gene of the isolated 12 strains viruses were sequenced and analyzed. The results showed that the isolated 12 strains of avian influenza virus of different regions had high homology. The nucleotide homology was between the 91.5-99.9% and the amino acid homology was between 90.6%-100%. HA gene sequence comparison showed that the 12 isolated strains had high homology with strain isolated from Fujian in 2007 and Shandong isolates in 1999. And the nucleotide homology was 93.2%-96.9% and 92.4%-99.5%, the amino acid sequence homology were 91.2%-98.6% and 87.8%-99.1%. They had distantly genetic relationship with foreign isolates, such as Korean isolates in 2000, 2003 and 2008, Iran and Saudi Arabia isolates in 2002 and North Indian and West Indian isolates in 2004. Among them they had the farthest genetic relationship with Korean isolates in 2008. The nucleotide homology was 81.1%-83.3%, the amino acid sequence homology was 74.5%-85.1%. Meanwhile, the homologous analysis of 12 isolates and Shandong EU939143 H9N2 subtype influenza virus isolates which had 93.4% homology with representative strains of Eurasian C/BJ/1/94 in 2007 showed that the nucleotide homology and amino acid homology were 90.2%-94.5% and 87.3%-94.8%. Phylogenetic tree analysis showed that 12 isolates and 07 Shandong isolates are both belonged to Eurasian germline classes CKBJ1/94 subline. The cleavage site (335-341 aa) of the amino acid sequences of 12 strains are RSSR↓GLF, which are consistent with the features of cleavage site of amino acid sequence of low pathogenic avian influenza. However nucleotide analysis showed that 337 site of 3 of the 12 isolates were serine, the corresponding nucleotide were respectively AGT and AGC, which were likely to mutate into AGA that was the serine change into arginine, So that the cleavage sites mutated into the signature sequence (RPK-XK/RR) of the highly pathogenic avian influenza virus. The 234 receptor binding site of 5 of the 12 isolates mutated from Q into L, which amino acid was the same with humanized H9N2 strain in Hong Kong. But the 226 receptor binding site was Q, the viruses were difficult to infect humanbeings. 2 Immune function of H9N2 subtype avian influenza virus on chickensStrain SD06, one of the isolated H9N2 subtype avian influenza virus was selected to challenge SPF chickens to see what kind of effection strain SD06 would do to the chickens'immune organs. First 200 chickens were randomly divided into A and B two groups, isolated feeding. Group A was conteracting virus, while group B was not. Group A in eight day-old was infected artificially H9N2 avian influenza virus subtype SD06 strains by nose and eye, tapping toxic dose 0.4 mL/one (EID50 10 - 7.38/0.1 mL). After 7, 14, 21, 28 and 35d, 10 chickens were randomly selected and killed, acquainted trachea, lung, spleen from the two groups. The cindoruk and copy of AIV H9N2 different time and organizations in the infection chicken was detected using fluorescence organizations such as quantitative RT - PCR. After tapping poison 5 chicken were killed every 4 days. Lung, spleen, method surname bag, kidneys, pancreas and brain were collected and fixed. By the conventional H.E immune organs pathological changes were observed and by using indirect immunofluorescence fluorescence the virus were stained to antigens positioning. At the same time in the attack poison after 7, 14, 21, 28, 35d randomly 10 plume says, gathering the blood samples taken live weight, Annexin V - using flow cytometric FITC marker, the detection instrument T achroacyte subgroup and cell apoptosis, P53 and Bcl-2 mRNA expression level; Cutting inspection separation thymus, method surname bag, spleen and weighing, calculation of immuneobserveorgans index. And then the another 200 chickens were randomly divided into C (C1, C2, C3), D four groups .On the 14th day and 21st day they all were immured by the Newcastle disease vaccine. 7, 14, 21, 28 and 35 days after the Challenging we choose 10 chickens randomly .We took their live weight, gathered the blood samples, marked with Annexin V-FITC and then we tested the detection instrument of T achroacyte subgroup and cell apoptosis, P53 and Bcl-2 mRNA expression level with Streaming cells apparatus.At last we Separated the Thymus, sac and spleen from the bodies and weighed them, then we Calculated the Index. C1, C2, C3 groups were different doses of virus attacked. The agents of virus attacked were respectively 0.2 mL, 0.4 mL, 0.6 mL at the 8th day. After challenging on the 7th ,14th ,21st ,28th ,35th day,the 5 blood samples were taken randomly to separate the serum. By determining serum hemagglutination inhibition of anti-NDV antibody titers to draw antibody curve, then the level messages of influenza virus against Newcastle disease vaccine antibody were got.3 days after the SPF chickens conteracting toxic substances, they showed some symptoms like fever, depression, serous with foam on the flow of tears, later yellow-white purulent discharge flow and so on. Some of the chickens'fat of heart-crowm, lung, spleen and other visible bleeding. 7 d after conteracting toxic substances (15 days old) and 14 d (22 days old), especially decreased immune organ index, tissue sections showed that the thymus, spleen and bursa of degradation and bleeding bodies of the congestion. After staining, flow cytometry showed that the chickens H9N2 subtype of influenza virus after 14 days, CD4 + T cells and CD8 + T cells lower than the control group, CD4 + / CD8 + ratio was significantly decreased compared with the control group statistical significance; Annexin V-FITC/PI double staining showed apoptosis after virus attack to promote apoptosis of lymphocytes in the earlier stage; challenged group of HI antibody titers against NDV were markedly decreased. Above all, the last 3 years H9N2 influenza virus HA gene sequence did not have much variations, but view from the nucleotide variations there is a trend of increasing virulence of H9N2 influenza virus. SPF chickens experimental results show that the correlation, H9N2 infection of SPF chickens can cause atrophy of immune organs, humoral immune function; also causing imbalance of cytokines, lymphocyte apoptosis increased, decreased immune function, causing severe immune suppression, which leads failure of Newcastle disease and other vaccine.
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