Isolation And Identification Of H9 Subtype AIV And Sequence Analysis Of HA Gene In Rizhao Shandong Province

Avian influenza is caused by influenza virus type A with the clinical manifestations respiratory infection, egg dropping, alvi profluvium and so on. H9 subtype avian influenza is low-pathogenicity avian influenza. It can result in egg dropping, growth retardation and a small quantity death. The first H9 avian influenza virus was isolated from chickens in southern China in 1994, since then it was prevalent in Southeast Asia including China and many other countries worldwide for several years. However, the pandemic of H9 subtype AI, which broke out in summer, 1998, spread very rapidly to more than 20 provinces within several months. Since the AIVs of H9 subtype in poultry have been confirmed worldwide, they have the characteristic of high efficiency, spread and wide range in China after 1998, and AIV of H9 subtype in human had been discoverd in 1999, it raised great concerns for its economic importance to poultry industry and potential threat to the public health.Four stains of H9 hypotype AIV separated from the sick chicken internal organs from the clinical manifestation symptom, and biology characteristic and HA gene sequence analysis was studied. The purpose of the research is to understand the separation of 4 stains of H9 subtype AIV's toxicity and the variation of HA gene, thus provides certain rationale for better prevention and control of H9 subtype AIV.Part I: Isolation and identification of H9 subtype AIV of 4 stains of H9 hypotype AIVFour stains of H9 hypotype AIV separated from the sick chicken internal organs from the clinical manifestation symptom, which the names of are SDRZ1, SDRZ2, SDRZ3, SDRZ4. In the first experiment, through the determination of hemagglutination properties, infected chicken embryo, the average time of death of chicken embryo and the ICPI, the virulence was analyzed and compared. The results show that the virulences of there virus were weak and there were small disparity in antigenicity. The virulence of SDRZ2 was much stronger than the others. The HA potency is between 2⁸ and 2⁹. The EID₅₀ is between 10^-6.15/0.2mL and 10^-8.69/0.2mL. The EID₅₀ of the SDRZ2 is 10^-8.69/0.2mL. There are some difference in MDT is between 90.0 hours and 103.2 hours. And the SDRZ2 poisonous chicken embryo average death time most is short is 90.0h, explained that this viral the pathogenicity is strong; But other virus's chicken embryo average death time above 90h, showing that their pathogenicity is weak. Attacks the chickling which the poisonous chicken embryo hatches and attacks the poisonous chickling to appear obviously has split examines the symptom.Part II: Cloning and and Sequence analysis of complete HA gene of 4 stains of H9 subtype AIVThis research used the RT-PCR method to increase 4 H9 hypotype AIV HA gene fragment. The sequential analysis result indicated that amplificated the gene fragment length which is 549 bp. The nucleotide and the amino acid sequence homology analysis indicated that the relation of these 4 stains of H9 subtype AIV are close, the homology above 93.7%, H9 in 2003 separates with Shandong Province the hypotype AIV homology is 94.5%. Difference has not the big variation along with the time and the area. With domestic other province H9 the hypotype AIV homology is 78.9%⁹6.9%. The system cladogram analysis result indicated that these 4 stains of with Shandong, Guangzhou, Nanjing, the Hebei H9 hypotype AIV poisonous blood relationship is near, belongs to the identical branch. It might be indicated that H9 subtype AIV occurred in Shandong are closely related to those popular in Guangzhou, Nanjing, Hebei.