Isolation And Identification Of H9Subtype AIV From Weifang Shandong Province And Sequence Analysis Of HA Gene

Avian influenza is caused by influenza virus type A with the clinical manifestationsrespiratory infection, egg dropping, alvi profluvium and so on. H9subtype avian influenza islow-pathogenicity avian influenza. It can result in egg dropping, growth retardation and asmall quantity death. The first H9avian influenza virus was isolated from chickens insouthern China in1994, since then it was prevalent in Southeast Asia including China andmany other countries worldwide for several years. However, the pandemic of H9subtype AI,which broke out in summer,1998, spread very rapidly to more than20provinces withinseveral months. Since the AIV of H9subtype in poultry have been confirmed worldwide, theyhave the characteristic of high efficiency, spread and wide range in China after1998, and AIVof H9subtype in human had been discovered in1999, it raised great concerns for itseconomic importance to poultry industry and potential threat to the public health.Four stains of H9hypotype AIV separated from the sick ducks internal organs from theclinical manifestation symptom, and biology characteristic and HA gene sequence analysiswas studied. The purpose of the research is to understand the separation of4stains of H9subtype AIV’s toxicity and the variation of HA gene, thus provides certain rationale forbetter prevention and control of H9subtype AIV.Part I: Isolation and identification of H9subtype AIV of4stains of H9hypotype AIVFour stains of H9hypotype AIV separated from the sick ducks internal organs from theclinical manifestation symptom, which the names of are SDWF1, SDWF2, SDWF3, SDWF4.In the first experiment, through the determination of hemagglutination properties, infectedchicken embryo, the average time of death of chicken embryo and the ICPI, the virulence wasanalyzed and compared. The results show that the virulences of there virus were weak andthere were small disparity in antigenicity. The virulence of SDWF2was much stronger thanthe others. The HA potency is between28and29. The EID50is between10-6.25/0.2mL and10-7.69/0.2mL. The EID50of the SDWF2is10-7.69/0.2mL. There are some difference in MDTis between90.0hours and105.0hours. And the SDWF2poisonous chicken embryo averagedeath time most is short is90.0h, explained that this viral the pathogenicity is strong; But other virus’s chicken embryo average death time above90h, showing that their pathogenicityis weak. Attacks the duckling which the poisonous chicken embryo hatches and attacks thepoisonous duckling to appear obviously has split examines the symptom.Part II: Cloning and Sequence analysis of complete HA gene of4stains of H9subtypeAIVThis research used the RT-PCR method to increase4H9hypotype AIV HA genefragment. The sequential analysis result indicated that amplificated the gene fragment lengthwhich is1003bp. The nucleotide and the amino acid sequence homology analysis indicatedthat the relation of these4stains of H9subtype AIV are close, the homology above93.7%,H9in2003separates with Shandong Province the hypotype AIV homology is94.5%.Difference has not the big variation along with the time and the area. With domestic otherprovince H9the hypotype AIV homology is78.9%~96.9%. The system cladogram analysisresult indicated that these4stains of with Shandong, Hebei, Nanjing, the Guangzhou H9hypotype AIV poisonous blood relationship is near, belongs to the identical branch. It mightbe indicated that H9subtype AIV occurred in Shandong are closely related to those popular inHebei, Nanjing, Guangzhou.