Isolation And Identification Of Low Pathogenicity Avian Influenza (H9) Virus In Henan And Study On The Biological Charactistics

Avian Influenza(AI) was an infection disease caused by type A avian influenza virus(AIV), and it can be divided into highly pathogenic and low pathogenic according to their pathogenicity. A variety of disease syndromes range from sub-clinical to mild upper respiratory disease to loss of egg production caused by low pathogenic avian influenza virus.H9 subtype avian influenza virus was the main subtype viruses of low pathogenicity which affecting our poultry industry.In recent years a number of farms were suspected to infecte AIV after immuning the H9 subtype vaccine in Henan Province. AI caused amount of loss in economic and brought difficulties in the epidemic.180 samples were collected from different regions of Zhengzhou, Nanyang, Jiaozuo etal in Henan Province and viruses were isolated by carring out in embryonated eggs. Viruses were isolated by inoculated in 9-11 day embryonated eggs via the allantoic cavity and collected the allantoic fluid and identified by HA and HI tests.The result suggested that ten H9 subtype avian influenza were isolated. According to the standard body of influenza virus named them as respectively: A/chicken/Luohe/01/2009(H9 Subtype),A/chicken/Luohe/02/2010(H9 Subtype),A/chicken/Xinxiang/04/2009(H9 Subtype),A/chicken/Xingyang/18/2009(H9 Subtype),A/chicken/Xinzheng/22/2009(H9 Subtype),A/duck/Luohe/37/2009(H9 Subtype),A/chicken/Kaifeng/44/2009(H9 Subtype),A/chicken/Zhengzhou/45/2009(H9 Subtype),A/chicken/ Zhengzhou /131/2010(H9 Subtype),A/chicken/Xinxiang/132/2010(H9 Subtype),abbreviated for LH01,LH02,XX04,XY18,XZ22,LH37,KF44,ZZ45,ZZ131,XX132. Which four H9 subtype avian influenza viruses for studying on biological characteristics of physical and chemical properties, hemagglutination , EID50, MDT, ICPI. The results were that their physical and chemical properties meet the basic characteristics of the avian influenza viruse. Their results of EID50 were 10-8.68, 10-8.5,10-9.5, 10-7.83 EID50/0.1ml;Their results of MDT were 85.6 h,107.2 h,117.6 h,110h;Their results of ICPI were17/80=0.2125, 13/80=0.1625,14/80=0.175,11/80=0.1375.In order to understand the molecular epidemiology of H9 subtype avian influenza in Henan Province, we have been sequencing whole genome nucleotide sequence of four isolated strains. Then comparated and analyzed nucleotide and deduced amino acid sequences of these four H9 subtype of avian influenza virus to the representatives of H9 subtype strains in GenBank , and analyzed HA and NA of the deduced amino acid cleavage site, receptor binding sites and potential glycosylation sites and HB sites with analysis software to learn the variation and the phylogenetic position . The date indicated that the open reading frame of PB2, PB1, PA, HA, NP, NA, M, NS gene of these viruses were constitute of 2280, 2274, 2151, 1683, 1497, 1401, 759, 864bp, and separately coded 759,757,716,560,498,466,252/97,217/121 amino acids.The HA cleavage site sequence of these four viruses both were RSSR↓GLF, which was a typical characteristics of low-pathogenic avian influenza virus.The stalks of NA were lacked of I,T,E amino acid residues in sites of 62,63,64. Homology analysis showed that the eight fragments gene of two strains XX132 ,XZ22 were highly homologous to more than 99%, and their evolutionary relations were also in the same evolutionary branch. The four isolated viruses were closed to most of the isolates in mainland China in the 1997-2009 years, and homology of their NA, NP and three polymerase genes were close related to partial sequence of different subtypes of avian influenza virus. Phylogenetic relationships showed that the two surface protein genes and non-structural genes belong to the G1 group; and two internal genes NP, M and PA polymerase genes can be divided into two groups, of which LH01 belong to the Y280 group ,or XZ22, ZZ131 and XX132 belong to the Y280 group; and two polymerase genes PB1 and PB2 are broadly divided into two groups, of which LH01 and ZZ131 belong to the Y280group , or XZ22 and XX132 belong to the G1 group. These results provided the technical foundation for the molecular epidemiology, differential diagnosis and prevention research of H9 subtype avian influenza viruse in Henan.