Genomic Characterization Of H9N2AIV Epidemic Strains And Chicken Alpha Interferon And Interieukin-18Expression Products Antiviral Research

The H9N2subtype of avian influenza virus is widespread in the worldwide, not only thepoultry industry has caused tremendous economic losses, but also pose a threat to humanhealth and life safety. Our country isolated from a chicken farm in Guangdong Province to thefirst strains of H9N2subtype avian influenza virus in1994, From then on, which the subtypeof avian influenza virus quickly spread to neighboring provinces. In this study, carried out inShandong H9N2subtype of avian influenza molecular epidemiology work, to grasp the H9N2subtype of avian influenza virus Variation in Shandong Province in recent years, and studiedthe antiviral activity of the H9N2subtype of avian influenza virus in chicken interferon alphaand interleukin-18(IL-18).In this study, the biological characteristics of laboratory isolated and identified in2012,three from chicken H9N2subtype of avian influenza. The three virus in SPF chicken embryosvalue-added generation, The HA potency is between27to29，The EID50is between10-7.75/0.1mL and10-8.5/0.1mL, TCID50is between10-8.00/0.1mL and10-8.50/0.1mL. Inaccordance with the genome sequence in GenBank H9N2subtype of avian influenza virus(AIV) design primers and make use of RT-PCR amplification, PCR product is send tosequencing Company sequencing, completed three of H9N2AIV Shandong2012localisolates CK/SD/SG/2012the CK/SD/JN/2012, CK/SD/SG/2012of the completegenome sequencing of three H9N2viruses eight gene segments (PB2, PB1, PA, HA, NP, NA,M and NS) phylogenetic analysis, make the phylogenetic tree. HA gene phylogenetic tree, thethree viruses in the same evolutionary position, are CK/SH/Y2/2008-like pedigree, NA, NP,M, NS, PB2, PB1, PA gene phylogenetic tree are the same as HA gene phylogenetic tree,suggesting that the three H9N2isolates may be derived from CK/SH/Y2/2008-like.This study using the Bac-to-Bac baculovirus/insect cell expression system, theencoding interferon the alpha (mChIFN-alpha) and chicken white interleukin18(mChIL-18)mature protein gene was inserted into the donor plasmid pFastBacDual, They were placedunder the control of pH and P10promoter, the introduction of honeybee melittin signalpeptide (HBM) gene instead of chicken interferon alpha and interleukin-18gene of the original signal peptide to achieve secretive expression, and C-terminal fusion of six histidinetag to facilitate purification, Constructed plasmid was transformed into DH10competent cellsby homologous recombination to obtain the recombinant shuttle plasmid Bacmid recombinantbaculovirus transfected Sf9insect cells in logarithmic growth phase. Recombinantbaculovirus infected insect cells and tested activity of recombinant protein. Recombinant ofthe interferon alpha and the interleukin-18has been confirmed for having the inhibitory effectof the H9N2subtype of avian influenza virus on the MDCK cells.It lay good foundation forthe study of cytokines fight against avian influenza.