Generation And Immunogenicity Of Recombinant H5N2Avain Inlfuenza Virus

Avian Influenza（AI） is one of fatal infectious diseases by avian influenza Avirus. At present,the inactivated oil emμlsion vaccine with whole viruses for preventing avian influenza is thecommon vaccine in the world. However, using this vaccine has a serious result from interfering theidentification with vaccinated birds and infected birds by field virus during surveillance.“Markervaccine” could solved this problem with the stratigy that is differatiating infected and vaccinatedassay（DIVA）. In this paper, there are including:: rescuing the Recombinant Avian Influenza Strain（rgH5N2/PR8）, idetifying some biological characteristics of rgH5N2/PR8, the effectiveness ofrgH5N2/PR8inactivated vaccine inoculated in SPF chicken, detecting the NA antibody of avianinfluenza virus（H5N1） in sera.（1） Rescuing the Recombinant Avian Influenza Strain （rgH5N2/PR8）.Recombinant virus, rgH5N2/PR8was generated with8-plasmids reverse genetic system,whichwas involved in HA gene of H5N1, NA gene of H9N2and6internal genes of A/PR8/34（H1N1）. Toreduce the high pathogenicity linked to the series of basic residues in HA, the motifs of thecleavage site（PLRERRRKR↓GL） was replaced with PLIETR↓GL. rH5N2/PR8was seriallypropagated in9-day SPF chicken embryos to10-generation times. After transfection, cells wereseeded9to10-day-old SPF chick embryo Fan poison, after its activity by hemagglutination assaysequencing. The resμlts show that, rescue the virus genome is completely derived from therecombinant plasmid.（2） Idetifying some biological characteristics of rgH5N2/PR8.rgH5N2/PR8was serially propagated in9-10day SPF chicken embryos to10generation times.The highest titer of hemagglutination assay was1:2048and EID8.7750was10-/0.1ml. The virμlenceof rH5N2/PR8was greatly declined and the ELD₅₀ was10^-5/0.1ml.（3）Effectiveness of rgH5N2/PR8inactivated vaccine inoculated in SPF chicken.rgH5N2/PR8inactivated with formaldehyde, and emμlsified oil adjuvants to prepareinactivated vaccines.Three groups with106-week chickens were respectively inocμlated with0.1ml,0.2ml and0.3ml of inactivated rH5N2/PR8emμlsified with oil per chicken. After21days,all inocμlated chickens with5negative chickens were oronasally attacked with A/Chichen/SD/2010（H5N1）,0.1ml per chicken. The number of survival in Group I inocμlated with0.1ml were five of10chickens and Group III were obtained completely protection. The resμlts showed that thevaccine has a good immune effect.（4） detecting the NA antibody of avian influenza virus（H5N1） in sera.In order to determine the SPF chickens inocμlated with marker vaccines infection popμlarwild strain A/Chicken/SD/2010（H5N1）, Its NA antibodies by indirect immunofluorescence assay.Method is as follows: Sf9cells were seeded in6-well plates at1×10⁶cells/well in thelogarithmic growth phase, Was placed in a27℃incubator, and cμltured to50%to60%abundance, inocμlated to the cells with the recombinant bacμlovirus titer determined15μl/well,indirect immunofluorescence test operation to train120h. The resμlts showed that the NA-IFAdetection methods can only react H5N1subtype of avian influenza virus NA antibody, rather thanreact with recombinant H5N2subtype of avian influenza virus NA antibody. The method is able todistinguish between rgH5N2/PR8marker vaccine produces antibodies and natural infection withH5N1avian influenza virus antibodies. The statistical results show immunohistochemistry chickens（30chickens） by the the wild poisonous A/Chicken/SD/02/08（H5N1） after the attack,25chickenstested positive for NA-IFA, and cell surfaceyellow-green fluorescence, so infected with the wild virus,25chickens body in addition to produce N2antibody N1antibodies; another five chickensNA-IFA detection is negative, the cell surface does not appear yellow-green fluorescence,thereforethey are not infected with the wild virus, their body only N2antibody.The results showthat the vaccine can not completely prevent the virus infection, but to prevent the chickens someclinical symptoms.Thus, this method is able to identify a vaccinated with rgH5N2/PR8"marker"inactivated vaccine chickens infected with the H5N1subtype of avian influenza virus, provides aviable technical means to purify the H5N1subtype of avian influenza virus in immune flocks.In summary, the recombinant virus obtained in this experiment is a vaccine marker, breedinghigh titer and virμlence low and good immune effectiveness, very suitable as a marker vaccinecandidate strain.