Construction Of H5N9 Recombinant Influenza Virus And Analysis Of Its Infection Characteristics

Objective:The main threaten from the influenza A virus to human is because of the virus pool in bird and viral gene reassortment.In recent year,the H5 subtype high pathogenic avian influenza virus,causing pandemic in bird and poultry,displayed spreading tendency to human and other animals.Many of the H5 subtype viruses including H5N1,H5N2,H5N6,H5N8,H5N9 were isolated from birds and fowl of China and other countries in Asia and European.Besides the bird flu outbreaks,its transmission risk to mammal is concerned worldwidely.Although,the H5N9 has not make severe damages to the human,which was reported similar to the H5N1 and H5N8.According to the H5N9 outbreak in fowl in 1966 and many sporadic in recent years,remaining the main pathogens in bird,the high possibility of its mutation and reassorting with other subtype may have the potency of spreading to human.Meanwhile,because of the antigen shift/drift,the resistant to the antiviral drug limit the vaccine efficacy.Therefore,this study focused on the infectious characteristics of the H5N9 recombinant influenza virus by reverse genetics methods.Methods:In order to rescue the H5N9 strain,full length gene of A/Beijing/01/2003(H5N1)virus HA gene and A/Zhejiang/DTID-ZJU10/2013(H7N9)virus NA were synthesized and inserted into pHW2000 plasmid,then transfected to 293T,subsequently mixed with MDCK cells together with recombinant pHW2000 plasmids containing the other 6 A/Puerto Rico/8/34(H1N1)virus gene segments(M,NS,NP,PA,PB1,PB2).The pathogenic effect of H5N9 virus was observed.The expression of HA and NA proteins was verified by western blot and immunofluorescence.Hemagglutination test was used to verify the hemagglutination characteristics of influenza virus HA protein.The morphological structure of recombinant virus was observed by transmission electron microscopy.Thecombinant virus H5N9 Infection features were studied in the BALB/cmiceinoculated via intranasal route..Results:The results of cytopathic,nucleic acid sequencing,HA,and NA proteins western blot,immunofluorescence,demonstrated using the reverse genetics system can successfully rescued virus H5N9,Our findings showed that recombinant H5N9 virus replicating ability in MDCK cells is lower than the H1N1 rescued in the same experimental condition.There were no obvious clinical features in mice infected by H5N9,compared with H1N1-infected mice.The HI antibody level induced by the H5N9 infection is up to 1:160 is lower than the HIN1 virus(1:320)..Conclusion:In this study,H5N9 recombinant influenza virus was successfully constructed by reverse genetics techonology,the proliferation of this virus was verified passaged in MDCK cells.The infection of characteristics of this virus was revealed with Balb/c mice model.