The Mechanism Of Interferon Antagonism By The H5N1 Subtype Avian Influenza Virus With Partial Deletion In NA Gene

H5 subtype avian influenza virus(AIV)is an important zoonotic pathogen,which becomes the dominant strain with partial deletion in neuraminidase(NA)gene under the natural selection.Since it was isolated from a sick goose flock during an outbreak in Guangdong Province,China,in 1996,the NA gene of the H5N1 subtype avian influenza virus had different forms of deletion,and the deletion of 20 amino acids in the 49-68 position of the stalk became prevailing strains worldwide.Although previous studies initially found that NA stalk deletion was required for the interferon resistance of H5N1 subtype AIVs,however the relevant mechanism is unclear.In this study,the H5 subtype AIV with patial deletion of NA stalk was used as female parent,according to the existing deletion mode of AIV with NA gene stalk or addition different lengths of amino acids to the NA stalk,a series of recombinant mutants(only mutations in NA stalk,other genes consistent with the parental virus backbone)were constructed,the biological characteristics and anti-interferon ability of H5N1 subtype avian influenza virus with partial deletion in NA gene were evaluated,and the key functional position in NA stalk was determined.Our research has a great academic significance for analyzing the evolution of H5N1 subtype AIV.1 Construction and biological characteristics of the H5N1 subtype avian influenza virus with partial deletion in the stalk of NA geneFive mutant viruses were constructed,a H5N1 subtype AIV strain(A/mallard/Huadong/S/2005)with a deletion of 20 amino acid(aa)at position 49-68 of NA gene stalk and a deletion of 5 aa at position 80-84 of NS1 gene was used as female parent virus,20 aa,15 aa,10 aa,5 aa,and 0 aa was inserted into the NA stalk,respectively(named rSNA+,rSNA-5,rSNA-10,rSNA-15,rSNA-20).The results from biological characteristics showed that there was no significant difference in TCID50,EID50,and MDT between the five mutant viruses.The growth curve of these mutants showed that the NA stalk insertion could enhance the replication ability of H5N1 subtype AIV on MDCK and CEF cells,while NA stalk deletion could increase the replication ability of H5N1 subtype AIV on Vero cells.The neuraminidase assay showed that the longer the NA stalk was,the higher the NA activity was.The plaque test showed that the plaque diameter of the mutant virus with insertion in NA stalk was longer than that with a deletion of 20 aa in NA stalk.CEF or MDCK cells was infected by five mutant viruses with MOI=0.001,the mRNA levels of IFN-α and IFN-β were detected by relative fluorescence quantitative PCR(GAPDH gene as internal reference)at different time points(6h,12h,and 24 h).The results showed that,with the shortening of NA stalk the mRNA expression of IFN-α and IFN-β was increased within 12 hours but decreased at 24 h in both cells.In summary,NA stalk with partial deletion could affect the viral replication capacity in vitro,neuraminidase activity and the mRNA expression of IFN-α/β.2 Effects of NA gene stalk deletion on the antagonistic ability of H5N1 subtype AIV against interferonFirstly,eukaryotic plasmids with different lengths of NA stalk were transfected into Vero cells,then IFN-β-pretreated Vero cells were infected by five strains of mutant virus respectively infected,the TCID50 of the supernatant was measured after 72 h infection.The results showed that antagonism interference ability of virus on Vero cell all were promoted by five eukaryotic plasmids expressed different lengths of NA stalk.And the eukaryotic plasmids pcDNA3.1(＋)－NA-15 and pcDNA3.1(+)-NA-20 obviously enhance the antagonism interference ability.Then Vero cells were pretreated with different concentrations of IFN-β,and then infected by five mutant strains with MOI=0.001 respectively,and the TCID50 of the supernatant was measured after 72 h infection.The results showed that the titer of rSNA +,rSNA-5,and rSNA-10 was 0 respectively when the interferon concentration reached 1600U;rSNA-15 and rSNA-20 could still be replicated when the interferon concentration reached up to 10000U,suggesting that 15 and 20aa deletion in NA stalk improved the ability of H5N1 subtype AIV antagonist interferon.3 Effects of NA gene stalk deletion on NS1 expressionFirstly,in order to obtain the antibodies for detecting the expression of NA or NS 1 protein,the NA-20 or NS1 genes were cloned into the prokaryotic vector PET-32a(+).The BABL/c mice were immunized by purified NA or NS 1 protein for obtaining polyclonal antibodies aganist NA-20 or NS1 respectively.The results showed that the polyclonal antibodies of NA-20 or NS1 had good reactivity,against not only could detect their own proteins but also could detect the NA+,NA-5,NA-10,and NA-15 protein.Then,CEF cells were infected five mutant viruses with MOI = 0.001,the expression levels of NA and NS1 protein were determined at 3 h,6 h,9 h,12 h,and 24 h by Western-blot.The results showed that with prolonged NA stalk,the expression of NA protein was decreased gradually and while the expression of NS1 protein was increased gradually at different time points.Finally,Vero cells were pretreated with 400 U IFN-β and then were infected by mutant viruses,the mRNA levels of NA and NS1 were detected by relative fluorescence quantitative PCR(M gene as internal reference).The results showed that the mRNA expression of NS1 of the mutant with 20 aa deletion in NA stalk was higher than that of other mutants at different time points(6h,12h,and 24 h),and with the increase of NA stalk,the expression of NS1 mRNA was gradually decreased,however,the expression of NA mRNA was not significantly different.In conclusion,H5N1 AIVs with 15 aa or 20 aa deletion in the NA stalk had more antagonism role against type I interferon,which may due to the higher expression of NS1 protein in the mutant viruses.