The Effect Of Neuraminidase Stalk Region With Deletion Or Glycosylation Modulation On The Pathogenicity Of The H5N1 Subtype Avian Influenza Virus

The highly pathogenic avian influenza(HPAI)of the H5N1 subtype has devastating effects on the poultry industries and has a significance in public health.Avian influenza virus(AIV)undergoes genetic mutations to adapt to environment changes and broader their hosts.The H5N1 subtype AIV with 20 amino acids deletion in the stalk region of neuraminidase(NA)protein has become the dominant strain.Most researchers believe that deletion of 20 amino acids from 49th to 68th in the NA stalk may be an adaptive molecular marker for the evolution of the H5N1 subtype AIV from waterfowl to poultry.Sequencing analysis showed that 4 glycosylation sites(50NQS,58NNT,63NQT,68NIS)are included in the 20-amino-acid deletion region.In order to understand the effect of the deletion of amino acid as well as glycosylation sites of NA protein on the pathogenicity,we used site-directed mutagenesis to insert 20 amino acids and at same time modify potential glycosylation sites of parental virus A/mallard/Huadong/S/2005(S)and compared their pathogenicity together with viruses with shortened stalk in NA.1 Construction and biological characteristics of H5N1 subtype AIV with the deletion of different glycosylation sites in the stalk of the NA proteinSeven recombinant viruses with different glycosylation patterns in the stalk of the NA protein(rSNA-?50,rSNA-?50-?58,rSNA-?50-?58-?63,rSNA-?50-?58-?63-?68,rSNA-?68,rSNA-?63-?68 and rSNA-?58-?63-?68)was constructed by using S as the parental virus with 20 aa insertion in the stalk of the NA protein,and their biological characteristics were determined together with four recombinant viruses with shortened stalk in NA protein(rSNA-5,rSNA-10,rSNA-15 and rSNA-20)and rSNA+.Western blot results confirmed that all NA protein expression were correct.The titers of TCID50 of all 12 recombinant viruses on CEF cells ranged from 7.61 to 8.34 log10/0.1mL,and their titers of EID50 on SPF chicken embryos was higher than 8.0 log10/0.2mL.The plaque diameter of the rSNA-A50-A58-A63-A68 and rSNA+ were significantly larger than that of other strains.The results of the thermal stability test and the low pH stability test showed that the deletion of NA stalk region can improve the thermal stability and the low pH stability.Neuraminidase activity assay showed that both deletion of amino acids and deletion of glycosylation sites resulted in decreased neuraminidase activity.The above results showed that the deletion of NA stalk region improved the thermal stability and the low pH stability and made the viruses more stable.2 The effect of NA protein stalk deletion and glycosylation on the pathogenicity of H5N1 subtype AIVNine strains(rSNA-?50,rSNA-?50-?58,rSNA-?50-?58-?63,rSNA-?50-A58-?63-?68,rSNA-5,rSNA-10,rSNA-15,rSNA-20 and rSNA+)infected mallard ducks by intranasal route.The results showed that ducks in the rSNA-5,rSNA-10,rSNA-15 and rSNA-20 infection groups had clinical symptoms and the other groups had no clinical symptoms.All the strains can replicate in the heart,spleen,lung,kidney and brain,and viruses with deletion of NA stalk region had higher replication titers in the brain.At 3 dpi,the replication titers of viruses with deletion of NA stalk region and deletion of glycosylation sites were higher than that of rSNA+ in detected organs.At 5 dpi,the replication titers of recombinant viruses with deletion of glycosylation sites and rSNA+ were lower than that of viruses with deletion of NA stalk region.Therefore,we hypothesized that both deletion of NA stalk region and deletion of glycosylation sites can increase the replication efficiency of virus.Deletion of NA stalk region had more effect than the deletion of glycosylation sites on pathogenicity.Compared with the mallard ducks infected with rSNA+and viruses with deletion of glycosylation sites,the pathological changes of the mallard ducks infected with viruses with deletion of NA stalk region were more serious.The pathological changes in the brain and heart tissues of the mallard ducks infected with viruses with deletion of NA stalk region and glycosylation sites were more serious than that of rSNA+.The pathological changes in the lung and spleen tissues of the mallard ducks infected with viruses with deletion of NA stalk region and rSNA+ were more serious than that of viruses with deletion of glycosylation sites.All strains can shed the progeny virus through the oropharynx and cloacae,and oropharynx was the main virus shedding route.Deletion of glycosylation sites and NA stalk region can increase the virus shedding rate.All of the 9 strains could up-regulate the expression of immune-related factors in the lung of mallard ducks.IFN-? and Mx1 were significantly up-regulated among the immune-related factors tested.The rSNA-20 can up-regulate IFN-?,Mx1 and IL-8 significantly.In summary,deletion of the NA stalk region can enhance the virulence and the replication titer of AIV,enhance the innate immune response,and trigger host clinical symptoms.The above effect of NA stalk deletion is not entirely dependent on the absence of glycosylation sites in the stalk region.Therefore,the strain with deletion of 20 amino acids in the stalk of the NA becomes a pandemic strain,other than strains with deletion of glycosylation sites in NA stalk region.