Effect Of Glycosylation Modification Of HA/NA Protein On Pathogenicity And Antigenicity Of H5 Subtype Avian Influenza Virus

The H5 subtype of avian influenza virus(AIV)is prevalent in regions such as Asia,Africa,and Europe.Over the past decade,the H5N6 subtype of AIV represented by clade 2.3.4.4 and the H5N1 subtype of AIV represented by clade 2.3.2.1 have been widespread in Asian countries,causing severe economic losses to the poultry industry.To prevent and control the spread of H5 subtype AIVs,China implements vaccine immunization policies.Re-5,Re-8,and Re-11 were used against clade 2.3.4 AIVs and Re-6,Re-10,and Re-12 were used against clade 2.3.2.1 AIVs,respectively.With the tremendous success of Chinese vaccine control of H5 subtype AIV,countries in Southeast Asia such as Bangladesh and Vietnam have also introduced some Chinese vaccines.However,vaccine-induced immune pressure could drive the evolution of influenza virus,and the surface proteins Hemagglutinin(HA)and Neuraminidase(NA)are particularly affected by antibody pressure.In addition to amino acid mutations,HA and NA can also adapt to antibody pressure by changing glycosylation modifications.Based on epidemiology,this study explored the changes in glycosylation on HA and NA with clade 2.3.4.4 H5N6 subtype AIV(B,W,and HJ strains)and clade 2.3.2.1 H5N1 subtype AIV(DT and YZ strains)as viral models,reverse genetic technology was used to delete or add glycosylation to explore the effect of HA/NA protein glycosylation on the biological characteristics,pathogenicity,and antigenicity of H5 subtype AIV.Furthermore,the glycosylation level of HA also affected the binding of H5 subtype AIV to C-type lectin receptors.The constructed HA glycosylation mutant phenotype and the clade 2.3.4 H5N1 subtype AIV(SY strain)previously constructed in the laboratory were used as viral models to explore the differences between DC/L-SIGN and LSECtin receptors in recognizing different H5 subtype AIV HA glycosylated viruses.1.Biological characteristics and antigenic effects of glycosylation sites of HA protein at residues 63 and 129 on H5N6 AIVThe worldwide occurrence,distribution,and frequency of glycosylated strains of the H5N6 subtype AIV at residues 63 and 129 on HA protein were analyzed.The B(clade 2.3.4.4e,WT-HA-63-129-)and W(clade 2.3.4.4h.WT-HA-63+129+)strains were used as parental strains,with the addition or deletion of glycosylation sites at residues HA-63 and HA-129,and the biological characteristics and antigenicity of the glycosylation mutant viruses were measured.The results showed that H5N6 AIV with glycosylation at residue HA-129 appeared in clades 2.3.4.4d and 2.3.4.4h,first appeared in China and subsequently became prevalent.while H5N6 AIV with glycosylation at residue HA-63 appeared in clade 2.3.4.4h.In contrast.H5N6 subtype AIV with HA-63 glycosylation site appeared only in clade 2.3.4.4h.Glycosylation at residue HA-129 enhanced the thermal stability of the HA protein in H5N6 AIV.Further experiments showed that rB escaped neutralization by Re-8 serum after acquiring the HA-129 glycosylation site.while rW was completely neutralized by Re-8 serum after the HA-129 glycosylation site was deleted,and the commercial Re-I 1 serum contained antibodies against the HA-129 glycosylation site.Overall.the study highlighted that H5N6 subtype AIV with glycosylation at HA-129 is an immune adaptation strain to escape the antibody pressure induced by Re-8 vaccine in poultry in China.2.The effect of simultaneous evolution of glycosylation sites at 129 of HA and 86 of NA protein on the biological characteristics of clade 2.3.4.4 H5N6 AIVThe simultaneous evolution of glycosylation sites at residues 129 on HA protein(HA129)and 86 on NA protein(NA-86)of H5N6 subtype AIV in China were analyzed.The biological characteristics of glycosylated viruses were determined by adding or deleting HA129 and NA-86 glycosylation sites in the parental strains HJ(clade 2.3.4.4f.WT-HA129NA86+)and W(clade 2.3.4.4h,WT-HA129+NA86-),respectively.Results showed that the addition of HA-129 glycosylation and the loss of NA-86 glycosylation in H5N6 subtype AIV were highly epidemiologically related and widely occurred in clade 2.3.4.4h.However,NA86 glycosylation remained conserved in H6N6 subtype avian influenza virus,and the deletion of NA-88 was specific to the H5 subtype AIVs.HA-129 glycosylation stabilized viral binding affinity to α-2,3 and α-2,6 sialic acid receptors,while its addition increased viral binding affinity to α-2.3 sialic acid receptors.Both the addition of HA-129 glycosylation and the deglycosylation of NA-86 decreased neuraminidase activity of the virus.Animal experiments revealed that the addition of HA-129 glycosylation of H5 AIV increased its pathogenicity in chicken and mice,while the removal of NA-86 glycosylation of H5 AIV decreased its pathogenicity,when compared with wild-type viruses.In conclusion,HA-129 glycosylation and NA-86 deglycosylation of H5N6 subtype AIV is generated under antibody pressure,which enhances the virus pathogenicity to chickens and mice.3.The effect of simultaneous evolution of glycosylation sites at 158 of HA and 88 of NA protein on the antigenicity and pathogenicity of clade 2.3.2.1 H5N1 subtype avian influenza virusThe simultaneous evolution of the glycosylation sites at 158 of HA(HA-158)and NA88(NA-88)of global H5N1 subtype AIVs were analyzed.The biological characteristics of glycosylated viruses by adding or removing the glycosylation sites at HA-158 and NA-88 of YZ and DT strains were measured.The results showed that the addition of HA-158 glycosylation and the loss of NA-88 glycosylation of clade 2.3.2.1 H5N1 subtype AIV occurred in China and Bangladesh,and were highly correlated with the use of Re-6 vaccine in these regions.Hemagglutination inhibition and microneutralization tests showed that the deletion of HA-158 glycosylation site endowed DT of clade 2.3.2.1e the ability of complete neutralization by Re-6 antiserum.The neuraminidase activity of H5N1 subtype AIV and the viral pathogenicity to chicken and mice were increased when add glycosylation sites at residue of HA-158,while the removal of glycosylation at residue of NA-88 weakened the characteristics,when compared with wild-type viruses.In conclusion,the addition of glycosylation at HA-158 enables H5N1 subtype AIV to escape the antibody pressure induced by Re-6 vaccine,the clade 2.3.2.1e virus with HA158+NA88-pattern shows the enhanced pathogenicity to chickens and mice.4.The role of C-type lectin receptors DC/L-SIGN and LSECtin in recognition of H5 viruses with different HA glycosylation sitesTo investigate the differences in recognition of H5 AIVs with different levels of HA glycosylation by lectin receptors,the binding affinity of C-type lectin receptor proteins DC/LSIGN and LSECtin with different HA glycosylation mutant viruses of clade 2.3.4.4 H5N6(rW-HA129+NA86-and rW-HA129-NA86-),clade 2.3.2.1 H5N1(YZ-HA158+NA88-and YZ-HA158-NA88-),and clade 2.3.4 H5N1(rS-144-/158+/169+,rS-144-/158-/169+,and rS144-/158+/169-)viruses were measured by ELISA.The A549 cells stably expressing DC/LSIGN and LSECtin were constructed,treated with inhibitor of the sialic acid receptors and were infected with glycosylated mutated viruses.The results of ELISA showed that rS-144/158-/169+and rS-144-/158+/169-could bind better to DC/L-SIGN and LSECtin.Moreover,the expression of DC/L-SIGN and LSECtin in A549 cells promoted the infection of H5 subtype AIV when the sialic acid receptors were suppressed.In LSECtin knock-out mice,the strains with low glycosylation levels(rS-144-/158-/169+and rS-144-/158+/169-)caused more severe infections than the wild-type strain rS.The PR8 with lower glycosylation in the head domain of HA showed an increase mortality in mice.In summary,DC/L-SIGN and LSECtin expressed in A549 epithelial cells can promot the infection of H5 subtype AIV.The viruses with low glycosylation levels of HA such as H5 subtype AIV with deletion of glycosylation and PR8 increase their pathogenicity to LSECtin gene knock-out mice in vivo.Therefore,H5N6 and H5N1 subtype AIVs obtained the HA-129 glycosylation or HA158 glycosylation sites under vaccination antibody pressure,respectively,and evolved simultaneously to delete NA-86 glycosylation site or NA-88 glycosylation sites,which enhanced the pathogenicity of H5 subtype AIV to chickens and mice.The receptor LSECtin mediated the pathogenicity of H5 subtype AIV with different glycosylation level on HA protein in mice.