Mutations In Envelope Proteins Of H9N2 Influenza Virus Under Neuraminidase Antibody Pressure

H9N2 subtype avian influenza virus(AIV)belongs to the low pathogenic influenza virus,which is the most wide-spread influenza virus in China domestic poultry.H9N2 AIV can infect birds,mammals and humans.Furthermore,H9N2 AIV can provide internal genes for H5 and H7 highly pathogenic AIV as well as novel subtypes AIV like H10,which promotes the emerging of reassortant virus with potential pandemic risk.The H9N2 inactivated vaccine has been widely used in China domestic poultry since the late 1990s.However,the selective pressure of the vaccine drove antigenic drifts in field strains for escape from antibodies induced by the vaccine.Many researchers have proved that antigenic drifts in hemagglutinin(HA)of H9N2 AIV,which resulted in the antigenic variations between the vaccine strains and field strains,led to the immune failure of the vaccination.While it is still unknown if antigenic variations in the neuraminidase(NA)are also involved in helping H9N2 field strains evade from antibodies induced by vaccination.HA and NA are the most abundant glycoproteins on the envelope of AIV.HA binds with the sialic acid receptors on the cell membrane,and NA cleaves the linkage of the receptors.The functional balance of HA-NA plays an important role in viral production,mobility and pathogenicity.The neutralizing antibodies targeting the receptor binding pocket in HA or the active center in NA are the most important protective antibodies.However,the virus can escape from these neutralizing antibodies with amino acid substitution,deletion and glycosylation in or around the antigenic epitopes.To identify key mutant residues in NA of H9N2 AIV crucial for escape from protective antibodies,we prepared a panel of 22 monoclonal antibodies(mAbs)against NA of A/Chicken/Jiangsu/XXM/1999(XXM)H9N2.The field strains isolated from 1999 to 2019 were used to react with 22 mAbs for determination of potential residues involved in antibodies binding.The mutations in HA and NA under NA antibody pressure were also identified and further analyzed.1.Generation and characterization of mAbs against NA of H9N2 AIVAlthough amino acid substitutions in NA can be detected in conventional surveillance of H9N2 AIVs,it is still unknown if any antigenic drift take place in NA by amino acid sequence alignment or phylogenetic analysis.To further map antigenic epitopes in NA of H9N2 AIV and their changes,a large panel of mAbs against NA is necessary.In our research,XXM H9N2 virus with a vaccine-strain like NA was used to immunize the mice.The spleen lymphocytes of the immunized mice were isolated and fused with the SP2/0 myeloma cells.A total of 22 hybridoma cell lines which secrete mAbs against NA of XXM virus were screened from fused cells and individually named as A1C4,A1F1,A1G1,A2A3,A3C9,A3H3,A4C6,A5C9,A5D12,A6A7,A6D1,A6D6,A7E6,A7G7,A7G8,B1A11,B2B3,B2B6,B2G2,B4D6,B5B3 and B6G5.All mAbs have kappa isotype light chains and IgG isotype heavy chains.The ascitic fluid of each mAb was prepared in 8-week-old BALB/c mice and further tested in indirect immunofluorescence assay(IFA).Most mAbs had over 3000-fold IFA titers,while A7E6 and B5B3 had lower IFA titers.The 6 of 22 mAbs,A2A3,A3C9,A4C6,A5D12,A6A7 and B4D6,were proved to be able to neutralize XXM virus by micro-neutralization(MN)assay in vitro.All the mAbs cannot react with the A/Chicken/Jiangsu/YZ/1999 H9N1 virus,while 9 of 22 mAbs can cross-react with A/Canine/Jiangsu/06/2010 H3N2 virus and another 8 mAbs can cross-react with A/Eurasian teal/Jiangxi/49/2018 H6N2 virus.These mAbs are good tools to map antigenic changes in NA of H9N2 virus and identify the transspecies transmission of N2 subtype influenza virus.To map antigenic drifts in NA of H9N2 AIVs in China domestic poultry,a total of 19 H9N2 field strains isolated from 1999 to 2019 were tested with 22 mAbs in IFA.The NA sequences of these 19 H9N2 virus can be divided into 3 branches in phylogenetic tree.The viruses in the branch I had classical vaccine strain like NAs.The branch Ⅱ consisted of the viruses isolated around 2010.The branch Ⅲ was made up of the viruses since 2012,which circulated in the current poultry.According to the IFA results,the 22 mAbs can be assigned into 3 groups.The mAbs A1F1,A7E6,B2B3 and B5B3 in the group Ⅰ can only react with the viruses in the branch Ⅰ.The mAbs A2A3,A4C6,A5D12 and B4D6 in the group Ⅱ can react the viruses in branch Ⅰ and branch Ⅱ,but all cannot react with the viruses in branch Ⅲ.The other mAbs in group Ⅲ can almost react with all of the H9N2 viruses.2.Identification of NA mutations under NA antibody pressureThe amino acid sequence alignment result showed that mutation N356D is the only difference in NAs of A/Chicken/Jiangsu/C1/2002 H9N2 virus and viruses of other branches.The plasmid pCAGGS-NA(D356)was constructed by site-directed mutagenesis and transfected into COS-1 cells.The IFA result showed that only mAb B5B3 can still well recognize the mutant NA.The others mAbs in group Ⅰ cannot or very weakly bind with the mutant NA.These results indicated that N356D is a crucial mutation involved in escape from group Ⅰ mAbs.The group Ⅱ mAbs all have neutralization ability.Key residues in NA of H9N2 AIV for antibody binding were identified by escape mutant selection.Mutations at positions 344,368,369 and 400 were selected by incubation with group Ⅱ mAbs.Especially the mutation at position 369 can be selected by each mAb in group Ⅱ,which indicated these mAbs may share overlapped epitopes centered residue 369.The neuraminidase inhibition(NI)activity of each group II mAb to WT XXM H9N2 virus and escape mutants was measured by enzyme-linked lectin assay(ELLA).The NI result showed that mutations at residue 344 had more profound impact on the inhibition by mAb A2A3 than by other mAbs,while mutation S400R resulted in more complete loss of inhibition by mAbs A4C6 and A5D12 than by A2A3 and B4D6.Furthermore,mutations at residues 368 and 369 impacted the inhibition by all group Ⅱ mAbs,with significant reduction in inhibition by antibodies A4C6,A5D12 and B4D6.The mAbs A3C9 and A6A7 in group Ⅲ also have neutralization ability.Mutations G125D/K296N,K296N and R253K were identified in mutants selected by mAb A3C9,a mutant with the single mutation G248E in NA was selected with mAb A6A7.The NI result showed that these mutations except the single mutation K296N resulted in the NI decrease of these two mAbs.Especially the R253K mutation resulted in significant NI decreases of both mAbs A3C9 and A6A7,which indicated the epitopes bound by mAbs A3C9 and A6A7 are overlapping to some extent.3.Identification of HA mutations under NA antibody pressureThe function balance of HA and NA plays an important role in influenza virus genesis.However,whether and how NA-specific antibodies may impact the HA attributes remain to be investigated.In this study,we examined the presence of amino acid mutations in the HA of these variants and compared the HA properties to that of the parent viral HA.The NI ability of the six mAbs with neutralizing ability was further determined with 2’(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid(Mu-NANA)assay.The mAbs A2A3 and A3C9 with higher NI ability in Mu-NANA assay were found to result in greater proportions of mutations at residues 166,198 and 234 in HA.Similar mutations also identified in escape mutants of mAb A2A3 and mAb A3C9.The receptor binding assay showed that these combined mutations or the single T220I mutations in HA switched the binding preferences from α2,6-linked sialic acid receptor toα2,3-linked sialic acid receptor.Moreover,these mutations also promoted viral growth in MDCK cells and mice lungs.These HA mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition(HI)titers.Our data demonstrate that HA mutations caused by NA-specific antibodies affect receptor binding preference and may facilitate virus escape from protective antibodies.The evolutionary analysis also shows that some mutations in HA of H9N2 virus may highly be caused by NA antibody pressure.Although these HA mutations in H9N2 AIV field strains did not switch the viral receptor preferences,these HA mutations may contribute to the increasing H9N2 infections in the human in recent years.In this study,22 mAbs against NA of H9N2 AIV were prepared and reacted with H9N2 AIVs isolated in China from 1999 to 2019,which posed antigenic variations in NAs of H9N2 AIVs.Key residues for antibody binding were also identified and residues as D356,N368 and G369 in NA of H9N2 AIV were evolutionary markers under NA antibody pressure.In addition,we also found HA mutations under NA antibody pressure.These HA mutations switched receptor preference from α2,6-linked sialic acid to α2,3-linked sialic acid receptor and caused the antigenic drift in HA,indicating that the NA antibody pressure has potential impact on the HA evolution.