The Mechanism Of Interspecies Transmission Of Avian H7N9 Influenza Virus Hemagglutinin

Influenza viruses are enveloped and segmented negative-strand RNA viruses,belonging to the family of Orthomyxoviridae,which can cause severe zoonotic diseases.According to the antigenicity of nucleoprotein and matrix protein,they are typically classified into four types:A,B,C and D.Among them,influenza A viruses(IAVs)are the most pathogenic with it's a broad spectrum of host tropism.New influenza viruses can emrege through genomic reassortment and mutations,which has resulted in multiple large-scale pandemics and seasonal epidemics,posing an enormous threat to public health and economy.Basically,influenza viruses display remarkable host specificity.Typically,AIVs only bind to avian receptor.However,in recent years,human-infections with various subtypes of AIVs have emerged continuously,such as H5,H6,H7,H9 and H10 subtypes.Among them,the motality for H5N1 and H7N9 AIV infections is up to-60%and 30%,respectively,leading to a great panic in human polulations.There are many factors,in both virus and host aspects,contributing to the cross-species transmission of AIVs.One of the most crucial determinants is the variation of viral hemagglutinin(HA)glycoprotein which determines the receptor binding property of influenza viruses.The first step of influenza virus infection relies on HA molecules to recognize the sialic acid receptors on host cell surface and enable virion attachment.The amino acid substitutions in HA may alter its receptor binding property,which offers the possibility of cross-species transmission of influenza viruses.Thus,elucidating the molecular basis for the receptor binding properties of influenza viruses are very important prevention and control of potential influenza epidemics or pandemics.Since 2013,the H7N9 AIVs have caused more than 1,600 human-infections and several highly pathogenic viral strains have emerged in 2017.Previous studies have shown that human-infecting H7N9 influenza viruses can bind to both avian and human receptors(dual-receptor tropism),and residues A138/V186/P221/L226 or V186/L226 are the key determinants for H7 subtype HA to obtain human receptor binding property.However,the contribution of each specific residue for determining the receptor-binding property of HA is unclear,and the evolving process of avian-specific H7N9 influenza viruses to acquire human receptor binding property is not well understood.Therefore,in this study,we generated various H7N9-HA mutant proteins with different amino acid combinations in the receptor binding site(RBS),and evaulated the receptor binding property,protein stability and virus replication kenetics to identify the key determinant residue for obtaining human receptor binding capacity and investigate the possible route of H7N9 virus evolving.We found that the currently circulating H7N9 viruses are still dual-receptor tropic,similar to the property of AH1-H7N9 strain in 2013.Mutagenesis studies showed that the G186V single mutation is sufficient for H7N9 virus to obtain human receptor binding capacity,while the well defined signature in other influenza subtypes,Q226L substitution,is not the key determinant,which instead may render its effect in the later stage for further mammalian adaption.In addition,the four hydrophobic residue substitutions in the RBS can reduce the stability of HA molecule,suggesting the acquisition of human receptor binding capacity by H7 subtype HA was achieved by sacrificing its stability as the evolutionary cost.Thus,the evolving route of H7N9 AIVs is determined by the cooperative effect of receptor binding property and stability of HA molecules.In the early stage of H7N9 influenza virus outbreak in 2013,residues in 135 and 226 were dominated by alanine and leucine respectively.However,in the recent epedimic in 2017,the residue in 135 was replaced by valine and the frequency of Q226 displayed a slight increase.The effect of these substitutions was unclear.To investigate the question,we analyzed the receptor binding properties of HAs before and after A135V and L226Q substitutions and compared their reactivity with human H7N9 influenza virus targeted antibodies(4A-14,3A-44).We found that A135V and L226Q single or double substitutions did not alter the dual-receptor binding property of H7N9 HA,but reduced its binding affinity to 3A-44 antibody.In addition,the glycans on residue 158 in GD-8H324 HA also interfered with the binding between H7N9 HA and 4A-14 antibody.These results indicated that the newly emerged H7N9 influenza viruses have accumulated certain mutations to escape the host immunity and cannot be neutralized by the antibodies isolated in the early stage of H7N9 outbreak in 2013.In summary,the evolution of H7N9 AIVs is affected by the selection pressure of antibodies,receptor binding properties,protein stability,and other possible factors.Our studies would intensify our understanding for the molecular basis of interspecies transmission of H7 subtype AIVs,and provide important guidance for the prevention and control of potential influenza epidemics.