Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Alterations to influenza virus hemagglutinin cytoplasmic tail modulate virus infectivity.

The influenza virus hemagglutinin (HA) contains a cytoplasmic domain that consists of 10 to 11 amino acids, of which five residues have sequence identity for 10 of 13 HA subtypes. To investigate properties of these conserved residues, oligonucleotide-directed mutagenesis was performed, using an HA cDNA of influenza virus A/Udorn/72 (H3N2) to substitute the conserved cysteine residues with other residues, to delete the three C-terminal conserved residues, or to remove the entire cytoplasmic domain. The altered HAs were expressed in eukaryotic cells, and the rates of intracellular transport were examined. It was found that substitution of either conserved cysteine residue within the cytoplasmic domain did not affect the rate of intracellular transport, whereas deletion of residues within the C-terminal domain resulted in delayed cell surface expression. All the altered HAs were biologically active in hemadsorption and fusion assays. To investigate whether the wild-type HA and HAs with altered cytoplasmic tails could complement the influenza virus temperature-sensitive transport-defective HA mutant A/WSN/33 ts61S, the HA cDNAs were expressed by using a transient expression system and released virus was assayed by plaque analysis. The wild-type HA expression resulted in a release of approximately 10(3) PFU of virus per ml. Antibody neutralization of complemented virus indicated that the infectivity was due to incorporation of wild-type H3 HA into ts61S virions. Sucrose density gradient analysis of released virions showed that each of the HA cytoplasmic domain mutants was incorporated into virus particles. Virions containing HAs with substitution of the cysteine residues in the cytoplasmic domain were found to be infectious. However, no infectivity could be detected from virions containing HAs that had deletions in their cytoplasmic domains. Possible roles of the HA cytoplasmic domain in forming protein-protein interactions in virions and their involvement in the initiation of the infection process in cells are discussed.     

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.     

Recognition of cloned influenza virus hemagglutinin gene products by cytotoxic T lymphocytes.

The influenza A virus hemagglutinin (HA) is an integral membrane glycoprotein expressed in large quantities on infected cell surfaces and is known to serve as a target antigen for influenza virus-specific cytotoxic T lymphocytes (CTL). Despite the fact that HAs derived from different influenza A virus subtypes are serologically non-cross-reactive, the HA has been implicated by previous experiments to be a target antigen for the subset of T cells capable of lysing cells infected with any human influenza A subtype (cross-reactive CTL). To directly determine whether the HA is recognized by cross-reactive CTL, we used vaccinia virus recombinants containing DNA copies of the PR8 (A/Puerto Rico/8/34) (H1N1) or JAP (A/JAP/305) (H2N2) HA genes. When these viruses were used to stimulate HA-specific CTL and to sensitize target cells for lysis by HA-specific CTL, we found no evidence for HA recognition by cross-reactive CTL aside from a relatively small degree of cross-reactivity between H1 and H2 HAs. Results of unlabeled target inhibition studies were consistent with the conclusion that the HA is, at most, only a minor target antigen for cross-reactive CTL.     

Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine.     

A recombinant vaccine of H5N1 HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against divergent H5N1 viruses

Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI) caused by influenza A virus (IAV) subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1) fragment of A/Anhui/1/2005(H5N1) to either Fc of human IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc), and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3) and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4) of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus.     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Effects of receptor binding specificity of avian influenza virus on the human innate immune response

Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.     

V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor

Human signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been shown to be a cellular receptor for measles virus (MV). Chinese hamster ovary cells transfected with a mouse SLAM cDNA were not susceptible to MV and the vesicular stomatitis virus pseudotype bearing MV envelope proteins alone, indicating that mouse SLAM cannot act as an MV receptor. To determine the functional domain of the receptor, we tested the abilities of several chimeric SLAM proteins to function as MV receptors. The ectodomain of SLAM comprises the two immunoglobulin superfamily domains (V and C2). Various chimeric transmembrane proteins possessing the V domain of human SLAM were able to act as MV receptors, whereas a chimera consisting of human SLAM containing the mouse V domain instead of the human V domain no longer acted as a receptor. To examine the interaction between SLAM and MV envelope proteins, recombinant soluble forms of SLAM were produced. The soluble molecules possessing the V domain of human SLAM were shown to bind to cells expressing the MV hemagglutinin (H) protein but not to cells expressing the MV fusion protein or irrelevant envelope proteins. These results indicate that the V domain of human SLAM is necessary and sufficient to interact with the MV H protein and allow MV entry.     

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.     

<Emphasis Type="Italic">Trichoplusia ni</Emphasis> cells (High Five<Superscript>TM</Superscript>) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines

Virus-like particles (VLPs) consisting of the influenza A virus proteins haemagglutinin (HA) and matrix protein (M1) represent a new alternative approach for vaccine design against influenza virus. Influenza VLPs can be fast and easily produced in sufficient amounts in insect cells using the baculovirus expression system. Up to now, influenza VLPs have been produced in the Spodoptera frugiperda cell line Sf9. We compared VLP production in terms of yield and quality in two insect cell lines, namely Sf9 and the Trichoplusia ni cell line BTI-TN5B1-4 (High Five^TM). Additionally we compared VLP production with three different HAs and two different M1s from influenza H1 and H3 strains including one swine-origin pandemic H1N1 strain. Comparison of the two cell lines showed dramatic differences in baculovirus background as well as in yield and particle density. Taken together, we consider the establishment of the BTI-TN5B1-4 cell line advantageous as production cell line for influenza VLPs.     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.     

A non-neutralizing antibody broadly protects against influenza virus infection by engaging effector cells

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HA_mg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HA_fg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HA_mg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.     

Characterization of domains of herpes simplex virus type 1 glycoprotein E involved in Fc binding activity for immunoglobulin G aggregates.

Herpes simplex virus type 1 glycoproteins gE and gI form receptors for the Fc domain of immunoglobulin G (IgG) which are expressed on the surface of infected cells and on the virion envelope and which protect the virus from immune attack. Glycoprotein gE-1 is a low-affinity Fc receptor (FcR) that binds IgG aggregates, while gE-1 and gI-1 form a complex which serves as a higher-affinity FcR capable of binding IgG monomers. In this study, we describe two approaches used to map an Fc binding domain on gE-1 for IgG aggregates. First, we constructed nine plasmids encoding gE-1/gD-1 fusions proteins, each containing a large gE-1 peptide inserted into the ectodomain of gD-1. Fusion proteins were tested for FcR activity with IgG-sensitized erythrocytes in a rosetting assay. Three of the fusion proteins containing overlapping gE-1 peptides demonstrated FcR activity; the smallest peptide that retained Fc binding activity includes gE-1 amino acids 183 to 402. These results indicate that an Fc binding domain is located between gE-1 amino acids 183 and 402. To more precisely map the Fc binding domain, we tested a panel of 21 gE-1 linker insertion mutants. Ten mutants with insertions between gE-1 amino acids 235 and 380 failed to bind IgG-sensitized erythrocytes, while each of the remaining mutants demonstrated wild-type Fc binding activity. Taken together, these results indicate that the region of gE-1 between amino acids 235 and 380 forms an FcR domain. A computer-assisted analysis of the amino acid sequence of gE-1 demonstrates an immunoglobulin-like domain contained within this region (residues 322 to 359) which shares homology with mammalian FcRs.     

The hemagglutinins of duck and human H1 influenza viruses differ in sequence conservation and in glycosylation.

We determined the deduced amino acid sequences of two H1 duck influenza A virus hemagglutinins (HAs) and found that the consensus sequence of the HA, determined directly from virus recovered from the intestinal tract, remains unchanged through many generations of growth in MDCK cells and chicken embryos. These two duck viruses differ from each other by 5 amino acids and from A/Dk/Alberta/35/1976 (F. J. Austin, Y. Kawaoka, and R. G. Webster, J. Gen. Virol. 71:2471-2474, 1990) by 9 and 12 amino acids, most of which are in the HA1 subunit. They are antigenically similar to each other but different from the Alberta virus. We compared these H1 duck HAs with the HAs of human isolates to identify structural properties of this viral glycoprotein that are associated with host range. By comparison to the human H1 HAs, the duck virus HA sequences are highly conserved as judged by the small fraction of nucleotide differences between strains which result in amino acid substitutions. However, the most striking difference between these duck and human HAs is in the number and distribution of glycosylation sites. Whereas duck and swine viruses have four and five conserved glycosylation sites per HA1 subunit, none of which are on the tip of the HA, all human viruses have at least four additional sites, two or more of which are on the tip of the HA. These findings stress the role of glycosylation in the control of host range and suggest that oligosaccharides on the tip of the HA are important to the survival of H1 viruses in humans but not in ducks or swine.     

Structural Analysis of the Hemagglutinin from the Recent 2013 H7N9 Influenza Virus

In March 2013, the Chinese Center for Disease Control and Prevention reported human infections with an H7N9 influenza virus, and by 20 July 2013, the numbers of laboratory-confirmed cases had climbed to 134, including 43 fatalities and 127 hospitalizations. The newly emerging H7N9 viruses constitute an obvious public health concern because of the apparent severity of this outbreak. Here we focus on the hemagglutinins (HAs) of these viruses and assess their receptor binding phenotype in relation to previous HAs studied. Glycan microarray and kinetic analyses of recombinant A(H7N9) HAs were performed to compare the receptor binding profile of wild-type receptor binding site variants at position 217, a residue analogous to one of two positions known to switch avian to human receptor preference in H2N2 and H3N2 viruses. Two recombinant A(H7N9) HAs were structurally characterized, and a mutational study of the receptor binding site was performed to analyze important residues that can affect receptor preference and affinity. Results highlight a weak human receptor preference of the H7N9 HAs, suggesting that these viruses require further adaptation in order to adapt fully to humans.     

An Eight-Segment Swine Influenza Virus Harboring H1 and H3 Hemagglutinins Is Attenuated and Protective against H1N1 and H3N2 Subtypes in Pigs

Swine influenza virus (SIV) infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method of controlling SIV is through vaccination. The killed SIV vaccines currently in use must be closely matched to the challenge virus, and their protective efficacy is limited. Live attenuated influenza vaccines (LAIV) provide strong, long-lived cell-mediated and humoral immunity against different influenza virus subtypes with no need for antigen matching. Here we report the generation of a new potential LAIV, an eight-segment SIV harboring two different SIV hemagglutinins (HAs), H1 and H3, in the genetic background of H1N1 SIV. This mutant SIV was generated by fusing the H3 HA ectodomain from A/Swine/Texas/4199-2/98 (H3N2) to the cytoplasmic tail, transmembrane domain, and stalk region of neuraminidase (NA) from A/Swine/Saskatchewan/18789/02 (H1N1) SIV. While this H1-H3 chimeric SIV, when propagated in vitro in the presence of exogenous neuraminidase, showed kinetics and growth properties similar to those of the parental wild-type virus, in vivo it was highly attenuated in pigs, demonstrating a great potential for serving as a dual LAIV. Furthermore, vaccination with the H1-H3 virus elicited robust immune responses, which conferred complete protection against infections with both H1 and H3 SIV subtypes in pigs.     

Infection of cultured human airway epithelial cells by influenza A virus

The lack of an adequate in vitro model has hampered study of the cellular basis by which influenza A virus causes disease in the human airway. We report in vitro infection of human airway epithelial cells by influenza A virus. Fetal and adult human tracheal and bronchial epithelial cells cultured from explants and SV40 transformed adult human tracheal epithelial cells were exposed to a recently isolated strain of influenza A virus (H1N1) and a laboratory passaged strain (WSN) of influenza A virus at similar multiplicity of infection. All cultures derived from explants showed hemadsorption (approximately 30% of the cells) with the H1N1 virus. No hemadsorption was detected with the WSN virus. One of two transformed cell lines showed a 5-10% hemadsorption to cells after H1N1 exposure and none following exposure to WSN. Immunofluorescent staining for influenza A-specific antigens in virus-exposed, explant-derived cells indicated viral infection and replication in these cells. Hemagglutinating material in the growth medium of infected, explant-derived cell lines, increased as a function of time, indicating the production of virus proteins. Exposure of rhesus monkey kidney cells and new human tracheal epithelial cultures to supernatant from these cells resulted in hemadsorption, indicating the presence of infectious virus in the supernatant. Light microscopic examination of virally infected bronchial epithelial cells demonstrated that the common types of cytopathic changes were rarely seen while cell proliferation continued over time. The data indicate that influenza A virus can infect, replicate, and produce infectious virus in cultured human tracheal and bronchial epithelial cells.     

Adaptation of avian influenza A (H6N1) virus from avian to human receptor‐binding preference

The receptor‐binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor‐binding properties of Taiwan‐isolated H6 HAs from 1972 to 2013. We propose that the receptor‐binding properties of Taiwan‐isolated H6 HAs have undergone three major stages: initially avian receptor‐binding preference, secondarily obtaining human receptor‐binding capacity, and recently human receptor‐binding preference, which has been confirmed by receptor‐binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor‐binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor‐binding site of HA determines the receptor‐binding preference change. We conclude that the human‐infecting H6N1 evolved into a human receptor preference.     

Crystal structure of unliganded influenza B virus hemagglutinin

Here we report the crystal structure of hemagglutinin (HA) from influenza B/Hong Kong/8/73 (B/HK) virus determined to 2.8 Å. At a sequence identity of ~25% to influenza A virus HAs, B/HK HA shares a similar overall structure and domain organization. More than two dozen amino acid substitutions on influenza B virus HAs have been identified to cause antigenicity alteration in site-specific mutants, monoclonal antibody escape mutants, or field isolates. Mapping these substitutions on the structure of B/HK HA reveals four major epitopes, the 120 loop, the 150 loop, the 160 loop, and the 190 helix, that are located close in space to form a large, continuous antigenic site. Moreover, a systematic comparison of known HA structures across the entire influenza virus family reveals evolutionarily conserved ionizable residues at all regions along the chain and subunit interfaces. These ionizable residues are likely the structural basis for the pH dependence and sensitivity to ionic strength of influenza HA and hemagglutinin-esterase fusion proteins.