An avian influenza H5N1 virus that binds to a human-type receptor

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.     

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.     

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.     

The Mechanism of Poly-Galloyl-Glucoses Preventing Influenza A Virus Entry into Host Cells

Hemagglutinin (HA) is essential for Influenza A virus infection, but its diversity of subtypes presents an obstacle to developing broad-spectrum HA inhibitors. In this study, we investigated the molecular mechanisms by which poly-galloyl glucose (pGG) analogs inhibit influenza hemagglutinin (HA) in vitro and in silico. We found that (1) star-shaped pGG analogs exhibit HA-inhibition activity by interacting with the conserved structural elements of the receptor binding domain (RBD); (2) HA inhibition depends on the number of galloyl substituents in a pGG analog; the best number is four; and when PGG binds with two HA trimers at their conserved receptor binding domains (loop 130, loop 220, and 190-α-helix), PGG acts as a molecular glue by aggregating viral particles so as to prevent viral entry into host cells (this was revealed via an in silico simulation on the binding of penta-galloyl-glucose (PGG) with HA). pGGs are also effective on a broad-spectrum influenza A subtypes (including H1, H3, H5, H7); this suggests that pGG analogs can be applied to most influenza A subtypes as a prophylactic against influenza viral infections.     

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.     

Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets

A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to α2,3-linked sialosides (α2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both α2,6SAL and α2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both α2,3SAL and α2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its α2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.Influenza A viruses have the potential to cause pandemics of various severities. The emergence of new influenza virus strains to which the general population has low or no immunity, such as the 2009 swine-origin influenza A H1N1 viruses, will continue to challenge public health authorities and the scientific community to develop quick and efficient mitigation responses (18). Highly pathogenic avian influenza A (HPAI) H5N1 viruses pose a serious pandemic threat due to their virulence and high mortality in humans, and their increasingly expanding host reservoir and significant ongoing evolution could enhance their human-to-human transmissibility (8). Currently, the case fatality rate of HPAI H5N1 viruses in humans is estimated to be approximately 60% (30).Although HPAI H5N1 viruses are now endemic in several countries (2), direct transmission of influenza viruses from avian species to humans remains a relatively rare event. The hemagglutinin (HA) protein''s affinity for cell surface sialic acid-containing molecules is one of the determinants of influenza A virus host range restriction. Human and avian influenza virus isolates differ in their recognition of host cell receptors; human strains mainly bind α2,3-linked sialosides (α2,6SAL), whereas the avian strains have a high affinity to α2,3SAL (15, 32). The influenza pandemics of the last century have been suggested to result from switching of HA receptor-binding specificity from α2,3SAL to α2,6SAL receptors (6, 26, 31).The receptor-binding specificity of the HA protein can be influenced by several critical residues. For influenza H3 subtype viruses, substitutions of Q226L and G228S could completely reverse receptor-binding specificity from α2,3SAL to α2,6SAL (4, 21). For the H1 subtype viruses, the E190D and D225G residues switch virus receptor binding specificity from α2,3SAL to α2,6SAL for the 1918 pandemic H1N1 viruses (6, 25). However, based on glycan microarray analysis, the 190E and 225D residues cannot alter the HA binding preference from α2,3SAL to α2,6SAL for H5N1 viruses (26).Vaccination is considered a preferred approach to prevent influenza-related illness in the community. A pandemic influenza vaccine should stimulate protective immunity in the target population using the smallest amount of antigen possible, thus enabling availability of maximal vaccine doses. The inactivated H5N1 VN04 vaccines have been found to be poorly immunogenic in humans, and adjuvants are needed to enhance vaccine immunogenicity (13). Live attenuated influenza vaccines (LAIV) have several desirable attributes: the stimulation of a durable mucosal and systemic immunity, broad efficacy against homologous and drifted strains, and efficient production (17).Several H5N1 LAIV vaccines possessing a modified HA and neuraminidase (NA) of an H5N1 virus and the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) of the A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) master donor virus were previously generated and evaluated for their immunogenicity and efficacy in mice and ferrets (29). A single dose of A/Vietnam/1203/2004 (VN04 ca) LAIV elicited very low levels of serum neutralizing antibodies against homologous and heterologous wild-type (wt) H5N1 viruses 4 weeks after administration to mice and ferrets. In contrast, a single dose of A/Hong Kong/213/2003 (H5N1) (HK03 ca) LAIV was more immunogenic (29). A specific amino acid residue at position 227 in the HK03 HA has been reported to be responsible for the greater immunogenicity of HK03 (9). VN04 and HK03 also differ in their receptor binding specificities. The VN04 HA mainly recognizes α2,3SAL, while the HK03 HA recognizes both α2,3SAL and α2,6SAL (7, 14, 22, 36). Sequence alignment of the two H5 HA proteins revealed nine amino acid differences in their HA1 region (9). The current analysis evaluates the impact of these amino acid differences on H5N1 VN ca vaccine strain replication and immunogenicity. In addition, adaptive mutations selected from MDCK passage of the H5N1 VN04 ca virus and introduction of known receptor binding sites were evaluated for their effect on antigenicity and immunogenicity of the H5N1 VN04 ca virus.     

Two glycosylation sites in H5N1 influenza virus hemagglutinin that affect binding preference by computer-based analysis

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.     

Inefficient transmission of H5N1 influenza viruses in a ferret contact model

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two na?ve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other na?ve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.     

Decreased neuraminidase activity is important for the adaptation of H5N1 influenza virus to human airway epithelium

Highly pathogenic avian H5N1 influenza viruses remain a pandemic threat. Antiviral drugs such as neuraminidase (NA) inhibitors will be crucial for disease control in the event of a pandemic. Should drug-resistant H5N1 viruses develop, all defense strategies will be compromised. To determine the likelihood and mechanisms of emergence of NA inhibitor-resistant H5N1 variants in humans, we serially passaged two H5N1 viruses, A/Hong Kong/213/03 and A/Turkey/65-1242/06, in normal human bronchial epithelial (NHBE) cells in the presence of oseltamivir, zanamivir, or peramivir. To monitor the emergence of changes associated with the adaptation of H5N1 viruses to humans, we passaged the strains in the absence of drugs. Under pressure of each NA inhibitor, A/Turkey/65-1242/06 developed mutations in the hemagglutinin (HA) (H28R and P194L/T215I) and NA (E119A) proteins that reduced virus binding to α2,3-sialyl receptor and NA activity. Oseltamivir pressure selected a variant of A/Hong Kong/213/03 virus with HA P194S mutation that decreased viral binding to α2,6 receptor. Under peramivir pressure, A/Hong Kong/213/03 virus developed a novel NA mutation, R156K, that reduced binding to all three drugs, caused about 90% loss of NA activity, and compromised replication in NHBE cells. Both strains were eliminated in NHBE cells when they were cultivated in the absence of drugs. Here, we show for the first time that decreased NA activity mediated through NA inhibitors is essential for the adaptation of pandemic H5N1 influenza virus to humans. This ability of decreased NA activity to promote H5N1 infection underlines the necessity to optimize management strategies for a plausible H5N1 pandemic.     

Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

The receptor binding specificity of the live attenuated influenza H2 and H6 vaccine viruses contributes to vaccine immunogenicity and protection in ferrets

To prepare for influenza pandemics that may be caused by the H2 and H6 subtype influenza viruses, live attenuated influenza virus (LAIV) H2 and H6 vaccines are being developed and evaluated. The H2 and H6 vaccine candidates with different receptor binding preferences specified by amino acid substitutions at residues 226 and 228 were generated and evaluated for their growth in embryonated chicken eggs and their immunogenicity and protection against wild-type virus challenge in the ferret model. The viruses containing Q226 and G228 in the hemagglutinin (HA) protein bound to the avian-like α2,3-sialic acid (SA) receptor and replicated efficiently in chicken eggs. The viruses with L226 and G228 bound preferentially to the human-like α2,6-SA receptor. The viruses containing L226 and S228 displayed dual binding to both α2,3-SA and α2,6-SA receptors and replicated efficiently in eggs. The strains containing L226/G228 or L226/S228 that preferentially bound to α2,6-SA receptors replicated efficiently in the upper respiratory tract of ferrets, induced high levels of neutralizing antibody, and conferred a high level of protection against wild-type virus challenge infection compared to the strain with the Q226/G228 residues. Our data suggest that pandemic vaccines with receptor binding preference to both avian- and human-like receptors might be desired for efficient viral replication in eggs and for inducing protective immune responses in humans.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

In silico characterization of the functional and structural modules of the hemagglutinin protein from the swine-origin influenza virus A (H1N1)-2009

The 2009 swine-origin influenza virus (S-OIV,H1N1 subtype) has developed into a new pandemic influenza as announced by the World Health Organization.In order to uncover clues about the determinants for virulence and pathogenicity of the virus,we characterized the functional modules of the surface glycoprotein hemagglutinin (HA),the most important protein in molecular epidemiology and pathogenesis of influenza viruses.We analyzed receptor binding sites,basic patch,neutralization antibody epitopes and T cell epitopes in the HA protein of the current S-OIV according to the corresponding functional and structural modules previously characterized in other H1 HA molecules or HA molecules of other subtypes.We compared their differences and similarities systematically.Based on the amino acids defined as the functional and structural modules,the HA protein of 2009 S-OIV should specifically bind to the human 2,6-receptor.The D225G/E mutation in HA,which is found in some isolates,may confer dual binding specificity to the 2,3and 2,6-receptor based on previously reported work.This HA variant contains two basic patches,one of which results in increased basicity,suggesting enhanced membrane fusion function.The 2009 S-OIV HA also has an extra glycosylation site at position 276.Four of the five antibody neutralization epitopes identified in A/RP/8/34(H1N1) were exposed,but the other was hidden by a glycosylation site.The previously identified cytotoxic T cell epitopes in various HA molecules were summarized and their corresponding sequences in 2009 S-OIV HA were defined.These results are critical for understanding the pathogenicity of the virus and host immune response against the virus.     

<Emphasis Type="Italic">In silico</Emphasis> characterization of the functional and structural modules of the hemagglutinin protein from the swine-origin influenza virus A (H1N1)-2009

The 2009 swine-origin influenza virus (S-OIV, H1N1 subtype) has developed into a new pandemic influenza as announced by the World Health Organization. In order to uncover clues about the determinants for virulence and pathogenicity of the virus, we characterized the functional modules of the surface glycoprotein hemagglutinin (HA), the most important protein in molecular epidemiology and pathogenesis of influenza viruses. We analyzed receptor binding sites, basic patch, neutralization antibody epitopes and T cell epitopes in the HA protein of the current S-OIV according to the corresponding functional and structural modules previously characterized in other H1 HA molecules or HA molecules of other subtypes. We compared their differences and similarities systematically. Based on the amino acids defined as the functional and structural modules, the HA protein of 2009 S-OIV should specifically bind to the human 2,6-receptor. The D225G/E mutation in HA, which is found in some isolates, may confer dual binding specificity to the 2,3- and 2,6-receptor based on previously reported work. This HA variant contains two basic patches, one of which results in increased basicity, suggesting enhanced membrane fusion function. The 2009 S-OIV HA also has an extra glycosylation site at position 276. Four of the five antibody neutralization epitopes identified in A/RP/8/34(H1N1) were exposed, but the other was hidden by a glycosylation site. The previously identified cytotoxic T cell epitopes in various HA molecules were summarized and their corresponding sequences in 2009 S-OIV HA were defined. These results are critical for understanding the pathogenicity of the virus and host immune response against the virus.     

Residue Y161 of influenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.     

Sialic acid recognition by Vibrio cholerae neuraminidase

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid from higher order gangliosides to unmask GM1, the receptor for cholera toxin. We previously showed that the structure of VCNA is composed of a central beta-propeller catalytic domain flanked by two lectin-like domains; however the nature of the carbohydrates recognized by these lectin domains has remained unknown. We present here structures of the enzyme in complex with two substrates, alpha-2,3-sialyllactose and alpha-2,6-sialyllactose. Both substrate complexes reveal the alpha-anomer of N-acetylneuraminic acid (Neu5Ac) bound to the N-terminal lectin domain, thereby revealing the role of this domain. The large number of interactions suggest a relatively high binding affinity for sialic acid, which was confirmed by calorimetry, which gave a Kd approximately 30 microm. Saturation transfer difference NMR using a non-hydrolyzable substrate, Neu5,9Ac2-2-S-(alpha-2,6)-GlcNAcbeta1Me, was also used to map the ligand interactions at the VCNA lectin binding site. It is well known that VCNA can hydrolyze both alpha-2,3- and alpha-2,6-linked sialic acid substrates. In this study using alpha-2,3-sialyllactose co-crystallized with VCNA it was revealed that the inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) was bound at the catalytic site. This observation supports the notion that VCNA can produce its own inhibitor and has been further confirmed by 1H NMR analysis. The discovery of the sialic acid binding site in the N-lectin-like domain suggests that this might help target VCNA to sialic acid-rich environments, thereby enhancing the catalytic efficiency of the enzyme.     

Evaluation of Live Attenuated H7N3 and H7N7 Vaccine Viruses for Their Receptor Binding Preferences,Immunogenicity in Ferrets and Cross Reactivity to the Novel H7N9 Virus

Live attenuated influenza vaccine (LAIV) candidates of the H7 subtype, A/Netherlands/219/03 (H7N7, NL03 ca) and A/chicken/British Columbia/CN-6/2004 (H7N3, BC04 ca), were evaluated for their receptor binding specificity and immunogenicity in ferrets. The BC04 ca virus exhibited α2,3-SA and α2,6-SA dual receptor binding preference while the NL03 ca virus preferentially bound to α2,3-SA. Substitution of the Q226 and G228 (Q-G) by the L226 and S228 (L-S) residues in the HA improved binding to α2,6-SA for NL03 ca. The vaccine viruses with L-S retained the attenuation phenotype. NL03 L-S ca replicated more efficiently than the original NL03 ca virus in the upper respiratory tract of ferrets, and induced higher levels of humoral and cellular immune responses. Prior vaccination with seasonal LAIV reduced H7-specific antibody responses, but did not reduce the H7N7 vaccine mediated protection against a heterologous H7N3 BC04 wt virus infection in ferrets. In addition, the H7N3 and H7N7 vaccine immunized ferret sera cross reacted with the newly emerged H7N9 virus. These data, in combination with the safety data from previously conducted Phase 1 studies, suggest that these vaccines may have a role in responding to the threat posed by the H7N9 virus.     

唾液酸受体并非流感病毒各亚型在雪貂组织中播散分布的决定因子

目的探讨流感病毒在雪貂组织中的分布与唾液酸受体的关系。方法用病毒分离的方法分析流感病毒H5N1（SZ406H,A/VN/1203/04）,SH1N1,H3N2（Brisbane/09,HK/09）在雪貂各组织中分布,用直接免疫荧光法分析雪貂各组织的唾液酸受体的分布,并通过体外实验证实活病毒与组织上受体的结合。结果 H5N1（SZ）和H5N1（A/VN/1203/04）在雪貂的肝、脾、肺、肠中有分布,H5N1（A/VN/1203/04）在脑组织中也有分布,而SH1N1、H3N2（Brisbane/09,HK/09）只分布于肠组织。而唾液酸受体SAα2,6Gal和SAα2,3Gal的I型受体分布于脾、心、肺、肠、脑组织中,和SAα2,3Gal II型受体分布于肝、脾、心、肺、肠、脑组织。SH1N1病毒与SAα2,6Gal能结合,而H5N1与SAα2,3Gal结合。结论 H5N1能在雪貂的多器官组织组织中分布和繁殖,而H3N2和SH1N1仅能在肠组织中分布繁殖。SAα2,6Gal和SAα2,3Gal受体在雪貂多器官组织中均有表达,说明唾液酸受体是病毒进入的门户,但不是病毒分布的决定因子。     

Key Molecular Factors in Hemagglutinin and PB2 Contribute to Efficient Transmission of the 2009 H1N1 Pandemic Influenza Virus

Animal influenza viruses pose a clear threat to public health. Transmissibility among humans is a prerequisite for a novel influenza virus to cause a human pandemic. A novel reassortant swine influenza virus acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. However, the molecular aspects of influenza virus transmission remain poorly understood. Here, we show that an amino acid in hemagglutinin (HA) is important for the 2009 H1N1 influenza pandemic virus (2009/H1N1) to bind to human virus receptors and confer respiratory droplet transmissibility in mammals. We found that the change from glutamine (Q) to arginine (R) at position 226 of HA, which causes a switch in receptor-binding preference from human α-2,6 to avian α-2,3 sialic acid, resulted in a virus incapable of respiratory droplet transmission in guinea pigs and reduced the virus's ability to replicate in the lungs of ferrets. The change from alanine (A) to threonine (T) at position 271 of PB2 also abolished the virus's respiratory droplet transmission in guinea pigs, and this mutation, together with the HA Q226R mutation, abolished the virus's respiratory droplet transmission in ferrets. Furthermore, we found that amino acid 271A of PB2 plays a key role in virus acquisition of the mutation at position 226 of HA that confers human receptor recognition. Our results highlight the importance of both the PB2 and HA genes on the adaptation and transmission of influenza viruses in humans and provide important insights for monitoring and evaluating the pandemic potential of field influenza viruses.