Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-22 (of 22 Records) |
Query Trace: Carney PJ [original query] |
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Use of biolayer interferometry to identify dominant binding epitopes of influenza hemagglutinin protein of A(H1N1)pdm09 in the antibody response to 2010-2011 influenza seasonal vaccine
Guo Z , Lu X , Carney PJ , Chang J , Tzeng WP , York IA , Levine MZ , Stevens J . Vaccines (Basel) 2023 11 (8) The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010-2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines. |
Genetically and antigenically divergent influenza A(H9N2) viruses exhibit differential replication and transmission phenotypes in mammalian models.
Belser JA , Sun X , Brock N , Pappas C , Pulit-Penaloza JA , Zeng H , Jang Y , Jones J , Carney PJ , Chang J , Van Long N , Diep NT , Thor S , Di H , Yang G , Cook PW , Creager HM , Wang D , McFarland J , Van Dong P , Wentworth DE , Tumpey TM , Barnes JR , Stevens J , Davis CT , Maines TR . J Virol 2020 94 (17) Low pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Co-circulation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world, and for over twenty years, have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported and was found to exhibit properties of adaptation to humans and, importantly, show similarities to strains isolated from the live bird markets of Vietnam. |
Molecular characterization and three-dimensional structures of avian H8, H11, H14, H15 and swine H4 influenza virus hemagglutinins.
Yang H , Carney PJ , Chang JC , Stevens J . Heliyon 2020 6 (6) e04068 Of the eighteen hemagglutinin (HA) subtypes (H1-H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. |
Standard-dose intradermal influenza vaccine elicits cellular immune responses similar to those of intramuscular vaccine in men with and those without HIV infection
Amoah S , Mishina M , Praphasiri P , Cao W , Kim JH , Liepkalns JS , Guo Z , Carney PJ , Chang JC , Fernandez S , Garg S , Beacham L , Holtz TH , Curlin ME , Dawood F , Olsen SJ , Gangappa S , Stevens J , Sambhara S . J Infect Dis 2019 220 (5) 743-751 BACKGROUND: Human immunodeficiency virus (HIV)-infected persons are at a higher risk of severe influenza. Although we have shown that a standard-dose intradermal influenza vaccine versus a standard-dose intramuscular influenza vaccine does not result in differences in hemagglutination-inhibition titers in this population, a comprehensive examination of cell-mediated immune responses remains lacking. METHODS: Serological, antigen-specific B-cell, and interleukin 2-, interferon gamma-, and tumor necrosis factor alpha-secreting T-cell responses were assessed in 79 HIV-infected men and 79 HIV-uninfected men. RESULTS: The route of vaccination did not affect the immunoglobulin A and immunoglobulin G (IgG) plasmablast or memory B-cell response, although these were severely impaired in the group with a CD4+ T-cell count of <200 cells/muL. The frequencies of IgG memory B cells measured on day 28 after vaccination were highest in the HIV-uninfected group, followed by the group with a CD4+ T-cell count of >/=200 cells/muL and the group with a CD4+ T-cell count of <200 cells/muL. The route of vaccination did not affect the CD4+ or CD8+ T-cell responses measured at various times after vaccination. CONCLUSIONS: The route of vaccination had no effect on antibody responses, antibody avidity, T-cell responses, or B-cell responses in HIV-infected or HIV-uninfected subjects. With the serological and cellular immune responses to influenza vaccination being impaired in HIV-infected individuals with a CD4+ T-cell count of <200 cells/muL, passive immunization strategies need to be explored to protect this population. CLINICAL TRIALS REGISTRATION: NCT01538940. |
The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain
Wan H , Gao J , Yang H , Yang S , Harvey R , Chen YQ , Zheng NY , Chang J , Carney PJ , Li X , Plant E , Jiang L , Couzens L , Wang C , Strohmeier S , Wu WW , Shen RF , Krammer F , Cipollo JF , Wilson PC , Stevens J , Wan XF , Eichelberger MC , Ye Z . Nat Microbiol 2019 4 (12) 2216-2225 A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines. |
Structural and Molecular Characterization of the Hemagglutinin from the Fifth Epidemic Wave A(H7N9) Influenza Viruses.
Yang H , Carney PJ , Chang JC , Guo Z , Stevens J . J Virol 2018 92 (16) The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that when compared to the 2013 virus HAs, the fifth wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9)HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth wave H7 HAs analyzed in this study.IMPORTANCE The A(H7N9) viruses circulating in China are of great public health concern. Herein, we report a molecular and structural study of the major surface proteins from several recent A(H7N9) influenza viruses. Our results improve the understanding of these evolving viruses and provide important information on their receptor preference that is central to ongoing pandemic risk assessment. |
The molecular characterizations of surface proteins hemagglutinin and neuraminidase from recent H5Nx avian influenza viruses.
Yang H , Carney PJ , Mishin VP , Guo Z , Chang JC , Wentworth DE , Gubareva LV , Stevens J . J Virol 2016 90 (12) 5770-5784 During 2014, a subclade 2.3.4.4 HPAI A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015 the virus was detected in wild birds in Canada and the U.S. and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North America lineages. In particular, viruses were found with N1, N2 and N8 neuraminidase vRNAs, and are collectively referred to as H5Nx viruses. In the U. S., more than 48 million domestic birds have been affected. Here, we present a detailed structural and biochemical analysis of the surface antigens from H5N1, H5N2 and H5N8 in addition to a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses, and to continually assess future changes that may increase their pandemic potential. IMPORTANCE: The H5Nx viruses emerging in North America, Europe, and Asia are of great public health concern. Herein, we report a molecular and structural study of the major surface proteins from several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preference and susceptibility to antivirals, which is central to pandemic risk assessment. |
Structure and receptor binding preferences of recombinant hemagglutinins from avian and human h6 and h10 influenza A virus subtypes
Yang H , Carney PJ , Chang JC , Villanueva JM , Stevens J . J Virol 2015 89 (8) 4612-23 During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. IMPORTANCE: Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses. |
Assessment of transmission, pathogenesis and adaptation of H2 subtype influenza viruses in ferrets
Pappas C , Yang H , Carney PJ , Pearce MB , Katz JM , Stevens J , Tumpey TM . Virology 2015 477c 61-71 After their disappearance from the human population in 1968, influenza H2 viruses have continued to circulate in the natural avian reservoir. The isolation of this virus subtype from multiple bird species as well as swine highlights the need to better understand the potential of these viruses to spread and cause disease in humans. Here we analyzed the virulence, transmissibility and receptor-binding preference of two avian influenza H2 viruses (H2N2 and H2N3) and compared them to a swine H2N3 (A/swine/Missouri/2124514/2006 [swMO]), and a human H2N2 (A/England/10/1967 [Eng/67]) virus using the ferret model as a mammalian host. Both avian H2 viruses possessed the capacity to spread efficiently between cohoused ferrets, and the swine (swMO) and human (Eng/67) viruses transmitted to naive ferrets by respiratory droplets. Further characterization of the swMO hemagglutinin (HA) by x-ray crystallography and glycan microarray array identified receptor-specific adaptive mutations. As influenza virus quasispecies dynamics during transmission have not been well characterized, we sequenced nasal washes collected during transmission studies to better understand experimental adaptation of H2 HA. The avian H2 viruses isolated from ferret nasal washes contained mutations in the HA1, including a Gln226Leu substitution, which is a mutation associated with alpha2,6 sialic acid (human-like) binding preference. These results suggest that the molecular structure of HA in viruses of the H2 subtype continue to have the potential to adapt to a mammalian host and become transmissible, after acquiring additional genetic markers. |
Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers
Wan H , Yang H , Shore DA , Garten RJ , Couzens L , Gao J , Jiang L , Carney PJ , Villanueva J , Stevens J , Eichelberger MC . Nat Commun 2015 6 6114 A(H1N1)pdm09 influenza A viruses predominated in the 2013-2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses. |
Structure and receptor binding preferences of recombinant human A(H3N2) virus hemagglutinins
Yang H , Carney PJ , Chang JC , Guo Z , Villanueva JM , Stevens J . Virology 2015 477c 18-31 A(H3N2) influenza viruses have circulated in humans since 1968, and antigenic drift of the hemagglutinin (HA) protein continues to be a driving force that allows the virus to escape the human immune response. Since the major antigenic sites of the HA overlap into the receptor binding site (RBS) of the molecule, the virus constantly struggles to effectively adapt to host immune responses, without compromising its functionality. Here, we have structurally assessed the evolution of the A(H3N2) virus HA RBS, using an established recombinant expression system. Glycan binding specificities of nineteen A(H3N2) influenza virus HAs, each a component of the seasonal influenza vaccine between 1968 and 2012, were analyzed. Results suggest that while its receptor-binding site has evolved from one that can bind a broad range of human receptor analogs to one with a more restricted binding profile for longer glycans, the virus continues to circulate and transmit efficiently among humans. |
Structural and functional analysis of surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals
Yang H , Nguyen HT , Carney PJ , Guo Z , Chang JC , Jones J , Davis CT , Villanueva JM , Gubareva LV , Stevens J . J Virol 2014 89 (5) 2801-12 In late 2011, an A(H3N8) influenza virus infection resulted in the deaths of 162 New England harbor seals. Virus sequence analysis and virus receptor binding studies highlighted potential markers responsible for mammalian adaptation and a mixed receptor binding preference (Anthony et al. ; MBio 3: e00166-00112). Here we present a detailed structural and biochemical analysis of the surface antigens of this virus. Results obtained with recombinant proteins for both the hemagglutinin and neuraminidase, indicate a true avian receptor binding preference. Although the detection of this virus in new species highlights an increased potential for cross-species transmission with these viruses, our results indicate that the A(H3N8) virus currently poses a low risk to humans. IMPORTANCE: Cross-species transmission of zoonotic influenza viruses increases public health concerns. Here we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. Results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses, as these zoonotic influenza viruses continue to circulate and adapt to new hosts. |
Improved specificity and reduced subtype cross-reactivity for antibody detection by ELISA using globular head domain recombinant hemagglutinin
Li ZN , Carney PJ , Lin SC , Li J , Chang JC , Veguilla V , Stevens J , Miller JD , Levine M , Katz JM , Hancock K . J Virol Methods 2014 209 121-5 The relative performance of ELISA using globular head domain (GH) and ectodomain hemagglutinins (HAs) as antigens to detect influenza A virus IgG antibody responses was assessed. Assay sensitivity and subtype cross-reactivity were evaluated using sera collected from recipients of monovalent H5N1 vaccine and A(H1N1)pdm09 virus-infected persons. Assay specificity was determined using collections of sera from either individuals unexposed to either H5N1 or A(H1N1)pdm09 viruses or exposed to H5N1 or A(H1N1)pdm09 viruses through vaccination or infection, respectively. ELISA using GH HA showed a similar degree of sensitivity, significantly higher specificity, and significantly lower subtype cross-reactivity compared to ELISA using ectodomain HA. |
IgM, IgG, and IgA antibody responses to influenza A(H1N1)pdm09 hemagglutinin in infected persons during the first wave of the 2009 pandemic in the United States
Li ZN , Lin SC , Carney PJ , Li J , Liu F , Lu X , Liu M , Stevens J , Levine M , Katz JM , Hancock K . Clin Vaccine Immunol 2014 21 (8) 1054-60 Novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as an indicator of primary virus infection were mainly detected in young patient groups (≤5 yrs and 6-15 yrs), not in older age group, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons aged 17-80 years, paired acute and convalescent serum samples demonstrated a four-fold or greater increase in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes suggesting that infections with subtypes other than A(H1N1)pdm09 could result in false positives by ELISA. Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawback for application of ELISA in influenza serologic studies. |
Structural stability of influenza A(H1N1)pdm09 virus hemagglutinins
Yang H , Chang JC , Guo Z , Carney PJ , Shore DA , Donis RO , Cox NJ , Villanueva JM , Klimov AI , Stevens J . J Virol 2014 88 (9) 4828-38 The non-covalent interactions that mediate trimerization of the influenza hemagglutinin (HA) are important determinants of its biological activities. Recent studies have demonstrated that mutations in the HA trimer interface affect the thermal and pH sensitivities of HA, suggesting a possible impact on vaccine stability (Farnsworth et al. 2011. Vaccine 29:: 1529-1533). We used size exclusion chromatography analysis of recombinant HA ectodomain to compare the differences among recombinant trimeric HA proteins from early 2009 pandemic H1N1 viruses, which dissociate to monomers, with those of more recent virus HAs that can be expressed as trimers. We analyzed differences amongst the HA sequences and identified inter-molecular interactions mediated by the residue at position 374 (HA0 numbering) of the HA2 sub-domain as critical for HA trimer stability. Crystallographic analyses of HA from the recent H1N1 virus A/Washington/5/2011 highlight the structural basis for this observed phenotype. It remains to be seen whether more recent viruses with this mutation will yield more stable vaccines in the future. IMPORTANCE: Hemagglutinins from the early 2009 H1N1 pandemic viruses are unable to maintain a trimeric complex when expressed in a recombinant system. However HAs from 2010 and 2011 strains are more stable and our work highlights the improvement in stability can be attributed to an E47K substitution in the HA2 subunit of the stalk that emerged naturally in the circulating viruses. |
The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.
Mishin VP , Sleeman K , Levine M , Carney PJ , Stevens J , Gubareva LV . Antiviral Res 2013 101 93-6 Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that approximately 90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star(R) substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence. |
Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice
Belser JA , Gustin KM , Pearce MB , Maines TR , Zeng H , Pappas C , Sun X , Carney PJ , Villanueva JM , Stevens J , Katz JM , Tumpey TM . Nature 2013 501 (7468) 556-9 On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like alpha2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus. |
Crystal structures of two subtype N10 neuraminidase-like proteins from bat influenza A viruses reveal a diverged putative active site
Zhu X , Yang H , Guo Z , Yu W , Carney PJ , Li Y , Chen LM , Paulson JC , Donis RO , Tong S , Stevens J , Wilson IA . Proc Natl Acad Sci U S A 2012 109 (46) 18903-8 Recently, we reported a unique influenza A virus subtype H17N10 from little yellow-shouldered bats. Its neuraminidase (NA) gene encodes a protein that appears to be highly divergent from all known influenza NAs and was assigned as a new subtype N10. To provide structural and functional insights on the bat H17N10 virus, X-ray structures were determined for N10 NA proteins from influenza A viruses A/little yellow-shouldered bat/Guatemala/164/2009 (GU09-164) in two crystal forms at 1.95 A and 2.5 A resolution and A/little yellow-shouldered bat/Guatemala/060/2010 (GU10-060) at 2.0 A. The overall N10 structures are similar to each other and to other known influenza NA structures, with a single highly conserved calcium binding site in each monomer. However, the region corresponding to the highly conserved active site of influenza A N1-N9 NA subtypes and influenza B NA differs substantially. In particular, most of the amino acid residues required for NA activity are substituted, and the putative active site is much wider because of displacement of the 150-loop and 430-loop. These structural features and the fact that the recombinant N10 protein exhibits no, or extremely low, NA activity suggest that it may have a different function than the NA proteins of other influenza viruses. Accordingly, we propose that the N10 protein be termed an NA-like protein until its function is elucidated. |
Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus
Yang H , Carney PJ , Donis RO , Stevens J . J Virol 2012 86 (16) 8645-8652 Recurrence of highly pathogenic avian influenza (HPAI) subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in the Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than non-lethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild type recombinant NL219 HA to a variant with a Threonine to Alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. Results suggest that the additional glycosylation sequon increases binding affinity to avian-type alpha2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens which calls attention to the need for further studies to fully understand the unique properties of these viruses. |
Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site
Yang H , Chen LM , Carney PJ , Donis RO , Stevens J . PLoS Pathog 2010 6 (9) e1001081 Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (alpha2-3) receptor binding profile, with only moderate binding to human-type (alpha2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential. |
Flexible label-free quantitative assay for antibodies to influenza virus hemagglutinins
Carney PJ , Lipatov AS , Monto AS , Donis RO , Stevens J . Clin Vaccine Immunol 2010 17 (9) 1407-16 During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization, provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. F-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature indicating that the same reagent lots could be used in different laboratories to minimize inter-laboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities. |
Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs
Stevens J , Chen LM , Carney PJ , Garten R , Foust A , Le J , Pokorny BA , Manojkumar R , Silverman J , Devis R , Rhea K , Xu X , Bucher DJ , Paulson J , Cox NJ , Klimov A , Donis RO . J Virol 2010 84 (16) 8287-99 Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into 2 groups. Group 1 cell isolates displayed binding to a restricted set of alpha2-6 and alpha2-3 sialoglycans whereas Group 2 cell isolates revealed broader receptor specificity relative to their egg counterparts. Egg isolates from Group 1 showed similar binding specificity as cell isolates, whereas Group 2 egg isolates showed a significantly reduced binding to alpha2-6 and alpha2-3-type receptors but retained substantial binding to specific O- and N-linked alpha2-3 glycans, including alpha2-3GalNAc and fucosylated alpha2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses; with distinct patterns of amino acid substitution in the HA upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with Group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines. |
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