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ABSTRACT: The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic virus, however it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06) displayed increased virulence in mice. The addition of glycosylation sites to the 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan-receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 virus also caused changes in viral antigenicity based on cross hemagglutinin-inhibition antibody titers with antisera from mice infected with wild type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 virus directly contributes to differences in virulence and is partially responsible for their distinct antigenicity.
Journal of Virology 06/2013; · 5.40 Impact Factor
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Rafael A Medina,
Silke Stertz,
Balaji Manicassamy,
Petra Zimmermann,
Xiangjie Sun,
Randy A Albrecht,
Hanni Uusi-Kerttula,
Osvaldo Zagordi,
Robert B Belshe,
Sharon E Frey, Terrence M Tumpey,
Adolfo García-Sastre
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ABSTRACT: With the global spread of the 2009 pandemic H1N1 (pH1N1) influenza virus, there are increasing worries about evolution through antigenic drift. One way previous seasonal H1N1 and H3N2 influenza strains have evolved over time is by acquiring additional glycosylations in the globular head of their hemagglutinin (HA) proteins; these glycosylations have been believed to shield antigenically relevant regions from antibody immune responses. We added additional HA glycosylation sites to influenza A/Netherlands/602/2009 recombinant (rpH1N1) viruses, reflecting their temporal appearance in previous seasonal H1N1 viruses. Additional glycosylations resulted in substantially attenuated infection in mice and ferrets, whereas deleting HA glycosylation sites from a pre-pandemic virus resulted in increased pathogenicity in mice. We then more directly investigated the interactions of HA glycosylations and antibody responses through mutational analysis. We found that the polyclonal antibody response elicited by wild-type rpH1N1 HA was likely directed against an immunodominant region, which could be shielded by glycosylation at position 144. However, rpH1N1 HA glycosylated at position 144 elicited a broader polyclonal response able to cross-neutralize all wild-type and glycosylation mutant pH1N1 viruses. Moreover, mice infected with a recent seasonal virus in which glycosylation sites were removed elicited antibodies that protected against challenge with the antigenically distant pH1N1 virus. Thus, acquisition of glycosylation sites in the HA of H1N1 human influenza viruses affected not only their pathogenicity and ability to escape from polyclonal antibodies elicited by previous influenza virus strains but also their ability to induce cross-reactive antibodies against drifted antigenic variants.
Science translational medicine 05/2013; 5(187):187ra70. · 7.80 Impact Factor
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ABSTRACT: Influenza viruses pose a major public health burden to communities around the world by causing respiratory infections that can be highly contagious and spread rapidly through the population. Despite extensive research on influenza viruses, the modes of transmission occurring most often among humans are not entirely clear. Contributing to this knowledge gap is the lack of an understanding of the levels of infectious virus present in respirable aerosols exhaled from infected hosts. Here, we use the ferret model to evaluate aerosol shedding patterns and measure the amount of infectious virus present in exhaled respirable aerosols. By comparing these parameters among a panel of human and avian influenza viruses exhibiting diverse respiratory droplet transmission efficiencies, we are able to report that ferrets infected by highly transmissible influenza viruses exhale a greater number of aerosol particles and more infectious virus within respirable aerosols compared to ferrets infected by influenza viruses that do not readily transmit. Our findings improve our understanding of the ferret transmission model and provide support for the potential for influenza virus aerosol transmission.
Journal of Virology 05/2013; · 5.40 Impact Factor
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ABSTRACT: Avian influenza H5, H7 and H9 viruses top the World Health Organization's (WHO) list of subtypes with the greatest pandemic potential. Here we describe a recombinant virus-like particle (VLP) that co-localizes hemagglutinin (HA) proteins derived from H5N1, H7N2, and H9N2 viruses as an experimental vaccine against these viruses. A baculovirus vector was configured to co-express the H5, H7, and H9 genes from A/Viet Nam/1203/2004 (H5N1), A/New York/107/2003 (H7N2) and A/Hong Kong/33982/2009 (H9N2) viruses, respectively, as well as neuraminidase (NA) and matrix (M1) genes from A/Puerto Rico/8/1934 (H1N1) virus. Co-expression of these genes in Sf9 cells resulted in production of triple-subtype VLPs containing HA molecules derived from the three influenza viruses. The triple-subtype VLPs exhibited hemagglutination and neuraminidase activities and morphologically resembled influenza virions. Intranasal vaccination of ferrets with the VLPs resulted in induction of serum antibody responses and efficient protection against experimental challenges with H5N1, H7N2, and H9N2 viruses.
Virology 04/2013; · 3.35 Impact Factor
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Jessica A Belser,
C Todd Davis,
Amanda Balish,
Lindsay E Edwards,
Hui Zeng,
Taronna R Maines,
Kortney M Gustin,
Irma López Martínez,
Rodrigo Fasce,
Nancy J Cox,
Jacqueline M Katz, Terrence M Tumpey
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ABSTRACT: H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus [A/Mexico/InDRE7218/2012 (MX/7218)] and related avian H7 subtype viruses in mouse and ferret models. Several high and low pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tract of ferrets, however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naïve ferrets in a direct contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naïve contact ferrets, while concurrently retaining the ability to replicate within ocular tissue allowing the eye to serve as a portal of entry.
Journal of Virology 03/2013; · 5.40 Impact Factor
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ABSTRACT: SUMMARY Respiratory viruses (including adenovirus, influenza virus, respiratory syncytial virus, coronavirus, and rhinovirus) cause a broad spectrum of disease in humans, ranging from mild influenza-like symptoms to acute respiratory failure. While species D adenoviruses and subtype H7 influenza viruses are known to possess an ocular tropism, documented human ocular disease has been reported following infection with all principal respiratory viruses. In this review, we describe the anatomical proximity and cellular receptor distribution between ocular and respiratory tissues. All major respiratory viruses and their association with human ocular disease are discussed. Research utilizing in vitro and in vivo models to study the ability of respiratory viruses to use the eye as a portal of entry as well as a primary site of virus replication is highlighted. Identification of shared receptor-binding preferences, host responses, and laboratory modeling protocols among these viruses provides a needed bridge between clinical and laboratory studies of virus tropism.
Microbiology and molecular biology reviews: MMBR 03/2013; 77(1):144-56. · 12.59 Impact Factor
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ABSTRACT: Nonspecific anti-inflammatory drugs have been purported to reduce the burden of severe influenza disease. We demonstrate that, unlike oseltamivir administration, simvastatin administration did not reduce morbidity, mortality, or viral load of mice infected with H1N1 or H5N1 viruses. No added benefit to the efficacy of oseltamivir therapy was observed when mice were treated in combination with simvastatin. Modest reductions in lung cytokine production in H5N1 but not H1N1 virus-infected simvastatin-treated mice indicate a potential benefit for statin use in mitigating disease following severe virus infection.
Virology 02/2013; · 3.35 Impact Factor
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ABSTRACT: H5N1 influenza viruses are capable of causing severe disease and death in humans, and represent a potential pandemic subtype should they acquire a transmissible phenotype. Due to the expanding host and geographic range of this virus subtype, there is an urgent need to better understand the contribution of both virus and host responses following H5N1 virus infection to prevent and control human disease. The use of mammalian models, notably the mouse and ferret, has enabled the detailed study of both complex virus-host interactions as well as the contribution of individual viral proteins and point mutations which influence virulence. In this review, we describe the behavior of H5N1 viruses which exhibit high and low virulence in numerous mammalian species, and highlight the contribution of inoculation route to virus pathogenicity. The involvement of host responses as studied in both inbred and outbred mammalian models is discussed. The roles of individual viral gene products and molecular determinants which modulate the severity of H5N1 disease in vivo are presented. This research contributes not only to our understanding of influenza virus pathogenesis, but also identifies novel preventative and therapeutic targets to mitigate the disease burden caused by avian influenza viruses.
Virus Research 02/2013; · 2.94 Impact Factor
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ABSTRACT: Highly pathogenic influenza A viruses including avian H5N1 viruses and the 1918 pandemic virus cause severe respiratory disease in humans and animals. Virus infection is followed by intense pulmonary congestion due to an extensive influx of macrophages and neutrophils which can release large quantities of reactive oxygen species potentially contributing to the pathogenesis of lung disease. Here the role of nitric oxide (NO), a potent signaling molecule in inflammation was evaluated following highly pathogenic influenza virus challenge in mice. We observed higher levels of NO in mice infected with H5N1 and 1918 viruses compared to a seasonal H1N1 virus. Mice deficient in inducible nitric oxide synthase (NOS2(-/-)) exhibited reduced morbidity, mortality and diminished cytokine production in lung tissue following H5N1 and 1918-virus challenge compared with wild-type control mice. Furthermore, systemic treatment of mice with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) delayed weight loss and death among 1918 virus infected mice compared to untreated control animals. This study demonstrates that NO contributes to the pathogenic outcome of H5N1 and 1918 viral infections in the mouse model.
The Journal of Infectious Diseases 02/2013; · 6.41 Impact Factor
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ABSTRACT: While influenza viruses are typically considered respiratory pathogens, the ocular system represents a secondary entry point for virus to establish a productive respiratory infection and the location for rare instances of virus-induced conjunctivitis. We used the ferret model to conduct a side-by-side comparison of virus infectivity, kinetics of viral replication, and induction of host responses following inoculation by either the intranasal or ocular routes with two viruses, A/Netherlands/230/03 (H7N7) and A/Panama/2007/99 (H3N2). We show that ocular inoculation resulted in delayed virus replication and reduced levels of proinflammatory cytokine and chemokine transcript in respiratory tract but not ocular tissues compared with intranasally inoculated animals. We identified numerous proinflammatory mediators with known roles in ocular disease elicited in ferret eye tissue following influenza virus infection. These findings provide a greater understanding of the modulation of host responses following different inoculation routes and underscore the risk associated with ocular exposure to influenza viruses.
Virology 02/2013; · 3.35 Impact Factor
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Ron A M Fouchier,
Adolfo García-Sastre,
Yoshihiro Kawaoka,
Wendy S Barclay,
Nicole M Bouvier,
Ian H Brown,
Ilaria Capua,
Hualan Chen,
Richard W Compans,
Robert B Couch, [......],
Kanta Subbarao,
David E Swayne,
Toru Takimoto,
Masato Tashiro,
Jeffery K Taubenberger,
Paul G Thomas,
Ralph A Tripp, Terrence M Tumpey,
Richard J Webby,
Robert G Webster
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ABSTRACT: In January 2012, influenza virus researchers from around the world announced a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. Now that the aims of the voluntary moratorium have been met in some countries and are close to being met in others, we declare an end to the voluntary moratorium on avian flu transmission studies.
Science 01/2013; · 31.20 Impact Factor
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ABSTRACT: Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and non-ciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on non-ciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas, a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within non-ciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea, and at a higher temperature (37°C) compared with a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes, cell death and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and pathogenesis of influenza viruses.
Journal of Virology 12/2012; · 5.40 Impact Factor
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ABSTRACT: BACKGROUND: The 2009 pandemic H1N1 influenza virus emerged in swine and quickly became a major global health threat. In mouse, non-human primate, and swine infection models, the pH1N1 virus efficiently replicates in the lung and induces pro-inflammatory host responses; however, whether similar or different cellular pathways were impacted by pH1N1 virus across independent infection models remains to be further defined. To address this we have performed a comparative transcriptomic analysis of acute phase responses to a single pH1N1 influenza virus, A/California/04/2009 (CA04), in the lung of mice, macaques and swine. RESULTS: Despite similarities in the clinical course, we observed differences in inflammatory molecules elicited, and the kinetics of their gene expression changes across all three species. We found genes associated with the retinoid X receptor (RXR) signaling pathway known to control pro-inflammatory and metabolic processes that were differentially regulated during infection in each species, though the heterodimeric RXR partner, pathway associated signaling molecules, and gene expression patterns varied among the three species. CONCLUSIONS: By comparing transcriptional changes in the context of clinical and virological measures, we identified differences in the host transcriptional response to pH1N1 virus across independent models of acute infection. Antiviral resistance and the emergence of new influenza viruses have placed more focus on developing drugs that target the immune system. Underlying overt clinical disease are molecular events that suggest therapeutic targets identified in one host may not be appropriate in another.
BMC Genomics 11/2012; 13(1):627. · 4.07 Impact Factor
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ABSTRACT: The recent increase in human cases with influenza A H3N2 variant virus [A(H3N2)v] highlights the need to assess whether seasonal influenza vaccination provides cross-protection against A(H3N2)v virus. Our data demonstrate that the 2011-2012 trivalent inactivated influenza vaccine (TIV) protected ferrets against homologous H3N2 virus challenge, but provided minimal to no protection against A(H3N2)v virus. The complete absence of specific hemagglutination-inhibition antibody response to A(H3N2)v is consistent with the poor cross-protection observed among TIV-immune animals.
Journal of Virology 10/2012; · 5.40 Impact Factor
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ABSTRACT: The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.
Journal of Virology 05/2012; 86(13):7118-25. · 5.40 Impact Factor
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ABSTRACT: Influenz a viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Improved vaccines that can
overcome the decline in immune function with aging and/or can induce broader immunity to novel pandemic strains are a high
priority. To design improved vaccines for the elderly, we need to better understand the effects of age on both innate and
adaptive immunity. In a murine model, we have determined that defects in antigen-presenting cell (APC) expression of pattern-recognition
molecules, costimulatory molecules, and cytokine production may play an important role in the reduced clonal expansion of
T cells in aging. The use of immunomodulators such as adjuvants may overcome some of the defects of aging immunity and may
also be, useful in the development of improved vaccines for avian influenza A subtypes that pose a pandemic threat. Several
novel strategies including the use of ISCOM-formulated vaccines, mucosal delivery, or DNA vaccination provided cross-subtype
protection that could provide an important component of immunity in the event of a pandemic.
Immunologic Research 04/2012; 29(1):113-124. · 3.03 Impact Factor
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ABSTRACT: While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.
Journal of Virology 04/2012; 86(13):7192-206. · 5.40 Impact Factor
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ABSTRACT: Respiratory viruses represent one of the most substantial infectious disease burdens to the human population today, and in particular, seasonal and pandemic influenza viruses pose a persistent threat to public health worldwide. In recent years, advances in techniques used in experimental research have provided the means to better understand the mechanisms of pathogenesis and transmission of respiratory viruses, and thus more accurately model these infections in the laboratory. Here, we briefly review the model systems used to study influenza virus infections, and focus particularly on recent advances that have increased our knowledge of these formidable respiratory pathogens.
Trends in Microbiology 04/2012; 20(6):275-81. · 7.91 Impact Factor
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Jens C Krause,
Tshidi Tsibane, Terrence M Tumpey,
Chelsey J Huffman,
Randy Albrecht,
David L Blum,
Irene Ramos,
Ana Fernandez-Sesma,
Kathryn M Edwards,
Adolfo García-Sastre,
Christopher F Basler,
James E Crowe
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ABSTRACT: Investigation of the human antibody response to the 1957 pandemic H2N2 influenza A virus has been largely limited to serologic studies. We generated five influenza virus hemagglutinin (HA)-reactive human monoclonal antibodies (MAbs) by hybridoma technology from the peripheral blood of healthy donors who were born between 1950 and 1968. Two MAbs reacted with the pandemic H2N2 virus, two recognized the pandemic H3N2 virus, and remarkably, one reacted with both the pandemic H2N2 and H3N2 viruses. Each of these five naturally occurring MAbs displayed hemagglutination inhibition activity, suggesting specificity for the globular head domain of influenza virus HA. When incubated with virus, MAbs 8F8, 8M2, and 2G1 each elicited H2N2 escape mutations immediately adjacent to the receptor-binding domain on the HA globular head in embryonated chicken eggs. All H2N2-specific MAbs were able to inhibit a 2006 swine H2N3 influenza virus. MAbs 8M2 and 2G1 shared the V(H)1-69 germ line gene, but these antibodies were otherwise not genetically related. Each antibody was able to protect mice in a lethal H2N2 virus challenge. Thus, even 43 years after circulation of H2N2 viruses, these subjects possessed peripheral blood B cells encoding potent inhibiting antibodies specific for a conserved region on the globular head of the pandemic H2 HA.
Journal of Virology 03/2012; 86(11):6334-40. · 5.40 Impact Factor
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Melissa B Pearce,
Akila Jayaraman,
Claudia Pappas,
Jessica A Belser,
Hui Zeng,
Kortney M Gustin,
Taronna R Maines,
Xiangjie Sun,
Rahul Raman,
Nancy J Cox,
Ram Sasisekharan,
Jaqueline M Katz, Terrence M Tumpey
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ABSTRACT: Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6-sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.
Proceedings of the National Academy of Sciences 03/2012; 109(10):3944-9. · 9.68 Impact Factor
