scholarly journals Antibody Neutralization of an Influenza Virus that Uses Neuraminidase for Receptor Binding

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 597 ◽  
Author(s):  
Lauren E. Gentles ◽  
Hongquan Wan ◽  
Maryna C. Eichelberger ◽  
Jesse D. Bloom
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Viral Entry ◽  
Active Site ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Viral Escape ◽  
Single Cycle ◽  
Escape Mutants

Influenza virus infection elicits antibodies against the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Because HA is essential for viral entry, antibodies targeting HA often potently neutralize the virus in single-cycle infection assays. However, antibodies against NA are not potently neutralizing in such assays, since NA is dispensable for single-cycle infection. Here we show that a modified influenza virus that depends on NA for receptor binding is much more sensitive than a virus with receptor-binding HA to neutralization by some anti-NA antibodies. Specifically, a virus with a receptor-binding G147R N1 NA and a binding-deficient HA is completely neutralized in single-cycle infections by an antibody that binds near the NA active site. Infection is also substantially inhibited by antibodies that bind NA epitopes distant from the active site. Finally, we demonstrate that this modified virus can be used to efficiently select mutations in NA that escape antibody binding, a task that can be laborious with typical influenza viruses that are not well neutralized by anti-NA antibodies. Thus, viruses dependent on NA for receptor binding allow for sensitive in vitro detection of antibodies binding near the catalytic site of NA and enable the selection of viral escape mutants.

scholarly journals Antibody neutralization of an influenza virus that uses neuraminidase for receptor binding

2020 ◽  
Author(s):  
Lauren E. Gentles ◽  
Hongquan Wan ◽  
Maryna C. Eichelberger ◽  
Jesse D. Bloom
Keyword(s):  
Influenza Virus ◽  
Receptor Binding ◽  
Viral Entry ◽  
Active Site ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Viral Escape ◽  
Single Cycle ◽  
Escape Mutants

AbstractInfluenza virus infection elicits antibodies against the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Because HA is essential for viral entry, antibodies targeting HA often potently neutralize the virus in single-cycle infection assays. But antibodies against NA are not potently neutralizing in such assays, since NA is dispensable for single-cycle infection. Here we show that a modified influenza virus that depends on NA for receptor binding is much more sensitive than a virus with receptor-binding HA to neutralization by some anti-NA antibodies. Specifically, virus with a receptor-binding G147R N1 NA and a binding-deficient HA is completely neutralized in single-cycle infections by an antibody that binds near the NA active site. Infection is also substantially inhibited by antibodies that bind NA epitopes distant from the active site. Finally, we demonstrate that this modified virus can be used to efficiently select mutations in NA that escape antibody binding, a task that can be laborious with typical influenza viruses that are not well-neutralized by anti-NA antibodies. Thus, viruses dependent on NA for receptor binding allow for sensitive in vitro detection of antibodies binding near the catalytic site of NA and enable selection of viral escape mutants.

scholarly journals Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Hui Zeng ◽  
Cynthia S. Goldsmith ◽  
Amrita Kumar ◽  
Jessica A. Belser ◽  
Xiangjie Sun ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Avian Influenza ◽  
H5n1 Virus ◽  
H1n1 Virus ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Host Interactions ◽  
Culture Model

ABSTRACTFerrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridgein vivoandin vitrostudies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCEAlthough ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlledin vitroenvironmental setting.

scholarly journals Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses

2016 ◽  
Vol 91 (2) ◽  
Author(s):  
Catherine M. Crosby ◽  
William E. Matchett ◽  
Stephanie S. Anguiano-Zarate ◽  
Christopher A. Parks ◽  
Eric A. Weaver ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Viral Vectors ◽  
Rhesus Macaques ◽  
Influenza Virus Infection ◽  
Single Cycle ◽  
Syrian Hamsters ◽  
Hemagglutinin Protein

ABSTRACT Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro. SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines. IMPORTANCE Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.

scholarly journals Mouse Saliva Inhibits Transit of Influenza Virus to the Lower Respiratory Tract by Efficiently Blocking Influenza Virus Neuraminidase Activity

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Brad Gilbertson ◽  
Wy Ching Ng ◽  
Simon Crawford ◽  
Jenny L. McKimm-Breschkin ◽  
Lorena E. Brown
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Respiratory Tract ◽  
Active Site ◽  
Lower Respiratory Tract ◽  
Mdck Cells ◽  
Influenza Virus Infection ◽  
Natural Form ◽  
Influenza Virus Neuraminidase

ABSTRACT We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location, and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge, mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a possible mechanism through which the species may not experience severe influenza virus infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza virus neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection.

scholarly journals Perforin and Fas Cytolytic Pathways Coordinately Shape the Selection and Diversity of CD8+-T-Cell Escape Variants of Influenza Virus

2005 ◽  
Vol 79 (13) ◽  
pp. 8545-8559 ◽  
Author(s):  
Graeme E. Price ◽  
Lei Huang ◽  
Rong Ou ◽  
Menghua Zhang ◽  
Demetrius Moskophidis
Keyword(s):  
Influenza Virus ◽  
T Cell ◽  
Vaccine Development ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Viral Escape ◽  
Viral Control ◽  
Ctl Escape ◽  
Selection Of

ABSTRACT Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.

scholarly journals Ethanol Extract of Caesalpinia decapetala Inhibits Influenza Virus Infection In Vitro and In Vivo

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 557 ◽  
Author(s):  
Li Zhang ◽  
Jungang Chen ◽  
Chang Ke ◽  
Haiwei Zhang ◽  
Shoujun Zhang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Antiviral Agents ◽  
Influenza Virus Infection ◽  
Virus Titer ◽  
Ethanol Extract ◽  
Influenza Viruses ◽  
Virus Infections

Influenza virus infections can lead to viral pneumonia and acute respiratory distress syndrome in severe cases, causing significant morbidity and mortality and posing a great threat to human health. Because of the diversity of influenza virus strains and drug resistance to the current direct antiviral agents, there have been no effective drugs as yet to cure all patients infected by influenza viruses. Natural products from plants contain compounds with diverse structures that have the potential to interact with multiple host and virus factors. In this study, we identified the ethanol extract of Caesalpinia decapetala (Roth) Alston (EEC) as an inhibitor against the replication of a panel of influenza A and B viruses both on human pulmonary epithelial A549 and human monocytic U937 cells. The animal study revealed that EEC administration reduces the weight loss and improves the survival rate of mice infected with lethal influenza virus. Also, EEC treatment attenuated lung injury and reduced virus titer significantly. In conclusion, we showed that EEC has antiviral activity both in vitro and in vivo, suggesting that the plant C. decapetala has the potential to be further developed as a resource of new anti-influenza drugs.

scholarly journals Cross-Protection of Chicken Immunoglobulin Y Antibodies against H5N1 and H1N1 Viruses Passively Administered in Mice

2011 ◽  
Vol 18 (7) ◽  
pp. 1083-1090 ◽  
Author(s):  
Michael G. Wallach ◽  
Richard J. Webby ◽  
Fakhrul Islam ◽  
Stephen Walkden-Brown ◽  
Eva Emmoth ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Pandemic ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Antigenic Drift ◽  
Virus Infectivity ◽  
Lethal Challenge ◽  
H5n1 Influenza

ABSTRACTInfluenza viruses remain a major threat to global health due to their ability to undergo change through antigenic drift and antigenic shift. We postulated that avian IgY antibodies represent a low-cost, effective, and well-tolerated approach that can easily be scaled up to produce enormous quantities of protective antibodies. These IgY antibodies can be administered passively in humans (orally and intranasally) and can be used quickly and safely to help in the fight against an influenza pandemic. In this study, we raised IgY antibodies against H1N1, H3N2, and H5N1 influenza viruses. We demonstrated that, using whole inactivated viruses alone and in combination to immunize hens, we were able to induce a high level of anti-influenza virus IgY in the sera and eggs, which lasted for at least 2 months after two immunizations. Furthermore, we found that by use ofin vitroassays to test for the ability of IgY to inhibit hemagglutination (HI test) and virus infectivity (serum neutralization test), IgYs inhibited the homologous as well as in some cases heterologous clades and strains of viruses. Using anin vivomouse model system, we found that, when administered intranasally 1 h prior to infection, IgY to H5N1 protected 100% of the mice against lethal challenge with H5N1. Of particular interest was the finding that IgY to H5N1 cross-protected against A/Puerto Rico/8/34 (H1N1) bothin vitroandin vivo. Based on our results, we conclude that anti-influenza virus IgY can be used to help prevent influenza virus infection.

scholarly journals Neuraminidase Inhibitor-Resistant Recombinant A/Vietnam/1203/04 (H5N1) Influenza Viruses Retain Their Replication Efficiency and Pathogenicity In Vitro and In Vivo

2007 ◽  
Vol 81 (22) ◽  
pp. 12418-12426 ◽  
Author(s):  
Hui-Ling Yen ◽  
Natalia A. Ilyushina ◽  
Rachelle Salomon ◽  
Erich Hoffmann ◽  
Robert G. Webster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Pandemic ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Wild Type Virus ◽  
H5n1 Influenza Virus ◽  
Wild Type ◽  
Replication Efficiency ◽  
H5n1 Influenza

ABSTRACT Effective antiviral drugs are essential for early control of an influenza pandemic. It is therefore crucial to evaluate the possible threat posed by neuraminidase (NA) inhibitor-resistant influenza viruses with pandemic potential. Four NA mutations (E119G, H274Y, R292K, and N294S) that have been reported to confer resistance to NA inhibitors were each introduced into recombinant A/Vietnam/1203/04 (VN1203) H5N1 influenza virus. For comparison, the same mutations were introduced into recombinant A/Puerto Rico/8/34 (PR8) H1N1 influenza virus. The E119G and R292K mutations significantly compromised viral growth in vitro, but the H274Y and N294S mutations were stably maintained in VN1203 and PR8 viruses. In both backgrounds, the H274Y and N294S mutations conferred resistance to oseltamivir carboxylate (50% inhibitory concentration [IC50] increases, >250-fold and >20-fold, respectively), and the N294S mutation reduced susceptibility to zanamivir (IC50 increase, >3.0-fold). Although the H274Y and N294S mutations did not compromise the replication efficiency of VN1203 or PR8 viruses in vitro, these mutations slightly reduced the lethality of PR8 virus in mice. However, the VN1203 virus carrying either the H274Y or N294S mutation exhibited lethality similar to that of the wild-type VN1203 virus. The different enzyme kinetic parameters (V max and Km ) of avian-like VN1203 NA and human-like PR8 NA suggest that resistance-associated NA mutations can cause different levels of functional loss in NA glycoproteins of the same subtype. Our results suggest that NA inhibitor-resistant H5N1 variants may retain the high pathogenicity of the wild-type virus in mammalian species. Patients receiving NA inhibitors for H5N1 influenza virus infection should be closely monitored for the emergence of resistant variants.

scholarly journals Structural and Functional Analysis of Surface Proteins from an A(H3N8) Influenza Virus Isolated from New England Harbor Seals

2014 ◽  
Vol 89 (5) ◽  
pp. 2801-2812 ◽  
Author(s):  
Hua Yang ◽  
Ha T. Nguyen ◽  
Paul J. Carney ◽  
Zhu Guo ◽  
Jessie C. Chang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
New England ◽  
Receptor Binding ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
Harbor Seals ◽  
Binding Studies ◽  
Binding Preference ◽  
Major Surface

ABSTRACTIn 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 (S. J. Anthony, J. A. St Leger, K. Pugliares, H. S. Ip, J. M. Chan, Z. W. Carpenter, I. Navarrete-Macias, M. Sanchez-Leon, J. T. Saliki, J. Pedersen, W. Karesh, P. Daszak, R. Rabadan, T. Rowles, W. I. Lipkin, MBio 3:e00166-00112, 2012,http://dx.doi.org/10.1128/mBio.00166-12). Here, we present a detailed structural and biochemical analysis of the surface antigens of the 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, our results indicate that the A(H3N8) virus currently poses a low risk to humans.IMPORTANCECross-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. The 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.

scholarly journals Oseltamivir-Ribavirin Combination Therapy for Highly Pathogenic H5N1 Influenza Virus Infection in Mice

2008 ◽  
Vol 52 (11) ◽  
pp. 3889-3897 ◽  
Author(s):  
Natalia A. Ilyushina ◽  
Alan Hay ◽  
Neziha Yilmaz ◽  
Adrianus C. M. Boon ◽  
Robert G. Webster ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Virus Infection ◽  
Antiviral Effect ◽  
Influenza Viruses ◽  
Oseltamivir Carboxylate ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Pathogenic H5n1

ABSTRACT We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC50] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC50 = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used.

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