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Sample records for proline-based neuraminidase inhibitor

  1. Synthesis of influenza neuraminidase inhibitors.

    PubMed

    Abdel-Magid, A F; Maryanoff, C A; Mehrman, S J

    2001-11-01

    Influenza neuraminidase inhibitors provide a means to combat flu, a potentially very serious disease. For the first time, there is a way to treat influenza by blocking the influenza enzyme neuraminidase to stop or slow the progression of infection. The diverse structures and synthesis of several influenza neuraminidase inhibitors are discussed. Contributions from chemical process development groups are highlighted for those compounds that have reached the market, such as zanamivir and oseltamivir phosphate.

  2. Neuraminidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  3. Neuraminidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Pharmaceutical companies have been developing drugs that can inhibit the function of neuraminidase hoping to create an effective weapon against the flu. Researchers from the pharmaceutical industry and from the Center for Macromolecular Crystallography have grown crystals of neuraminidase in space. These improved, space-grown crystals have provided information that have helped design drugs which form a stronger interaction with the enzyme. These drugs inhibit neuraminidase by attaching themselves to the enzyme. Since the drugs are less likely to detach from the enzyme, they are more effective, require smaller dosages, and have fewer side effects. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  4. Bacterial neuraminidase rescues influenza virus replication from inhibition by a neuraminidase inhibitor.

    PubMed

    Nishikawa, Tomoko; Shimizu, Kazufumi; Tanaka, Torahiko; Kuroda, Kazumichi; Takayama, Tadatoshi; Yamamoto, Tatsuo; Hanada, Nobuhiro; Hamada, Yoshiki

    2012-01-01

    Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may

  5. Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

    PubMed Central

    Nishikawa, Tomoko; Shimizu, Kazufumi; Tanaka, Torahiko; Kuroda, Kazumichi; Takayama, Tadatoshi; Yamamoto, Tatsuo; Hanada, Nobuhiro; Hamada, Yoshiki

    2012-01-01

    Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may

  6. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  7. Influenza treatment and prophylaxis with neuraminidase inhibitors: a review

    PubMed Central

    Kamali, Amanda; Holodniy, Mark

    2013-01-01

    Influenza virus is a pathogen that causes morbidity and mortality worldwide. Whereas vaccination is important for prevention of disease, given its limitations, antiviral therapy is at the forefront of treatment and also plays a role in prevention. Currently, two classes of antiviral medications, the adamantanes and the neuraminidase inhibitors, are approved for treatment. Given the resistance patterns of circulating influenza, adamantanes are not recommended. Within the US, two neuraminidase inhibitors are currently approved for both treatment and prevention, while worldwide there are four available. In this review, we will briefly discuss the epidemiology and pathology of influenza and then discuss neuraminidase inhibitors: their mechanism of action, resistance, development, and future applications. PMID:24277988

  8. Resistance to anti-influenza drugs: adamantanes and neuraminidase inhibitors.

    PubMed

    Hurt, Aeron C; Ho, Hui-Ting; Barr, Ian

    2006-10-01

    Development of effective drugs for the treatment or prevention of epidemic and pandemic influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs available for influenza therapy currently. However, emergence of resistance to these drugs has been detected, which raises concerns regarding their widespread use. In this review, resistance to the adamantanes and neuraminidase inhibitors will be discussed in relation to both epidemic and pandemic influenza viruses.

  9. Influenza virus resistance to neuraminidase inhibitors.

    PubMed

    Samson, Mélanie; Pizzorno, Andrés; Abed, Yacine; Boivin, Guy

    2013-05-01

    In addition to immunization programs, antiviral agents can play a major role for the control of seasonal influenza epidemics and may also provide prophylactic and therapeutic benefits during an eventual pandemic. The purpose of this article is to review the mechanism of action, pharmacokinetics and clinical indications of neuraminidase inhibitors (NAIs) with an emphasis on the emergence of antiviral drug resistance. There are two approved NAIs compounds in US: inhaled zanamivir and oral oseltamivir, which have been commercially available since 1999-2000. In addition, two other NAIs, peramivir (an intravenous cyclopentane derivative) and laninamivir (a long-acting NAI administered by a single nasal inhalation) have been approved in certain countries and are under clinical evaluations in others. As for other antivirals, the development and dissemination of drug resistance is a significant threat to the clinical utility of NAIs. The emergence and worldwide spread of oseltamivir-resistant seasonal A(H1N1) viruses during the 2007-2009 seasons emphasize the need for continuous monitoring of antiviral drug susceptibilities. Further research priorities should include a better understanding of the mechanisms of resistance to existing antivirals, the development of novel compounds which target viral or host proteins and the evaluation of combination therapies for improved treatment of severe influenza infections, particularly in immunocompromised individuals. This article forms part of a symposium in Antiviral Research on "Treatment of influenza: targeting the virus or the host."

  10. Neuraminidase Inhibitors from the Fermentation Broth of Phellinus linteus

    PubMed Central

    Hwang, Byung Soon; Lee, Myeong-Seok; Lee, Seung Woong; Lee, In-Kyoung; Seo, Geon-Sik; Choi, Hwa Jung

    2014-01-01

    During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 µM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections. PMID:25071390

  11. Neuraminidase Inhibitors from the Fruiting Body of Phellinus igniarius

    PubMed Central

    Kim, Ji-Yul; Kim, Dae-Won; Hwang, Byung Soon; Woo, E-Eum; Lee, Yoon-Ju; Jeong, Kyeong-Woon; Lee, In-Kyoung

    2016-01-01

    During our ongoing investigation of neuraminidase inhibitors from medicinal fungi, we found that the fruiting bodies of Phellinus igniarius exhibited significant inhibitory activity against neuraminidase from recombinant H3N2 influenza viruses. Two active compounds were isolated from the methanolic extract of P. igniarius through solvent partitioning and Sephadex LH-20 column chromatography. The active compounds were identified as phelligridins E and G on proton nuclear magnetic resonance (1H NMR) and electrospray ionization mass measurements. These compounds inhibited neuraminidases from recombinant rvH1N1, H3N2, and H5N1 influenza viruses, with IC50 values in the range of 0.7~8.1 µM. PMID:27433123

  12. Neuraminidase Inhibitors from the Fermentation Broth of Phellinus linteus.

    PubMed

    Hwang, Byung Soon; Lee, Myeong-Seok; Lee, Seung Woong; Lee, In-Kyoung; Seo, Geon-Sik; Choi, Hwa Jung; Yun, Bong-Sik

    2014-06-01

    During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 µM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.

  13. Inhibition of neuraminidase by Ganoderma triterpenoids and implications for neuraminidase inhibitor design

    PubMed Central

    Zhu, Qinchang; Bang, Tran Hai; Ohnuki, Koichiro; Sawai, Takashi; Sawai, Ken; Shimizu, Kuniyoshi

    2015-01-01

    Neuraminidase (NA) inhibitors are the dominant antiviral drugs for treating influenza in the clinic. Increasing prevalence of drug resistance makes the discovery of new NA inhibitors a high priority. Thirty-one triterpenoids from the medicinal mushroom Ganoderma lingzhi were analyzed in an in vitro NA inhibition assay, leading to the discovery of ganoderic acid T-Q and TR as two inhibitors of H5N1 and H1N1 NAs. Structure-activity relationship studies revealed that the corresponding triterpenoid structure is a potential scaffold for the design of NA inhibitors. Using these triterpenoids as probes we found, through further in silico docking and interaction analysis, that interactions with the amino-acid residues Arg292 and/or Glu119 of NA are critical for the inhibition of H5N1 and H1N1. These findings should prove valuable for the design and development of NA inhibitors. PMID:26307417

  14. Proline-Based Macrocyclic Inhibitors of the Hepatitis C Virus: Stereoselective Synthesis and Biological Activity

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Vibulbhan, Bancha; Prongay, Andrew; Pichardo, John; Madison, Vincent; Buevich, Alexei; Chan, Tze-Ming

    2008-06-30

    Macrocyclization through a Mitsunobu reaction was used to synthesize a 17-membered macrocycle. The bicyclic acetal core was prepared completely diastereoselectively. The macrocyclic peptidomimetic surrogate of the P2-P3 dipeptide moiety was designed to function as a hepatitis C virus (HCV) NS3 serine protease inhibitor, and the pentapeptide {alpha}-ketoamides derived from the macrocycle were shown to be potent HCV inhibitors.

  15. Discovery and Characterization of Diazenylaryl Sulfonic Acids as Inhibitors of Viral and Bacterial Neuraminidases.

    PubMed

    Hoffmann, Anja; Richter, Martina; von Grafenstein, Susanne; Walther, Elisabeth; Xu, Zhongli; Schumann, Lilia; Grienke, Ulrike; Mair, Christina E; Kramer, Christian; Rollinger, Judith M; Liedl, Klaus R; Schmidtke, Michaela; Kirchmair, Johannes

    2017-01-01

    Viral neuraminidases are an established drug target to combat influenza. Severe complications observed in influenza patients are primarily caused by secondary infections with e.g., Streptococcus pneumoniae. These bacteria engage in a lethal synergism with influenza A viruses (IAVs) and also express neuraminidases. Therefore, inhibitors with dual activity on viral and bacterial neuraminidases are expected to be advantageous for the treatment of influenza infections. Here we report on the discovery and characterization of diazenylaryl sulfonic acids as dual inhibitors of viral and Streptococcus pneumoniae neuraminidase. The initial hit came from a virtual screening campaign for inhibitors of viral neuraminidases. For the most active compound, 7-[2-[4-[2-[4-[2-(2-hydroxy-3,6-disulfo-1-naphthalenyl)diazenyl]-2-methylphenyl]diazenyl]-2-methylphenyl]diazenyl]-1,3-naphthalenedisulfonic acid (NSC65847; 1), the Ki-values measured in a fluorescence-based assay were lower than 1.5 μM for both viral and pneumococcal neuraminidases. The compound also inhibited N1 virus variants containing neuraminidase inhibitor resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, 1 was suggested to impair the viral neuraminidases and pneumococcal neuraminidase with a mixed-type inhibition mode. Given its antiviral and antipneumococcal activity, 1 was identified as a starting point for the development of novel, dual-acting anti-infectives.

  16. Discovery and Characterization of Diazenylaryl Sulfonic Acids as Inhibitors of Viral and Bacterial Neuraminidases

    PubMed Central

    Hoffmann, Anja; Richter, Martina; von Grafenstein, Susanne; Walther, Elisabeth; Xu, Zhongli; Schumann, Lilia; Grienke, Ulrike; Mair, Christina E.; Kramer, Christian; Rollinger, Judith M.; Liedl, Klaus R.; Schmidtke, Michaela; Kirchmair, Johannes

    2017-01-01

    Viral neuraminidases are an established drug target to combat influenza. Severe complications observed in influenza patients are primarily caused by secondary infections with e.g., Streptococcus pneumoniae. These bacteria engage in a lethal synergism with influenza A viruses (IAVs) and also express neuraminidases. Therefore, inhibitors with dual activity on viral and bacterial neuraminidases are expected to be advantageous for the treatment of influenza infections. Here we report on the discovery and characterization of diazenylaryl sulfonic acids as dual inhibitors of viral and Streptococcus pneumoniae neuraminidase. The initial hit came from a virtual screening campaign for inhibitors of viral neuraminidases. For the most active compound, 7-[2-[4-[2-[4-[2-(2-hydroxy-3,6-disulfo-1-naphthalenyl)diazenyl]-2-methylphenyl]diazenyl]-2-methylphenyl]diazenyl]-1,3-naphthalenedisulfonic acid (NSC65847; 1), the Ki-values measured in a fluorescence-based assay were lower than 1.5 μM for both viral and pneumococcal neuraminidases. The compound also inhibited N1 virus variants containing neuraminidase inhibitor resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, 1 was suggested to impair the viral neuraminidases and pneumococcal neuraminidase with a mixed-type inhibition mode. Given its antiviral and antipneumococcal activity, 1 was identified as a starting point for the development of novel, dual-acting anti-infectives. PMID:28261167

  17. Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor

    SciTech Connect

    Warner, T.G.; Louie, A.; Potier, M. )

    1990-11-30

    Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.

  18. Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance

    PubMed Central

    McKimm‐Breschkin, Jennifer L.

    2012-01-01

    Please cite this paper as: McKimm‐Breschkin (2012) Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza and Other Respiratory Viruses 7(Suppl. 1), 25–36. There are two major classes of antivirals available for the treatment and prevention of influenza, the M2 inhibitors and the neuraminidase inhibitors (NAIs). The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A(H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long‐acting NAI, laninamivir. This review focuses on resistance to the NAIs. Because of differences in their chemistry and subtle differences in NA structures, resistance can be both NAI‐ and subtype specific. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity. In the winter of 2007–2008, an oseltamivir‐resistant seasonal influenza A(H1N1) strain with an H274Y mutation emerged in the northern hemisphere and spread rapidly around the world. In contrast to earlier evidence of such resistant viruses being unfit, this mutant virus remained fully transmissible and pathogenic and became the major seasonal A(H1N1) virus globally within a year. This resistant A(H1N1) virus was displaced by the sensitive A(H1N1)pdm09 virus. Approximately 0·5–1·0% of community A(H1N1)pdm09 isolates are currently resistant to oseltamivir. It is now apparent that variation in non‐active site amino acids can affect the fitness of the enzyme and compensate for mutations that confer high‐level oseltamivir resistance resulting in minimal impact on enzyme function. PMID:23279894

  19. RWJ-270201 (BCX-1812): a novel neuraminidase inhibitor for influenza.

    PubMed Central

    Young, D; Fowler, C; Bush, K

    2001-01-01

    The influenza virus neuraminidase (NA) is important in the pathogenesis of infection and, thus, is an attractive target for agents used in the treatment and prophylaxis of influenza. This article describes preclinical and early clinical data related to RWJ-270201 (BCX-1812), a novel, orally active NA inhibitor that was rationally designed for having potent and selective activity against influenza A and B viruses. RWJ-270201 is a unique NA inhibitor with a cyclopentane ring structure and high selectivity for the influenza NA. RWJ-270201 has efficacy comparable to or better than earlier NA inhibitors against a wide range of influenza A and B isolates, including recently emerged and avian strains, both in vitro and in a lethal murine model of influenza. Based on the high selectivity and efficacy of RWJ-270201 against both type A and B influenza strains in preclinical studies as well as murine pharmacodynamic studies supporting the potential for once-daily administration, clinical trials were initiated in order to determine the tolerability and antiviral activity of RWJ-270201 in humans. To date, clinical studies have indicated that RWJ-270201 is well tolerated and has antiviral activity in human experimental influenza models when administered orally once daily. PMID:11779391

  20. RWJ-270201 (BCX-1812): a novel neuraminidase inhibitor for influenza.

    PubMed

    Young, D; Fowler, C; Bush, K

    2001-12-29

    The influenza virus neuraminidase (NA) is important in the pathogenesis of infection and, thus, is an attractive target for agents used in the treatment and prophylaxis of influenza. This article describes preclinical and early clinical data related to RWJ-270201 (BCX-1812), a novel, orally active NA inhibitor that was rationally designed for having potent and selective activity against influenza A and B viruses. RWJ-270201 is a unique NA inhibitor with a cyclopentane ring structure and high selectivity for the influenza NA. RWJ-270201 has efficacy comparable to or better than earlier NA inhibitors against a wide range of influenza A and B isolates, including recently emerged and avian strains, both in vitro and in a lethal murine model of influenza. Based on the high selectivity and efficacy of RWJ-270201 against both type A and B influenza strains in preclinical studies as well as murine pharmacodynamic studies supporting the potential for once-daily administration, clinical trials were initiated in order to determine the tolerability and antiviral activity of RWJ-270201 in humans. To date, clinical studies have indicated that RWJ-270201 is well tolerated and has antiviral activity in human experimental influenza models when administered orally once daily.

  1. Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor.

    PubMed

    Zhao, Haiyan; Chen, Zilin

    2014-05-02

    A simple and effective neuraminidase-immobilized capillary microreactor was fabricated by glutaraldehyde cross-linking technology for screening the neuraminidase inhibitors from traditional Chinese medicines. The substrate and product were separated by CE in short-end injection mode within 2 min. Dual-wavelength ultraviolet detection was employed to eliminate the interference from the screened compounds. The parameters relating to the separation efficiency and the activity of immobilized neuraminidase were systematically evaluated. The activity of the immobilized neuraminidase remained 90% after 30 days storage at 4°C. The immobilized NA microreactor could be continuously used for more than 200 runs. The Michaelis-Menten constant of neuraminidase was determined by the microreactor as 136.6 ± 10.8 μM. In addition, six in eighteen natural products were found as potent inhibitors and the inhibition potentials were ranked in the following order: bavachinin>bavachin>baicalein>baicalin>chrysin and vitexin. The half-maximal inhibitory concentrations were 59.52 ± 4.12, 65.28 ± 1.07, 44.79 ± 1.21 and 31.62 ± 2.04 for baicalein, baicalin, bavachin and bavachinin, respectively. The results demonstrated that the neuraminidase-immobilized capillary microreactor was a very effective tool for screening neuraminidase inhibitors from traditional Chinese medicines.

  2. Neuraminidase inhibitors for influenza B virus infection: efficacy and resistance

    PubMed Central

    Burnham, Andrew J.; Baranovich, Tatiana; Govorkova, Elena A.

    2013-01-01

    Many aspects of the biology and epidemiology of influenza B viruses are far less studied than for influenza A viruses, and one of these aspects is effectiveness and resistance to the clinically available antiviral drugs, the neuraminidase (NA) inhibitors (NAIs). Acute respiratory infections are one of the leading causes of death in children and adults, and influenza is among the few respiratory infections that can be prevented and treated by vaccination and antiviral treatment. Recent data has suggested that influenza B virus infections are of specific concern to pediatric patients because of the increased risk of severe disease. Treatment of influenza B is a challenging task for the following reasons: NAIs (e.g., oseltamivir and zanamivir) are the only FDA-approved class of antivirals available for treatment;the data suggest that oseltamivir is less effective than zanamivir in pediatric patients;zanamivir is not prescribed to patients younger than 7;influenza B viruses are less susceptible than influenza A viruses to NAIs in vitro;although the level of resistance to NAIs is low, the number of different molecular markers of resistance is higher than for influenza A viruses, and they are not well defined;the relationship between levels of NAI phenotypic resistance and known molecular markers, frequency of emergence, transmissibility, and fitness of NAI-resistant variants are not well established. This review presents current knowledge of the effectiveness of NAIs for influenza B virus and antiviral resistance in clinical, surveillance, and experimental studies. PMID:24013000

  3. Economics of neuraminidase inhibitor stock piling for pandemic influenza, Singapore.

    PubMed

    Lee, Vernon J; Phua, Kai Hong; Chenm, Mark I; Chow, Angela; Ma, Stefan; Goh, Kee Tai; Leo, Yee Sin

    2006-01-01

    We compared strategies for stock piling neuraminidase inhibitors to treat and prevent influenza in Singapore. Cost-benefit and cost-effectiveness analyses, with Monte Carlo simulations, were used to determine economic outcomes. A pandemic in a population of 4.2 million would result in an estimated 525-1,775 deaths, 10,700-38,600 hospitalization days, and economic costs of 0.7 dollars to 2.2 billion Singapore dollars. The treatment-only strategy had optimal economic benefits: stock piles of antiviral agents for 40% of the population would save an estimated 418 lives and 414 million dollars, at a cost of 52.6 million dollars per shelf-life cycle of the stock pile. Prophylaxis was economically beneficial in high-risk subpopulations, which account for 78% of deaths, and in pandemics in which the death rate was >0.6%. Prophylaxis for pandemics with a 5% case-fatality rate would save 50,000 lives and 81 billion dollars. These models can help policymakers weigh the options for pandemic planning.

  4. Citations alone were enough to predict favorable conclusions in reviews of neuraminidase inhibitors.

    PubMed

    Zhou, Xujuan; Wang, Ying; Tsafnat, Guy; Coiera, Enrico; Bourgeois, Florence T; Dunn, Adam G

    2015-01-01

    To examine the use of supervised machine learning to identify biases in evidence selection and determine if citation information can predict favorable conclusions in reviews about neuraminidase inhibitors. Reviews of neuraminidase inhibitors published during January 2005 to May 2013 were identified by searching PubMed. In a blinded evaluation, the reviews were classified as favorable if investigators agreed that they supported the use of neuraminidase inhibitors for prophylaxis or treatment of influenza. Reference lists were used to identify all unique citations to primary articles. Three classification methods were tested for their ability to predict favorable conclusions using only citation information. Citations to 4,574 articles were identified in 152 reviews of neuraminidase inhibitors, and 93 (61%) of these reviews were graded as favorable. Primary articles describing drug resistance were among the citations that were underrepresented in favorable reviews. The most accurate classifier predicted favorable conclusions with 96.2% accuracy, using citations to only 24 of 4,574 articles. Favorable conclusions in reviews about neuraminidase inhibitors can be predicted using only information about the articles they cite. The approach highlights how evidence exclusion shapes conclusions in reviews and provides a method to evaluate citation practices in a corpus of reviews. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses.

    PubMed

    Hurt, Aeron C; Holien, Jessica K; Barr, Ian G

    2009-10-01

    To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.

  6. Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals.

    PubMed

    Leang, Sook-Kwan; Hurt, Aeron C

    2017-04-15

    The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

  7. Novel α- and β-Amino Acid Inhibitors of Influenza Virus Neuraminidase

    PubMed Central

    Kati, Warren M.; Montgomery, Debra; Maring, Clarence; Stoll, Vincent S.; Giranda, Vincent; Chen, Xiaoqi; Laver, W. Graeme; Kohlbrenner, William; Norbeck, Daniel W.

    2001-01-01

    In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 α- and β-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited Ki values in the 50 μM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the α-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the β-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the α- or β-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design. PMID:11502530

  8. Peramivir: an intravenous neuraminidase inhibitor for the treatment of 2009 H1N1 influenza.

    PubMed

    Mancuso, Carissa E; Gabay, Michael P; Steinke, Leah M; Vanosdol, Sherilyn J

    2010-01-01

    To review the efficacy and safety of peramivir, an unapproved neuraminidase inhibitor recently granted an Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) for the treatment of 2009 H1N1 influenza in select patients. Literature was accessed via MEDLINE (1950-April 2010) using the search terms peramivir, BCX-1812, RWJ 270201, influenza H1N1, swine influenza, and neuraminidase inhibitors. The manufacturer of peramivir, BioCryst Pharmaceuticals, was contacted for unpublished data and information presented at recent scientific meetings. Information was obtained from the Centers for Disease Control and Prevention (CDC) and FDA Web sites. The mandatory requirements for the EUA for peramivir were also consulted. Available English-language literature was reviewed and selected based on relevance, as was information from the CDC, FDA, and the drug manufacturer. The 2009 H1N1 influenza pandemic has necessitated the selective use of intravenous peramivir, an unapproved neuraminidase inhibitor. Intravenous peramivir has been studied in 4 clinical trials, 2 of which compared the drug to oseltamivir. Dose adjustments are required in pediatric patients and in those with impaired renal function. Clinicians wishing to use peramivir must request authorization from the CDC to confirm patient characteristics warranting its use and to verify the prescriber's understanding of dosing considerations and unapproved status. Peramivir has shown efficacy for the treatment of 2009 H1N1 influenza; however, it has yet to receive FDA approval. Peramivir is used in hospitalized adult and pediatric patients with suspected or laboratory-confirmed 2009 H1N1 influenza meeting specific criteria, including those unable to receive inhaled or oral neuraminidase inhibitors, those who have not responded to other neuraminidase inhibitors, or when drug delivery by a route other than intravenous is not feasible.

  9. Pyrrolidinobenzoic Acid Inhibitors of Influenza Virus Neuraminidase: the Hydrophobic Side Chain Influences Type A Subtype Selectivity

    PubMed Central

    Li, Yanwu; Silamkoti, Arundutt; Kolavi, Gundurao; Mou, Liyuan; Gulati, Shelly; Air, Gillian M.

    2012-01-01

    Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in order to investigate structure-activity relationships. The results indicated that size and geometry of the C3-side chain are important for selectivity of inhibition against N1 vs N2 NA, important type A influenza variants that infect man, including the highly lethal avian influenza. PMID:22677529

  10. Identification of Selective Nanomolar Inhibitors of the Human Neuraminidase, NEU4

    PubMed Central

    2013-01-01

    The human neuraminidase enzymes (hNEU) play important roles in human physiology and pathology. The lack of potent and selective inhibitors toward these enzymes has limited our understanding of their function and the development of therapeutic applications. Here we report the evaluation of a panel of compounds against the four human neuraminidase isoenzymes. Among the compounds tested, we identified the first selective, nanomolar inhibitors of the human neuraminidase 4 enzyme (NEU4). The most potent NEU4 inhibitor (5-acetamido-9-[4-hydroxymethyl[1,2,3]triazol-1-yl]-2,3,5,9-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosonic acid) was found to have an inhibitory constant (Ki) of 30 ± 19 nM and was 500-fold selective for its target over the other hNEU isoenzymes tested in vitro (NEU1, NEU2, and NEU3). This is the first report of any inhibitor of hNEU with nanomolar potency, and this confirms that the 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) scaffold can be exploited to develop new, potent, and selective inhibitors that target this important family of human enzymes. PMID:24900705

  11. Neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis.

    PubMed

    Jagannath, Vanitha A; Asokan, G V; Fedorowicz, Zbys; Lee, Tim W R

    2016-02-24

    Cystic fibrosis is the most common, life-threatening, recessively inherited disease of Caucasian populations. It is a multisystem disorder caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein which is important in producing sweat, digestive juices and mucus.The impaired or absent function of this protein results in the production of viscous mucus within the lungs and an environment that is susceptible to chronic airway obstruction and pulmonary colonization by a range of pathogenic bacteria. Morbidity and mortality of cystic fibrosis is related to chronic pulmonary sepsis and its complications by these bacteria.Influenza can worsen the course of the disease in cystic fibrosis by increasing the risk of pneumonia and secondary respiratory complications. Antiviral agents form an important part of influenza management and include the neuraminidase inhibitors zanamivir and oseltamivir. These inhibitors can limit the infection and prevent the spread of the virus. To assess the effects of neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings.Most recent search: 02 November 2015. Randomised controlled trials and quasi-randomised controlled trials comparing neuraminidase inhibitors with placebo or other antiviral drugs. Two review authors had planned to independently screen studies, extract data and assess risk of bias using standard Cochrane methodologies. No studies were identified for inclusion. No relevant studies were retrieved after a comprehensive search of the literature. We were unable to identify any randomised controlled studies or quasi-randomised controlled studies on the efficacy of neuraminidase inhibitors for the

  12. Neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis.

    PubMed

    Jagannath, Vanitha A; Asokan, G V; Fedorowicz, Zbys; Lee, Tim W R

    2014-02-10

    Cystic fibrosis is the most common, life-threatening, recessively inherited disease of Caucasian populations. It is a multisystem disorder caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein which is important in producing sweat, digestive juices and mucus.The impaired or absent function of this protein results in the production of viscous mucus within the lungs and an environment that is susceptible to chronic airway obstruction and pulmonary colonization by a range of pathogenic bacteria. Morbidity and mortality of cystic fibrosis is related to chronic pulmonary sepsis and its complications by these bacteria.Influenza can worsen the course of the disease in cystic fibrosis by increasing the risk of pneumonia and secondary respiratory complications. Antiviral agents form an important part of influenza management and include the neuraminidase inhibitors zanamivir and oseltamivir. These inhibitors can limit the infection and prevent the spread of the virus. To assess the effects of neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings.Most recent search: 08 July 2013. Randomised controlled trials and quasi-randomised controlled trials comparing neuraminidase inhibitors with placebo or other antiviral drugs. Two review authors had planned to independently screen studies, extract data and assess risk of bias using standard Cochrane Collaboration methodologies. No studies were identified for inclusion. No relevant studies were retrieved after a comprehensive search of the literature. We were unable to identify any randomised controlled trials or quasi-randomised controlled trials on the efficacy of neuraminidase inhibitors for

  13. Neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis.

    PubMed

    Jagannath, Vanitha A; Asokan, G V; Fedorowicz, Zbys; Singaram, Jai Shanthini; Lee, Tim Wr

    2010-03-17

    Cystic fibrosis is the most common, life-threatening, recessively inherited disease of Caucasian populations. It is a multisystem disorder caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein which is important in producing sweat, digestive juices and mucus.The impaired or absent function of this protein results in the production of viscous mucus within the lungs and an environment that is susceptible to chronic airway obstruction and pulmonary colonization by a range of pathogenic bacteria. Morbidity and mortality of cystic fibrosis is related to chronic pulmonary sepsis and its complications by these bacteria.Influenza can worsen the course of the disease in cystic fibrosis by increasing the risk of pneumonia and secondary respiratory complications. Antiviral agents form an important part of influenza management and include the neuraminidase inhibitors zanamivir and oseltamivir. These inhibitors can limit the infection and prevent the spread of the virus. To assess the effects of neuraminidase inhibitors for the treatment of influenza infection in people with cystic fibrosis. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings.Most recent search: 12 August 2009. Randomised controlled trials and quasi-randomised controlled trials comparing neuraminidase inhibitors with placebo or other antiviral drugs. Two review authors had planned to independently screen studies, extract data and assess risk of bias using standard Cochrane Collaboration methodologies. No studies were identified for inclusion. No relevant studies were retrieved after a comprehensive search of the literature. We were unable to identify any randomised controlled trials or quasi-randomised controlled trials on the efficacy of neuraminidase inhibitors for

  14. Neuraminidase inhibitor R-125489 - A promising drug for treating influenza virus: Steered molecular dynamics approach

    SciTech Connect

    Mai, Binh Khanh; Li, Mai Suan

    2011-07-08

    Highlights: {yields} We study binding affinity of R-125489 and its prodrug CS-8958 to neuraminidase of pathogenic influenza viruses by molecular dynamics simulations. {yields} It is shown that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. {yields} We predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus. {yields} The high correlation between theoretical and experimental data implies that SMD is a very promising tool for drug design. -- Abstract: Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson-Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.

  15. Structural basis for a class of nanomolar influenza A neuraminidase inhibitors

    NASA Astrophysics Data System (ADS)

    Kerry, Philip S.; Mohan, Sankar; Russell, Rupert J. M.; Bance, Nicole; Niikura, Masahiro; Pinto, B. Mario

    2013-10-01

    The influenza virus neuraminidase (NA) is essential for the virus life cycle. The rise of resistance mutations against current antiviral therapies has increased the need for the development of novel inhibitors. Recent efforts have targeted a cavity adjacent to the catalytic site (the 150-cavity) in addition to the primary catalytic subsite in order to increase specificity and reduce the likelihood of resistance. This study details structural and in vitro analyses of a class of inhibitors that bind uniquely in both subsites. Crystal structures of three inhibitors show occupation of the 150-cavity in two distinct and novel binding modes. We believe these are the first nanomolar inhibitors of NA to be characterized in this way. Furthermore, we show that one inhibitor, binding within the catalytic site, offers reduced susceptibility to known resistance mutations via increased flexibility of a pendant pentyloxy group and the ability to pivot about a strong hydrogen-bonding network.

  16. Neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis

    PubMed Central

    Jefferson, Tom; Jones, Mark; Doshi, Peter

    2009-01-01

    Objectives To update a 2005 Cochrane review that assessed the effects of neuraminidase inhibitors in preventing or ameliorating the symptoms of influenza, the transmission of influenza, and complications from influenza in healthy adults, and to estimate the frequency of adverse effects. Search strategy An updated search of the Cochrane central register of controlled trials (Cochrane Library 2009, issue 2), which contains the Acute Respiratory Infections Group’s specialised register, Medline (1950-Aug 2009), Embase (1980-Aug 2009), and post-marketing pharmacovigilance data and comparative safety cohorts. Selection criteria Randomised placebo controlled studies of neuraminidase inhibitors in otherwise healthy adults exposed to naturally occurring influenza. Main outcome measures Duration and incidence of symptoms; incidence of lower respiratory tract infections, or their proxies; and adverse events. Data extraction Two reviewers applied inclusion criteria, assessed trial quality, and extracted data. Data analysis Comparisons were structured into prophylaxis, treatment, and adverse events, with further subdivision by outcome and dose. Results 20 trials were included: four on prophylaxis, 12 on treatment, and four on postexposure prophylaxis. For prophylaxis, neuraminidase inhibitors had no effect against influenza-like illness or asymptomatic influenza. The efficacy of oral oseltamivir against symptomatic laboratory confirmed influenza was 61% (risk ratio 0.39, 95% confidence interval 0.18 to 0.85) at 75 mg daily and 73% (0.27, 0.11 to 0.67) at 150 mg daily. Inhaled zanamivir 10 mg daily was 62% efficacious (0.38, 0.17 to 0.85). Oseltamivir for postexposure prophylaxis had an efficacy of 58% (95% confidence interval 15% to 79%) and 84% (49% to 95%) in two trials of households. Zanamivir performed similarly. The hazard ratios for time to alleviation of influenza-like illness symptoms were in favour of treatment: 1.20 (95% confidence interval 1.06 to 1.35) for

  17. Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.

    PubMed

    Sahasrabudhe, A; Lawrence, L; Epa, V C; Varghese, J N; Colman, P M; McKimm-Breschkin, J L

    1998-07-20

    We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

  18. Substituent effects on the binding of natural product anthocyanidin inhibitors to influenza neuraminidase with mass spectrometry.

    PubMed

    Swaminathan, Kavya; Müller, Patrick; Downard, Kevin M

    2014-05-30

    The binding of three closely related anthocyanins within the 430-cavity of influenza neuraminidase is studied using a combination of mass spectrometry and molecular docking. Despite their similar structures, which differ only in the number and position of the hydroxyl substituents on the phenyl group attached to the chromenylium ring, subtle differences in their binding characteristics are revealed by mass spectrometry and molecular docking that are in accord with their inhibitory properties by neuraminidase inhibition assays. The cyanidin and delphinidin, with the greatest number of hydroxyl groups, bind more strongly and are better inhibitors than pelargonidin that contains a lone hydroxyl group at the 4' position. The study demonstrates, for the first time, the sensitivity of the mass spectrometry based approach for investigating the molecular basis and relative affinity of antiviral inhibitors, with subtly different structures, to their target protein. It has broader application for the screening of other protein interactions more generally with reasonable high-throughput. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Aurintricarboxylic Acid Is a Potent Inhibitor of Influenza A and B Virus Neuraminidases

    PubMed Central

    Farnsworth, Aaron; Brown, Earl G.; Van Domselaar, Gary; He, Runtao; Li, Xuguang

    2009-01-01

    Background Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies. Methodology/Principal Findings We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus. Conclusions/Significance Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride. PMID:20020057

  20. Neuraminidase Inhibitor Susceptibility Testing in Human Influenza Viruses: A Laboratory Surveillance Perspective

    PubMed Central

    Okomo-Adhiambo, Margaret; Sleeman, Katrina; Ballenger, Kristina; Nguyen, Ha T.; Mishin, Vasiliy P.; Sheu, Tiffany G.; Smagala, James; Li, Yan; Klimov, Alexander I.; Gubareva, Larisa V.

    2010-01-01

    Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008–2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance. PMID:21994620

  1. A Novel Potent and Highly Specific Inhibitor against Influenza Viral N1-N9 Neuraminidases: Insight into Neuraminidase-Inhibitor Interactions.

    PubMed

    Sriwilaijaroen, Nongluk; Magesh, Sadagopan; Imamura, Akihiro; Ando, Hiromune; Ishida, Hideharu; Sakai, Miho; Ishitsubo, Erika; Hori, Takanori; Moriya, Setsuko; Ishikawa, Takeshi; Kuwata, Kazuo; Odagiri, Takato; Tashiro, Masato; Hiramatsu, Hiroaki; Tsukamoto, Kenji; Miyagi, Taeko; Tokiwa, Hiroaki; Kiso, Makoto; Suzuki, Yasuo

    2016-05-26

    People throughout the world continue to be at risk for death from influenza A virus, which is always creating a new variant. Here we present a new effective and specific anti-influenza viral neuraminidase (viNA) inhibitor, 9-cyclopropylcarbonylamino-4-guanidino-Neu5Ac2en (cPro-GUN). Like zanamivir, it is highly effective against N1-N9 avian and N1-N2 human viNAs, including H274Y oseltamivir-resistant N1 viNA, due to its C-6 portion still being anchored in the active site, different from the disruption of oseltamivir's C-6 anchoring by H274Y mutation. Unlike zanamivir, no sialidase inhibitory activity has been observed for cPro-GUN against huNeu1-huNeu4 enzymes. Broad efficacy of cPro-GUN against avian and human influenza viruses in cell cultures comparable to its sialidase inhibitory activities makes cPro-GUN ideal for further development for safe therapeutic or prophylactic use against both seasonal and pandemic influenza.

  2. Susceptibility of influenza viruses circulating in Western Saudi Arabia to neuraminidase inhibitors

    PubMed Central

    Tolah, Ahmed M.; Azhar, Esam I.; Hashem, Anwar M.

    2016-01-01

    Objectives: To investigate the sensitivity of circulating influenza viruses in Western Saudi Arabia to neuraminidase inhibitors (NAIs); mainly, zanamivir and oseltamivir. Methods: Respiratory samples were collected from patients presenting with respiratory symptoms to King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia (KSA) between September 2013 and October 2014. All samples were tested prospectively by real-time reverse-transcription polymerase chain reaction for influenza A and B viruses. Positive samples were then inoculated on Madin-Darby Canine Kidney (MDCK) cells and isolated viruses were examined for their sensitivity to NAIs using fluorescent neuraminidase inhibition assay. Results: Out of 406 tested samples, 25 samples (6.2%) were positive for influenza A/pdmH1N1 virus, one sample (0.25%) was positive for influenza A/H3N2 virus, and 7 samples (1.7%) were positive for influenza B Yamagata-like virus. Screening of isolated influenza A and B viruses (9 out of 33) for their sensitivity to NAIs showed no significant resistance to available NAIs. Conclusion: Our results show that circulating influenza viruses in Jeddah are still sensitive to NAIs. PMID:27052292

  3. Design and one-pot synthesis of 2-thiazolylhydrazone derivatives as influenza neuraminidase inhibitors.

    PubMed

    Yuan, Keyang; Xiao, Mengwu; Tan, Ying; Ye, Jiao; Xie, Yongle; Sun, Xiaoxiao; Hu, Aixi; Lian, Wenwen; Liu, Ailin

    2017-05-23

    Two series of novel 2-thiazolylhydrazone derivatives were designed and synthesized via one-pot reaction of benzaldehyde derivatives, [Formula: see text]-haloketones and thiosemicarbazide. The structures of compounds 1 and 2 were characterized by [Formula: see text] NMR and [Formula: see text] NMR, and compound 1g was further confirmed by X-ray crystallography. All of the target compounds were evaluated for their NA inhibitory activity against influenza viral neuraminidase (H1N1) in vitro, and the results showed that many compounds exhibited moderate to strong inhibitory activities against influenza viral neuraminidase (H1N1). Among them, compounds 1p, 1q and 2c showed the most potent inhibitory activities with [Formula: see text] values ranging from 10.50 to [Formula: see text]. Our structure-activity relationship analysis indicated that 2-thiazolylhydrazone is an effective scaffold for NA inhibitors and that introducing an ethoxycarbonyl group to the 5-position of thiazole ring could enhance inhibitory potency. Molecular docking was performed on the most active compounds 1p and 2c to provide more insight into their mechanism of interaction.

  4. Susceptibility of influenza viruses circulating in Western Saudi Arabia to neuraminidase inhibitors.

    PubMed

    Tolah, Ahmed M; Azhar, Esam I; Hashem, Anwar M

    2016-04-01

    To investigate the sensitivity of circulating influenza viruses in Western Saudi Arabia to neuraminidase inhibitors (NAIs); mainly, zanamivir and oseltamivir. Respiratory samples were collected from patients presenting with respiratory symptoms to King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia (KSA) between September 2013 and October 2014. All samples were tested prospectively by real-time reverse-transcription polymerase chain reaction for influenza A and B viruses. Positive samples were then inoculated on Madin-Darby Canine Kidney (MDCK) cells and isolated viruses were examined for their sensitivity to NAIs using fluorescent neuraminidase inhibition assay. Out of 406 tested samples, 25 samples (6.2%) were positive for influenza A/pdmH1N1 virus, one sample (0.25%) was positive for influenza A/H3N2 virus, and 7 samples (1.7%) were positive for influenza B Yamagata-like virus. Screening of isolated influenza A and B viruses (9 out of 33) for their sensitivity to NAIs showed no significant resistance to available NAIs. Our results show that circulating influenza viruses in Jeddah are still sensitive to NAIs.

  5. Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata.

    PubMed

    Dao, Trong Tuan; Nguyen, Phi Hung; Lee, Hong Sik; Kim, Eunhee; Park, Junsoo; Lim, Seong Il; Oh, Won Keun

    2011-01-01

    The emergence of highly pathogenic influenza A virus strains, such as the new H1N1 swine influenza (novel influenza), represents a serious threat to global human health. During our course of an anti-influenza screening program on natural products, one new licochalcone G (1) and seven known (2-8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, the efficacy of oseltamivir with the presence of compound 3 (5 μM) was increased against H274Y neuraminidase. This evidence of synergistic effect makes this inhibitor to have a potential possibility for control of pandemic infection by oseltamivir-resistant influenza virus. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Financial competing interests were associated with favorable conclusions and greater author productivity in nonsystematic reviews of neuraminidase inhibitors.

    PubMed

    Dunn, Adam G; Zhou, Xujuan; Hudgins, Joel; Arachi, Diana; Mandl, Kenneth D; Coiera, Enrico; Bourgeois, Florence T

    2016-12-01

    To characterize the conclusions and production of nonsystematic reviews about neuraminidase inhibitors relative to financial competing interests held by the authors. We searched for articles about neuraminidase inhibitors and influenza (January 2005 to April 2015), identifying nonsystematic reviews and grading them according to the favorable/nonfavorable presentation of evidence on safety and efficacy. We recorded financial competing interests disclosed in the reviews and from other articles written by their authors. We measured associations between competing interests, author productivity, and conclusions. Among 213 nonsystematic reviews, 138 (65%) presented favorable conclusions. Financial competing interests were identified for 26% (137/532) of authors; 51% (108/213) of reviews were associated with a financial competing interest. Reviews produced exclusively by authors with financial competing interests (33%; 71/213) were more likely to present favorable conclusions than reviews with no competing interests (risk ratio 1.27; 95% confidence interval 1.03-1.55). Authors with financial competing interests published more articles about neuraminidase inhibitors than their counterparts. Half of nonsystematic reviews about neuraminidase inhibitors included an author with a financial competing interest. Reviews produced exclusively by these authors were more likely to present favorable conclusions, and authors with financial competing interests published a greater number of reviews. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Polyphenolic glycosides isolated from Pogostemon cablin (Blanco) Benth. as novel influenza neuraminidase inhibitors.

    PubMed

    Liu, Fang; Cao, Wei; Deng, Chao; Wu, Zhaoquan; Zeng, Guangyao; Zhou, Yingjun

    2016-01-01

    Influenza is historically an ancient disease that causes annual epidemics and, at irregular intervals, pandemics. At present, the first-line drugs (oseltamivir and zanamivir) don't seem to be optimistic due to the spontaneously arising and spreading of oseltamivir resistance among influenza virus. Pogostemon cablin (Blanco) Benth. (P. cablin) is an important traditional Chinese medicine herb that has been widely used for treatment on common cold, nausea and fever. In our previous study, we have identified an extract derived from P. cablin as a novel selective neuraminidase (NA) inhibitor. A series of polyphenolic compounds were isolated from P. cablin for their potential ability to inhibit neuraminidase of influenza A virus. Two new octaketides (1, 2), together with other twenty compounds were isolated from P. cablin. These compounds showed better inhibitory activity against NA. The significant potent compounds of this series were compounds 2 (IC50 = 3.87 ± 0.19 μ mol/ml), 11, 12, 14, 15, 19 and 20 (IC50 was in 2.12 to 3.87 μ mol/ml), which were about fourfold to doubled less potent than zanamivir and could be used to design novel influenza NA inhibitors, especially compound 2, that exhibit increased activity based on these compounds. With the help of molecular docking, we had a preliminary understanding of the mechanism of the two new compounds (1-2)' NA inhibitory activity. Fractions 6 and polyphenolic compounds isolated from fractions 6 showed higher NA inhibition than that of the initial plant exacts. The findings of this study indicate that polyphenolic compounds and fractions 6 derived from P. cablin are potential NA inhibitors. This work is one of the evidence that P. cablin has better inhibitory activity against influenza, which not only enriches the compound library of P. cablin, but also facilitates further development and promises its therapeutic potential for the rising challenge of influenza diseases.

  8. Virtual screening of Indonesian flavonoid as neuraminidase inhibitor of influenza a subtype H5N1

    NASA Astrophysics Data System (ADS)

    Parikesit, A. A.; Ardiansah, B.; Handayani, D. M.; Tambunan, U. S. F.; Kerami, D.

    2016-02-01

    Highly Pathogenic Avian Influenza (HPAI) H5N1 poses a significant threat to animal and human health worldwide. The number of H5N1 infection in Indonesia is the highest during 2005-2013, with a mortality rate up to 83%. A mutation that occurred in H5N1 strain made it resistant to commercial antiviral agents such as oseltamivir and zanamivir, so the more potent antiviral agent is needed. In this study, virtual screening of Indonesian flavonoid as neuraminidase inhibitor of H5N1 was conducted. Total 491 flavonoid compound obtained from HerbalDB were screened. Molecular docking was performed using MOE 2008.10. This research resulted in Guajavin B as the best ligand.

  9. From neuraminidase inhibitors to conjugates: a step towards better anti-influenza drugs?

    PubMed

    Cheng, Chung-Kai; Tsai, Chen-Hsuan; Shie, Jiun-Jie; Fang, Jim-Min

    2014-05-01

    For the treatment of seasonal flu and possible pandemic infections the development of new anti-influenza drugs that have good bioavailability against a broad spectrum of influenza viruses including the resistant strains is needed. In this review, we summarize previous methods for the structural modification of zanamivir, a potent neuraminidase inhibitor that has rare drug resistance, in order to develop effective anti-influenza drugs. We also report recent research into the design of multivalent zanamivir drugs and bifunctional zanamivir conjugates, some of which have shown better efficacy in animal experiments. As a step towards developing improved antivirals, conjugating anti-influenza drugs with anti-inflammatory agents can improve oral bioavailability and also exert synergistic effect in influenza therapy.

  10. An activity-integrated strategy of the identification, screening and determination of potential neuraminidase inhibitors from Radix Scutellariae

    PubMed Central

    Liu, Wei; Wang, Huilin; Zhu, Bo; Yin, Chengqian; Chen, Shuyang; Li, Jin; Yu, Xie-an; Azietaku, John Teye; An, Mingrui; Gao, Xiu-mei

    2017-01-01

    Small molecules isolated from herbal medicines (HMs) were identified as the potential neuraminidase inhibitors which are effective in influenza prevention and treatment. Unfortunately, current available screen methods of small molecules isolated from HMs are inefficient and insensitive. Here a novel Ultra Performance Liquid Chromatography coupled with diode-array detectors and auto-fraction collector / time-of-flight mass spectrometry (UPLC-DAD-FC/Q-TOF-MS) screening method with high efficiency was developed and validated to separate, collect, enrich, identify and quantify potential neuraminidase inhibitors from Radix Scutellariae. The results showed that 26 components with neuraminidase inhibitory activity were identified from Radix Scutellariae extracts. It was also found that the influence of origins on the quality of RS was more than that of cultivated time on the basis of the concentration of the effective components. These results brought novel insights into quality evaluation of Radix Scutellariae. It was demonstrated that new activity-integrated strategy was a suitable technique for the identification, screening and determination of potential neuraminidase inhibitors in herbal medicine and will provide novel potential strategies in other drug screening from herbal medicine. PMID:28486473

  11. Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

    PubMed

    Jia, Feng; Howlader, Md Amran; Cairo, Christopher W

    2016-09-01

    Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

    PubMed Central

    Shichinohe, Shintaro; Nakayama, Misako; Igarashi, Manabu; Ishii, Akihiro; Ishigaki, Hirohito; Ishida, Hideaki; Kitagawa, Naoko; Sasamura, Takako; Shiohara, Masanori; Doi, Michiko; Tsuchiya, Hideaki; Nakamura, Shinichiro; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi

    2015-01-01

    The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors. PMID:26055368

  13. Glycosidase inhibitors (castanospermine and swainsonine) and neuraminidase inhibit pokeweed mitogen-induced B cell maturation.

    PubMed

    Karasuno, T; Kanayama, Y; Nishiura, T; Nakao, H; Yonezawa, T; Tarui, S

    1992-08-01

    Castanospermine (CSP), an inhibitor of alpha-glucosidase, enhanced immunoglobulin (Ig) release in a Staphylococcus aureus Cowan I (SAC)-induced lymphocyte culture (Scand. J. Immunol. 1990. 32: 529). In a pokeweed mitogen (PWM)-human lymphocyte culture, unlike the SAC-stimulated system, CSP strongly decreased the number of IgG-, IgA- and IgM-secreting cells as well as that of Ig-bearing cells. Peripheral blood lymphocytes treated with swainsonine, a mannosidase II inhibitor, or with neuraminidase also showed a reduced response to PWM. In cross-culture experiments, only a mixture of B cells pretreated with either agent and untreated T cells showed such a suppressive effect. Adhesion was decreased between B cells treated with either agent and untreated T cells, but not between treated T cells and untreated B cells. These results demonstrate that a certain alteration in B cell membrane oligosaccharides inhibited the T cell-B cell adhesion in the PWM culture, leading to an arrest of B cell maturation. Considering that these inhibitors eventually prevent terminal sialic acid addition, the present study provides evidence that sialic acids on B cell surface oligosaccharides play a biological role in the T cell-B cell interaction.

  14. Potent and Long-Acting Dimeric Inhibitors of Influenza Virus Neuraminidase Are Effective at a Once-Weekly Dosing Regimen

    PubMed Central

    Macdonald, Simon J. F.; Watson, Keith G.; Cameron, Rachel; Chalmers, David K.; Demaine, Derek A.; Fenton, Rob J.; Gower, David; Hamblin, J. Nicole; Hamilton, Stephanie; Hart, Graham J.; Inglis, Graham G. A.; Jin, Betty; Jones, Haydn T.; McConnell, Darryl B.; Mason, Andy M.; Nguyen, Van; Owens, Ian J.; Parry, Nigel; Reece, Phillip A.; Shanahan, Stephen E.; Smith, Donna; Wu, Wen-Yang; Tucker, Simon P.

    2004-01-01

    Dimeric derivatives (compounds 7 to 9) of the influenza virus neuraminidase inhibitor zanamivir (compound 2), which have linking groups of 14 to 18 atoms in length, are approximately 100-fold more potent inhibitors of influenza virus replication in vitro and in vivo than zanamivir. The observed optimum linker length of 18 to 22 Å, together with observations that the dimers cause aggregation of isolated neuraminidase tetramers and whole virus, indicate that the dimers benefit from multivalent binding via intertetramer and intervirion linkages. The outstanding long-lasting protective activities shown by compounds 8 and 9 in mouse influenza infectivity experiments and the extremely long residence times observed in the lungs of rats suggest that a single low dose of a dimer would provide effective treatment and prophylaxis for influenza virus infections. PMID:15561823

  15. Characterization of Neuraminidase Inhibitors in Korean Papaver rhoeas Bee Pollen Contributing to Anti-Influenza Activities In Vitro.

    PubMed

    Lee, In-Kyoung; Hwang, Byung Soon; Kim, Dae-Won; Kim, Ji-Yul; Woo, E-Eum; Lee, Yoon-Ju; Choi, Hwa Jung; Yun, Bong-Sik

    2016-04-01

    The active constituents of Korean Papaver rhoeas bee pollen conferring neuraminidase inhibitory activities (H1N1, H3N2, and H5N1) were investigated. Six flavonoids and one alkaloid were isolated and characterized by nuclear magnetic resonance and mass spectrometry data. These included kaempferol-3-sophoroside (1), kaempferol-3-neohesperidoside (2), kaempferol-3-sambubioside (3), kaempferol-3-glucoside (4), quercetin-3-sophoroside (5), luteolin (6), and chelianthifoline (7). All compounds showed neuraminidase inhibitory activities with IC50 values ranging from 10.7 to 151.1 µM. The most potent neuraminidase inhibitor was luteolin, which was the dominant content in the ethyl acetate fraction. All tested compounds displayed noncompetitive inhibition of H3N2 neuraminidase. Furthermore, compounds 1-7 all reduced the severity of virally induced cytopathic effects as determined by the Madin-Darby canine kidney cell-based assay showing antiviral activity with IC50 values ranging from 10.7 to 33.4 µM (zanamivir: 58.3 µM). The active compounds were quantified by high-performance liquid chromatography, and the total amount of compounds 1-7 made up about 0.592 g/100 g bee pollen, contributing a rich resource of a natural antiviral product.

  16. Prognosis of hospitalized patients with 2009 H1N1 influenza in Spain: influence of neuraminidase inhibitors

    PubMed Central

    Delgado-Rodríguez, Miguel; Castilla, Jesús; Godoy, Pere; Martín, Vicente; Soldevila, Nuria; Alonso, Jordi; Astray, Jenaro; Baricot, Maretva; Cantón, Rafael; Castro, Ady; Gónzález-Candelas, Fernando; Mayoral, José María; Quintana, José María; Pumarola, Tomás; Tamames, Sonia; Sáez, Marc; Domínguez, Angela

    2012-01-01

    Background The H1N1 influenza pandemic strain has been associated with a poor prognosis in hospitalized patients. The present report evaluates the factors influencing prognosis. Methods A total of 813 patients hospitalized with H1N1 influenza in 36 hospitals (nationwide) in Spain were analysed. Detailed histories of variables preceding hospital admission were obtained by interview, validating data on medications and vaccine with their attending physicians. Data on treatment and complications during hospital stay were recorded. As definition of poor outcome, the endpoints of death and admission to intensive care were combined; and as a further outcome, length of stay was used. Results The mean age was 38.5 years (SD 22.8 years). There were 10 deaths and 79 admissions to intensive care (combined, 88). The use of neuraminidase inhibitors was reported by 495 patients (60.9%). The variables significantly associated with a poor outcome were diabetes (OR = 2.21, 95% CI = 1.21–4.02), corticosteroid therapy (OR = 3.37, 95% CI = 1.39–8.20) and use of histamine-2 receptor antagonists (OR = 2.68, 95% CI = 1.14–6.36), while the use of neuraminidase inhibitors (OR = 0.57, 95% CI = 0.34–0.94) was protective. Neuraminidase inhibitors within the first 2 days after the influenza onset reduced hospital stay by a mean of 1.9 days (95% CI = 4.7–6.6). Conclusions The use of neuraminidase inhibitors decreases the length of hospital stay and admission to intensive care and/or death. PMID:22467633

  17. Increased virulence of neuraminidase inhibitor-resistant pandemic H1N1 virus in mice

    PubMed Central

    Song, Min-Suk; Hee Baek, Yun; Kim, Eun-Ha; Park, Su-Jin; Kim, Semi; Lim, Gyo-Jin; Kwon, Hyeok-il; Pascua, Philippe Noriel Q; Decano, Arun G; Lee, Byeong-Jae; Kim, Young-Il; Webby, Richard J; Choi, Young-Ki

    2013-01-01

    Pandemic H1N1 2009 (A[H1N1]pdm09) variants associated with oseltamivir resistance have emerged with a histidine-to-tyrosine substitution in the neuraminidase(NA) at position 274 (H274Y). To determine whether the H274Y variant has increased virulence potential, A(H1N1)pdm09 virus, with or without the H274Y mutation, was adapted by serial lung-to-lung passages in mice. The mouse-adapted H274Y (maCA04H274Y) variants showed increased growth properties and virulence in vitro and in vivo while maintaining high NA inhibitor resistance. Interestingly, most maCA04H274Y and maCA04 viruses acquired common mutations in HA (S183P and D222G) and NP (D101G), while only maCA04H274Y viruses had consensus additional K153E mutation in the HA gene, suggesting a potential association with the H274Y substitution. Collectively, our findings highlight the potential emergence of A(H1N1)pdm09 drug-resistant variants with increased virulence and the need for rapid development of novel antiviral drugs. PMID:23924955

  18. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014-2015.

    PubMed

    Hurt, Aeron C; Besselaar, Terry G; Daniels, Rod S; Ermetal, Burcu; Fry, Alicia; Gubareva, Larisa; Huang, Weijuan; Lackenby, Angie; Lee, Raphael T C; Lo, Janice; Maurer-Stroh, Sebastian; Nguyen, Ha T; Pereyaslov, Dmitriy; Rebelo-de-Andrade, Helena; Siqueira, Marilda M; Takashita, Emi; Tashiro, Masato; Tilmanis, Danielle; Wang, Dayan; Zhang, Wenqing; Meijer, Adam

    2016-08-01

    The World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza (WHO CCs) tested 13,312 viruses collected by WHO recognized National Influenza Centres between May 2014 and May 2015 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Ninety-four per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.5% (n = 68) of viruses showed either highly reduced inhibition (HRI) or reduced inhibition (RI) (n = 56) against at least one of the four NAIs. Of the twelve viruses with HRI, six were A(H1N1)pdm09 viruses, three were A(H3N2) viruses and three were B/Yamagata-lineage viruses. The overall frequency of viruses with RI or HRI by the NAIs was lower than that observed in 2013-14 (1.9%), but similar to the 2012-13 period (0.6%). Based on the current analysis, the NAIs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections.

  19. Peramivir: A Novel Intravenous Neuraminidase Inhibitor for Treatment of Acute Influenza Infections

    PubMed Central

    Alame, Malak M.; Massaad, Elie; Zaraket, Hassan

    2016-01-01

    Peramivir is a novel cyclopentane neuraminidase inhibitor of influenza virus. It was approved by the Food and Drug Administration in December 2014 for treatment of acute uncomplicated influenza in patients 18 years and older. For several months prior to approval, the drug was made clinically available under Emergency Use authorization during the 2009 H1N1 influenza pandemic. Peramivir is highly effective against human influenza A and B isolates as well as emerging influenza virus strains with pandemic potential. Clinical trials demonstrated that the drug is well-tolerated in adult and pediatric populations. Adverse events are generally mild to moderate and similar in frequency to patients receiving placebo. Common side effects include gastrointestinal disorders and decreased neutrophil counts but are self-limiting. Peramivir is administered as a single-dose via the intravenous route providing a valuable therapeutic alternative for critically ill patients or those unable to tolerate other administration routes. Successful clinical trials and post-marketing data in pediatric populations in Japan support the safety and efficacy of peramivir in this population where administration of other antivirals might not be feasible. PMID:27065996

  20. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014.

    PubMed

    Takashita, Emi; Meijer, Adam; Lackenby, Angie; Gubareva, Larisa; Rebelo-de-Andrade, Helena; Besselaar, Terry; Fry, Alicia; Gregory, Vicky; Leang, Sook-Kwan; Huang, Weijuan; Lo, Janice; Pereyaslov, Dmitriy; Siqueira, Marilda M; Wang, Dayan; Mak, Gannon C; Zhang, Wenqing; Daniels, Rod S; Hurt, Aeron C; Tashiro, Masato

    2015-05-01

    Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases (n=3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 2% (n=172) showed highly reduced inhibition (HRI) against at least one of the four NAIs, commonly oseltamivir, while 0.3% (n=32) showed reduced inhibition (RI). Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=169), A(H3N2) with NA E119V (n=1), B/Victoria-lineage with NA E117G (n=1) and B/Yamagata-lineage with NA H273Y (n=1); amino acid position numbering is A subtype and B type specific. Although approximately 98% of circulating viruses tested during the 2013-2014 period were sensitive to all four NAIs, a large community cluster of A(H1N1)pdm09 viruses with the NA H275Y substitution from patients with no previous exposure to antivirals was detected in Hokkaido, Japan. Significant numbers of A(H1N1)pdm09 NA H275Y viruses were also detected in China and the United States: phylogenetic analyses showed that the Chinese viruses were similar to those from Japan, while the United States viruses clustered separately from those of the Hokkaido outbreak, indicative of multiple resistance-emergence events. Consequently, global surveillance of influenza antiviral susceptibility should be continued from a public health perspective. Copyright © 2015 The Authors. Published by Elsevier B.V. All

  1. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012-2013.

    PubMed

    Meijer, Adam; Rebelo-de-Andrade, Helena; Correia, Vanessa; Besselaar, Terry; Drager-Dayal, Renu; Fry, Alicia; Gregory, Vicky; Gubareva, Larisa; Kageyama, Tsutomu; Lackenby, Angie; Lo, Janice; Odagiri, Takato; Pereyaslov, Dmitriy; Siqueira, Marilda M; Takashita, Emi; Tashiro, Masato; Wang, Dayan; Wong, Sun; Zhang, Wenqing; Daniels, Rod S; Hurt, Aeron C

    2014-10-01

    Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012-2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  2. In silico modification of oseltamivir as neuraminidase inhibitor of influenza A virus subtype H1N1

    PubMed Central

    Tambunan, Usman Sumo Friend; Rachmania, Rizky Archintya; Parikesit, Arli Aditya

    2015-01-01

    Abstract This research focused on the modification of the functional groups of oseltamivir as neuraminidase inhibitor against influenza A virus subtype H1N1. Interactions of three of the best ligands were evaluated in the hydrated state using molecular dynamics simulation at two different temperatures. The docking result showed that AD3BF2D ligand (N-[(1S,6R)-5-amino-5-{[(2R,3S,4S)-3,4-dihydroxy-4-(hydroxymethyl) tetrahydrofuran-2-yl]oxy}-4-formylcyclohex-3-en-1-yl]acetamide-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate) had better binding energy values than standard oseltamivir. AD3BF2D had several interactions, including hydrogen bonds, with the residues in the catalytic site of neuraminidase as identified by molecular dynamics simulation. The results showed that AD3BF2D ligand can be used as a good candidate for neuraminidase inhibitor to cope with influenza A virus subtype H1N1. PMID:25859271

  3. Influenza neuraminidase

    PubMed Central

    Air, Gillian M.

    2011-01-01

    Please cite this paper as: Air. (2012) Influenza neuraminidase. Influenza and Other Respiratory Viruses 6(4), 245–256. Influenza neuraminidase is the target of two licensed antivirals that have been very successful, with several more in development. However, neuraminidase has been largely ignored as a vaccine target despite evidence that inclusion of neuraminidase in the subunit vaccine gives increased protection. This article describes current knowledge on the structure, enzyme activity, and antigenic significance of neuraminidase. PMID:22085243

  4. Evidence synthesis and decision modelling to support complex decisions: stockpiling neuraminidase inhibitors for pandemic influenza usage.

    PubMed

    Watson, Samuel I; Chen, Yen-Fu; Nguyen-Van-Tam, Jonathan S; Myles, Puja R; Venkatesan, Sudhir; Zambon, Maria; Uthman, Olalekan; Chilton, Peter J; Lilford, Richard J

    2016-01-01

    Objectives: The stockpiling of neuraminidase inhibitor (NAI) antivirals as a defence against pandemic influenza is a significant public health policy decision that must be made despite a lack of conclusive evidence from randomised controlled trials regarding the effectiveness of NAIs on important clinical end points such as mortality. The objective of this study was to determine whether NAIs should be stockpiled for treatment of pandemic influenza on the basis of current evidence. Methods: A decision model for stockpiling was designed. Data on previous pandemic influenza epidemiology was combined with data on the effectiveness of NAIs in reducing mortality obtained from a recent individual participant meta-analysis using observational data. Evidence synthesis techniques and a bias modelling method for observational data were used to incorporate the evidence into the model. The stockpiling decision was modelled for adults (≥16 years old) and the United Kingdom was used as an example. The main outcome was the expected net benefits of stockpiling in monetary terms. Health benefits were estimated from deaths averted through stockpiling. Results: After adjusting for biases in the estimated effectiveness of NAIs, the expected net benefit of stockpiling in the baseline analysis was £444 million, assuming a willingness to pay of £20,000/QALY ($31,000/QALY). The decision would therefore be to stockpile NAIs. There was a greater probability that the stockpile would not be utilised than utilised. However, the rare but catastrophic losses from a severe pandemic justified the decision to stockpile. Conclusions: Taking into account the available epidemiological data and evidence of effectiveness of NAIs in reducing mortality, including potential biases, a decision maker should stockpile anti-influenza medication in keeping with the postulated decision rule.

  5. Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling ▿

    PubMed Central

    Jonges, Marcel; Liu, Wai Ming; van der Vries, Erhard; Jacobi, Ronald; Pronk, Inge; Boog, Claire; Koopmans, Marion; Meijer, Adam; Soethout, Ernst

    2010-01-01

    Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans. PMID:20089763

  6. Evidence synthesis and decision modelling to support complex decisions: stockpiling neuraminidase inhibitors for pandemic influenza usage

    PubMed Central

    Watson, Samuel I.; Chen, Yen-Fu; Nguyen-Van-Tam, Jonathan S.; Myles, Puja R.; Venkatesan, Sudhir; Zambon, Maria; Uthman, Olalekan; Chilton, Peter J.; Lilford, Richard J.

    2017-01-01

    Objectives: The stockpiling of neuraminidase inhibitor (NAI) antivirals as a defence against pandemic influenza is a significant public health policy decision that must be made despite a lack of conclusive evidence from randomised controlled trials regarding the effectiveness of NAIs on important clinical end points such as mortality. The objective of this study was to determine whether NAIs should be stockpiled for treatment of pandemic influenza on the basis of current evidence. Methods: A decision model for stockpiling was designed. Data on previous pandemic influenza epidemiology was combined with data on the effectiveness of NAIs in reducing mortality obtained from a recent individual participant meta-analysis using observational data. Evidence synthesis techniques and a bias modelling method for observational data were used to incorporate the evidence into the model. The stockpiling decision was modelled for adults (≥16 years old) and the United Kingdom was used as an example. The main outcome was the expected net benefits of stockpiling in monetary terms. Health benefits were estimated from deaths averted through stockpiling. Results: After adjusting for biases in the estimated effectiveness of NAIs, the expected net benefit of stockpiling in the baseline analysis was £444 million, assuming a willingness to pay of £20,000/QALY ($31,000/QALY). The decision would therefore be to stockpile NAIs. There was a greater probability that the stockpile would not be utilised than utilised. However, the rare but catastrophic losses from a severe pandemic justified the decision to stockpile. Conclusions: Taking into account the available epidemiological data and evidence of effectiveness of NAIs in reducing mortality, including potential biases, a decision maker should stockpile anti-influenza medication in keeping with the postulated decision rule. PMID:28413608

  7. Safety of the long-acting neuraminidase inhibitor laninamivir octanoate hydrate in post-marketing surveillance.

    PubMed

    Kashiwagi, Seizaburo; Yoshida, Sanae; Yamaguchi, Hiroki; Niwa, Shinpei; Mitsui, Noriko; Tanigawa, Masatoshi; Shiosakai, Kazuhito; Yamanouchi, Naoki; Shiozawa, Tomoo; Yamaguchi, Fumie

    2012-11-01

    Laninamivir octanoate hydrate (laninamivir) is a long-acting neuraminidase inhibitor (NAI) that completes treatment with only a single inhalation. It was launched in Japan in October 2010 as an anti-influenza agent. A post-marketing surveillance study was conducted in the 2010/2011 influenza season to assess the safety of this drug in clinical settings. Adverse drug reactions (ADRs) were observed in 50 patients (59 events) out of 3542 patients subjected to safety evaluation (incidence 1.41%). Commonly reported ADRs were psychiatric disorders (abnormal behaviour, etc.), gastrointestinal disorders (diarrhoea, nausea, etc.) and nervous system disorders (dizziness, etc.), with incidences of 0.48% (n=17), 0.45% (n=16) and 0.17% (n=6), respectively. No serious ADRs occurred. ADRs usually emerged on the day on which laninamivir was inhaled (52.5%) and ADRs emerged within 3 days after inhalation in >90% of adversely affected patients. ADRs resolved or improved within 3 days in >85% of patients. The incidence of adverse events involving abnormal behaviour was 3.1% (30/959) among patients <10 years of age, 0.7% (8/1088) among patients aged 10-19 years, 0.1% (2/1431) among adult patients aged 20-64 years and 0.0% (0/64) among patients aged ≥65 years. It was confirmed that laninamivir is unlikely to cause delayed ADRs or a prolonged duration of ADRs despite this drug being a long-acting NAI. Furthermore, the incidence of ADRs was not found to have increased compared with that observed during clinical trials, and the types of ADR observed during this study were similar to those previously observed. Thus, laninamivir octanoate hydrate was confirmed to have no noticeable problem with safety. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  8. Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue.

    PubMed

    Gupta, Deepak; Varghese Gupta, Sheeba; Dahan, Arik; Tsume, Yasuhiro; Hilfinger, John; Lee, Kyung-Dall; Amidon, Gordon L

    2013-02-04

    showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.

  9. [Susceptibility of human influenza A (H3N2) viruses to neuraminidase inhibitors isolated during 2011-2012 in China].

    PubMed

    Huang, Weijuan; Tan, Minju; Zhao, Xiang; Cheng, Yanhui; Li, Xiyan; Guo, Junfeng; Wei, Hejiang; Xiao, Ning; Wang, Zhao; Wang, Dayan; Shu, Yuelong

    2015-06-01

    To analyze the susceptibility of influenza A (H3N2) viruses to neuraminidase inhibitors during 2011-2012 in Mainland China. All the tested viruses were obtained from the Chinese National Influenza Surveillance Network, which covers 31 provinces in mainland China, including 408 network laboratories and 554 sentinel hospitals. In total 1 903 viruses were selected with isolation date from January 1, 2011 to December 31, 2012 in Mainland China, among these viruses, 721 were confirmed to be influenza A (H3N2) virus by Chinese National Influenza Center and tested for the susceptibility to oseltamivir and zanamivir using chemiluminescence-based assay. The neuraminidase inhibitor sensitive reference virus A/Washington/01/2007 (119E) and oseltamivir resistant virus A/Texas/12/2007 (E119V) were used as control in this study. The t -test was used to compare the difference of NAI susceptibility of viruses isolated from different years. The half maximal inhibitory concentration (IC₅₀) of A/Washington/01/2007 for oseltamivir and zanamivir was (0.10 ± 0.02) and (0.30 ± 0.05) nmol/L, respectively. The IC₅₀ of A/Texas/12/2007 for oseltamivir and zanamivir was (4.27 ± 1.60) and (0.20 ± 0.03) nmol/L, respectively. Among the 721 influenza A (H3N2) viruses, 132 influenza A (H3N2) viruses were isolated in 2011 and 589 influenza A (H3N2) viruses were isolated in 2012. The IC50 for oseltamivir ranged from 0.04 to 0.62 nmol/L for viruses isolated in 2011 and ranged from 0.02 to 0.95 nmol/L for viruses in 2012, and the IC₅₀ of all the viruses tested was within 10-fold IC₅₀ (1.0 nmol/L) of the neuraminidase inhibitor sensitive reference virus A/Washington/01/2007. The IC50 of zanamivir ranged from 0.12 to 0.80 nmol/L for viruses in 2011 and ranged from 0.04 to 0.72 nmol/L for viruses in 2012, and was within 10-fold IC₅₀ (3.0 nmol/L) of the neuraminidase inhibitor sensitive reference virus A/Washington/01/2007. The influenza A(H3N2) viruses isolated during 2011-2012 in

  10. Structure Optimization of Neuraminidase Inhibitors as Potential Anti-Influenza (H1N1Inhibitors) Agents Using QSAR and Molecular Docking Studies.

    PubMed

    Inamdar, Poonam; Bhandari, Shashikant; Sonawane, Bhagyashri; Hole, Asha; Jadhav, Chintamani

    2014-01-01

    The urgent need of neuraminidase inhibitors (NI) has provided an impetus for understanding the structure requisite at molecular level. Our search for selective inhibitors of neuraminidase has led to the identification of pharmacophoric requirements at various positions around acyl thiourea pharmacophore. The main objective of present study is to develop selective NI, with least toxicity and drug like ADMET properties. Electronic, Steric requirements were defined using kohnone nearest neighbour- molecular field analysis (kNN-MFA) model of 3D-QSAR studies. Results generated by QSAR studies showed that model has good internal as well as external predictivity. Such defined requirements were used to generate new chemical entities which exhibit higher promising predicted activities. To check selective binding of designed NCE's docking studies were carried out using the crystal structure of the neuraminidase enzyme having co-crystallized ligand Oseltamivir. Thus, molecular modelling provided a good platform to optimize the acyl thiourea pharmacophore for designing its derivatives having potent anti-viral activity.

  11. Lack of pharmacokinetic interaction between the oral anti-influenza neuraminidase inhibitor prodrug oseltamivir and antacids

    PubMed Central

    Snell, Paul; Oo, Charles; Dorr, Al; Barrett, Joanne

    2002-01-01

    Aims Oseltamivir is an oral ester prodrug of its active metabolite Ro 64–0802, a potent and selective neuraminidase inhibitor of the influenza virus. The object of this study was to evaluate whether the oral absorption of oseltamivir was reduced in the presence of two main classes of antacid, Maalox® suspension (containing magnesium hydroxide and aluminium hydroxide) and Titralac® tablets (containing calcium carbonate). Methods Twelve healthy volunteers completed a randomized, single dose, three-period crossover study. Each volunteer received in a fasted state, 150 mg oseltamivir alone (Treatment A), 150 mg oseltamivir with a 20 ml Maalox® suspension (Treatment B), and 150 mg oseltamivir with four Titralac® tablets (Treatment C), with 7–10 days washout in between treatments. Plasma and urine concentrations of oseltamivir and Ro 64–0802 were measured using a validated h.p.l.c./MS/MS assay. Pharmacokinetic parameters were calculated for oseltamivir and Ro 64–0802. Since antacids are locally acting drugs and generally not expected to be absorbed substantially into the systemic system, no plasma or urine concentrations of antacids were measured. Results Bioequivalence was achieved for the primary pharmacokinetic parameters Cmax and AUC(0,∞) of Ro 64–0802 following administration of oseltamivir with either Maalox® suspension or Titralac® tablets vs administration of oseltamivir alone. The bioavailability (90% confidence intervals) of Ro 64–0802 following administration of oseltamivir together with Maalox® suspension vs administration of oseltamivir alone, was 90% (83.6, 96.9%) for Cmax and 94.1% (91.4, 96.9%) for AUC(0,∞); similarly, for Titralac® tablets, the equivalent values were 95.1% (88.3, 102%) for Cmax and 94.7% (91.9, 97.5%) for AUC(0,∞). Conclusions The coadministration of either Maalox® suspension or Titralac® tablets with oseltamivir has no effect on the pharmacokinetics of either oseltamivir or Ro 64–0802, and conversely, there

  12. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

    PubMed

    Gubareva, Larisa V; Besselaar, Terry G; Daniels, Rod S; Fry, Alicia; Gregory, Vicki; Huang, Weijuan; Hurt, Aeron C; Jorquera, Patricia A; Lackenby, Angie; Leang, Sook-Kwan; Lo, Janice; Pereyaslov, Dmitriy; Rebelo-de-Andrade, Helena; Siqueira, Marilda M; Takashita, Emi; Odagiri, Takato; Wang, Dayan; Zhang, Wenqing; Meijer, Adam

    2017-08-10

    Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC50) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs. Of the viruses tested by WHO CCs 93% were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.8% (n = 113) exhibited either RI or HRI by at least one of four NAIs. As in previous seasons, the most common NA AAS was H275Y in A(H1N1)pdm09 viruses, which confers HRI by oseltamivir and peramivir. Two A(H1N1)pdm09 viruses carried a rare NA AAS, S247R, shown in this study to confer RI/HRI by the four NAIs. The overall frequency of A(H1N1)pdm09 viruses containing NA AAS associated with RI/HRI was approximately 1.8% (125/6915), which is slightly higher than in the previous 2014-15 season (0.5%). Three B/Victoria-lineage viruses contained a new AAS, NA H134N, which conferred HRI by zanamivir and laninamivir, and borderline HRI by peramivir. A single B/Victoria-lineage virus harboured NA G104E, which was associated with HRI by all four NAIs. The overall frequency of RI/HRI phenotype among type B viruses was approximately 0.6% (43/7677), which is lower than that in the previous season. Overall, the vast majority (>99%) of the viruses tested by WHO CCs were susceptible to all four NAIs, showing normal inhibition (NI). Hence, NAIs remain the recommended antivirals for treatment of

  13. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    PubMed Central

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K.V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-01-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors. PMID:27995961

  14. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay.

    PubMed

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K V; Kamarulzaman, Ezatul E; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A

    2016-12-20

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  15. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    NASA Astrophysics Data System (ADS)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-12-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  16. Neuraminidase inhibitors for influenza: a systematic review and meta-analysis of regulatory and mortality data.

    PubMed Central

    Heneghan, Carl J; Onakpoya, Igho; Jones, Mark A; Doshi, Peter; Del Mar, Chris B; Hama, Rokuro; Thompson, Matthew J; Spencer, Elizabeth A; Mahtani, Kamal R; Nunan, David; Howick, Jeremy; Jefferson, Tom

    2016-01-01

    BACKGROUND Neuraminidase inhibitors (NIs) are stockpiled and recommended by public health agencies for treating and preventing seasonal and pandemic influenza. They are used clinically worldwide. OBJECTIVES To (1) describe the potential benefits and harms of NIs for influenza in all age groups by reviewing all clinical study reports (CSRs) of published and unpublished randomised, placebo-controlled trials and regulatory comments; and (2) determine the effect of oseltamivir (Tamiflu(®), Roche) treatment on mortality in patients with 2009A/H1N1 influenza. METHODS We searched trial registries, electronic databases and corresponded with regulators and sponsors to identify randomised trials of NIs. We requested full CSRs and accessed regulators' comments. We included only those trials for which we had CSRs. To examine the effects of oseltamivir on 2009A/H1N1 influenza mortality, we requested individual patient data (IPD) from corresponding authors of all included observational studies. RESULTS Effect of oseltamivir and zanamivir (Relenza®, GlaxoSmithKline) in the prevention and treatment of influenza: Oseltamivir reduced the time to first alleviation of symptoms in adults by 16.8 hours [95% confidence interval (CI) 8.4 to 25.1 hours]. Zanamivir reduced the time to first alleviation of symptoms in adults by 0.60 days (95% CI 0.39 to 0.81 days). Oseltamivir reduced unverified pneumonia in adult treatment [risk difference (RD) 1.00%, 95% CI 0.22% to 1.49%]; similar findings were observed with zanamivir prophylaxis in adults (RD 0.32%, 95% CI 0.09% to 0.41%). Oseltamivir treatment of adults increased the risk of nausea (RD 3.66%, 95% CI 0.90% to 7.39%) and vomiting (RD 4.56%, 95% CI 2.39% to 7.58%). In the treatment of children, oseltamivir induced vomiting (RD 5.34%, 95% CI 1.75% to 10.29%). Both oseltamivir and zanamivir prophylaxis reduced the risk of symptomatic influenza in individuals (oseltamivir RD 3.05%, 95% CI 1.83% to 3.88%; zanamivir RD 1.98%, 95% CI 0.98% to

  17. QSAR analyses on avian influenza virus neuraminidase inhibitors using CoMFA, CoMSIA, and HQSAR

    NASA Astrophysics Data System (ADS)

    Zheng, Mingyue; Yu, Kunqian; Liu, Hong; Luo, Xiaomin; Chen, Kaixian; Zhu, Weiliang; Jiang, Hualiang

    2006-09-01

    The recent wide spreading of the H5N1 avian influenza virus (AIV) in Asia, Europe and Africa and its ability to cause fatal infections in human has raised serious concerns about a pending global flu pandemic. Neuraminidase (NA) inhibitors are currently the only option for treatment or prophylaxis in humans infected with this strain. However, drugs currently on the market often meet with rapidly emerging resistant mutants and only have limited application as inadequate supply of synthetic material. To dig out helpful information for designing potent inhibitors with novel structures against the NA, we used automated docking, CoMFA, CoMSIA, and HQSAR methods to investigate the quantitative structure-activity relationship for 126 NA inhibitors (NIs) with great structural diversities and wide range of bioactivities against influenza A virus. Based on the binding conformations discovered via molecular docking into the crystal structure of NA, CoMFA and CoMSIA models were successfully built with the cross-validated q 2 of 0.813 and 0.771, respectively. HQSAR was also carried out as a complementary study in that HQSAR technique does not require 3D information of these compounds and could provide a detailed molecular fragment contribution to the inhibitory activity. These models also show clearly how steric, electrostatic, hydrophobicity, and individual fragments affect the potency of NA inhibitors. In addition, CoMFA and CoMSIA field distributions are found to be in well agreement with the structural characteristics of the corresponding binding sites. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing novel potent NA inhibitors.

  18. Neuraminidase Ribbon Diagram

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Ribbons is a program developed at UAB used worldwide to graphically depict complicated protein structures in a simplified format. The program uses sophisticated computer systems to understand the implications of protein structures. The Influenza virus remains a major causative agent for a large number of deaths among the elderly and young children and huge economic losses due to illness. Finding a cure will have a general impact both on the basic research of viral pathologists of fast evolving infectious agents and clinical treatment of influenza virus infection. The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  19. Neuraminidase Ribbon Diagram

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Ribbons is a program developed at UAB used worldwide to graphically depict complicated protein structures in a simplified format. The program uses sophisticated computer systems to understand the implications of protein structures. The Influenza virus remains a major causative agent for a large number of deaths among the elderly and young children and huge economic losses due to illness. Finding a cure will have a general impact both on the basic research of viral pathologists of fast evolving infectious agents and clinical treatment of influenza virus infection. The reproduction process of all strains of influenza are dependent on the same enzyme neuraminidase. Shown here is a segmented representation of the neuraminidase inhibitor compound sitting inside a cave-like contour of the neuraminidase enzyme surface. This cave-like formation present in every neuraminidase enzyme is the active site crucial to the flu's ability to infect. The space-grown crystals of neuraminidase have provided significant new details about the three-dimensional characteristics of this active site thus allowing researchers to design drugs that fit tighter into the site. Principal Investigator: Dr. Larry DeLucas

  20. Synthesis of C-4-modified zanamivir analogs as neuraminidase inhibitors and their anti-AIV activities.

    PubMed

    Ye, Deju; Shin, Woo-Jin; Li, Ning; Tang, Wei; Feng, Enguang; Li, Jian; He, Pei-Lan; Zuo, Jian-Ping; Kim, Hanjo; Nam, Ky-Youb; Zhu, Weiliang; Seong, Baik-Lin; No, Kyoung Tai; Jiang, Hualiang; Liu, Hong

    2012-08-01

    With the introduction of bioisosteres of the guanidinium group together with scaffold hopping, 35 zanamivir analogs with C-4-modification were synthesized, and their inhibitory activities against both group-1 and group-2 neuraminidase (H5N1 and H3N2) were determined. Compound D26 exerts the most potency, with IC(50) values of 0.58 and 2.72 μM against N2 and N1, respectively. Further preliminary anti-avian influenza virus (AIV, H5N1) activities against infected MDCK cells were evaluated, and D5 exerts ∼58% protective against AIV infection, which was comparable to zanamivir (∼67%). In a rat pharmacokinetic study, compound D5 showed an increased plasma half-life (t(1/2)) compared to zanamivir following either intravenous or oral administration. This study may represent a new start point for the future development of improved anti-AIV agents. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  1. Identification of Suitable Natural Inhibitor against Influenza A (H1N1) Neuraminidase Protein by Molecular Docking

    PubMed Central

    Sahoo, Maheswata; Jena, Lingaraja; Rath, Surya Narayan

    2016-01-01

    The influenza A (H1N1) virus, also known as swine flu is a leading cause of morbidity and mortality since 2009. There is a need to explore novel anti-viral drugs for overcoming the epidemics. Traditionally, different plant extracts of garlic, ginger, kalmegh, ajwain, green tea, turmeric, menthe, tulsi, etc. have been used as hopeful source of prevention and treatment of human influenza. The H1N1 virus contains an important glycoprotein, known as neuraminidase (NA) that is mainly responsible for initiation of viral infection and is essential for the life cycle of H1N1. It is responsible for sialic acid cleavage from glycans of the infected cell. We employed amino acid sequence of H1N1 NA to predict the tertiary structure using Phyre2 server and validated using ProCheck, ProSA, ProQ, and ERRAT server. Further, the modelled structure was docked with thirteen natural compounds of plant origin using AutoDock4.2. Most of the natural compounds showed effective inhibitory activity against H1N1 NA in binding condition. This study also highlights interaction of these natural inhibitors with amino residues of NA protein. Furthermore, among 13 natural compounds, theaflavin, found in green tea, was observed to inhibit H1N1 NA proteins strongly supported by lowest docking energy. Hence, it may be of interest to consider theaflavin for further in vitro and in vivo evaluation. PMID:27729839

  2. A randomized, controlled trial comparing traditional herbal medicine and neuraminidase inhibitors in the treatment of seasonal influenza.

    PubMed

    Nabeshima, Shigeki; Kashiwagi, Kenichiro; Ajisaka, Kazuhiko; Masui, Shinta; Takeoka, Hiroaki; Ikematsu, Hideyuki; Kashiwagi, Seizaburo

    2012-08-01

    The herbal medicine, maoto, has been traditionally prescribed to patients with influenza in Japan. To better understand the efficacy of maoto for the treatment of influenza, a randomized trial was conducted for comparison with oseltamivir or zanamivir. Adult patients with influenza symptoms, including fever, positive for quick diagnostic kit for influenza within 48 h of fever onset were assessed for enrollment. The data of 28 patients randomly assigned to maoto (n = 10), oseltamivir (n = 8), or zanamivir (n = 10) were analyzed for the duration of fever (>37.5°C) and total symptom score from symptom cards recorded by the patient. Viral isolation and serum cytokine measurements were also done on days 1, 3, and 5. Maoto granules, a commercial medical dosage form, are made from four plants: Ephedra Herb, Apricot Kernel, Cinnamon Bark, and Glycyrrhiza Root. Median durations of fever of patients assigned maoto, oseltamivir, or zanamivir were 29, 46, or 27 h, respectively, significantly different for maoto and oseltamivir. No significant between-group differences were found in total symptom score among three groups. Viral persistent rates and serum cytokine levels (IFN-α, IL-6, IL-8, IL-10, and TNF-α) during the study period showed no differences among three groups. The administration of oral maoto granules to healthy adults with seasonal influenza was well tolerated and associated with equivalent clinical and virological efficacy to neuraminidase inhibitors.

  3. Detection of influenza B viruses with reduced sensitivity to neuraminidase inhibitor in Morocco during 2014/15 season.

    PubMed

    Elfalki, F; Ihazmad, H; Bimouhen, A; Regragui, Z; Benkaroum, S; Bakri, Y; Barakat, A

    2016-10-02

    We monitored phenotypic and genotypic susceptibility of influenza viruses circulating in Morocco during 2014-2015 to oseltamivir and zanamivir. Throat and nasal swab specimens were collected from outpatients (with influenza-like illness) and inpatients (with severe acute respiratory illness) and tested for influenza viruses using real-time reverse transcription polymerase chain reaction. Positive samples were inoculated in MDCK cells and virus phenotypic susceptibility to neuraminidase inhibitors (NAIs) was assessed using fluorescent NA inhibition. Of 440 specimens, 135 were positive for influenza B Yamagata-like virus, 38 were A(H1N1)pdm09 and 25 were A(H3N2). Sixty influenza B viruses isolated from MDCK cells showed no significant resistance to NAIs. However, two of these strains, B/Morocco/176H/2015 and B/Morocco/CP10/2015, showed reduced susceptibility to oseltamivir. The two influenza B viruses with reduced susceptibility to oseltamivir show that ongoing NAI susceptibility surveillance is essential.

  4. Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

    PubMed Central

    Eshaghi, Alireza; Shalhoub, Sarah; Rosenfeld, Paul; Li, Aimin; Higgins, Rachel R.; Stogios, Peter J.; Savchenko, Alexei; Bastien, Nathalie; Li, Yan; Rotstein, Coleman

    2014-01-01

    Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission. PMID:25246391

  5. Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6.

    PubMed

    Venkatramani, Lalitha; Johnson, Eric S; Kolavi, Gundurao; Air, Gillian M; Brouillette, Wayne J; Mooers, Blaine H M

    2012-05-06

    Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite. Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC50 for compound 2 remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit. We designed inhibitor 2 with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in 2 is tilted relative to 1 and the number of

  6. Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6

    PubMed Central

    2012-01-01

    Background Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite. Results Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC50 for compound 2 remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit. Conclusions We designed inhibitor 2 with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in 2 is tilted

  7. Effectiveness of neuraminidase inhibitors in preventing hospitalization during the H1N1 influenza pandemic in British Columbia, Canada

    PubMed Central

    Marra, Fawziah; Chong, Mei; Henry, Bonnie; Patrick, David M.; Kendall, Perry

    2014-01-01

    Objectives In British Columbia (BC), Canada, neuraminidase inhibitors (NIs) were publicly funded during the 2009 A(H1N1)pdm09 pandemic for treatment of high-risk patients and/or anyone with moderate-to-severe illness. We assessed antiviral effectiveness (AVE) against hospitalization in that context. Methods A population-based cohort study was conducted using linked administrative data. The cohort included all individuals living in BC during the study period (1 September to 31 December 2009) with a diagnostic code consistent with influenza or pandemic H1N1. The main study period pertained to the second-wave A(H1N1)pdm09 circulation (1 October to 31 December 2009), with sensitivity analyses around the more specific pandemic peak (18 October to 7 November). Exposure was defined by same-day NI prescription. The main outcome was all-cause hospitalization within 14 days of the outpatient influenza diagnosis. Cox proportional hazards models assessed AVE with 1 : 1 propensity-score matching and covariate adjustment. Results After matching, there were 304/58 061 NI-exposed and 345/58 061 unexposed patients hospitalized during the main study period. The very young [<6 months (35.0; 95% CI 16.7–73.4)], the old [65–79 years (13.7; 95% CI 10.1–18.6)] and the very old [≥80 years (38.7; 95% CI 26.6–56.5)] had the highest hospitalization rate per 1000 patients overall. Fully adjusted AVE against all-cause hospitalization during the main study period was 16% (95% CI 2%–28%), similar to the pandemic peak (15%; 95% CI −4%–30%). Conclusions The use of NIs was associated with modest protection against hospitalization during the 2009 pandemic, but appeared underutilized in affected age groups with the highest hospitalization risk. PMID:24346762

  8. Use of neuraminidase inhibitors in primary health care during pandemic and seasonal influenza between 2009 and 2013

    PubMed Central

    Blanchon, Thierry; Geffrier, Félicité; Turbelin, Clément; Daviaud, Isabelle; Laouénan, Cédric; Duval, Xavier; Lambert, Bruno; Hanslik, Thomas; Mosnier, Anne; Leport, Catherine

    2015-01-01

    Background In a context of controversy about influenza antiviral treatments, this study assessed primary health care physicians’ prescription of neuraminidase inhibitors (NIs) in France during pandemic and seasonal influenza between 2009 and 2013. Methods This observational study, using data recorded in three national databases, estimated the rate of NIs’ prescription among influenza like-illness (ILI) patients seen in GPs’ and paediatricians’ consultations, and determined factors associated with this prescription according to a multivariate analysis. NIs’ delivery by pharmacists was also evaluated. Results Rates of NIs’ prescription were estimated to 61.1% among ILI patients with a severe influenza risk factor seen in GPs’ consultation during the A(H1N1)pdm2009 pandemic versus an average rate of 25.9% during the three following seasonal influenza epidemics. Factors associated with NIs’ prescription were a chronic disease in patients under 65 years (OR, 14.85; 95%CI, 13.00–16.97) and in those aged ≥ 65 and older (OR, 7.54; 5.86–9.70), an age 65 years in patients without chronic disease (OR, 1.35; 1.04–1.74), a pregnancy (OR, 10.63; 7.67–15.76), obesity (OR, 4.67; 3.50–6.22), and a consultation during the pandemic A(H1N1)pdm2009 (OR, 3.19; 2.93–3.48). The number of antiviral treatments delivered by pharmacists during the A(H1N1)pdm2009 pandemic was 835 per 100 000 inhabitants, and an average of 275 per 100 000 inhabitants during the three following seasonal influenza epidemics. Conclusions Although physicians seem to follow the recommended indications for NIs in primary health care practice, this study confirms the low rate of NIs prescription to ILI patients with a severe influenza risk factor, especially during seasonal epidemics. PMID:25687219

  9. Anti-influenza neuraminidase inhibitor oseltamivir phosphate induces canine mammary cancer cell aggressiveness.

    PubMed

    de Oliveira, Joana T; Santos, Ana L; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J; Vasconcelos, M Helena; Oliveira, Maria José; Reis, Celso A; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness.

  10. Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

    PubMed Central

    de Oliveira, Joana T.; Santos, Ana L.; Gomes, Catarina; Barros, Rita; Ribeiro, Cláudia; Mendes, Nuno; de Matos, Augusto J.; Vasconcelos, M. Helena; Oliveira, Maria José; Reis, Celso A.; Gärtner, Fátima

    2015-01-01

    Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness. PMID:25850034

  11. Design, in silico studies, synthesis and in vitro evaluation of oseltamivir derivatives as inhibitors of neuraminidase from influenza A virus H1N1.

    PubMed

    Neri-Bazán, Rocío M; García-Machorro, Jazmín; Méndez-Luna, David; Tolentino-López, Luis E; Martínez-Ramos, Federico; Padilla-Martínez, Itzia I; Aguilar-Faisal, Leopoldo; Soriano-Ursúa, Marvin A; Trujillo-Ferrara, José G; Fragoso-Vázquez, M Jonathan; Barrón, Blanca L; Correa-Basurto, José

    2017-03-10

    Since the neuraminidase (NA) enzyme of the influenza A virus plays a key role in the process of release of new viral particles from a host cell, it is often a target for new drug design. The emergence of NA mutations, such as H275Y, has led to great resistance against neuraminidase inhibitors, including oseltamivir and zanamivir. Hence, we herein designed a set of derivatives by modifying the amine and/or carboxylic groups of oseltamivir. After being screened for their physicochemical (Lipinski's rule) and toxicological properties, the remaining compounds were submitted to molecular and theoretical studies. The docking simulations provided insights into NA recognition patterns, demonstrating that oseltamivir modified at the carboxylic moiety and coupled with anilines had higher affinity and a better binding pose for NA than the derivatives modified at the amine group. Based on these theoretical studies, the new oseltamivir derivatives may have higher affinity to mutant variants and possibly to other viral subtypes. Accordingly, two compounds were selected for synthesis, which together with their respective intermediates were evaluated for their cytotoxicity and antiviral activities. Their biological activity was then tested in cells infected with the A/Puerto Rico/916/34 (H1N1) influenza virus, and virus yield reduction assays were performed. Additionally, by measuring neuraminidase activity with the neuraminidase assay kit it was found that the compounds produced inhibitory activity on this enzyme. Finally, the infected cells were analysed with atomic force microscopy (AFM), observing morphological changes strongly suggesting that these compounds interfered with cellular release of viral particles.

  12. CS-8958, a Prodrug of the Novel Neuraminidase Inhibitor R-125489, Demonstrates a Favorable Long-Retention Profile in the Mouse Respiratory Tract▿

    PubMed Central

    Koyama, Kumiko; Takahashi, Makoto; Oitate, Masataka; Nakai, Naoko; Takakusa, Hideo; Miura, Shin-ichi; Okazaki, Osamu

    2009-01-01

    CS-8958 is a prodrug of the pharmacologically active form R-125489, a selective neuraminidase inhibitor, and has long-acting anti-influenza virus activity in vivo. In this study, the tissue distribution profiles after a single intranasal administration of CS-8958 (0.5 μmol/kg of body weight) to mice were investigated, focusing especially on the retention of CS-8958 in the respiratory tract by comparing it with R-125489 and a marketed drug, zanamivir. After administration of [14C]CS-8958, radioactivity was retained in the respiratory tract over long periods. At 24 h postdose, the radioactivity concentrations after administration of [14C]CS-8958 were approximately 10-fold higher in both the trachea and the lung than those of [14C]R-125489 and [14C]zanamivir. The [14C]CS-8958-derived radioactivity present in these two tissues consisted both of unchanged CS-8958 and of R-125489 at 1 h postdose, while only R-125489, and no other metabolites, was detected at 24 h postdose. After administration of unlabeled CS-8958, CS-8958 was rapidly eliminated from the lungs, whereas the lung R-125489 concentration reached a maximum at 3 h postdose and gradually declined, with an elimination half-life of 41.4 h. The conversion of CS-8958 to R-125489 was observed in mouse trachea and lung S9 fractions and was inhibited by esterase inhibitors, such as diisopropylfluorophosphate and bis-p-nitrophenylphosphate. These results demonstrated that CS-8958 administered intranasally to mice was efficiently converted to R-125489 by a hydrolase(s) such as carboxylesterase, and then R-125489 was slowly eliminated from the respiratory tract. These data support the finding that CS-8958 has potential as a long-acting neuraminidase inhibitor, leading to significant efficacy as an anti-influenza drug by a single treatment. PMID:19687241

  13. Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses.

    PubMed

    Barrett, Susan; Mohr, Peter G; Schmidt, Peter M; McKimm-Breschkin, Jennifer L

    2011-01-01

    The influenza neuraminidase (NA) inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA) was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50)s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50)s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50)s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50)s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50)s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50), and critically demonstrates the differential effect of incubation times on the IC(50) and K(i) values of wild type and mutant viruses for each of the inhibitors.

  14. Neuraminidase Inhibitor Sensitivity and Receptor-Binding Specificity of Cambodian Clade 1 Highly Pathogenic H5N1 Influenza Virus▿†

    PubMed Central

    Naughtin, M.; Dyason, J. C.; Mardy, S.; Sorn, S.; von Itzstein, M.; Buchy, P.

    2011-01-01

    The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat. PMID:21343450

  15. Efficacy of novel hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 against human parainfluenza viruses in vitro and in vivo.

    PubMed

    Alymova, Irina V; Taylor, Garry; Takimoto, Toru; Lin, Tsu-Hsing; Chand, Pooran; Babu, Y Sudhakara; Li, Chenghong; Xiong, Xiaoping; Portner, Allen

    2004-05-01

    Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK(2) cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 micro M in HA inhibition assays and from 0.02 to 20 micro M in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 micro M. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.

  16. Identification of selective inhibitors for human neuraminidase isoenzymes using C4,C7-modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) analogues.

    PubMed

    Zhang, Yi; Albohy, Amgad; Zou, Yao; Smutova, Victoria; Pshezhetsky, Alexey V; Cairo, Christopher W

    2013-04-11

    In the past two decades, human neuraminidases (human sialidases, hNEUs) have been found to be involved in numerous pathways in biology. The development of selective and potent inhibitors of these enzymes will provide critical tools for glycobiology, help to avoid undesired side effects of antivirals, and may reveal new small-molecule therapeutic targets for human cancers. However, because of the high active site homology of the hNEU isoenzymes, little progress in the design and synthesis of selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-modified analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar potency.

  17. E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient

    PubMed Central

    L'Huillier, Arnaud G.; Abed, Yacine; Petty, Tom J.; Cordey, Samuel; Thomas, Yves; Bouhy, Xavier; Schibler, Manuel; Simon, Audrey; Chalandon, Yves; van Delden, Christian; Zdobnov, Evgeny; Boquete-Suter, Patricia; Boivin, Guy; Kaiser, Laurent

    2015-01-01

    Background. An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Methods. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. Results. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. Conclusions. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies. PMID:25985905

  18. Computational modeling and validation studies of 3-D structure of neuraminidase protein of H1N1 influenza A virus and subsequent in silico elucidation of piceid analogues as its potent inhibitors.

    PubMed

    Gupta, Chhedi Lal; Akhtar, Salman; Bajpaib, Preeti; Kandpal, K N; Desai, G S; Tiwari, Ashok K

    2013-01-01

    Emergence of the drug resistant variants of the Influenza A virus in the recent years has aroused a great need for the development of novel neuraminidase inhibitors for controlling the pandemic. The neuraminidase (NA) protein of the influenza virus has been the most potential target for the anti-influenza. However, in the absence of any experimental structure of the drug targeting NA protein of H1N1 influenza A virus as zanamivir and oseltamivir, the comprehensive study of the interaction of the drug molecules with the target protein has been missing. Hence in this study a computational 3-D structure of neuraminidase of H1N1 influenza A virus has been developed using homology modeling technique, and the same was validated for its reliability by ProSA web server in term of energy profile & Z scores and PROCHECK program followed by Ramachandran plot. Further, the developed 3-D model had been employed for docking studies with the class of compounds as Piceid and its analogs. In this context, two novel compounds (ChemBank ID 2110359 and 3075417) were found to be more potent inhibitors of neuraminidase than control drugs as zanamivir and oseltamivir in terms of their robust binding energies, strong inhibition constant (Ki) and better hydrogen bond interactions between the protein-ligand complex. The interaction of these compounds with NA protein has been significantly studied at the molecular level.

  19. A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus.

    PubMed

    Qi, Yuhua; Fan, Huan; Qi, Xian; Zhu, Zheng; Guo, Xiling; Chen, Yin; Ge, Yiyue; Zhao, Kangchen; Wu, Tao; Li, Yan; Shan, Yunfeng; Zhou, Minghao; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao

    2014-01-22

    A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.

  20. Exploring virus release as a bottleneck for the spread of influenza A virus infection in vitro and the implications for antiviral therapy with neuraminidase inhibitors

    PubMed Central

    Kowal, Szymon; Cardenas, Daniel A.; Beauchemin, Catherine A. A.

    2017-01-01

    Mathematical models (MMs) have been used to study the kinetics of influenza A virus infections under antiviral therapy, and to characterize the efficacy of antivirals such as neuraminidase inhibitors (NAIs). NAIs prevent viral neuraminidase from cleaving sialic acid receptors that bind virus progeny to the surface of infected cells, thereby inhibiting their release, suppressing infection spread. When used to study treatment with NAIs, MMs represent viral release implicitly as part of viral replication. Consequently, NAIs in such MMs do not act specifically and exclusively on virus release. We compared a MM with an explicit representation of viral release (i.e., distinct from virus production) to a simple MM without explicit release, and investigated whether parameter estimation and the estimation of NAI efficacy were affected by the use of a simple MM. Since the release rate of influenza A virus is not well-known, a broad range of release rates were considered. If the virus release rate is greater than ∼0.1 h−1, the simple MM provides accurate estimates of infection parameters, but underestimates NAI efficacy, which could lead to underdosing and the emergence of NAI resistance. In contrast, when release is slower than ∼0.1 h−1, the simple MM accurately estimates NAI efficacy, but it can significantly overestimate the infectious lifespan (i.e., the time a cell remains infectious and producing free virus), and it will significantly underestimate the total virus yield and thus the likelihood of resistance emergence. We discuss the properties of, and a possible lower bound for, the influenza A virus release rate. PMID:28837615

  1. Molecular docking and QSAR studies on substituted acyl(thio)urea and thiadiazolo [2,3-alpha] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase.

    PubMed

    Sun, Jiaying; Cai, Shaoxi; Mei, Hu; Li, Jian; Yan, Ning; Wang, Qin; Lin, Zhihua; Huo, Danqun

    2010-09-01

    Surflex-Dock was employed to dock 36 thiourea and thiadiazolo [2,3-alpha] pyrimidine derivatives into neuraminidase 1a4g. Molecular docking results showed that hydrogen bonding, electrostatic, and hydrophobic features were important factors affecting inhibitory activities of these neuraminidase inhibitors. Moreover, there was a significant correlation between the predicted binding affinity (total scores) and experimental pIC50 values with correlation coefficient r=0.846 and p<0.0001. Hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis were used to develop quantitative structure-activity relationship models. Squared multiple correlation coefficients (r2) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.899, 0.878, and 0.865, respectively. Squared cross-validated correlation coefficient (q2) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models was in turn 0.628, 0.656, and 0.509. In addition, squared multiple correlation coefficients for test set (r2test) of hologram quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices analysis models were 0.558, 0.667, and 0.566, respectively. The most active sample ID 2 was taken as a template molecule to design new molecules. Based on the comparative molecular field analysis model, new compounds were designed by LeapFrog. Seven new compounds with improved binding energy and predicted activities were finally obtained.

  2. Identification of bioactivating enzymes involved in the hydrolysis of laninamivir octanoate, a long-acting neuraminidase inhibitor, in human pulmonary tissue.

    PubMed

    Koyama, Kumiko; Ogura, Yuji; Nakai, Daisuke; Watanabe, Mihoko; Munemasa, Toshiko; Oofune, Yuka; Kubota, Kazuishi; Shinagawa, Akira; Izumi, Takashi

    2014-06-01

    Laninamivir octanoate (LO) is an octanoyl ester prodrug of the neuraminidase inhibitor laninamivir. After inhaled administration, LO exhibits clinical efficacy for both treatment and prophylaxis of influenza virus infection, resulting from hydrolytic bioactivation into its pharmacologically active metabolite laninamivir in the pulmonary tissue. In this study, we focused on the identification of LO-hydrolyzing enzymes from human pulmonary tissue extract using proteomic correlation profiling-a technology integration of traditional biochemistry and proteomics. In a single elution step by gel-filtration chromatography, LO-hydrolyzing activity was separated into two distinct peaks, designated as peak I and peak II. By mass spectrometry, 1160 and 1003 proteins were identified and quantitated for peak I and peak II, respectively, and enzyme candidates were ranked based on the correlation coefficient between the enzyme activity and the proteomic profiles. Among proteins with a high correlation value, S-formylglutathione hydrolase (esterase D; ESD) and acyl-protein thioesterase 1 (APT1) were selected as the most likely candidates for peak I and peak II, respectively, which was confirmed by LO-hydrolyzing activity of recombinant proteins. In the case of peak II, LO-hydrolyzing activity was completely inhibited by treatment with a specific APT1 inhibitor, palmostatin B. Moreover, immunohistochemical analysis revealed that both enzymes were mainly localized in the pulmonary epithelia, a primary site of influenza virus infection. These findings demonstrate that ESD and APT1 are key enzymes responsible for the bioactivation of LO in human pulmonary tissue.

  3. Neuraminidase-resistant hemagglutination inhibitors: acrylamide copolymers containing a C-glycoside of N-acetylneuraminic acid.

    PubMed

    Sparks, M A; Williams, K W; Whitesides, G M

    1993-03-19

    Copolymers consisting of a polyacrylamide backbone with side chains terminated in C-glycosidic analogs of N-acetylneuraminic acid were synthesized by free radical copolymerization of alpha-2-C-[3-[[2-(N-acryloylamino)ethyl]thio]propyl]-N- acetylneuraminic acid (5) with acrylamide. Unlike natural and synthetic polyvalent materials that contain N-acetylneuraminic acid in O-glycosidic form, these C-glycosidic copolymers resist neuraminidase-catalyzed cleavage of the neuraminic acid residue from the copolymer backbone. Examination of these C-glycosidic copolymers in a hemagglutination inhibition assay indicated that they are as effective in vitro as polyvalent O-glycosidic copolymers in inhibiting agglutination of erythrocytes by influenza virus. The minimum value of the inhibition constant, calculated on the basis of the concentration of Neu5Ac groups in solution, is Ki(HAI) approximately 10(-7) M for both copolymers. The inhibitory potency of the C-glycoside-based copolymers becomes more significant at lower concentrations of Neu5Ac moieties in solution than does the inhibitory potency of the O-glycoside-based copolymer.

  4. Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

    PubMed

    Lee, Youngjin; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Kang, Jung Youn; Ryu, Young Bae; Jin, Mi Sun; Park, Ki Hun; Eom, Soo Hyun

    2017-03-16

    Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanICD) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer. Here, we report the crystal structure of CpNanICD in complex with CHES at 1.24 Å resolution. Amphipathic CHES binds to the catalytic site of CpNanICD similar to the substrate (Neu5Ac) binding site. The 2-aminoethanesulfonic acid moiety and cyclohexyl groups of CHES interact with the cluster of three arginine residues and with the hydrophobic pocket of the CpNanICD catalytic site. In addition, a structural comparison with other bacterial and human neuraminidases suggests that CHES could serve as a scaffold for the development of new anti-neuraminidase agents targeting CpNanI.

  5. The in vivo efficacy of neuraminidase inhibitors cannot be determined from the decay rates of influenza viral titers observed in treated patients

    NASA Astrophysics Data System (ADS)

    Palmer, John; Dobrovolny, Hana M.; Beauchemin, Catherine A. A.

    2017-01-01

    Antiviral therapy is a first line of defence against new influenza strains. Current pandemic preparations involve stock- piling oseltamivir, an oral neuraminidase inhibitor (NAI), so rapidly determining the effectiveness of NAIs against new viral strains is vital for deciding how to use the stockpile. Previous studies have shown that it is possible to extract the drug efficacy of antivirals from the viral decay rate of chronic infections. In the present work, we use a nonlinear mathematical model representing the course of an influenza infection to explore the possibility of extracting NAI drug efficacy using only the observed viral titer decay rates seen in patients. We first show that the effect of a time-varying antiviral concentration can be accurately approximated by a constant efficacy. We derive a relationship relating the true treatment dose and time elapsed between doses to the constant drug dose required to approximate the time- varying dose. Unfortunately, even with the simplification of a constant drug efficacy, we show that the viral decay rate depends not just on drug efficacy, but also on several viral infection parameters, such as infection and production rate, so that it is not possible to extract drug efficacy from viral decay rate alone.

  6. The Neuraminidase Inhibitor Oseltamivir Is Effective Against A/Anhui/1/2013 (H7N9) Influenza Virus in a Mouse Model of Acute Respiratory Distress Syndrome

    PubMed Central

    Baranovich, Tatiana; Burnham, Andrew J.; Marathe, Bindumadhav M.; Armstrong, Jianling; Guan, Yi; Shu, Yuelong; Peiris, Joseph Malik Sriyal; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2014-01-01

    Background. High mortality and uncertainty about the effectiveness of neuraminidase inhibitors (NAIs) in humans infected with influenza A(H7N9) viruses are public health concerns. Methods. Susceptibility of N9 viruses to NAIs was determined in a fluorescence-based assay. The NAI oseltamivir (5, 20, or 80 mg/kg/day) was administered to BALB/c mice twice daily starting 24, 48, or 72 hours after A/Anhui/1/2013 (H7N9) virus challenge. Results. All 12 avian N9 and 3 human H7N9 influenza viruses tested were susceptible to NAIs. Without prior adaptation, A/Anhui/1/2013 (H7N9) caused lethal infection in mice that was restricted to the respiratory tract and resulted in pulmonary edema and acute lung injury with hyaline membrane formation, leading to decreased oxygenation, all characteristics of human acute respiratory distress syndrome. Oseltamivir at 20 and 80 mg/kg protected 80% and 88% of mice when initiated after 24 hours, and the efficacy decreased to 70% and 60%, respectively, when treatment was delayed by 48 hours. Emergence of oseltamivir-resistant variants was not detected. Conclusions. H7N9 viruses are comparable to currently circulating influenza A viruses in susceptibility to NAIs. Based on these animal studies, early treatment is associated with improved outcomes. PMID:24133191

  7. The in vivo efficacy of neuraminidase inhibitors cannot be determined from the decay rates of influenza viral titers observed in treated patients

    PubMed Central

    Palmer, John; Dobrovolny, Hana M.; Beauchemin, Catherine A. A.

    2017-01-01

    Antiviral therapy is a first line of defence against new influenza strains. Current pandemic preparations involve stock- piling oseltamivir, an oral neuraminidase inhibitor (NAI), so rapidly determining the effectiveness of NAIs against new viral strains is vital for deciding how to use the stockpile. Previous studies have shown that it is possible to extract the drug efficacy of antivirals from the viral decay rate of chronic infections. In the present work, we use a nonlinear mathematical model representing the course of an influenza infection to explore the possibility of extracting NAI drug efficacy using only the observed viral titer decay rates seen in patients. We first show that the effect of a time-varying antiviral concentration can be accurately approximated by a constant efficacy. We derive a relationship relating the true treatment dose and time elapsed between doses to the constant drug dose required to approximate the time- varying dose. Unfortunately, even with the simplification of a constant drug efficacy, we show that the viral decay rate depends not just on drug efficacy, but also on several viral infection parameters, such as infection and production rate, so that it is not possible to extract drug efficacy from viral decay rate alone. PMID:28067324

  8. Post-marketing safety and effectiveness evaluation of the intravenous anti-influenza neuraminidase inhibitor peramivir (I): a drug use investigation.

    PubMed

    Komeda, Takuji; Ishii, Shingo; Itoh, Yumiko; Ariyasu, Yasuyuki; Sanekata, Masaki; Yoshikawa, Takayoshi; Shimada, Jingoro

    2014-11-01

    Peramivir is the only intravenous formulation among anti-influenza neuraminidase inhibitors currently available. Peramivir was approved for manufacturing and marketing in Japan in January 2010. We conducted a drug use investigation of peramivir from October 2010 to February 2012 and evaluated its safety and effectiveness under routine clinical settings. We collected data of 1309 patients from 189 facilities across Japan and examined safety in 1174 patients and effectiveness in 1158 patients. In total, 143 adverse events were observed with an incidence rate of 7.33% (86/1174). Of these, 78 events were adverse drug reactions (ADRs) with an incidence rate of 4.34% (51/1174). The most frequently reported ADRs were diarrhea, vomiting, and nausea, with incidence rates of 1.87% (22/1174), 0.85% (10/1174), and 0.68% (8/1174), respectively. Moreover, no ADR was reported as serious. ADR onset was within 3 days after the start of peramivir administration in 91.0% (71 events) of the 78 ADRs, and ADRs were resolved or improved within 7 days after onset in 96.2% (75 events) of the 78 ADRs. Neither patient characteristics nor treatment factors appeared to significantly affect drug safety. With regard to effectiveness, the median time to alleviation of both influenza symptoms and fever was 3 days, including the first day of administration. The present study demonstrates the safety and effectiveness of peramivir under routine clinical settings.

  9. Effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza A H1N1pdm09 virus infection: a meta-analysis of individual participant data.

    PubMed

    Muthuri, Stella G; Venkatesan, Sudhir; Myles, Puja R; Leonardi-Bee, Jo; Al Khuwaitir, Tarig S A; Al Mamun, Adbullah; Anovadiya, Ashish P; Azziz-Baumgartner, Eduardo; Báez, Clarisa; Bassetti, Matteo; Beovic, Bojana; Bertisch, Barbara; Bonmarin, Isabelle; Booy, Robert; Borja-Aburto, Victor H; Burgmann, Heinz; Cao, Bin; Carratala, Jordi; Denholm, Justin T; Dominguez, Samuel R; Duarte, Pericles A D; Dubnov-Raz, Gal; Echavarria, Marcela; Fanella, Sergio; Gao, Zhancheng; Gérardin, Patrick; Giannella, Maddalena; Gubbels, Sophie; Herberg, Jethro; Iglesias, Anjarath L Higuera; Hoger, Peter H; Hu, Xiaoyun; Islam, Quazi T; Jiménez, Mirela F; Kandeel, Amr; Keijzers, Gerben; Khalili, Hossein; Knight, Marian; Kudo, Koichiro; Kusznierz, Gabriela; Kuzman, Ilija; Kwan, Arthur M C; Amine, Idriss Lahlou; Langenegger, Eduard; Lankarani, Kamran B; Leo, Yee-Sin; Linko, Rita; Liu, Pei; Madanat, Faris; Mayo-Montero, Elga; McGeer, Allison; Memish, Ziad; Metan, Gokhan; Mickiene, Auksė; Mikić, Dragan; Mohn, Kristin G I; Moradi, Ahmadreza; Nymadawa, Pagbajabyn; Oliva, Maria E; Ozkan, Mehpare; Parekh, Dhruv; Paul, Mical; Polack, Fernando P; Rath, Barbara A; Rodríguez, Alejandro H; Sarrouf, Elena B; Seale, Anna C; Sertogullarindan, Bunyamin; Siqueira, Marilda M; Skręt-Magierło, Joanna; Stephan, Frank; Talarek, Ewa; Tang, Julian W; To, Kelvin K W; Torres, Antoni; Törün, Selda H; Tran, Dat; Uyeki, Timothy M; Van Zwol, Annelies; Vaudry, Wendy; Vidmar, Tjasa; Yokota, Renata T C; Zarogoulidis, Paul; Nguyen-Van-Tam, Jonathan S

    2014-05-01

    Neuraminidase inhibitors were widely used during the 2009-10 influenza A H1N1 pandemic, but evidence for their effectiveness in reducing mortality is uncertain. We did a meta-analysis of individual participant data to investigate the association between use of neuraminidase inhibitors and mortality in patients admitted to hospital with pandemic influenza A H1N1pdm09 virus infection. We assembled data for patients (all ages) admitted to hospital worldwide with laboratory confirmed or clinically diagnosed pandemic influenza A H1N1pdm09 virus infection. We identified potential data contributors from an earlier systematic review of reported studies addressing the same research question. In our systematic review, eligible studies were done between March 1, 2009 (Mexico), or April 1, 2009 (rest of the world), until the WHO declaration of the end of the pandemic (Aug 10, 2010); however, we continued to receive data up to March 14, 2011, from ongoing studies. We did a meta-analysis of individual participant data to assess the association between neuraminidase inhibitor treatment and mortality (primary outcome), adjusting for both treatment propensity and potential confounders, using generalised linear mixed modelling. We assessed the association with time to treatment using time-dependent Cox regression shared frailty modelling. We included data for 29,234 patients from 78 studies of patients admitted to hospital between Jan 2, 2009, and March 14, 2011. Compared with no treatment, neuraminidase inhibitor treatment (irrespective of timing) was associated with a reduction in mortality risk (adjusted odds ratio [OR] 0·81; 95% CI 0·70-0·93; p=0·0024). Compared with later treatment, early treatment (within 2 days of symptom onset) was associated with a reduction in mortality risk (adjusted OR 0·48; 95% CI 0·41-0·56; p<0·0001). Early treatment versus no treatment was also associated with a reduction in mortality (adjusted OR 0·50; 95% CI 0·37-0·67; p<0·0001). These

  10. Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

    PubMed Central

    Burnham, Andrew J.; Armstrong, Jianling; Lowen, Anice C.; Webster, Robert G.

    2015-01-01

    ABSTRACT Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. IMPORTANCE Influenza B viruses are important human respiratory pathogens contributing to a

  11. Impact of neuraminidase inhibitors on influenza A(H1N1)pdm09-related pneumonia: an individual participant data meta-analysis.

    PubMed

    Muthuri, Stella G; Venkatesan, Sudhir; Myles, Puja R; Leonardi-Bee, Jo; Lim, Wei Shen; Al Mamun, Abdullah; Anovadiya, Ashish P; Araújo, Wildo N; Azziz-Baumgartner, Eduardo; Báez, Clarisa; Bantar, Carlos; Barhoush, Mazen M; Bassetti, Matteo; Beovic, Bojana; Bingisser, Roland; Bonmarin, Isabelle; Borja-Aburto, Victor H; Cao, Bin; Carratala, Jordi; Cuezzo, María R; Denholm, Justin T; Dominguez, Samuel R; Duarte, Pericles A D; Dubnov-Raz, Gal; Echavarria, Marcela; Fanella, Sergio; Fraser, James; Gao, Zhancheng; Gérardin, Patrick; Giannella, Maddalena; Gubbels, Sophie; Herberg, Jethro; Higuera Iglesias, Anjarath L; Hoeger, Peter H; Hoffmann, Matthias; Hu, Xiaoyun; Islam, Quazi T; Jiménez, Mirela F; Kandeel, Amr; Keijzers, Gerben; Khalili, Hossein; Khandaker, Gulam; Knight, Marian; Kusznierz, Gabriela; Kuzman, Ilija; Kwan, Arthur M C; Lahlou Amine, Idriss; Langenegger, Eduard; Lankarani, Kamran B; Leo, Yee-Sin; Linko, Rita; Liu, Pei; Madanat, Faris; Manabe, Toshie; Mayo-Montero, Elga; McGeer, Allison; Memish, Ziad A; Metan, Gokhan; Mikić, Dragan; Mohn, Kristin G I; Moradi, Ahmadreza; Nymadawa, Pagbajabyn; Ozbay, Bulent; Ozkan, Mehpare; Parekh, Dhruv; Paul, Mical; Poeppl, Wolfgang; Polack, Fernando P; Rath, Barbara A; Rodríguez, Alejandro H; Siqueira, Marilda M; Skręt-Magierło, Joanna; Talarek, Ewa; Tang, Julian W; Torres, Antoni; Törün, Selda H; Tran, Dat; Uyeki, Timothy M; van Zwol, Annelies; Vaudry, Wendy; Velyvyte, Daiva; Vidmar, Tjasa; Zarogoulidis, Paul; Nguyen-Van-Tam, Jonathan S

    2016-05-01

    The impact of neuraminidase inhibitors (NAIs) on influenza-related pneumonia (IRP) is not established. Our objective was to investigate the association between NAI treatment and IRP incidence and outcomes in patients hospitalised with A(H1N1)pdm09 virus infection. A worldwide meta-analysis of individual participant data from 20 634 hospitalised patients with laboratory-confirmed A(H1N1)pdm09 (n = 20 021) or clinically diagnosed (n = 613) 'pandemic influenza'. The primary outcome was radiologically confirmed IRP. Odds ratios (OR) were estimated using generalised linear mixed modelling, adjusting for NAI treatment propensity, antibiotics and corticosteroids. Of 20 634 included participants, 5978 (29·0%) had IRP; conversely, 3349 (16·2%) had confirmed the absence of radiographic pneumonia (the comparator). Early NAI treatment (within 2 days of symptom onset) versus no NAI was not significantly associated with IRP [adj. OR 0·83 (95% CI 0·64-1·06; P = 0·136)]. Among the 5978 patients with IRP, early NAI treatment versus none did not impact on mortality [adj. OR = 0·72 (0·44-1·17; P = 0·180)] or likelihood of requiring ventilatory support [adj. OR = 1·17 (0·71-1·92; P = 0·537)], but early treatment versus later significantly reduced mortality [adj. OR = 0·70 (0·55-0·88; P = 0·003)] and likelihood of requiring ventilatory support [adj. OR = 0·68 (0·54-0·85; P = 0·001)]. Early NAI treatment of patients hospitalised with A(H1N1)pdm09 virus infection versus no treatment did not reduce the likelihood of IRP. However, in patients who developed IRP, early NAI treatment versus later reduced the likelihood of mortality and needing ventilatory support. © 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  12. Post-marketing safety and effectiveness evaluation of the intravenous anti-influenza neuraminidase inhibitor peramivir. II: a pediatric drug use investigation.

    PubMed

    Komeda, Takuji; Ishii, Shingo; Itoh, Yumiko; Ariyasu, Yasuyuki; Sanekata, Masaki; Yoshikawa, Takayoshi; Shimada, Jingoro

    2015-03-01

    Peramivir is the only intravenous formulation among anti-influenza neuraminidase inhibitors currently available. Peramivir was approved for manufacturing and marketing in Japan in January 2010. In October 2010, an additional indication for pediatric use was approved. We conducted a pediatric drug use investigation of peramivir from October 2010 to February 2012 and evaluated its real-world safety and effectiveness in pediatric patients. We collected the data of 1254 peramivir-treated pediatric patients from 161 facilities across Japan and examined the safety in 1199 patients and effectiveness in 1188 patients. In total, 245 adverse events were observed with an incidence rate of 14.01% (168/1199). Of these, 115 events were adverse drug reactions (ADRs) with an incidence rate of 7.67% (92/1199). Common ADRs were diarrhea and abnormal behavior, with incidence rates of 2.50% (30/1199) and 2.25% (27/1199), respectively. Fourteen serious ADRs were observed in 12 patients (1.00%), including 5 cases each of abnormal behavior and neutrophil count decreased. While 87.0% (100 events) of ADRs occurred within 3 days after the initiation of peramivir administration, 87.8% (101 events) resolved or improved within 7 days after onset. Multivariate analyses indicated that the presence or absence of underlying diseases/complications was significantly related to ADR incidence. With regard to effectiveness, the median time to alleviation of both influenza symptoms and fever was 3 days, including the first day of administration. Thus, this study confirms the pediatric safety of peramivir without any concerns about effectiveness under routine clinical settings.

  13. Post-marketing safety evaluation of the intravenous anti-influenza neuraminidase inhibitor peramivir: A drug-use investigation in patients with high risk factors.

    PubMed

    Komeda, Takuji; Ishii, Shingo; Itoh, Yumiko; Sanekata, Masaki; Yoshikawa, Takayoshi; Shimada, Jingoro

    2016-10-01

    Peramivir, the only injectable anti-influenza neuraminidase inhibitor medically available in Japan at present, is considered first-line treatment in patients with high risk factors for influenza exacerbation. We conducted a drug-use investigation of peramivir in inpatients with high risk factors (old age, pregnancy, and underlying disease such as chronic respiratory disease) from January 2010 to March 2013. Data of 772 patients from 124 facilities across Japan were collected; peramivir's safety in 770 patients and effectiveness in 688 patients were examined. In total, 412 adverse events were observed in 219 patients (28.4%). Of these, 155 events were adverse drug reactions (ADRs) observed in 98 patients (12.7%). Major ADRs (≥2%) were increased aspartate aminotransferase (5.1%), increased alanine aminotransferase (3.8%) and decreased white blood cell count (2.5%). Fourteen serious ADRs were observed in 12 patients (1.6%). All serious ADRs were resolved or improved except for two events for which outcomes were unknown. Multivariate analyses revealed that ADR incidences were significantly associated with these four backgrounds of patients: medical history, no influenza vaccination, renal impairment and other infection(s). With regard to its effectiveness, the median time to alleviation of both influenza symptoms and fever was 3 days, including the first day of administration, which was the same as in other previous surveillance studies. This surveillance study indicated the safety of peramivir in the treatment of influenza inpatients with high risk factors under routine clinical settings. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production

    PubMed Central

    Soong, Grace; Muir, Amanda; Gomez, Marisa I.; Waks, Jonathan; Reddy, Bharat; Planet, Paul; Singh, Pradeep K.; Kanetko, Yukihiro; Wolfgang, Matthew C.; Hsiao, Yu-Shan; Tong, Liang; Prince, Alice

    2006-01-01

    Many respiratory pathogens, including Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa, express neuraminidases that can cleave α2,3-linked sialic acids from glycoconjugates. As mucosal surfaces are heavily sialylated, neuraminidases have been thought to modify epithelial cells by exposing potential bacterial receptors. However, in contrast to neuraminidase produced by the influenza virus, a role for bacterial neuraminidase in pathogenesis has not yet been clearly established. We constructed a mutant of P. aeruginosa PAO1 by deleting the PA2794 neuraminidase locus (Δ2794) and tested its virulence and immunostimulatory capabilities in a mouse model of infection. Although fully virulent when introduced i.p., the Δ2794 mutant was unable to establish respiratory infection by i.n. inoculation. The inability to colonize the respiratory tract correlated with diminished production of biofilm, as assessed by scanning electron microscopy and in vitro assays. The importance of neuraminidase in biofilm production was further demonstrated by showing that viral neuraminidase inhibitors in clinical use blocked P. aeruginosa biofilm production in vitro as well. The P. aeruginosa neuraminidase has a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. Inhibiting bacterial neuraminidases could provide a novel mechanism to prevent bacterial pneumonia. PMID:16862214

  15. In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675.

    PubMed

    Molla, Akhteruzzaman; Kati, Warren; Carrick, Robert; Steffy, Kevin; Shi, Yan; Montgomery, Debra; Gusick, Nanette; Stoll, Vincent S; Stewart, Kent D; Ng, Teresa I; Maring, Clarence; Kempf, Dale J; Kohlbrenner, William

    2002-06-01

    With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold

  16. Susceptibility of highly pathogenic H5N1 influenza viruses to the neuraminidase inhibitor oseltamivir differs in vitro and in a mouse model.

    PubMed

    Govorkova, Elena A; Ilyushina, Natalia A; McClaren, Jennifer L; Naipospos, Tri S P; Douangngeun, Bounlom; Webster, Robert G

    2009-07-01

    While the neuraminidase (NA) inhibitor oseltamivir is currently our first line of defense against a pandemic threat, there is little information about whether in vitro testing can predict the in vivo effectiveness of antiviral treatment. Using a panel of five H5N1 influenza viruses (H5 clades 1 and 2), we determined that four viruses were susceptible to the drug in vitro (mean 50% inhibitory concentration [IC(50)], 0.1 to 4.9 nM), and A/Turkey/65-1242/06 virus was slightly less susceptible (mean IC(50), 10.8 nM). Two avian viruses showed significantly greater NA enzymatic activity (V(max)) than the human viruses, and the five viruses varied in their affinity for the NA substrate MUNANA (K(m), 64 to 300 muM) and for oseltamivir carboxylate (K(i), 0.1 to 7.9 nM). The protection of mice provided by a standard oseltamivir regimen (20 mg/kg/day for 5 days) also varied among the viruses used. We observed (i) complete protection against the less virulent A/chicken/Jogjakarta/BBVET/IX/04 virus; (ii) moderate protection (60 to 80% survival) against three viruses, two of which are neurotropic; and (iii) no protection against A/Turkey/65-1242/06 virus, which induced high pulmonary expression of proinflammatory mediators (interleukin-1alpha [IL-1alpha], IL-6, alpha interferon, and monocyte chemotactic protein 1) and contained a minor subpopulation of drug-resistant clones (I117V and E119A NA mutations). We found no correlation between in vitro susceptibility and in vivo protection (Spearman rank correlation coefficient rho = -0.1; P > 0.05). Therefore, the in vivo efficacy of oseltamivir against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing, and more prognostic ways to evaluate anti-influenza compounds must be developed. Multiple viral and host factors modulate the effectiveness of NA inhibitor regimens against such viruses and new, more consistently effective treatment options, including combination therapies, are needed.

  17. Modeling the paramyxovirus hemagglutinin-neuraminidase protein.

    PubMed

    Epa, V C

    1997-11-01

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein exhibits neuraminidase activity and has an active site functionally similar to that in influenza neuraminidases. Earlier work identified conserved amino acids among HN sequences and proposed similarity between HN and influenza neuraminidase sequences. In this work we identify the three-dimensional fold and develop a more detailed model for the HN protein, in the process we examine a variety of protein structure prediction methods. We use the known structures of viral and bacterial neuraminidases as controls in testing the success of protein structure prediction and modeling methods, including knowledge-based threading, discrete three-dimensional environmental profiles, hidden Markov models, neural network secondary structure prediction, pattern matching, and hydropathy plots. The results from threading show that the HN protein sequence has a 6 beta-sheet propellor fold and enable us to assign the locations of the individual beta-strands. The three-dimensional environmental profile and hidden Markov model methods were not successful in this work. The model developed in this work helps to understand better the biological function of the HN protein and design inhibitors of the enzyme and serves as an assessment of some protein structure prediction methods, especially after the x-ray crystallographic solution of its structure.

  18. Application of a seven-target pyrosequencing assay to improve the detection of neuraminidase inhibitor-resistant Influenza A(H3N2) viruses.

    PubMed

    Tamura, Daisuke; Okomo-Adhiambo, Margaret; Mishin, Vasiliy P; Guo, Zhu; Xu, Xiyan; Villanueva, Julie; Fry, Alicia M; Stevens, James; Gubareva, Larisa V

    2015-04-01

    National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis and pyrosequencing analysis of clinical specimens. A lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for moderately or highly reduced inhibition in the NI assay and/or for the presence of NA markers E119V, R292K, and N294S. In 2012 to 2013, three drug-resistant A(H3N2) viruses were detected by NI assay among isolates (n = 1,424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (n = 1,024) by a 3-target pyrosequencing assay. Overall, the frequency of NAI resistance was low (0.16% [4 of 2,448]). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, and deletions encompassing residues 245 to 248 (del245-248) and residues 247 to 250 (del247-250). The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136, and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (n = 2), E119V (n = 1), and del247-250 (n = 1). Recombinant NAs with del247-250 or del245-248 conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance-Associated Substitutions in Ferrets.

    PubMed

    Pascua, Philippe Noriel Q; Marathe, Bindumadhav M; Burnham, Andrew J; Vogel, Peter; Webby, Richard J; Webster, Robert G; Govorkova, Elena A

    2016-01-01

    Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)-generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg-wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6-4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A.

  20. Competitive Fitness of Influenza B Viruses Possessing E119A and H274Y Neuraminidase Inhibitor Resistance–Associated Substitutions in Ferrets

    PubMed Central

    Pascua, Philippe Noriel Q.; Marathe, Bindumadhav M.; Burnham, Andrew J.; Vogel, Peter; Webby, Richard J.; Webster, Robert G.; Govorkova, Elena A.

    2016-01-01

    Neuraminidase (NA) inhibitors (NAIs) are the only antiviral drugs recommended for influenza treatment and prophylaxis. Although NAI-resistant influenza B viruses that could pose a threat to public health have been reported in the field, their fitness is poorly understood. We evaluated in ferrets the pathogenicity and relative fitness of reverse genetics (rg)–generated influenza B/Yamanashi/166/1998-like viruses containing E119A or H274Y NA substitutions (N2 numbering). Ferrets inoculated with NAI-susceptible rg–wild-type (rg-WT) or NAI-resistant (rg-E119A or rg-H274Y) viruses developed mild infections. Growth of rg-E119A virus in the nasal cavities was delayed, but the high titers at 3 days post-inoculation (dpi) were comparable to those of the rg-WT and rg-H274Y viruses (3.6–4.1 log10TCID50/mL). No virus persisted beyond 5 dpi and replication did not extend to the trachea or lungs. Positive virus antigen-staining of the nasal turbinate epithelium was intermittent with the rg-WT and rg-H274Y viruses; whereas antigen-staining for the rg-E119A virus was more diffuse. Virus populations in ferrets coinoculated with NAI-susceptible and -resistant viruses (1:1 mixture) remained heterogeneous at 5 dpi but were predominantly rg-WT (>70%). Although the E119A substitution was associated with delayed replication in ferrets, the H274Y substitution did not measurably affect viral growth properties. These data suggest that rg-H274Y has undiminished fitness in single virus inoculations, but neither rg-E119A nor rg-H274Y gained a fitness advantage over rg-WT in direct competition experiments without antiviral drug pressure. Taken together, our data suggest the following order of relative fitness in a ferret animal model: rg-WT > rg-H274Y > rg-E119A. PMID:27466813

  1. Crystal Structures of Respiratory Pathogen Neuraminidases

    SciTech Connect

    Hsiao, Y.; Parker, D; Ratner, A; Prince, A; Tong, L

    2009-01-01

    Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 {angstrom} resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.

  2. Supply of neuraminidase inhibitors related to reduced influenza A (H1N1) mortality during the 2009-2010 H1N1 pandemic: summary of an ecological study.

    PubMed

    Miller, Paula E; Rambachan, Aksharananda; Hubbard, Roderick J; Li, Jiabai; Meyer, Alison E; Stephens, Peter; Mounts, Anthony W; Rolfes, Melissa A; Penn, Charles R

    2013-09-01

    When the influenza A (H1N1) pandemic spread across the globe from April 2009 to August 2010, many WHO Member States used antiviral drugs, specifically neuraminidase inhibitors (NAIs) oseltamivir and zanamivir, to treat influenza patients in critical condition. Antivirals have been found to be effective in reducing severity and duration of influenza illness, and likely reduce morbidity; however, it is unclear whether NAIs used during the pandemic reduced H1N1 mortality. To assess the association between antivirals and influenza mortality, at an ecologic level, country-level data on supply of oseltamivir and zanamivir were compared to laboratory-confirmed H1N1 deaths (per 100 000 people) from July 2009 to August 2010 in 42 WHO Member States. From this analysis, it was found that each 10% increase in kilograms of oseltamivir, per 100 000 people, was associated with a 1·6% reduction in H1N1 mortality over the pandemic period [relative rate (RR) = 0·84 per log increase in oseltamivir supply]. Each 10% increase in kilogram of active zanamivir, per 100 000, was associated with a 0·3% reduction in H1N1 mortality (RR = 0·97 per log increase). While limitations exist in the inference that can be drawn from an ecologic evaluation, this analysis offers evidence of a protective relationship between antiviral drug supply and influenza mortality and supports a role for influenza antiviral use in future pandemics. This article summarises the original study described previously, which can be accessed through the following citation: Miller PE, Rambachan A, Hubbard RJ, Li J, Meyer AE, et al. (2012) Supply of Neuraminidase Inhibitors Related to Reduced Influenza A (H1N1) Mortality during the 2009-2010 H1N1 Pandemic: An Ecological Study. PLoS ONE 7(9): e43491.

  3. Comparison of the clinical symptoms and the effectiveness of neuraminidase inhibitors for patients with pandemic influenza H1N1 2009 or seasonal H1N1 influenza in the 2007-2008 and 2008-2009 seasons.

    PubMed

    Kawai, Naoki; Ikematsu, Hideyuki; Tanaka, Osame; Matsuura, Shinro; Maeda, Tetsunari; Yamauchi, Satoshi; Hirotsu, Nobuo; Nishimura, Mika; Iwaki, Norio; Kashiwagi, Seizaburo

    2011-06-01

    The clinical symptoms and effectiveness of neuraminidase inhibitors (NAI) have not been adequately compared among pandemic H1N1 2009 patients, seasonal H1N1 patients, and patients with H1N1 with the H275Y mutation. The data of 68 seasonal H1N1 patients in 2007-2008, 193 seasonal H1N1 patients in 2008-2009, and 361 pandemic H1N1 2009 patients diagnosed by PCR who received an NAI were analyzed. The duration of fever (body temperature ≥ 37.5 ºC) after the first dose of NAI and from onset was calculated. The H275Y neuraminidase mutation status was determined for 166 patients. Significantly lower mean age (18.4 ± 13.2 years) and a higher percentage of teenagers (53.7%) were found for pandemic 2009 influenza than for seasonal influenza (P < 0.001). The peak body temperature was equivalent (mean, 39.0 ºC) in the three seasons, and the frequency of symptoms was the same or lower for pandemic influenza compared with seasonal H1N1. None of the 34 analyzed pandemic H1N1 virus isolates contained the H275Y mutation, which was commonly detected in the 2008-2009 season. The duration of fever after the start of oseltamivir therapy was significantly shorter for patients with pandemic (23.0 ± 11.6 h) than with seasonal H1N1 in both the 2008-2009 (49.7 ± 32.3 h) and 2007-2008 seasons (32.0 ± 18.9 h). The mean duration of fever after the first dose of zanamivir was not different among the three seasons (26.9-31.5 h). Clinical symptoms were the same or somewhat milder, and oseltamivir was more effective, for pandemic 2009 than for seasonal H1N1 influenza with or without H275Y mutation.

  4. The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness.

    PubMed

    Tu, Véronique; Abed, Yacine; Barbeau, Xavier; Carbonneau, Julie; Fage, Clément; Lagüe, Patrick; Boivin, Guy

    2017-01-01

    Emergence of pan neuraminidase inhibitor (NAI)-resistant variants constitutes a serious clinical concern. An influenza A(H1N1)pdm09 variant containing the I427T/Q313R neuraminidase (NA) substitutions was previously identified in a surveillance study. Although these changes are not part of the NA active site, the variant showed reduced susceptibility to many NAIs. In this study, we investigated the mechanism of resistance for the I427T/Q313R substitution and its impact on the NA enzyme and viral fitness. Recombinant wild-type (WT), I427T/Q313R and I427T A(H1N1)pdm09 viruses were generated by reverse genetics and tested for their drug susceptibilities, enzymatic properties and replication kinetics in vitro as well as their virulence in mice. Molecular dynamics (MD) simulations were performed for NA structural analysis. The I427T substitution, which was responsible for the resistance phenotype observed in the double (I427T/Q313R) mutant, induced 17-, 56-, 7-, and 14-fold increases in IC50 values against oseltamivir, zanamivir, peramivir and laninamivir, respectively. The I427T substitution alone or combined to Q313R significantly reduced NA affinity. The I427T/Q313R and to a lesser extent I427T recombinant viruses displayed reduced viral titers vs WT in vitro. In experimentally-infected mice, the mortality rates were 62.5%, 0% and 14.3% for the WT, I417T/Q313R and I427T viruses, respectively. There were about 2.5- and 2-Log reductions in mean lung viral titers on day 5 post-infection for the I427T/Q313R and I427T mutants, respectively, compared to WT. Results from simulations revealed that the I427T change indirectly altered the stability of the catalytic R368 residue of the NA enzyme causing its reduced binding to the substrate/inhibitor. This study demonstrates that the I427T/Q313R mutant, not only alters NAI susceptibility but also compromises NA properties and viral fitness, which could explain its infrequent detection in clinic.

  5. The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets.

    PubMed

    Yen, Hui-Ling; Zhou, Jie; Choy, Ka-Tim; Sia, Sin Fun; Teng, Ooiean; Ng, Iris H; Fang, Vicky J; Hu, Yunwen; Wang, Wei; Cowling, Benjamin J; Nicholls, John M; Guan, Yi; Peiris, Joseph Sriyal Malik

    2014-12-15

    Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. The R292K Mutation That Confers Resistance to Neuraminidase Inhibitors Leads to Competitive Fitness Loss of A/Shanghai/1/2013 (H7N9) Influenza Virus in Ferrets

    PubMed Central

    Yen, Hui-Ling; Zhou, Jie; Choy, Ka-Tim; Sia, Sin Fun; Teng, Ooiean; Ng, Iris H.; Fang, Vicky J.; Hu, Yunwen; Wang, Wei; Cowling, Benjamin J.; Nicholls, John M.; Guan, Yi; Peiris, Joseph Sriyal Malik

    2014-01-01

    Background Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. Methods We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. Results Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. Conclusions The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus. PMID:24951824

  7. Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

    PubMed

    Yen, H-L; McKimm-Breschkin, J L; Choy, K-T; Wong, D D Y; Cheung, P P H; Zhou, J; Ng, I H; Zhu, H; Webby, R J; Guan, Y; Webster, R G; Peiris, J S M

    2013-07-16

    We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which

  8. Neuraminidase production by Erysipelothrix rhusiopathiae.

    PubMed

    Wang, Qinning; Chang, Barbara J; Mee, Brian J; Riley, Thomas V

    2005-05-20

    In order to characterise neuraminidase activity by Erysipelothrix, 85 isolates of Erysipelothrix spp. from a variety of sources including human clinical, marine and terrestrial animals, and the environment were investigated for neuraminidase production. Neuraminidase activity was detected by a peanut lectin haemagglutination method. The effects of media, incubation conditions and pH on the production and activity of neuraminidase were also investigated. Enzyme activity was detected only in the supernatants of the isolates of Erysipelothrix rhusiopathiae which had been incubated in cooked meat broth and Todd Hewitt broth supplemented with horse serum after 16 and 36 h incubation at 37 degrees C. The maximum titres were reached at 40 h in cooked meat broth and 56 h in Todd Hewitt broth supplemented with horse serum. All 58 isolates and the type strain (ATCC 19414) of E. rhusiopathiae produced detectable neuraminidase activity with titres between 10 and 320. The optimal pH for the enzyme activity varied among the isolates with a pH between 6.0 and 7.0 covering the highest enzyme activity of the most. There was no statistically significant difference in the level of neuraminidase activity between isolates from different sources (p > 0.05). Neuraminidase activity was not detected in the non-pathogenic Erysipelothrix spp. such as E. tonsillarum. Neuraminidase was detected only in E. rhusiopathiae suggesting its possible role as a virulence factor. Enzyme production and activity were medium and pH dependent. The peanut lectin haemagglutination assay is a simple, rapid and sensitive method and is particularly useful for the analysis of multiple samples.

  9. Antipneumococcal activity of neuraminidase inhibiting artocarpin.

    PubMed

    Walther, E; Richter, M; Xu, Z; Kramer, C; von Grafenstein, S; Kirchmair, J; Grienke, U; Rollinger, J M; Liedl, K R; Slevogt, H; Sauerbrei, A; Saluz, H P; Pfister, W; Schmidtke, M

    2015-05-01

    Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 μM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 μM) and biofilm formation (MBIC: 1.15-2.97 μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

  10. Antiviral Drug–Resistant Influenza B Viruses Carrying H134N Substitution in Neuraminidase, Laos, February 2016

    PubMed Central

    Baranovich, Tatiana; Vongphrachanh, Phengta; Ketmayoon, Pakapak; Sisouk, Thongchanh; Chomlasack, Khampheng; Khanthamaly, Viengphone; Nguyen, Ha Thuy; Mishin, Vasiliy P.; Marjuki, Henju; Barnes, John R.; Garten, Rebecca J.; Stevens, James; Wentworth, David E.

    2017-01-01

    In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation. PMID:28322707

  11. Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

    PubMed

    Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

    2017-06-01

    Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

  12. Attenuation of TNF-α secretion by L-proline-based cyclic dipeptides produced by culture broth of Pseudomonas aeruginosa.

    PubMed

    Khan, Rukaiyya; Basha, Ameer; Goverdhanam, Ragavendra; Rao, Poorna Chandra; Tanemura, Yuhei; Fujimoto, Yoshinori; Begum, Ahil Sajeli

    2015-12-15

    To identify small molecule inhibitors of TNF-α, bioassay- and LC-MS-guided chemical investigation on EtOAc extract of Pseudomonas aeruginosa ABS-36 culture broth (EEPA) was performed, which yielded four proline-based cyclic dipeptides, cyclo(Gly-l-Pro) (1), cyclo(l-Pro-l-Phe) (2), cyclo(trans-4-hydroxy-l-Pro-l-Phe) (3) and cyclo(trans-4-hydroxy-l-Pro-l-Leu) (4). Compounds 1 and 3 exhibited potent inhibition of TNF-α release with IC50 values of 4.5 and 14.2μg/mL, respectively, while EEPA showed IC50 of 38.8μg/mL under lipopolysaccharide treated RAW 264.7 cell ELISA assay. Also, marked attenuation of mRNA-expression of TNF-α was shown by all compounds. In vivo testing in rats of EEPA and chemically synthesized 4 validated significant TNF-α reduction with 51% (500mg/kg) and 79% (50mg/kg), respectively. In addition, all compounds exhibited significant diminution of IL-1β and IL-6 mRNA-expression levels and NO production. All samples displayed only weak toxicity to lipopolysaccharide-induced RAW 264.7 cells.

  13. Neuraminidase activity of blue eye disease porcine rubulavirus: Specificity, affinity and inhibition studies.

    PubMed

    Santos-López, Gerardo; Borraz-Argüello, María T; Márquez-Domínguez, Luis; Flores-Alonso, Juan Carlos; Ramírez-Mendoza, Humberto; Priem, Bernard; Fort, Sébastien; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Herrera-Camacho, Irma

    2017-05-06

    Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km=0.029μM, Vmax=522.8nmolmin(-1)mg(-1)). When 3SL was used as substrate, typical cooperative kinetics were found (S50=0.15μM, Vmax=154.3nmolmin(-1)mg(-1)). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC50 of 0.24μM. PorPV neuraminidase was activated by Ca(2+) and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. L-proline-based deep eutectic solvents (DESs) for deep catalytic oxidative desulfurization (ODS) of diesel.

    PubMed

    Hao, Lingwan; Wang, Meiri; Shan, Wenjuan; Deng, Changliang; Ren, Wanzhong; Shi, Zhouzhou; Lü, Hongying

    2017-10-05

    A series of L-proline-based DESs was prepared through an atom economic reaction between L-proline (L-Pro) and four different kinds of organic acids. The DESs were characterized by Fourier transform infrared spectroscopy (FT-IR), H nuclear magnetic resonance ((1)HNMR), cyclic voltammogram (CV) and the Hammett method. The synthesized DESs were used for the oxidative desulfurization and the L-Pro/p-toluenesultonic acid (L-Pro/p-TsOH) system shows the highest catalytic activity that the removal of dibenzothiophene (DBT) reached 99% at 60°C in 2h, which may involve the dual activation of the L-Pro/p-TsOH. The acidity of four different L-proline-based DESs was measured and the results show that it could not simply conclude that the correlation between the acidity of DESs and desulfurization capability was positive or negative. The electrochemical measurements evidences and recycling experiment indicate a good stability performance of L-Pro/p-TsOH in desulfurization. This work will provide a novel and potential method for the deep oxidation desulfurization. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Catechin inhibition of influenza neuraminidase and its molecular basis with mass spectrometry.

    PubMed

    Müller, Patrick; Downard, Kevin M

    2015-01-01

    The molecular basis for the antiviral inhibitory properties of three catechins epigallocatechin gallate, epicatechin gallate and catechin-5-gallate derived from green tea was assessed in terms of their ability to interact with influenza neuraminidase. This was investigated using a molecular based MALDI mass spectrometry approach in conjunction with companion inhibition assays employing confocal microscopy. Together with computational molecular docking, all three catechins were found to bind to influenza neuraminidase in the vicinity of a structurally conserved cavity adjacent to residue 430 that has been suggested to be a secondary sialic acid binding site. In doing so, they were effective inhibitors of the enzyme preventing the release of progeny viruses from host cells at inhibitor concentrations (IC50 values) of between 100 and 173 μM. Importantly, their different binding profiles avoid the limitations of existing neuraminidase inhibitors manifested by the evolution of antiviral resistance strains.

  16. [Influenza A, pregnancy and neuraminidase inhibitors].

    PubMed

    Montané, Eva; Lecumberri, Josep; Pedro-Botet, María Luisa

    2011-05-28

    Following the explosion of the influenza A pandemic (H1N1) during the first semester of 2009, oseltamivir and zanamivir were used as the treatment of choice in the absence of rigorous clinical studies demonstrating their efficacy in the treatment and prophylaxis of this disease. Knowledge of seasonal influenza, flu pandemics and particularly the H1N1, which produces more severe infection and a higher mortality rate during pregnancy, led to the use of antiviral treatment despite the scarcity of clinical studies on their efficacy and effectiveness, mainly due to the influence of the media. This study reviewed the experimental and clinical studies performed on the safety of oseltamivir and zanamivir in pregnancy. Likewise, the recommendations made by the different health care and governmental authorities as well as other institutions and scientific and health care organizations on the therapeutic management and prophylaxis of influenza A 2009 in pregnant women were reviewed. Copyright © 2010 Elsevier España, S.L. All rights reserved.

  17. A proline-based aminophenol ligand: synthesis, iron complexation, magnetic, electronic and redox investigation.

    PubMed

    Sheykhi, Hamid; Safaei, Elham

    2014-01-24

    A new proline-based aminophenol ligand was synthesized by a convenient procedure. The ligand was characterized by (1)H NMR, (13)C NMR and IR spectroscopies, elemental analysis and optical activity measurements. Mononuclear iron(III) complex (FeL(Pro)) of this ligand was synthesized and characterized by IR, UV-vis, ESI-MS, magnetic susceptibility studies and cyclic voltammetry techniques. The equilibrium formation constant of FeL(Pro) and the pure UV-vis spectral profile of the complex was determined by multivariate hard modeling method. The molecular structure of FeL(Pro) determined by ESI-MS consist of two aminophenolate ligands. The variation of magnetic susceptibility with temperature indicates paramagnetic iron(III) in the monomeric complex. FeL(Pro) complex undergo metal-centered reduction, and ligand-centered oxidation. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

    PubMed

    Varghese, J N; Epa, V C; Colman, P M

    1995-06-01

    The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.

  19. Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

    PubMed Central

    Varghese, J. N.; Epa, V. C.; Colman, P. M.

    1995-01-01

    The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase. PMID:7549872

  20. A Virtual Screening Approach For Identifying Plants with Anti H5N1 Neuraminidase Activity

    PubMed Central

    2016-01-01

    Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources. PMID:25555059

  1. Selective CB2 receptor agonists. Part 2: Structure-activity relationship studies and optimization of proline-based compounds.

    PubMed

    Riether, Doris; Zindell, Renee; Wu, Lifen; Betageri, Raj; Jenkins, James E; Khor, Someina; Berry, Angela K; Hickey, Eugene R; Ermann, Monika; Albrecht, Claudia; Ceci, Angelo; Gemkow, Mark J; Nagaraja, Nelamangala V; Romig, Helmut; Sauer, Achim; Thomson, David S

    2015-02-01

    Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia.

  2. Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly.

    PubMed

    Hinek, Aleksander; Pshezhetsky, Alexey V; von Itzstein, Mark; Starcher, Barry

    2006-02-10

    We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.

  3. A Beneficiary Role for Neuraminidase in Influenza Virus Penetration through the Respiratory Mucus

    PubMed Central

    Yang, Xiaoyun; Steukers, Lennert; Forier, Katrien; Xiong, Ranhua; Braeckmans, Kevin

    2014-01-01

    Swine influenza virus (SIV) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. Sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. However, mucus which is also rich in sialic acids may serve as the first barrier of selection. It was investigated how influenza virus interacts with the mucus to infect epithelial cells. Two techniques were applied to track SIV H1N1 in porcine mucus. The microscopic diffusion of SIV particles in the mucus was analyzed by single particle tracking (SPT), and the macroscopic penetration of SIV through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. Furthermore, the effects of neuraminidase on SIV getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (NAI), and Arthrobacter ureafaciens neuraminidase. The distribution of the diffusion coefficient shows that 70% of SIV particles were entrapped, while the rest diffused freely in the mucus. Additionally, SIV penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. Both the microscopic diffusion and macroscopic penetration were largely diminished by NAI, while were clearly increased by the effect of exogenous neuraminidase. Moreover, the exogenous neuraminidase sufficiently prevented the binding of SIV to mucus which was reversely enhanced by effect of NAI. These findings clearly show that the neuraminidase helps SIV move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract. PMID:25333824

  4. A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus.

    PubMed

    Yang, Xiaoyun; Steukers, Lennert; Forier, Katrien; Xiong, Ranhua; Braeckmans, Kevin; Van Reeth, Kristien; Nauwynck, Hans

    2014-01-01

    Swine influenza virus (SIV) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. Sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. However, mucus which is also rich in sialic acids may serve as the first barrier of selection. It was investigated how influenza virus interacts with the mucus to infect epithelial cells. Two techniques were applied to track SIV H1N1 in porcine mucus. The microscopic diffusion of SIV particles in the mucus was analyzed by single particle tracking (SPT), and the macroscopic penetration of SIV through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. Furthermore, the effects of neuraminidase on SIV getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (NAI), and Arthrobacter ureafaciens neuraminidase. The distribution of the diffusion coefficient shows that 70% of SIV particles were entrapped, while the rest diffused freely in the mucus. Additionally, SIV penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. Both the microscopic diffusion and macroscopic penetration were largely diminished by NAI, while were clearly increased by the effect of exogenous neuraminidase. Moreover, the exogenous neuraminidase sufficiently prevented the binding of SIV to mucus which was reversely enhanced by effect of NAI. These findings clearly show that the neuraminidase helps SIV move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract.

  5. Trypsin inhibitors for the treatment of pancreatitis.

    PubMed

    Brandl, Trixi; Simic, Oliver; Skaanderup, Philip R; Namoto, Kenji; Berst, Frederic; Ehrhardt, Claus; Schiering, Nikolaus; Mueller, Irene; Woelcke, Julian

    2016-09-01

    Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

  6. Influence of Hofmeister Ions on the Structure of Proline-Based Peptide Models: A Combined Experimental and Molecular Modeling Study.

    PubMed

    Bröhl, Andreas; Albrecht, Benjamin; Zhang, Yong; Maginn, Edward; Giernoth, Ralf

    2017-02-23

    The influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are also able to stabilize the conformers over wide temperature ranges.

  7. The NanA Neuraminidase of Streptococcus pneumoniae Is Involved in Biofilm Formation▿

    PubMed Central

    Parker, Dane; Soong, Grace; Planet, Paul; Brower, Jonathan; Ratner, Adam J.; Prince, Alice

    2009-01-01

    Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccines and effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acid residues from glycoconjugates, is involved in host colonization in animal models of infection and may provide a novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase (NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role in pathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even though we show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formation incorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA block biofilm formation and may provide a novel target for preventative therapy. This work highlights the role played by the neuraminidase in pathogenesis and represents an important step in drug development for prevention of colonization of the respiratory tract by this important pathogen. PMID:19564377

  8. Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

    PubMed Central

    Yen, H.-L.; McKimm-Breschkin, J. L.; Choy, K.-T.; Wong, D. D. Y.; Cheung, P. P. H.; Zhou, J.; Ng, I. H.; Zhu, H.; Webby, R. J.; Guan, Y.; Webster, R. G.; Peiris, J. S. M.

    2013-01-01

    ABSTRACT We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2′-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. PMID:23860768

  9. Neuraminidase inhibitory terpenes from endophytic Cochliobolus sp.

    PubMed

    Zhang, Gao-Fei; Guo, Zhi-Kai; Wang, Wei; Cui, Jiang-Tao; Tan, Ren-Xiang; Ge, Hui-Ming

    2011-08-01

    The chemical study of endophytic fungus of Cochliobolus led to the isolation of 10 terpenes (1-10), including one new compound named isocochlioquinone B (1). Their structures were elucidated on the basis of spectroscopic methods, including 2D NMR techniques. Compounds 5-7 showed significant neuraminidase inhibitory activity with IC(50) values of 0.79-1.75 μM.

  10. Influenza neuraminidase as a vaccine antigen

    USDA-ARS?s Scientific Manuscript database

    The neuraminidase protein of influenza viruses is a surface glycoprotein that has enzymatic activity to remove sialic acid, the viral receptor, from both viral and host proteins. The removal of sialic acid from viral proteins plays a key role in the release of the virus from the cell by preventing ...

  11. Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility.

    PubMed

    Farrukee, Rubaiyea; Leang, Sook-Kwan; Butler, Jeff; Lee, Raphael T C; Maurer-Stroh, Sebastian; Tilmanis, Danielle; Sullivan, Sheena; Mosse, Jennifer; Barr, Ian G; Hurt, Aeron C

    2015-07-01

    The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of the monomeric interface were also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Characterization of neuraminidases produced by various serotypes of Pasteurella haemolytica.

    PubMed Central

    Straus, D C; Jolley, W L; Purdy, C W

    1993-01-01

    Neuraminidases produced by 16 strains of Pasteurella haemolytica (serotypes 1 to 16) were characterized by molecular weight, antigenic identity, and substrate specificity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. haemolytica serotype A1 (Ph A1) was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified Ph A1 neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of Ph A1 neuraminidase by 46%. This antiserum also reduced the activity of neuraminidase produced by the other serotypes by between 15 and 66%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Fifteen of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 150,000 to 200,000. One serotype (serotype 11) produced no material with neuraminidase activity. In addition, all 15 high-molecular-weight neuraminidases showed similar substrate specificities. That is, they were all most active against N-acetylneuramin lactose and least active against bovine submaxillary mucin. On the basis of these results, it appears that the high-molecular-weight neuraminidases produced by the different P. haemolytica serotypes are quite similar. PMID:8406865

  13. Combining Molecular Docking and Molecular Dynamics to Predict the Binding Modes of Flavonoid Derivatives with the Neuraminidase of the 2009 H1N1 Influenza A Virus

    PubMed Central

    Lu, Shih-Jen; Chong, Fok-Ching

    2012-01-01

    Control of flavonoid derivatives inhibitors release through the inhibition of neuraminidase has been identified as a potential target for the treatment of H1N1 influenza disease. We have employed molecular dynamics simulation techniques to optimize the 2009 H1N1 influenza neuraminidase X-ray crystal structure. Molecular docking of the compounds revealed the possible binding mode. Our molecular dynamics simulations combined with the solvated interaction energies technique was applied to predict the docking models of the inhibitors in the binding pocket of the H1N1 influenza neuraminidase. In the simulations, the correlation of the predicted and experimental binding free energies of all 20 flavonoid derivatives inhibitors is satisfactory, as indicated by R2 = 0.75. PMID:22605992

  14. Influence of Hofmeister ions on the structure of proline-based peptide models: A combined experimental and molecular modeling study

    DOE PAGES

    Brohl, Andreas; Albrecht, Benjamin; Zhang, Yong; ...

    2017-02-13

    Here, the influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are alsomore » able to stabilize the conformers over wide temperature ranges.« less

  15. Mutation-induced loop opening and energetics for binding of tamiflu to influenza N8 neuraminidase.

    PubMed

    Kar, Parimal; Knecht, Volker

    2012-05-31

    Tamiflu, also known as oseltamivir (OTV), binds to influenza A neuraminidase (H5N1) with very high affinity (0.32 nM). However, this inhibitor binds to other neuraminidases as well. In the present work, a systematic computational study is performed to investigate the mechanism underlying the binding of oseltamivir to N8 neuraminidase (NA) in "open" and "closed" conformations of the 150-loop through molecular dynamics simulations and the popular and well established molecular mechanics Poisson-Boltzmann (MM-PBSA) free energy calculation method. Whereas the closed conformation is stable for wild type N8, it transforms into the open conformation for the mutants Y252H, H274Y, and R292K, indicating that bound to oseltamivir these mutants are preferentially in the open conformation. Our calculations show that the binding of wild type oseltamivir to the closed conformation of N8 neuraminidase is energetically favored compared to the binding to the open conformation. We observe water mediated binding of oseltamivir to the N8 neuraminidase in both conformations which is not seen in the case of binding of the same drug to the H5N1 neuraminidase. The decomposition of the binding free energy reveals the mechanisms underlying the binding and changes in affinity due to mutations. Considering the mutant N8 variants in the open conformation adopted during the simulations, we observe a significant loss in the size of the total binding free energy for the N8(Y252H)-OTV, N8(H274Y)-OTV, and N8(R292K)-OTV complexes compared to N8(WT)-OTV, mainly due to the decrease in the size of the intermolecular electrostatic energy. For R292K, an unfavorable shift in the van der Waals interactions also contributes to the drug resistance. The mutations cause a significant expansion in the active site cavity, increasing its solvent accessible surface compared to the crystal structures of both the open and closed conformations. Our study underscores the need to consider dynamics in rationalizing the

  16. Zanamivir-resistant influenza viruses with a novel neuraminidase mutation.

    PubMed

    Hurt, Aeron C; Holien, Jessica K; Parker, Michael; Kelso, Anne; Barr, Ian G

    2009-10-01

    The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.

  17. Erysipelothrix rhusiopathiae neuraminidase and its role in pathogenicity.

    PubMed

    Abrashev, Ignat; Orozova, Petya

    2006-01-01

    The role of the enzyme neuraminidase in pathogenicity of the bacillus Erysipelothrix rhusiopathiae was studied. Different substances with low and high molecular weight were tested as inducers of E. rhusiopathiae neuraminidase biosynthesis. It was found that macromolecular complexes induce the secretion of the enzyme. K(M) values for different substrates showed that the affinity of the E. rhusiopathiae neuraminidase increases in parallel with the enlargement of the molecular weight of glycoproteins. Results from the rabbits skin test confirmed the role of E. rhusiopathiae neuraminidase as a factor of pathogenicity with spreading functions.

  18. C-Methylated Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase.

    PubMed

    Dao, Trong-Tuan; Tung, Bui-Thanh; Nguyen, Phi-Hung; Thuong, Phuong-Thien; Yoo, Sung-Sik; Kim, Eun-Hee; Kim, Sang-Kyum; Oh, Won-Keun

    2010-10-22

    As part of an ongoing study focused on the discovery of anti-influenza agents from plants, four new (1-4) and 10 known (5-14) C-methylated flavonoids were isolated from a methanol extract of Cleistocalyx operculatus buds using an influenza H1N1 neuraminidase inhibition assay. Compounds 4, 7, 8, and 14, with a chalcone skeleton, showed significant inhibitory effects on the viral neuraminidases from two influenza viral strains, H1N1 and H9N2. Compound 4 showed the strongest inhibitory activity against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y mutant) expressed in 293T cells with IC50 values of 8.15 ± 1.05 and 3.31 ± 1.34 μM, respectively. Compounds 4, 7, 8, and 14 behaved as noncompetitive inhibitors in the kinetic studies. These results indicate that C-methylated flavonoids from C. operculatus have the potential to be developed as neuraminidase inhibitors for novel influenza H1N1.

  19. Characterization of neuraminidases produced by various serotypes of Pasteurella multocida.

    PubMed Central

    Straus, D C; Jolley, W L; Purdy, C W

    1996-01-01

    Neuraminidases produced by 16 strains of Pasteurella multocida (serotypes 1 to 16) were characterized by molecular weight, substrate specificity, and antigenic identity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. multocida A:3 was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified P. multocida A:3 neuraminidase was employed to immunize rabbits, and the resulting antiserum reduced the activity of the P. multocida A:3 enzyme by 40.3%. This antiserum also reduced the activities of the neuraminidases produced by other serotypes by between 30.8 and 59.6%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Each of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 500,000. In addition, all 16 high-molecular-weight neuraminidases showed similar substrate specificities. On the basis of these data, it appears that the high-molecular-weight neuraminidases produced by different P. multocida serotypes are quite similar. PMID:8606116

  20. Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species.

    PubMed

    Berčič, Rebeka Lucijana; Cizelj, Ivanka; Benčina, Mateja; Narat, Mojca; Bradbury, Janet M; Dovč, Peter; Benčina, Dušan

    2012-03-23

    Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type A(T)) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14(T)), M. cynos (isolate 896) and M. molare (type strain H542(T)) proteins with NEAC had molecular masses of ∼130kDa, 105kDa and 110kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization.

  1. Immunocytochemical localization of neuraminidase in Trypanosoma cruzi.

    PubMed Central

    Souto-Padrón, T; Harth, G; de Souza, W

    1990-01-01

    A polyclonal antibody obtained against neuraminidase purified from Trypanosoma cruzi was used for the localization of the protein in whole cells by immunofluorescence microscopy and in thin sections of parasites (epimastigote, amastigote, and trypomastigote forms) embedded at a low temperature in Lowicryl K4M resin. The intensity of labeling, as evaluated by the number of gold particles associated with the parasite, varied according to the protozoan developmental stage. In the noninfective epimastigote forms, labeling of the cell surface was very weak. However, an intense labeling of some cytoplasmic vacuoles was observed. Labeling of the surfaces of most of the trypomastigote forms was weak, while gold particles were seen in association with the flagellar pockets of these forms, which suggests that the enzyme is secreted through this region. Intense labeling of the surfaces of many, but not all, transition forms between trypomastigote and amastigote forms was observed. Amastigote forms found in the supernatant of infected cell cultures had their surfaces intensely labeled, while few particles were seen on the surfaces of intracellular amastigotes. The results obtained are discussed in relation to the role played by T. cruzi neuraminidase in the process of parasite-host cell interaction. Images PMID:2407649

  2. Understanding of known drug-target interactions in the catalytic pocket of neuraminidase subtype N1.

    PubMed

    Malaisree, Maturos; Rungrotmongkol, Thanyada; Decha, Panita; Intharathep, Pathumwadee; Aruksakunwong, Ornjira; Hannongbua, Supot

    2008-06-01

    To provide detailed information and insight into the drug-target interaction, structure, solvation, and dynamic and thermodynamic properties, the three known-neuraminidase inhibitors-oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV)-embedded in the catalytic site of neuraminidase (NA) subtype N1 were studied using molecular dynamics simulations. In terms of ligand conformation, there were major differences in the structures of the guanidinium and the bulky groups. The atoms of the guanidinium group of PRV were observed to form many more hydrogen bonds with the surrounded residues and were much less solvated by water molecules, in comparison with the other two inhibitors. Consequently, D151 lying on the 150-loop (residues 147-152) of group-1 neuraminidase (N1, N4, N5, and N8) was considerably shifted to form direct hydrogen bonds with the --OH group of the PRV, which was located rather far from the 150-loop. For the bulky group, direct hydrogen bonds were detected only between the hydrophilic side chain of ZNV and residues R224, E276, and E277 of N1 with rather weak binding, 20-70% occupation. This is not the case for OTV and PRV, in which flexibility and steric effects due to the hydrophobic side chain lead to the rearrangement of the surrounded residues, that is, the negatively charged side chain of E276 was shifted and rotated to form hydrogen bonds with the positively charged moiety of R224. Taking into account all the ligand-enzyme interaction data, the gas phase MM interaction energy of -282.2 kcal/mol as well as the binding free energy (DeltaG(binding)) of -227.4 kcal/mol for the PRV-N1 are significantly lower than those of the other inhibitors. The ordering of DeltaG(binding) of PRV < ZNV < OTV agrees well with the ordering of experimental IC(50) value. (c) 2008 Wiley-Liss, Inc.

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

    PubMed Central

    Gilbertson, Brad; Ng, Wy Ching; Crawford, Simon; McKimm-Breschkin, Jenny L.

    2017-01-01

    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

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

    PubMed

    Gilbertson, Brad; Ng, Wy Ching; Crawford, Simon; McKimm-Breschkin, Jenny L; Brown, Lorena E

    2017-07-15

    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

  5. Neuraminidase-1: A novel therapeutic target in multistage tumorigenesis

    PubMed Central

    Haxho, Fiona; Neufeld, Ronald J.; Szewczuk, Myron R.

    2016-01-01

    Several of the growth factors and their receptor tyrosine kinases (RTK) such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and insulin are promising candidate targets for cancer therapy. Indeed, tyrosine kinase inhibitors (TKI) have been developed to target these growth factors and their receptors, and have demonstrated dramatic initial responses in cancer therapy. Yet, most patients ultimately develop TKI drug resistance and relapse. It is essential in the clinical setting that the targeted therapies are to circumvent multistage tumorigenesis, including genetic mutations at the different growth factor receptors, tumor neovascularization, chemoresistance of tumors, immune-mediated tumorigenesis and the development of tissue invasion and metastasis. Here, we identify a novel receptor signaling platform linked to EGF, NGF, insulin and TOLL-like receptor (TLR) activations, all of which are known to play major roles in tumorigenesis. The importance of these findings signify an innovative and promising entirely new targeted therapy for cancer. The role of mammalian neuraminidase-1 (Neu1) in complex with matrix metalloproteinase-9 and G protein-coupled receptor tethered to RTKs and TLRs is identified as a major target in multistage tumorigenesis. Evidence exposing the link connecting growth factor-binding and immune-mediated tumorigenesis to this novel receptor-signaling paradigm will be reviewed in its current relationship to cancer. PMID:27029067

  6. Site-specific indolation of proline-based peptides via copper(II)-catalyzed oxidative coupling of tertiary amine N-oxides.

    PubMed

    Wu, Xiaowei; Zhang, Dengyou; Zhou, Shengbin; Gao, Feng; Liu, Hong

    2015-08-14

    The first site-specific and purely chemical method for modifying proline-based peptides was developed via a convenient, copper-catalyzed oxidative coupling of tertiary amine N-oxides with indoles. This novel approach features high regioselectivity and diastereoselectivity, mild conditions, and compatibility with various functional groups. In addition, a simplified process was realized in one pot and two steps via in situ oxidative coupling of tertiary amine and indoles.

  7. [Effect of normal and specific immune sera on neuraminidase activity].

    PubMed

    Kotliar, T V; Zaikina, N A; Shataeva, L K

    1992-01-01

    We have got evidence that there is no antigenic relationship reflecting the structural similarity between neuraminidases synthesized by noncholera vibrios and Arthrobacter nicotianae. The cross-reactions between the enzymes and heterological antisera were not observed. Antibodies against the A. nicotianae neuraminidase inhibited the activity of the enzyme for a glycomacropeptide of milk whey and for components of the blood serum, and had no effect no the neuraminidase from noncholera vibrios. Antibodies against the neuraminidase of noncholera vibrios inhibited only the activity of the homologous enzyme. Upon gel-filtration on Sephadex G-200 the antibodies inhibiting the activity of the enzymes under study were found in the fraction of 7S-gamma-globulins.

  8. Red cells bound to influenza virus N9 neuraminidase are not released by the N9 neuraminidase activity.

    PubMed

    Air, G M; Laver, W G

    1995-08-01

    Influenza virus neuraminidase (NA) of the N9 subtype also possesses hemagglutinin activity and the hemagglutinating, or hemabsorbing (HB), site is distinct from the catalytic site. Previous results suggested that the NA was binding to sialic acid on the red cell surface, but we now report that the HB receptor is not sensitive to N9 influenza neuraminidase activity. Cell lines that constitutively express N9 or N2 neuraminidase have been used to further investigate the specificity of red blood cell binding to the HB site. The results suggest that the ligand is N-acetylneuraminic acid in a linkage or environment that is not sensitive to influenza virus neuraminidase, but which is released by the broadly specific bacterial sialidases from Micromonospora viridifaciens or Arthrobacter ureafaciens.

  9. One-pot synthesis of α,β-epoxy ketones through domino reaction between alkenes and aldehydes catalyzed by proline based chiral organocatalysts.

    PubMed

    Ashokkumar, Veeramanoharan; Siva, Ayyanar

    2017-03-22

    Proline based metal free organocatalysts were developed by using a new approach for the synthesis of epoxide derivatives through a domino reaction. This domino reaction (oxidative coupling) allows a direct access to epoxides from various alkenes and aldehydes through C-H functionalization and C-C/C-O bond formation. The catalytic efficiencies of the newly synthesized organocatalysts were also determined by domino reaction in the presence of various functional groups containing aldehyde and alkene derivatives with very good yields (up to 95%) and ee's (up to 99%).

  10. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans.

    PubMed

    Kilbourne, E D

    1976-10-01

    Groups of college students received either conventional A/England/42/72 (H3N2) vaccine (X-37), an antigenic hybrid (Heq1N2) vaccine (X-38) containing the same neuraminidase (and thus effectively neuraminidase-monospecific), or a placebo injection. The vaccines contained 798 and 643 chick cell-agglutinating units per dose, respectively, and equivalent immunogenic units of N2 as defined in antigenic extinction tests in rabbits. All subjects had antibody to N2 before immunization, and mean initial titers were comparable in both vaccine groups. Homotypic hemagglutination-inhibition response to vaccine hemagglutinin was slightly more frequent (77%) but of lower magnitude in the students vaccinated with X-38 than in those vaccinated with X-37. Significant antibody response to N2 was observed in 25% of those vaccinated with X-37 and in 69% of those vaccinated with X-38. Mean antibody response to N2 was twofold greater in those vaccinated with X-38. Heterotypic hemagglutination-inhibition was seen in 56% of those receiving X-38 vaccine. In preliminary plaque-inhibition titrations this heterotypic antibody did not have neuralizing activity. Testing of antibody response to N2 with earlier neuraminidase antigens demonstrated "original antigenic sin" from earlier priming. The superiority of the "neuraminidase-specific" X-38 (Heq1N2) vaccine as an immunogen for antibody to neuraminidase may reflect different processing of N2 when it is associated with a hemagglutinin to which the study population has not been previously exposed.

  11. New stilbenoid with inhibitory activity on viral neuraminidases from Erythrina addisoniae.

    PubMed

    Nguyen, Phi Hung; Na, MinKyun; Dao, Trong Tuan; Ndinteh, Derek Tantoh; Mbafor, Joseph Tanyi; Park, Jaeyoung; Cheong, Hyeonsook; Oh, Won Keun

    2010-11-15

    Influenza occurs with seasonal variations and reaches the peak prevalence in winter causing the death of many people worldwide. A few inhibitors of viral neuraminidase, including amantadine, rimantadine, zanamivir, and oseltamivir, have been used as influenza therapy. However, as drug-resistant influenza viruses are generated rapidly, there is a need to identify new agents for chemotherapy against influenza. Therefore, research on more effective drugs has been given high priority. During the course of an anti-influenza screening program on natural products, two new compounds (1 and 2) along with seven known flavonoid derivatives (3-9) were isolated as active principles from an EtOAc-soluble extract of the root bark of Erythrina addisoniae. The stilbenoid (2) and chalcone (3, 4, and 6) compounds of the isolates exhibited stronger activity than the isoflavone ones. Compound 2, which is a formylated stilbenoid derivative, exhibited strong inhibition of both influenza H1N1 and H9N2 neuraminidases with IC(50) values of 8.80±0.34 μg/mL and 7.19±0.40 μg/mL, respectively. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Neuroinflammation induced by intracerebroventricular injection of microbial neuraminidase.

    PubMed

    Granados-Durán, Pablo; López-Ávalos, María D; Grondona, Jesús M; Gómez-Roldán, María Del Carmen; Cifuentes, Manuel; Pérez-Martín, Margarita; Alvarez, Martina; Rodríguez de Fonseca, Fernando; Fernández-Llebrez, Pedro

    2015-01-01

    In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 μm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1β and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content.

  13. Neuroinflammation Induced by Intracerebroventricular Injection of Microbial Neuraminidase

    PubMed Central

    Granados-Durán, Pablo; López-Ávalos, María D.; Grondona, Jesús M.; Gómez-Roldán, María del Carmen; Cifuentes, Manuel; Pérez-Martín, Margarita; Alvarez, Martina; Rodríguez de Fonseca, Fernando; Fernández-Llebrez, Pedro

    2015-01-01

    In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 μm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1β and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content. PMID:25853134

  14. [Active neuraminidase constituents of Polygonum cuspidatum against influenza A(H1N1) influenza virus].

    PubMed

    Chen, Kao-Tan; Zhou, Wei-Ling; Liu, Jia-Wei; Zu, Mian; He, Zi-Ning; Du, Guan-Hua; Chen, Wei-Wen; Liu, Ai-Lin

    2012-10-01

    To isolate and identify active neuraminidase constituents of Polygonum cuspidatum against influenza A (H1N1) influenza virus. On the basis of the bioassay-guided fractionation,such chromatographic methods as silica gel, sephadex LH-20 and HPLC were adopted to isolate active constituents of extracts from Polygonum cuspidatum, and their molecular structures were identifiied on the basis of their spectral data such as NMR and MS and physico-chemical properties. Seven compounds were isolated from the ethyl acetate extract of P. cuspidatum and identified as 2-methoxystypandrone (1), emodin (2), resveratrol (3), polydatin (4), emodin-8-O-beta-D-glucopyranoside (5), (E)-3, 5, 12-trihydroxystilbene-3-O-beta-D-glucopyranoside-2'-(3", 4", 5"-trihydroxybenzoate) (6) and catechin-3-O-gallate (7), respectively. Among them, the NA test showed that compounds 3, 6 and 7 had inhibitory effect against NAs activity, with IC50 values of 129.8, 44.8 and 21.3 micromol x L(-1), respectively. Moreover, the further CPE test showed compounds 6 and 7 had significant inhibitory effect against H1N influenza virus (EC50 = 5.9, 0.9 micromol x L(-1), respectively), with very low cytotoxicity to the host cells, their therapeutic selective index(SI) in MDCK cells ranged from 56 to 269. The neuraminidase inhibitors against H1N1 anti-influenza virus isolated from extracts of P. cuspidatum on the basis of the bioassay-guided fractionation are significant in specifying their therapeutic material basis and drug R&D against influenza.

  15. Neuraminidase as an enzymatic marker for detecting airborne Influenza virus and other viruses.

    PubMed

    Turgeon, Nathalie; Toulouse, Marie-Josée; Ho, Jim; Li, Dongqing; Duchaine, Caroline

    2017-02-01

    Little information is available regarding the effectiveness of air samplers to collect viruses and regarding the effects of sampling processes on viral integrity. The neuraminidase enzyme is present on the surface of viruses that are of agricultural and medical importance. It has been demonstrated that viruses carrying this enzyme can be detected using commercial substrates without having to process the sample by methods such as RNA extraction. This project aims at evaluating the effects of 3 aerosol-sampling devices on the neuraminidase enzyme activity of airborne viruses. The purified neuraminidase enzymes from Clostridium perfringens, a strain of Influenza A (H1N1) virus, the FluMist influenza vaccine, and the Newcastle disease virus were used as models. The neuraminidase models were aerosolized in aerosol chambers and sampled with 3 different air samplers (SKC BioSampler, 3-piece cassettes with polycarbonate filters, and Coriolis μ) to assess the effect on neuraminidase enzyme activity. Our results demonstrated that Influenza virus and Newcastle disease virus neuraminidase enzymes are resistant to aerosolization and sampling with all air samplers tested. Moreover, we demonstrated that the enzymatic neuraminidase assay is as sensitive as RT-qPCR for detecting low concentrations of Influenza virus and Newcastle disease virus. Therefore, given the sensitivity of the assay and its compatibility with air sampling methods, viruses carrying the neuraminidase enzyme can be rapidly detected from air samples using neuraminidase activity assay without having to preprocess the samples.

  16. Proline-Based Cyclic Dipeptides from Korean Fermented Vegetable Kimchi and from Leuconostoc mesenteroides LBP-K06 Have Activities against Multidrug-Resistant Bacteria

    PubMed Central

    Liu, Rui; Kim, Andrew H.; Kwak, Min-Kyu; Kang, Sa-Ouk

    2017-01-01

    Lactobacillus plantarum and Leuconostoc mesenteroides play a prominent role as functional starters and predominant isolates in the production of various types of antimicrobial compound-containing fermented foods, especially including kimchi. In the case of the bioactive cyclic dipeptides, their racemic diastereomers inhibitory to bacteria and fungi have been suggested to come solely from Lactobacillus spp. of these strains. We previously demonstrated the antifungal and antiviral activities of proline-based cyclic dipeptides, which were fractionated from culture filtrates of Lb. plantarum LBP-K10 originated from kimchi. However, cyclic dipeptides have not been identified in the filtrates, either from cultures or fermented subject matter, driven by Ln. mesenteroides, which have been widely used as starter cultures for kimchi fermentation. Most importantly, the experimental verification of cyclic dipeptide-content changes during kimchi fermentation have also not been elucidated. Herein, the antibacterial fractions, including cyclo(Leu-Pro) and cyclo(Phe-Pro), from Ln. mesenteroides LBP-K06 culture filtrates, which exhibited a typical chromatographic retention behavior (tR), were identified by using semi-preparative high-performance liquid chromatography and gas chromatography-mass spectrometry. Based on this finding, the proline-based cyclic dipeptides, including cyclo(Ser-Pro), cyclo(Tyr-Pro), and cyclo(Leu-Pro), were additionally identified in the filtrates only when fermenting Chinese cabbage produced with Ln. mesenteroides LBP-K06 starter cultures. The detection and isolation of cyclic dipeptides solely in controlled fermented cabbage were conducted under the control of fermentation-process parameters concomitantly with strong CDP selectivity by using a two-consecutive-purification strategy. Interestingly, cyclic dipeptides in the filtrates, when using this strain as a starter, increased with fermentation time. However, no cyclic dipeptides were observed in the

  17. Sialidosis and galactosialidosis: chromosomal assignment of two genes associated with neuraminidase-deficiency disorders

    SciTech Connect

    Mueller, O.T.; Henry, W.M.; Haley, L.L.; Byers, M.G.; Eddy, R.L.; Shows, T.B.

    1986-03-01

    The inherited human disorders sialidosis and galactosialidosis are the result of deficiencies of glycoprotein-specific ..cap alpha..-neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18; sialidase) activity. Two genes were determined to be necessary for expression of neuraminidase by using human-mouse somatic cell hybrids segregating human chromosomes. A panel of mouse RAG-human hybrid cells demonstrated a single-gene requirement for human neuraminidase and allowed assignment of this gene to the (pter ..-->.. q23) region of chromosome 10. A second panel of mouse thymidine kinase (TK)-deficient LM/TK/sup -/-human hybrid cells demonstrated that human neuraminidase activity required both chromosomes 10 and 20 to be present. Analysis of human neuraminidase expression in interspecific hybrid cells or polykaryocytes formed from fusion of mouse RAG or LM/TK/sup -/ cell lines with human sialidosis or galactosialidosis fibroblasts indicated that the RAG cell line complemented the galactosialidosis defect, but the LM/TK/sup -/ cell line did not. This eliminates the requirement for this gene in RAG-human hybrid cells and explains the different chromosome requirements of these two hybrid panels. Fusion of LM/TK/sup -/ cell hybrids lacking chromosome 10 or 20 and neuraminidase-deficient fibroblasts confirmed by complementation analysis that the sialidosis disorder results from a mutation on chromosome 10, presumably encoding the neuraminidase structural gene. Galactosialidosis is caused by a mutation in a second gene required for neuraminidase expression located on chromosome 20.

  18. A point mutation in influenza B neuraminidase confers resistance to peramivir and loss of slow binding.

    PubMed

    Baum, Ellen Z; Wagaman, Pamela C; Ly, Linh; Turchi, Ignatius; Le, Jianhua; Bucher, Doris; Bush, Karen

    2003-06-01

    The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.

  19. Isolation, purification, and properties of neuraminidase from Propionibacterium acnes.

    PubMed

    von Nicolai, H; Höffler, U; Zilliken, F

    1980-06-01

    Neuraminidase activity was discovered in 32 of 38 strains of Propionibacterium acnes. Enzyme production was studied in yeast extract bouillon of different pH containing various amounts of human milk as neuraminidase inductor. Enzyme activity was found in the bacterial sediments as well as in the culture filtrates. Since neither ultrasonic treatment nor lysozyme incubation of bacterial sediments did release reasonable amounts of enzyme, culture filtrates were used for enzyme preparation. Neuraminidase was isolated by 40% ammonium sulfate precipitation, dialysis, concentration and repeated gel chromatography on Sephadex G-100. The enzyme posesses a molecular weight of about 33 000 and a pH-optimum around 5.0. The Michaelis constants are 1.8 x 10(-3) M for alpha 2 leads to 3 linked N-acetylneuraminic acid (NeuAc) in II3NeuAc-Lac, 3.7 x 10(-3) M for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 2.1 x 10(-3) M for the alpha 2 leads to 8 linkage of II3 (comes from 2 alpha NeuAc8)2-Lac, respectively. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. oligosaccharides, glycolipids and glycoproteins, the enzyme exhibits its highest activity towards low molecular weight oligosaccharides. Activity is considerably lower on glycoproteins. Glycolipids (gangliosides) are only little attacked under conditions used in the test. However, there is no remarkable specificity towards one of the different linkage types of N-acetylneuraminic acid. In general, the enzyme reveals a specificity pattern similar to that found in other bacteria of low pathogenicity towards man.

  20. Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity

    PubMed Central

    Adu-Gyamfi, Emmanuel; Kim, Lori S.; Jardetzky, Theodore S.

    2016-01-01

    ABSTRACT Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus

  1. Correlation between low neuraminidase blood levels and a predisposition toward family-related breast cancer.

    PubMed

    Rothenberg, R E; Laraja, R D; Imran-Ul-Hag; Saber, A A; Nadkarni, M

    1996-11-01

    It has been shown that high sialic acid levels are often found in conjunction with breast cancer, and these high concentrations are thought to be due to deficiency of the enzyme neuraminidase. The study proposes to elicit a relationship between low levels of blood neuraminidase and a family history of breast cancer. Neuraminidase blood levels were measured in 30 healthy women between the ages of 35 and 65 years with no evidence of a family history of breast cancer (control group), and in 33 healthy women between the ages of 35 to 65 years, all of whom had immediate members of their families with breast cancer (study group). The mean level of the blood neuraminidase was found to be 1.375 units in the control group. On the other hand, the mean level for the study group was 1.256 units. The difference between the two groups is statistically significant, (P value < 0.01). It is important to note that in the study group 20 of the 33 participants, 60.6 per cent, had neuraminidase levels below the mean of the study group, whereas only 3 of the 30, 10 per cent, in the control group had neuraminidase levels below the mean of the study group. Deficiency of the enzyme neuraminidase may suggest an elevated risk for breast cancer.

  2. Neuraminidase inhibitory activities of quaternary isoquinoline alkaloids from Corydalis turtschaninovii rhizome.

    PubMed

    Kim, Jang Hoon; Ryu, Young Bae; Lee, Woo Song; Kim, Young Ho

    2014-11-01

    Clostridium perfringens is a Gram-positive spore-forming bacterium that causes food poisoning. The neuraminidase (NA) protein of C. perfringens plays a pivotal role in bacterial proliferation and is considered a novel antibacterial drug target. Based on screens for novel NA inhibitors, a 95% EtOH extract of Corydalis turtschaninovii rhizome showed NA inhibitory activity (68% at 30 μg/ml), which resulted in the isolation of 10 isoquinoline alkaloids; namely, palmatine (1), berberine (2), coptisine (3), pseudodehydrocorydaline (4), jatrorrhizine (5), dehydrocorybulbine (6), pseudocoptisine (7), glaucine (8), corydaline (9) and tetrahydrocoptisine (10). Interestingly, seven quaternary isoquinoline alkaloids 1-7 (IC50 = 12.8 ± 1.5 to 65.2 ± 4.5 μM) showed stronger NA inhibitory activity than the tertiary alkaloids 8-10. In addition, highly active compounds 1 and 2 showed reversible non-competitive behavior based on a kinetic study. Molecular docking simulations using the Autodock 4.2 software increased our understanding of receptor-ligand binding of these compounds. In addition, we demonstrated that compounds 1 and 2 suppressed bacterial growth.

  3. Hydration of ligands of influenza virus neuraminidase studied by the fragment molecular orbital method.

    PubMed

    Tokuda, Kana; Watanabe, Chiduru; Okiyama, Yoshio; Mochizuki, Yuji; Fukuzawa, Kaori; Komeiji, Yuto

    2016-09-01

    The fragment molecular orbital (FMO) method was applied to quantum chemical calculations of neuramic acid, the natural substrate of the influenza virus neuraminidase, and two of its competitive inhibitors, Oseltamivir (Tamiful(®)) and Zanamivir (Relenza(®)), to investigate their hydrated structures and energetics. Each of the three ligands was immersed in an explicit water solvent, geometry-optimized by classical MM and QM/MM methods, and subjected to FMO calculations with 2-, 3-, and 4-body corrections under several fragmentation options. The important findings were that QM/MM optimization was preferable to obtain reliable hydrated structures of the ligands, that the 3-body correction was important for quantitative evaluation of the solvation energy, and that the dehydration effect was most remarkable near the hydrophobic sections of the ligands. In addition, the hydration energy calculated by the explicit solvent was compared with the hydration free energy calculated by the implicit solvent via the Poisson-Boltzmann equation, and the two showed a fairly good correlation. These findings will serve as useful information for rapid drug design.

  4. Purification and properties of rabbit spermatozoal acrosomal neuraminidase.

    PubMed Central

    Srivastava, P N; Abou-Issa, H

    1977-01-01

    Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C. Images PLATE 1 PMID:66917

  5. [The effects of complex platinum compounds on the neuraminidase activity of the Sendai virus].

    PubMed

    Repanovici, R; Călinoiu, A; Iliescu, R; Löber, G; Popa, L M

    1989-01-01

    The effect of di- and tetravalent cis-diaminoplatinum chlorides on Sendai virus envelop HN glycoprotein was investigated. The partial inhibition of neuraminidase activity was greater in the case of the divalent platinum complex derivative.

  6. Structural and functional studies of Streptococcus pneumoniae neuraminidase B: An intramolecular trans-sialidase.

    PubMed

    Gut, Heinz; King, Samantha J; Walsh, Martin A

    2008-10-15

    The human pathogen Streptococcus pneumoniae expresses neuraminidase proteins that cleave sialic acids from complex carbohydrates. The pneumococcus genome encodes up to three neuraminidase proteins that have been shown to be important virulence factors. Here, we report the first structure of a neuraminidase from S. pneumoniae: the crystal structure of NanB in complex with its reaction product 2,7-anhydro-Neu5Ac. Our structural data, together with biochemical analysis, establish NanB as an intramolecular trans-sialidase with strict specificity towards alpha2-3 linked sialic acid substrates. In addition, we show that NanB differs in its substrate specificity from the other pneumococcal neuraminidase NanA.

  7. Neuraminidase treatment of human T lymphocytes: effect on Fc receptor phenotype and function.

    PubMed Central

    Schulof, R S; Fernandes, G; Good, R A; Gupta, S

    1980-01-01

    Purified peripheral blood T cells or T mu cells from normal healthy donors were treated in vitro with neuraminidase and examined for the expression of IgM Fc and IgG Fc receptors. Increasing concentrations of neuraminidase selectively removed IgM Fc receptors, whereas the number of T cells expressing IgG Fc receptors was significantly increased. Following neuraminidase treatment, IgM Fc receptors could be regenerated by reincubation of T cells at 37 degrees C. The regeneration of IgM Fc receptors could be blocked by treatment with cycloheximide. Neuraminidase treatment of purified T mu cells resulted in the expression of IgG Fc receptors on a subpopulation of T mu lymphocytes. A small percentage of the neuraminidase-treated T cells expressed receptors for both IgG and IgM. Treatment of T cells with neuraminidase did not effect T cell-mediated spontaneous cytotoxicity (SLMC) or antibody-dependent cellular cytotoxicity (ADCC). Our results indicate that T cell Fc receptor phenotypes can be modulated in vitro without significantly altering their functional capacity. PMID:6968261

  8. Drug Susceptibility Evaluation of an Influenza A(H7N9) Virus by Analyzing Recombinant Neuraminidase Proteins.

    PubMed

    Gubareva, Larisa V; Sleeman, Katrina; Guo, Zhu; Yang, Hua; Hodges, Erin; Davis, Charles T; Baranovich, Tatiana; Stevens, James

    2017-09-15

    Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse. Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed. All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir. The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses.

  9. Survey of neuraminidase production by Clostridium butyricum, Clostridium beijerinckii, and Clostridium difficile strains from clinical and nonclinical sources.

    PubMed Central

    Popoff, M R; Dodin, A

    1985-01-01

    Neuraminidase production was investigated in 57 Clostridium butyricum strains, 16 Clostridium beijerinckii strains, and 25 Clostridium difficile strains. Neuraminidase activity was found only in C. butyricum strains originating from one human newborn with neonatal necrotizing enterocolitis, two newborns with hemorrhagic colitis, one infected placenta, and one adult with peritonitis, It was concluded that neuraminidase was not a major virulence factor in C. butyricum strains. PMID:4056013

  10. Influenza virus neuraminidase (NA): a target for antivirals and vaccines.

    PubMed

    Jagadesh, Anitha; Salam, Abdul Ajees Abdul; Mudgal, Piya Paul; Arunkumar, Govindakarnavar

    2016-08-01

    Influenza, the most common infectious disease, poses a great threat to human health because of its highly contagious nature and fast transmissibility, often leading to high morbidity and mortality. Effective vaccination strategies may aid in the prevention and control of recurring epidemics and pandemics associated with this infectious disease. However, antigenic shifts and drifts are major concerns with influenza virus, requiring effective global monitoring and updating of vaccines. Current vaccines are standardized primarily based on the amount of hemagglutinin, a major surface antigen, which chiefly constitutes these preparations along with the varying amounts of neuraminidase (NA). Anti-influenza drugs targeting the active site of NA have been in use for more than a decade now. However, NA has not been approved as an effective antigenic component of the influenza vaccine because of standardization issues. Although some studies have suggested that NA antibodies are able to reduce the severity of the disease and induce a long-term and cross-protective immunity, a few major scientific issues need to be addressed prior to launching NA-based vaccines. Interestingly, an increasing number of studies have shown NA to be a promising target for future influenza vaccines. This review is an attempt to consolidate studies that reflect the strength of NA as a suitable vaccine target. The studies discussed in this article highlight NA as a potential influenza vaccine candidate and support taking the process of developing NA vaccines to the next stage.

  11. Substrate specificity of the neuraminidase of different mumps virus strains.

    PubMed

    Klamm, H; Pollex, G

    1984-11-01

    The hydrolysis of neuraminlactose, fetuin, ovomucoid, kappa-caseinglycopeptide and mucine from the bovine submaxillary gland with the neuraminidase (NA) of mumps virus strains Jeryl Lynn, Enders and Berlin 9/76 was investigated to determine the pH-dependence of the reaction with the different substrates. The corresponding curves showed splitting into several peaks with reaction maxima ranging from pH 4.7 to pH 6.7. This occurred probably due to the heterogeneity of the virus samples. The NA of each virus strain had the best reaction affinity to neuraminlactose followed by decreasing affinities to ovomucoid, fetuin and kappa-caseinglycopeptide. The mucine from bovine submaxillary gland was not digested at all. The highest hydrolysis of each substrate was found with the Jeryl Lynn strain, which also possessed the lowest substrate specificity. It was followed by that of strain Enders and finally by isolate Berlin 9/76 which, in turn, had the highest substrate specificity. The lower substrate specificity of mumps virus NA seemed to correlate with a lower degree of its cytopathogenicity for hamster ependyma cells.

  12. I222 Neuraminidase Mutations Further Reduce Oseltamivir Susceptibility of Indonesian Clade 2.1 Highly Pathogenic Avian Influenza A(H5N1) Viruses

    PubMed Central

    McKimm-Breschkin, Jennifer L.; Barrett, Susan; Pudjiatmoko; Azhar, Muhammad; Wong, Frank Y. K.; Selleck, Paul; Mohr, Peter G.; McGrane, James; Kim, Mia

    2013-01-01

    We have tested the susceptibility to neuraminidase inhibitors of 155 clade 2.1 H5N1 viruses from Indonesia, isolated between 2006–2008 as well as 12 clade 1 isolates from Thailand and Cambodia from 2004–2007 using a fluorometric MUNANA-based enzyme inhibition assay. The Thailand and Cambodian clade 1 isolates tested here were all susceptible to oseltamivir and zanamivir, and sequence comparison indicated that reduced oseltamivir susceptibility we observed previously with clade 1 Cambodian isolates correlated with an S246G neuraminidase mutation. Eight Indonesian viruses (5%), all bearing I222 neuraminidase mutations, were identified as mild to extreme outliers for oseltamivir based on statistical analysis by box plots. IC50s were from 50 to 500-fold higher than the reference clade 1 virus from Viet Nam, ranging from 43–75 nM for I222T/V mutants and from 268–349 nM for I222M mutants. All eight viruses were from different geographic locales; all I222M variants were from central Sumatra. None of the H5N1 isolates tested demonstrated reduced susceptibility to zanamivir (IC50s all <5 nM). All I222 mutants showed loss of slow binding specifically for oseltamivir in an IC50 kinetics assay. We identified four other Indonesian isolates with higher IC50s which also demonstrated loss of slow binding, including one virus with an I117V mutation. There was a minimal effect on the binding of zanamivir and peramivir for all isolates tested. As H5N1 remains a potential pandemic threat, the incidence of mutations conferring reduced oseltamivir susceptibility is concerning and emphasizes the need for greater surveillance of drug susceptibility. PMID:23776615

  13. In vivo production of neuraminidase by Pasteurella haemolytica A1 in goats after transthoracic challenge.

    PubMed Central

    Straus, D C; Purdy, C W

    1994-01-01

    Nine goats were injected transthoracically with Pasteurella haemolytica A1 to determine if an extracellular bacterial enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 9) each received 1 ml of 7.25 x 10(5) live P. haemolytica A1 cells in polyacrylate beads transthoracically in the left lung on days 0 and 21. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 21. Serum was obtained from all animals on days -4, 3, 7, 14, 21, 24, and 32. Preimmune serum from all animals showed no detectable antibody to P. haemolytica A1 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals possessed a significant antibody concentration in response to the P. haemolytica A1 neuraminidase. On day 32, serum samples from the nine infected animals possessed enzyme neutralizing activity that ranged from 62% to 100%. Anti-neuraminidase antibody could be detected as early as day 14 by the enzyme neutralization assay. These data demonstrate that the enzyme neuraminidase is produced in vivo during an active P. haemolytica A1 lobar infection. PMID:7927740

  14. Mutation effects of neuraminidases and their docking with ligands: a molecular dynamics and free energy calculation study

    NASA Astrophysics Data System (ADS)

    Yang, Zhiwei; Yang, Gang; Zhou, Lijun

    2013-11-01

    A systematic study has been performed on neuraminidase (NA) mutations and NA-inhibitor docked complexes, with the aim to understand protein-ligand interactions and design broad-spectrum antiviral drugs with minimal resistances. The catalytic D151 residue is likely to mutate while others are relatively conserved. The NA active-site conformations are altered by mutations, but more alterations do not necessarily result in larger deviations to the binding properties. The effects of all related mutations have been discussed; e.g., for the arginine triad (R118, R292 and R371), it is found that residue R118 plays the most significant role during ligand binding. Generally, the calculated binding free energies agree well with the experimental observations. Susceptibility of influenza virus to NA inhibitors can be reinforced by some mutations; e.g., the binding free energies of ligands with N2 subtype increase from -18.0 to -42.1 kcal mol-1 by the E119D mutation. Mutations of the various NA subtypes often cause similar conformational and binding changes, explaining the occurrence of cross resistances; nonetheless, differences can be detected in some cases that correspond to subtype-specific resistances. For all NA subtypes, the electrostatic contributions are the major driving force for ligand binding and largely responsible for the binding differences between the wild-type and mutated NA proteins.

  15. Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses.

    PubMed

    Wu, Yan; Bi, Yuhai; Vavricka, Christopher J; Sun, Xiaoman; Zhang, Yanfang; Gao, Feng; Zhao, Min; Xiao, Haixia; Qin, Chengfeng; He, Jianhua; Liu, Wenjun; Yan, Jinghua; Qi, Jianxun; Gao, George F

    2013-12-01

    An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.

  16. Evolutionary interactions between haemagglutinin and neuraminidase in avian influenza

    PubMed Central

    2013-01-01

    Background Reassortment between the RNA segments encoding haemagglutinin (HA) and neuraminidase (NA), the major antigenic influenza proteins, produces viruses with novel HA and NA subtype combinations and has preceded the emergence of pandemic strains. It has been suggested that productive viral infection requires a balance in the level of functional activity of HA and NA, arising from their closely interacting roles in the viral life cycle, and that this functional balance could be mediated by genetic changes in the HA and NA. Here, we investigate how the selective pressure varies for H7 avian influenza HA on different NA subtype backgrounds. Results By extending Bayesian stochastic mutational mapping methods to calculate the ratio of the rate of non-synonymous change to the rate of synonymous change (dN/dS), we found the average dN/dS across the avian influenza H7 HA1 region to be significantly greater on an N2 NA subtype background than on an N1, N3 or N7 background. Observed differences in evolutionary rates of H7 HA on different NA subtype backgrounds could not be attributed to underlying differences between avian host species or virus pathogenicity. Examination of dN/dS values for each subtype on a site-by-site basis indicated that the elevated dN/dS on the N2 NA background was a result of increased selection, rather than a relaxation of selective constraint. Conclusions Our results are consistent with the hypothesis that reassortment exposes influenza HA to significant changes in selective pressure through genetic interactions with NA. Such epistatic effects might be explicitly accounted for in future models of influenza evolution. PMID:24103105

  17. Regulation of phagocytosis in macrophages by neuraminidase 1.

    PubMed

    Seyrantepe, Volkan; Iannello, Alexandre; Liang, Feng; Kanshin, Evgeny; Jayanth, Preethi; Samarani, Suzanne; Szewczuk, Myron R; Ahmad, Ali; Pshezhetsky, Alexey V

    2010-01-01

    The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA(S190A)) and a double CathA/Neu1 deficiency (CathA(S190A-Neo)). Macrophages of CathA(S190A-Neo) mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA(S190A) mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 degrees C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcgammaR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcgammaR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.

  18. Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

    PubMed

    Singh, Anirudh K; Woodiga, Shireen A; Grau, Margaret A; King, Samantha J

    2017-03-01

    Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. Copyright © 2017 American Society for Microbiology.

  19. Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets

    PubMed Central

    Singh, Anirudh K.; Woodiga, Shireen A.; Grau, Margaret A.

    2016-01-01

    ABSTRACT Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors. PMID:27993975

  20. On the activation of plasma membrane ecto-enzymes by treatment with neuraminidase.

    PubMed

    Trams, E G; Lauter, C J; Banfield, W G

    1976-11-01

    It had been proposed that sialyl-residues on the surface of the cell control the activity of certain plasma membrane ecto-enzymes. We have tested the effects of several established (or presumptive) ecto-enzymes in tissue cultures of CNS-derived cells. Application of neuraminidases to cultured mouse neuroblastoma (N-18), neonatal Syrian hamster astrocytes (NN), human astrocytoma (Cox clone) and two lines of primary mouse astroblasts failed to change the activity of ecto-ATPase and 5'-nucleotidase. Only two of the seven neuraminidase preparations produced marked or moderate increases in inorganic pyrophosphatase, p-nitrophenylphosphatase and cholinesterase. We have concluded that the stimulation of these enzymes was not due to removal of sialyl-residues. We suggest that contaminants (haemolysins?) in neuraminidase preparations of Clostridium perfringens increased membrane permeability and facilitated substrate-product translocation.

  1. Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient.

    PubMed

    Pontoriero, Andrea; Avaro, Martín; Benedetti, Estefania; Russo, Mara; Czech, Andrea; Periolo, Natalia; Campos, Ana; Zamora, Ana; Baumeister, Elsa

    2016-12-01

    Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA) protein of influenza A(H3N2) viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78%) were A(H3N2) strains, 244 (21.3%) were type B strains, and 8 (0.7%) were A(H1N1)pdm09 strains. Out of 888 A(H3N2) samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2) samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients.

  2. Surveillance of antiviral resistance markers in Argentina: detection of E119V neuraminidase mutation in a post-treatment immunocompromised patient

    PubMed Central

    Pontoriero, Andrea; Avaro, Martín; Benedetti, Estefania; Russo, Mara; Czech, Andrea; Periolo, Natalia; Campos, Ana; Zamora, Ana; Baumeister, Elsa

    2016-01-01

    Although vaccines are the best means of protection against influenza, neuraminidase inhibitors are currently the main antiviral treatment available to control severe influenza cases. One of the most frequent substitutions in the neuraminidase (NA) protein of influenza A(H3N2) viruses during or soon after oseltamivir administration is E119V mutation. We describe the emergence of a mixed viral population with the E119E/V mutation in the NA protein sequence in a post-treatment influenza sample collected from an immunocompromised patient in Argentina. This substitution was identified by a real-time reverse transcriptase polymerase chain reaction (RT-PCR) protocol and was confirmed by direct Sanger sequencing of the original sample. In 2014, out of 1140 influenza samples received at the National Influenza Centre, 888 samples (78%) were A(H3N2) strains, 244 (21.3%) were type B strains, and 8 (0.7%) were A(H1N1)pdm09 strains. Out of 888 A(H3N2) samples, 842 were tested for the E119V substitution by quantitative RT-PCR: 841 A(H3N2) samples had the wild-type E119 genotype and in one sample, a mixture of viral E119/ V119 subpopulations was detected. Influenza virus surveillance and antiviral resistance studies can lead to better decisions in health policies and help in medical treatment planning, especially for severe cases and immunocompromised patients. PMID:27849220

  3. The discovery of (2R,4R)-N-(4-chlorophenyl)-N- (2-fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-dicarboxamide (PD 0348292), an orally efficacious factor Xa inhibitor.

    PubMed

    Kohrt, Jeffrey T; Bigge, Christopher F; Bryant, John W; Casimiro-Garcia, Agustin; Chi, Liguo; Cody, Wayne L; Dahring, Tawny; Dudley, Danette A; Filipski, Kevin J; Haarer, Staci; Heemstra, Ron; Janiczek, Nancy; Narasimhan, Lakshmi; McClanahan, Thomas; Peterson, J Thomas; Sahasrabudhe, Vaisheli; Schaum, Robert; Van Huis, Chad A; Welch, Kathleen M; Zhang, Erli; Leadley, Robert J; Edmunds, Jeremy J

    2007-08-01

    Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.

  4. Extracellular neuraminidase production by a Pasteurella multocida A:3 strain associated with bovine pneumonia.

    PubMed Central

    White, D J; Jolley, W L; Purdy, C W; Straus, D C

    1995-01-01

    The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme. PMID:7729875

  5. Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid

    PubMed Central

    Ruangrung, Kanyarat; Suptawiwat, Ornpreya; Maneechotesuwan, Kittipong; Boonarkart, Chompunuch; Chakritbudsabong, Warunya; Assawabhumi, Jirawatna; Bhattarakosol, Parvapan; Uiprasertkul, Mongkol; Puthavathana, Pilaipan; Wiriyarat, Witthawat; Jongkaewwattana, Anan

    2016-01-01

    ABSTRACT Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the

  6. The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin

    PubMed Central

    1991-01-01

    Trypanosoma cruzi expresses a developmentally regulated neuraminidase (TCNA) implicated in parasite invasion of cells. We isolated full- length DNA clones encoding TCNA. Sequence analysis demonstrated an open reading frame coding for a polypeptide of 1,162 amino acids. In the N- terminus there is a cysteine-rich domain containing a stretch of 332 amino acids nearly 30% identical to the Clostridium perfringens neuraminidase, three repeat motifs highly conserved in bacterial and viral neuraminidases, and two segments with similarity to the YWTD repeats found in the low density lipoprotein (LDL) receptor and in other vertebrate and invertebrate proteins. This domain is connected by a structure characteristic of type III modules of fibronectin to a long terminal repeat (LTR) consisting of 44 full length copies of twelve amino acids rich (75%) in serine, threonine, and proline. LTR is unusual in that it contains at least 117 potential phosphorylation sites. At the extreme C-terminus is a hydrophobic segment of 35 amino acids, which could mediate anchorage of TCNA to membranes via a glycosylphosphatidylinositol linkage. This is the first time a protozoan protein has been found to contain a YWTD repeat and a fibronectin type III module. The domain structure of TCNA suggests that the enzyme may have functions additional to its catalytic activity such as in protein-protein interaction, which could play a role in T. cruzi binding to host cells. PMID:1711561

  7. Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.

    PubMed

    Triana-Baltzer, Gallen B; Gubareva, Larisa V; Klimov, Alexander I; Wurtman, David F; Moss, Ronald B; Hedlund, Maria; Larson, Jeffrey L; Belshe, Robert B; Fang, Fang

    2009-11-06

    Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

  8. Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro

    PubMed Central

    Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

    2014-01-01

    Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-l-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. PMID:25339691

  9. Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila.

    PubMed

    Huang, Kun; Wang, Mao M; Kulinich, Anna; Yao, Hong L; Ma, Hong Y; Martínez, Juana E R; Duan, Xu C; Chen, Huan; Cai, Zhi P; Flitsch, Sabine L; Liu, Li; Voglmeir, Josef

    2015-10-13

    Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu(2+) and Zn(2+), and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.

  10. Quantitative Predictions of Binding Free Energy Changes in Drug-Resistant Influenza Neuraminidase

    DTIC Science & Technology

    2012-08-30

    Energy Changes in Drug-Resistant Influenza Neuraminidase. PLoS Comput Biol 8(8): e1002665. doi:10.1371/journal.pcbi.1002665 Editor: Alex Mackerell ...Calculation of protein-ligand binding affinities. Annu Rev Biophys Biomol Struct 36: 21–42. 17. Guvench O, MacKerell Jr AD (2009) Computational evaluation

  11. Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

    PubMed

    Bellini, T; Pasti, C; Manfrinato, M C; Tomasi, M; Dallocchio, F

    1999-08-27

    The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase. Copyright 1999 Academic Press.

  12. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers

    PubMed Central

    Wan, Hongquan; Yang, Hua; Shore, David A.; Garten, Rebecca J.; Couzens, Laura; Gao, Jin; Jiang, Lianlian; Carney, Paul J.; Villanueva, Julie; Stevens, James; Eichelberger, Maryna C.

    2015-01-01

    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. PMID:25668439

  13. Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process.

    PubMed

    Cobaleda, C; Muñoz-Barroso, I; Sagrera, A; Villar, E

    2002-04-01

    Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.

  14. Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence

    PubMed Central

    Kahya, Hasan F.; Andrew, Peter W.

    2017-01-01

    Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia. PMID:28257499

  15. Comparative effect and fate of non-entrapped and liposome-entrapped neuraminidase injected into rats

    PubMed Central

    Gregoriadis, Gregory; Putman, Daphne; Louis, Loizos; Neerunjun, Diane

    1974-01-01

    Non-entrapped and liposome-entrapped Clostridium perfringens neuraminidase (0.5–0.6 unit) was injected into rats and its fate as well as its effect on plasma and erythrocyte N-acetylneuraminic acid was investigated. The following observations were made. (1) Although removal of both non-entrapped and liposome-entrapped neuraminidase from the circulation was completed within 5h after injection, their recovery in tissues was distinctly different; 7–10% of the injected non-entrapped enzyme was found in the liver and none in the liver lysosomal fraction or the spleen. In contrast, 20–26% of the liposome-entrapped enzyme was found in the liver of which 60–69% was in the lysosomal fraction. Spleen contained 3.6–5.0% of the enzyme. (2) The presence of the non-entrapped neuraminidase in blood led to the extensive desialylation of plasma and to a decrease in the concentration or total removal from the circulation of some of the plasma glycoproteins. (3) Injection of non-entrapped neuraminidase also led to the partial desialylation of erythrocytes the life span of which was diminished and their uptake by the liver and spleen augmented. (4) Entrapment of neuraminidase in liposomes before its injection prevented the enzyme from acting on its substrate in plasma or on the erythrocyte surface, and values obtained for plasma glycoproteins and erythrocyte survival were similar to those observed in control rats. (5) Entrapment in liposomes of therapeutic hydrolases intended for the degradation of substances stored within the tissue lysosomes of patients with storage diseases could prevent the potentially hazardous enzymic action of hydrolases in blood and at the same time direct the enzymes to the intracellular sites where they are needed. ImagesPLATE 1PLATE 2 PMID:4375965

  16. Effect of poly-L-lysine and neuraminidase on the infectivity of Trypanosoma cruzi in cultured HeLa cells.

    PubMed

    Gamarro, F; Castanys, S; Ruiz-Perez, L M; Adroher, F J; Osuna, A

    1985-01-01

    The percentage of parasitisation and index of adherence of Trypanosoma cruzi has been studied when host HeLa cells or metacyclic forms were pretreated with neuraminidase or with poly-L-lysine. The percentage of parasitisation was significatively reduced (P less than or equal to 0.001) when cells were pretreated with poly-L-lysine while pretreatment with neuraminidase caused no apparent effects. On the other hand, the adherence of the metacyclic forms pretreated with poly-L-lysine or neuraminidase was significantly higher than that of the control group.

  17. Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus.

    PubMed

    Quiliano, Miguelmiguel; Valdivia-Olarte, Hugo; Olivares, Carlos; Requena, David; Gutiérrez, Andrés H; Reyes-Loyola, Paola; Tolentino-Lopez, Luis E; Sheen, Patricia; Briz, Verónica; Muñoz-Fernández, Maria A; Correa-Basurto, José; Zimic, Mirko

    2013-01-01

    The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents.

  18. Molecular distribution of amino acid substitutions on neuraminidase from the 2009 (H1N1) human influenza pandemic virus

    PubMed Central

    Quiliano, MiguelMiguel; Valdivia-Olarte, Hugo; Olivares, Carlos; Requena, David; Gutiérrez, Andrés H; Reyes-Loyola, Paola; Tolentino-Lopez, Luis E; Sheen, Patricia; Briz, Verónica; Muñoz-Fernández, Maria A; Correa-Basurto, José; Zimic, Mirko

    2013-01-01

    The pandemic influenza AH1N1 (2009) caused an outbreak of human infection that spread to the world. Neuraminidase (NA) is an antigenic surface glycoprotein, which is essential to the influenza infection process, and is the target of anti-flu drugs oseltamivir and zanamivir. Currently, NA inhibitors are the pillar pharmacological strategy against seasonal and global influenza. Although mutations observed after NA-inhibitor treatment are characterized by changes in conserved amino acids of the enzyme catalytic site, it is possible that specific amino acid substitutions (AASs) distant from the active site such as H274Y, could confer oseltamivir or zanamivir resistance. To better understand the molecular distribution pattern of NA AASs, we analyzed NA AASs from all available reported pandemic AH1N1 NA sequences, including those reported from America, Africa, Asia, Europe, Oceania, and specifically from Mexico. The molecular distributions of the AASs were obtained at the secondary structure domain level for both the active and catalytic sites, and compared between geographic regions. Our results showed that NA AASs from America, Asia, Europe, Oceania and Mexico followed similar molecular distribution patterns. The compiled data of this study showed that highly conserved amino acids from the NA active site and catalytic site are indeed being affected by mutations. The reported NA AASs follow a similar molecular distribution pattern worldwide. Although most AASs are distributed distantly from the active site, this study shows the emergence of mutations affecting the previously conserved active and catalytic site. A significant number of unique AASs were reported simultaneously on different continents. PMID:23930018

  19. Characterizing Loop Dynamics and Ligand Recognition in Human- and Avian-Type Influenza Neuraminidases via Generalized Born Molecular Dynamics and End-Point Free Energy Calculations

    SciTech Connect

    Amaro, Rommie E; Cheng, Xiaolin; Ivanov, Ivaylo N; Xu, Dong; McCammon, Jonathan

    2009-01-01

    The comparative dynamics and inhibitor binding free energies of group-1 and group-2 pathogenic influenza A subtype neuraminidase (NA) enzymes are of fundamental biological interest and relevant to structure-based drug design studies for antiviral compounds. In this work, we present seven generalized Born molecular dynamics simulations of avian (N1)- and human (N9)-type NAs in order to probe the comparative flexibility of the two subtypes, both with and without the inhibitor oseltamivir bound. The enhanced sampling obtained through the implicit solvent treatment suggests several provocative insights into the dynamics of the two subtypes, including that the group-2 enzymes may exhibit similar motion in the 430-binding site regions but different 150-loop motion. End-point free energy calculations elucidate the contributions to inhibitor binding free energies and suggest that entropic considerations cannot be neglected when comparing across the subtypes. We anticipate the findings presented here will have broad implications for the development of novel antiviral compounds against both seasonal and pandemic influenza strains.

  20. Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

    PubMed

    Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

    2008-09-01

    The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

  1. Purification and characterization of the hemagglutinin-neuraminidase of Porcine rubulavirus LPMV.

    PubMed

    Reyes-Leyva, J; Espinosa, B; Santos, G; Zenteno, R; Hernández, J; Vallejo, V; Zenteno, E

    1999-09-01

    The Hemagglutinin-Neuraminidase (HN) from the LPMV strain of Porcine rubulavirus was purified from virions by ultracentrifugation in a continuous 20-60% sucrose gradient and by ion exchange chromatography. The HN is a glycoprotein of 66 kDa constituted by 50.5, 13.3 and 13.6% of non polar, uncharged polar, and charged polar amino acids, respectively. The HN contains 4% of carbohydrates, its glycannic portion is constituted by Man, Gal, GlcNAc, GalNAc, and Neu5Ac in 3:3:4:1:1 molar ratios. The HN possesses hemagglutinating activity in the presence of erythrocytes from several animal species, including human ABO, and treating the erythrocytes with neuraminidase or pronase abolishes this activity. The binding specificity of the purified HN was determined by hapten inhibition assays, indicating that the hemagglutinating activity of the HN is specific for sialic acid and Neu5Acalpha2,3Gal-containing structures.

  2. Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III.

    PubMed

    Lawrence, Michael C; Borg, Natalie A; Streltsov, Victor A; Pilling, Patricia A; Epa, V Chandana; Varghese, Joseph N; McKimm-Breschkin, Jennifer L; Colman, Peter M

    2004-01-30

    The three-dimensional structure of the haemagglutinin-neuraminidase (HN) from a human parainfluenza virus is described at ca 2.0 A resolution, both in native form and in complex with three substrate analogues. In support of earlier work on the structure of the homologous protein from the avian pathogen Newcastle disease virus (NDV), we observe a dimer of beta-propellers and find no evidence for spatially separated sites performing the receptor-binding and neuraminidase functions of the protein. As with the NDV HN, the active site of the HN of parainfluenza viruses is structurally flexible, suggesting that it may be able to switch between a receptor-binding state and a catalytic state. However, in contrast to the NDV structures, we observe no ligand-induced structural changes that extend beyond the active site and modify the dimer interface.

  3. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

    PubMed

    Sato, H; Aono, S; Semba, R; Kashiwamata, S

    1987-11-15

    Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.

  4. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

    SciTech Connect

    Wu, Zhengliang L.; Zhou, Hui; Ethen, Cheryl M.; Reinhold, Vernon N.

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain

  5. Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

    PubMed Central

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection. PMID:22025518

  6. H1N1 influenza A virus neuraminidase modulates infectivity in mice.

    PubMed

    Ferraris, Olivier; Escuret, Vanessa; Bouscambert, Maude; Casalegno, Jean-Sébastien; Jacquot, Frédéric; Raoul, Hervé; Caro, Valérie; Valette, Martine; Lina, Bruno; Ottmann, Michèle

    2012-03-01

    In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays.

    PubMed

    Kongkamnerd, Jarinrat; Milani, Adelaide; Cattoli, Giovanni; Terregino, Calogero; Capua, Ilaria; Beneduce, Luca; Gallotta, Andrea; Pengo, Paolo; Fassina, Giorgio; Monthakantirat, Orawan; Umehara, Kaoru; De-Eknamkul, Wanchai; Miertus, Stanislav

    2011-08-01

    Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

  8. Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2.

    PubMed

    Khedri, Zahra; Li, Yanhong; Cao, Hongzhi; Qu, Jingyao; Yu, Hai; Muthana, Musleh M; Chen, Xi

    2012-08-14

    Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

  9. A surface plasmon resonance assay for measurement of neuraminidase inhibition, sensitivity of wild-type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir.

    PubMed

    Somasundaram, Balaji; Fee, Conan J; Fredericks, Rayleen; Watson, Andrew J A; Fairbanks, Antony J; Hall, Richard J

    2015-09-01

    Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half-life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild-type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High-five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6-hexanediamine (HDA)) was conjugated to the 7-hydroxyl group of zanamivir, and the construct (HDA-zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA-zanamivir comprised a bio-specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC50-spr ), using a log dose-response curve fit. Although both NA isoforms had almost identical IC50-spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC50-spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs.

  10. Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge

    PubMed Central

    Cardoso, Francisco Miguel; Ibañez, Lorena Itatí; Van den Hoecke, Silvie; De Baets, Sarah; Smet, Anouk; Roose, Kenny; Schepens, Bert; Descamps, Francis J.; Fiers, Walter; Muyldermans, Serge

    2014-01-01

    ABSTRACT Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses

  11. Zanamivir immobilized magnetic beads for voltammetric measurement of neuraminidase at gold-modified boron doped diamond electrode

    SciTech Connect

    Wahyuni, Wulan Tri; Ivandini, Tribidasari A.; Saepudin, Endang; Einaga, Yasuaki

    2016-04-19

    Biomolecule modified magnetic beads has been widely used in separation and sensing process. This study used streptavidin modified magnetic beads to immobilize biotin modified zanamivir. Biotin-streptavidin affinity facilitates immobilization of zanamivir on magnetic beads. Then interaction of zanamivir and neuraminidase was adopted as basic for enzyme detection. Detection of neuraminidase was performed at gold modified BDD using cyclic voltammetry technique. The measurement was carried out based on alteration of electrochemical signals of working electrode as neuraminidase response. The result showed that zanamivir was successfully immobilized on magnetic beads. The optimum amount of magnetic beads for zanamivir immobilization was 120 ug. Linear responses of neuraminidase were detected in concentration range of 0-15 mU. Detection limit (LOD) of measurement was 2.32 mU (R2 = 0.959) with precision as % RSD of 1.41%. Measurement of neuraminidase on magnetic beads could be also performed in the presence of mucin matrix. The linearity range was 0-8 mU with LOD of 0.64 mU (R2 = 0.950) and % RSD of 7.25%.

  12. A heterologous neuraminidase subtype strategy for the differentiation of infected and vaccinated animals (DIVA) for avian influenza virus using an alternative neuraminidase inhibition test.

    PubMed

    Avellaneda, Gloria; Sylte, Matt J; Lee, Chang-Won; Suarez, David L

    2010-03-01

    The option of vaccinating poultry against avian influenza (AI) as a control tool is gaining greater acceptance by governments and the poultry industry worldwide. One disadvantage about vaccination with killed whole-virus vaccines is the resulting inability to use common serologic diagnostic tests for surveillance to identify infected flocks. There has been considerable effort to develop a reliable test for the differentiation of infected from vaccinated animals (DIVA). The heterologous neuraminidase (NA) subtype DIVA approach has been used with some success in the field accompanied by an ad hoc serologic test. The traditional NA inhibition (NI) test can be used for all nine NA subtypes, but it is time consuming, and it is not designed to screen large numbers of samples. In this study, a quantitative NI test using MUN (2'-[4-methylumbelliferyl]-alpha-D-Nacetylneuraminic acid sodium salt hydrate) as an NA substrate was investigated as an alternative to the traditional fetuin-based NI test in a heterologous neuraminidase DIVA strategy. Serum NI activity was determined in chickens administered different vaccines containing different H5 and NA subtypes and challenged with a highly pathogenic avian influenza (HPAI) H5N2 virus. Prior to challenge, the NI DIVA test clearly discriminated between chickens receiving vaccines containing different antigens (e.g., N8 or N9) from control birds that had no NA antibody. Some birds began to seroconvert 1 wk postchallenge, and 100% of the vaccinated birds had significant levels of N2 NI activity. This activity did not interfere with the presence of vaccine-induced NI activity against N8 or N9 subtypes. The level of N2-specific NI activity continued to increase to the last sampling date, 4 wk postchallenge, indicating the potential use for the heterologous NA-based DIVA strategy in the field.

  13. Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin

    PubMed Central

    Rajendran, Madhusudan; Ermler, Megan E.; Bunduc, Paul; Amanat, Fatima; Izikson, Ruvim; Cox, Manon; Palese, Peter; Eichelberger, Maryna

    2017-01-01

    ABSTRACT Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for “original antigenic sin.” Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.) PMID:28325769

  14. Sensitivity of molecular docking to induced fit effects in influenza virus neuraminidase

    NASA Astrophysics Data System (ADS)

    Birch, Louise; Murray, Christopher W.; Hartshorn, Michael J.; Tickle, Ian J.; Verdonk, Marcel L.

    2002-12-01

    Many proteins undergo small side chain or even backbone movements on binding of different ligands into the same protein structure. This is known as induced fit and is potentially problematic for virtual screening of databases against protein targets. In this report we investigate the limits of the rigid protein approximation used by the docking program, GOLD, through cross-docking using protein structures of influenza neuraminidase. Neuraminidase is known to exhibit small but significant induced fit effects on ligand binding. Some neuraminidase crystal structures caused concern due to the bound ligand conformation and GOLD performed poorly on these complexes. A `clean' set, which contained unique, unambiguous complexes, was defined. For this set, the lowest energy structure was correctly docked (i.e. RMSD < 1.5 Å away from the crystal reference structure) in 84% of proteins, and the most promiscuous protein (1mwe) was able to dock all 15 ligands accurately including those that normally required an induced fit movement. This is considerably better than the 70% success rate seen with GOLD against general validation sets. Inclusion of specific water molecules involved in water-mediated hydrogen bonds did not significantly improve the docking performance for ligands that formed water-mediated contacts but it did prevent docking of ligands that displaced these waters. Our data supports the use of a single protein structure for virtual screening with GOLD in some applications involving induced fit effects, although care must be taken to identify the protein structure that performs best against a wide variety of ligands. The performance of GOLD was significantly better than the GOLD implementation of ChemScore and the reasons for this are discussed. Overall, GOLD has shown itself to be an extremely good, robust docking program for this system.

  15. Immunobiological properties of influenza A (H7N9) hemagglutinin and neuraminidase proteins.

    PubMed

    Jiang, Li; Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Masamae, Wanibtisam; Noisumdaeng, Pirom; Jongkaewwattana, Anan; Puthavathana, Pilaipan

    2016-10-01

    Recombinant vaccinia viruses harboring the complete hemagglutinin (HA) or neuraminidase (NA) genes from the influenza A/Anhui/1/2013 (H7N9) virus were constructed (rVac-H7 HA and rVac-N9 NA viruses). The HA and NA proteins were expressed in the cytoplasm and on the plasma membrane of thymidine-kinase-negative (TK(-)) cells infected with these recombinant viruses. Only one form of the HA protein was expressed in infected TK(-) cells, with a molecular weight (MW) of 75 kDa, but three forms were found when the culture medium was supplemented with trypsin (MWs of 75, 50 and 27 kDa), which was similar to what was found in Madin-Darby canine kidney (MDCK) cells infected with reverse genetic (rg) influenza viruses carrying HA genes of H7N9 virus origin. One form of hyperglycosylated NA protein with a MW of 75 kDa was produced in rVac-N9-NA-virus-infected TK(-) or MDCK cells. The MW decreased to 55 kDa after deglycosylation. The hyperglycosylated recombinant NA protein demonstrated sialidase activity in a fetuin-based neuraminidase assay. The rVac-H7 HA and rVac-N9 NA viruses elicited significantly higher anti-HA and anti-NA antibody titers in BALB/c mice that were immunized once than in ICR mice. The anti-HA and anti-NA antibodies showed activity against homosubtypic HA or NA, but not against heterosubtypic HA or NA, as determined by hemagglutination-inhibition and microneutralization assays for anti-HA antibodies and neuraminidase-inhibition and replication-inhibition assays for anti-NA antibodies. Taken together, our data demonstrated immunobiological properties of recombinant HA and NA proteins that might be useful for vaccine development.

  16. Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

    PubMed Central

    Kalthoff, Donata; Röhrs, Susanne; Höper, Dirk; Hoffmann, Bernd; Bogs, Jessica; Stech, Jürgen

    2013-01-01

    Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins, referred to as “antigenic drift” and “antigenic shift.” To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged 50 times in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum. The resulting mutant acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected, in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity. PMID:24109212

  17. Changes in the Length of the Neuraminidase Stalk Region Impact H7N9 Virulence in Mice

    PubMed Central

    Xiao, Haixia; Chen, Quanjiao; Wu, Yan; Fu, Lifeng; Quan, Chuansong; Wong, Gary; Liu, Jun; Haywood, Joel; Liu, Yingxia; Zhou, Boping; Yan, Jinghua; Liu, Wenjun

    2015-01-01

    The neuraminidase stalk of the newly emerged H7N9 influenza virus possesses a 5-amino-acid deletion. This study focuses on characterizing the biological functions of H7N9 with varied neuraminidase stalk lengths. Results indicate that the 5-amino-acid deletion had no impact on virus infectivity or replication in vitro or in vivo compared to that of a virus with a full-length stalk, but enhanced virulence in mice was observed for H7N9 encoding a 19- to 20-amino-acid deletion, suggesting that N9 stalk length impacts virulence in mammals, as N1 stalk length does. PMID:26656694

  18. Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR.

    PubMed

    Barb, Adam W; Glushka, John N; Prestegard, James H

    2011-01-01

    The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques.

  19. Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein.

    PubMed

    Santos-López, Gerardo; Scior, Thomas; Borraz-Argüello, María del Tránsito; Vallejo-Ruiz, Verónica; Herrera-Camacho, Irma; Tapia-Ramírez, José; Reyes-Leyva, Julio

    2009-02-01

    A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

  20. Detection and comparison of neuraminidase activities in human and bovine group B streptococci.

    PubMed

    Ekin, Ismail Hakki; Gurturk, Kemal; Ilhan, Ziya; Ekin, Suat; Borum, Ayse Ebru; Arabaci, Cigdem; Yesilova, Abdullah

    2016-12-01

    Human and bovine group B streptococcus (GBS) isolates were serotyped and amounts of released N-acetylneuraminic acid from N-acetylneuraminyl-lactose by extracellular neuraminidase were colorimetrically assessed. According to serotyping by co-agglutination method, 30 of bovine GBS and 43 of human GBS could be serotyped (ST) by monospecific antisera coated with protein A. The remaining GBS strains were designated as nontypeable (NT). The released N-acetylneuraminic acid was determined in 90.9% of bovine GBS and 47.1% of human GBS isolates. The differences between the total bovine and human GBS isolates were statistically significant (p < 0.001). In comparison with detected N-acetylneuraminic acid level in bovine and human groups, significant decrease was observed in the bovine NT group according to increased human NT (p < 0.01) and bovine ST groups (p < 0.01). However, N-acetylneuraminic acid level in bovine ST and bovine total groups significantly (p < 0.001) increased with respect to the human ST group and human total group. Neuraminidase activity was detected more frequently in bovine GBS isolates. Considerable differentiations were observed between typeable and nontypeable isolates.

  1. Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR

    PubMed Central

    Barb, Adam W.; Glushka, John N.; Prestegard, James H.

    2011-01-01

    The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques. PMID:22058570

  2. Secondary structure prediction of the hemagglutinin-neuraminidase from a porcine rubulavirus.

    PubMed

    Zenteno-Cuevas, R; Hernández, J; Espinosa, B; Reyes, J; Zenteno, E

    1998-01-01

    The Hemagglutinin-Neuraminidase (HN) from 'La Piedad, Michoacan' porcine rubulavirus (LPMV) interacts specifically with NeuAc alpha 2,3 lactose residues on the target cell. In this work we report the secondary structure of this protein, determined with five different theoretical algorithms. Results indicate that the HN protein is organized in: an intracellular region (from amino acid 1 to 25); in a beta-strand transmembrane region (residue 26 to 47), typically hydrophobic, rigid and solvent inaccessible; and extracellular region (48 to 576), which possesses hemagglutinating and neuraminidase activity. The secondary structure in this region is organized in a beta-loop-beta alternated with few alpha-helices. Regions with structural and functional implications were determined by pattern search and multiple alignment of the HN from LPM with 12 rubulaviruses and paramyxoviruses HN sequences. The low diversity observed among the HN sequences evaluated indicates that in general the structural organization of the protein, and in particular its sugar binding domain, is closely related among both genera, thus suggesting that the sugar binding domain is well preserved through evolution.

  3. Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

    PubMed Central

    Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

    2013-01-01

    The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

  4. Persistence of Antibodies to Influenza Hemagglutinin and Neuraminidase Following One or Two Years of Influenza Vaccination.

    PubMed

    Petrie, Joshua G; Ohmit, Suzanne E; Johnson, Emileigh; Truscon, Rachel; Monto, Arnold S

    2015-12-15

    Antibody titers to influenza hemagglutinin (HA) and neuraminidase (NA) surface antigens increase in the weeks after infection or vaccination, and decrease over time thereafter. However, the rate of decline has been debated. Healthy adults participating in a randomized placebo-controlled trial of inactivated (IIV) and live-attenuated (LAIV) influenza vaccines provided blood specimens immediately prior to vaccination and at 1, 6, 12, and 18 months postvaccination. Approximately half had also been vaccinated in the prior year. Rates of hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) titer decline in the absence of infection were estimated. HAI and NAI titers decreased slowly over 18 months; overall, a 2-fold decrease in antibody titer was estimated to take >600 days for all HA and NA targets. Rates of decline were fastest among IIV recipients, explained in part by faster declines with higher peak postvaccination titer. IIV and LAIV recipients vaccinated 2 consecutive years exhibited significantly lower HAI titers following vaccination in the second year, but rates of persistence were similar. Antibody titers to influenza HA and NA antigens may persist over multiple seasons; however, antigenic drift of circulating viruses may still necessitate annual vaccination. Vaccine seroresponse may be impaired with repeated vaccination. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    NASA Astrophysics Data System (ADS)

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-04-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

  6. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    PubMed Central

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-01-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors. PMID:27125351

  7. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type

    USDA-ARS?s Scientific Manuscript database

    The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and re...

  8. A B-lymphoma cell line that forms rosettes with neuraminidase-treated sheep erythrocytes through monoclonal surface immunoglobulin.

    PubMed

    Tsutsumi, Y; Suzuki, S; Mikata, A; Suzuki, H; Kageyama, K; Watanabe, S; Minato, K; Shimoyama, M

    1982-06-01

    Undifferentiated lymphoma from a 39-year-old female became serially xenotransplantable to preirradiated nude mice. The tumor cells (KT) possessed a monoclonal surface immunoglobulin (SIg mu, kappa) and formed rosettes with neuraminidase-treated sheep erythrocytes (SEn). Precise characterizations of the SEn rosette, however, revealed the following facts: (1) Neuraminidase-untreated or 2-aminoethylisothiuronium bromide (AET) treated sheep erythrocytes were not bound to the KT cells. (2) SEn rosettes on the KT cells did not show a temperature dependency. (3) Neuraminidase-treated erythrocytes from man, horse, mouse, and rabbit were not bound to the KT cells. (4) Preincubation of the KT cells with antipolyvalent immunoglobulin or anti-kappa-chain serum abolished the SEn rosette formation. (5) Trypsinization decreased both SEn rosettes and SIg on the KT cells. (6) SEn rosettes on the KT cells were too loose to be separated from nonrosetting cells by a Percoll gradient centrifugation method. Summarizing these results, the monoclonal SIg on the KT cells recognized sheep erythrocyte antigen(s) that were exposed only after the neuraminidase treatment. Therefore, this was considered to be a case with peculiar B-lymphoma cells that bound SEn through their SIg.

  9. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  10. Neuraminidase-1 contributes significantly to the degradation of neuronal B-series gangliosides but not to the bypass of the catabolic block in Tay-Sachs mouse models.

    PubMed

    Timur, Z K; Akyildiz Demir, S; Marsching, C; Sandhoff, R; Seyrantepe, V

    2015-09-01

    Tay–Sachs disease is a severe lysosomal storage disorder caused by mutations in the HEXA gene coding for α subunit of lysosomal β-Hexosaminidase A enzyme, which converts GM2 to GM3 ganglioside. HexA(−/−) mice, depleted of the β-Hexosaminidase A iso-enzyme, remain asymptomatic up to 1 year of age because of a metabolic bypass by neuraminidase(s). These enzymes remove a sialic acid residue converting GM2 to GA2, which is further degraded by the still intact β-Hexosaminidase B iso-enzyme into lactosylceramide. A previously identified ganglioside metabolizing neuraminidase, Neu4, is abundantly expressed in the mouse brain and has activity against gangliosides like GM2in vitro. Neu4(−/−) mice showed increased GD1a and decreased GM1 ganglioside in the brain suggesting the importance of the Neu4 in ganglioside catabolism. Mice with targeted disruption of both HexA and Neu4 genes showed accumulating GM2 ganglioside and epileptic seizures with 40% penetrance, indicating that the neuraminidase Neu4 is a modulatory gene, but may not be the only neuraminidase contributing to the metabolic bypass in HexA(−/−) mice. Therefore, we elucidated the biological role of neuraminidase-1 in ganglioside degradation in mouse. Analysis of HexA(−/−) Neu1(−/−) and HexA(−/−) Neu4(−/−) Neu1(−/−) mice models showed significant contribution of neuraminidase-1 on B-series ganglioside degradation in the brain. Therefore, we speculate that other neuraminidase/neuraminidases such as Neu2 and/or Neu3 might be also involved in the ganglioside degradation pathway in HexA(−/−) mice.

  11. Droplet digital PCR to investigate quasi-species at codons 119 and 275 of the A(H1N1)pdm09 neuraminidase during zanamivir and oseltamivir therapies.

    PubMed

    Abed, Yacine; Carbonneau, Julie; L'Huillier, Arnaud G; Kaiser, Laurent; Boivin, Guy

    2017-04-01

    The H275Y and E119D neuraminidase (NA) mutations constitute important molecular markers of resistance to NA inhibitors in A(H1N1) pdm09 viruses. We used reverse transcriptase-droplet digital PCR amplification (RT-ddPCR) to analyze quasi-species at codons 275 and 119 of the NA in A(H1N1) pdm09 viruses recovered from an immuncompromised patient who received oseltamivir and zanamivir therapies. RT-ddPCR assays detected and quantified H275Y and E119D mutations with an efficiency that was comparable to that of high throughput sequencing (HiSeq 2500 Illumina, San Diego, CA) technology. With its sensitivity and reproducibility, RT-ddPCR could be a reliable method for accurate detection and quantification of major NAI-resistance mutations in clinical settings. J. Med. Virol. 89:737-741, 2017. © 2016 Wiley Periodicals, Inc.

  12. The inhibition of Clostridium chauvoei (jakari strain) neuraminidase activity by methanolic extracts of the stem barks of Tamarindus indicus and Combretum fragrans.

    PubMed

    Useh, N M; Nok, A J; Ambali, S F; Esievo, K A N

    2004-08-01

    The inhibition of neuraminidase from Clostridium chauvoei (jakari strain) with partially purified methanolic extracts of some plants used in Ethnopharmacological practice was evaluated. Extracts of two medicinal plants, Tamarindus indicus and Combretum fragrans at 100-1000 microg/ml, both significantly reduced the activity of the enzyme in a dose-dependent fashion (P < 0.001). The estimated IC50 values for Tamarindus indicus and Combretum fragrans were 100 and 150 microg/ml respectively. Initial velocity studies conducted, using fetuin as substrate revealed a non-competitive inhibition with the Vmax significantly altered from 500 micromole min(-1) mg(-1) to 240 micromole min(-1) mg(-1) and 340 micromole min(-1) mg(-1) in the presence of Tamarindus indicus and Combretum fragrans respectively. The KM remained unchanged at 0.42 mM. The computed Index of physiological efficiency was reduced from 1.19min(-1) to 0.57min(-1) and 0.75min(-1) with Tamarindus indicus and Combretum fragrans as inhibitors respectively.

  13. Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring.

    PubMed

    Little, Karen; Leang, Sook-Kwan; Butler, Jeff; Baas, Chantal; Harrower, Bruce; Mosse, Jenny; Barr, Ian G; Hurt, Aeron C

    2015-01-01

    Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue Q136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.

  14. Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

    PubMed Central

    Holland, P C; Pena, S D; Guerin, C W

    1984-01-01

    Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6712625

  15. Quantification of Influenza Neuraminidase Activity by Ultra-High Performance Liquid Chromatography and Isotope Dilution Mass Spectrometry.

    PubMed

    Solano, Maria I; Woolfitt, Adrian R; Williams, Tracie L; Pierce, Carrie L; Gubareva, Larisa V; Mishin, Vasiliy; Barr, John R

    2017-03-07

    Mounting evidence suggests that neuraminidase's functionality extends beyond its classical role in influenza virus infection and that antineuraminidase antibodies offer protective immunity. Therefore, a renewed interest in the development of neuraminidase (NA)-specific methods to characterize the glycoprotein and evaluate potential advantages for NA standardization in influenza vaccines has emerged. NA displays sialidase activity by cleaving off the terminal N-acetylneuraminic acid on α-2,3 or α-2,6 sialic acid containing receptors of host cells. The type and distribution of these sialic acid containing receptors is considered to be an important factor in transmission efficiency of influenza viruses between and among host species. Changes in hemagglutinin (HA) binding and NA specificity in reassortant viruses may be related to the emergence of new and potentially dangerous strains of influenza. Current methods to investigate neuraminidase activity use small derivatized sugars that are poor models for natural glycoprotein receptors and do not provide information on the linkage specificity. Here, a novel approach for rapid and accurate quantification of influenza neuraminidase activity is achieved utilizing ultra-high performance liquid chromatography (UPLC) and isotope dilution mass spectrometry (IDMS). Direct LC-MS/MS quantification of NA-released sialic acid provides precise measurement of influenza neuraminidase activity over a range of substrates. The method provides exceptional sensitivity and specificity with a limit of detection of 0.38 μM for sialic acid and the capacity to obtain accurate measurements of specific enzyme activity preference toward α-2,3-sialyllactose linkages, α-2,6-sialyllactose linkages, or whole glycosylated proteins such as fetuin.

  16. The neuraminidases of MDCK grown human influenza A(H3N2) viruses isolated since 1994 can demonstrate receptor binding.

    PubMed

    Mohr, Peter G; Deng, Yi-Mo; McKimm-Breschkin, Jennifer L

    2015-04-22

    The neuraminidases (NAs) of MDCK passaged human influenza A(H3N2) strains isolated since 2005 are reported to have dual functions of cleavage of sialic acid and receptor binding. NA agglutination of red blood cells (RBCs) can be inhibited by neuraminidase inhibitors (NAIs), thus distinguishing it from haemagglutinin (HA) binding. We wanted to know if viruses prior to 2005 can demonstrate this property. Pairs of influenza A(H3N2) isolates ranging from 1993-2008 passaged in parallel only in eggs or in MDCK cells were tested for inhibition of haemagglutination by various NAIs. Only viruses isolated since 1994 and cultured in MDCK cells bound chicken RBCs solely through their NA. NAI inhibition of agglutination of turkey RBCs was seen for some, but not all of these same MDCK grown viruses. Efficacy of inhibition of enzyme activity and haemagglutination differed between NAIs. For many viruses lower concentrations of oseltamivir could inhibit agglutination compared to zanamivir, although they could both inhibit enzyme activity at comparable concentrations. An E119V mutation reduced sensitivity to oseltamivir and 4-aminoDANA for both the enzyme assay and inhibition of agglutination. Sequence analysis of the NAs and HAs of some paired viruses revealed mutations in the haemagglutinin of all egg passaged viruses. For many of the paired egg and MDCK cultured viruses we found no differences in their NA sequences by Sanger sequencing. However, deep sequencing of MDCK grown isolates revealed low levels of variant populations with mutations at either D151 or T148 in the NA, suggesting mutations at either site may be able to confer this property. The NA active site of MDCK cultured human influenza A(H3N2) viruses isolated since 1994 can express dual enzyme and receptor binding functions. Binding correlated with either D151 or T148 mutations. The catalytic and receptor binding sites do not appear to be structurally identical since relative concentrations of the NAIs to inhibit

  17. Hemagglutination and the closest distance of approach of normal, neuraminidase- and papain-treated erythrocytes.

    PubMed

    van Oss, C J; Absolom, D R

    1984-01-01

    By means of potential energy versus distance diagrams, derived from electrophoretic and surface tension data, the minimum distances of approach of normal (NOR) and of neuraminidase-treated (NEU) and papain-treated (PAP) human erythrocytes could be determined. The minimum distances between the actual cell membranes of two opposing red cells are: 184 A (NOR), 111 A (NEU), and 113 A (PAP), which agrees well with the fact that anti-D (Rho) antibodies of the IgG-class (which have a maximum distance of approximately 120 A between the two antibody-active sites) can hemagglutinate NEU and PAP cells, but are incapable of hemagglutinating normal D (Rho)-positive erythrocytes.

  18. Influenza A virus hemagglutinin and neuraminidase act as novel motile machinery

    PubMed Central

    Sakai, Tatsuya; Nishimura, Shin I.; Naito, Tadasuke; Saito, Mineki

    2017-01-01

    Influenza A virus (IAV) membrane proteins hemagglutinin (HA) and neuraminidase (NA) are determinants of virus infectivity, transmissibility, pathogenicity, host specificity, and major antigenicity. HA binds to a virus receptor, a sialoglycoprotein or sialoglycolipid, on the host cell and mediates virus attachment to the cell surface. The hydrolytic enzyme NA cleaves sialic acid from viral receptors and accelerates the release of progeny virus from host cells. In this study, we identified a novel function of HA and NA as machinery for viral motility. HAs exchanged binding partner receptors iteratively, generating virus movement on a receptor-coated glass surface instead of a cell surface. The virus movement was also dependent on NA. Virus movement mediated by HA and NA resulted in a three to four-fold increase in virus internalisation by cultured cells. We concluded that cooperation of HA and NA moves IAV particles on a cell surface and enhances virus infection of host cells. PMID:28344335

  19. The universal epitope of influenza A viral neuraminidase fundamentally contributes to enzyme activity and viral replication.

    PubMed

    Doyle, Tracey M; Jaentschke, Bozena; Van Domselaar, Gary; Hashem, Anwar M; Farnsworth, Aaron; Forbes, Nicole E; Li, Changgui; Wang, Junzhi; He, Runtao; Brown, Earl G; Li, Xuguang

    2013-06-21

    The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

  20. Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

    SciTech Connect

    Yang, Hua; Carney, Paul J.; Mishin, Vasiliy P.; Guo, Zhu; Chang, Jessie C.; Wentworth, David E.; Gubareva, Larisa V.; Stevens, James; Schultz-Cherry, S.

    2016-04-06

    ABSTRACT

    During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (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 United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of 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.

    IMPORTANCEThe H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

  1. Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

    PubMed

    Creskey, Marybeth C; Li, Changgui; Wang, Junzhi; Girard, Michel; Lorbetskie, Barry; Gravel, Caroline; Farnsworth, Aaron; Li, Xuguang; Smith, Daryl G S; Cyr, Terry D

    2012-07-06

    Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 μg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current

  2. Neuraminidase production by a Pasteurella haemolytica A1 strain associated with bovine pneumonia.

    PubMed Central

    Straus, D C; Unbehagen, P J; Purdy, C W

    1993-01-01

    The properties of an extracellular neuroaminidase produced by a Pasteurella haemolytica A1 strain (isolated from a case of bovine pneumonia) during growth in a defined medium were examined in this investigation. This enzyme, isolated from concentrated culture supernatants of P. haemolytica A1, was active against N-acetylneuramin lactose, human alpha 1-acid glycoprotein, fetuin, and bovine submaxillary mucin. Neuraminidase production paralleled bacterial growth in a defined medium and was maximal in the stationary phase of growth. The enzyme was purified to homogeneity by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 100.62 mumol of sialic acid released per min per mg of protein against human alpha 1-acid glycoprotein. The Km value for this enzyme with human alpha 1-acid glycoprotein as the substrate was 1.1 mg/ml, and the enzyme possessed a pH optimum of 6.5. The P. haemolytica A1 neuraminidase had a molecular weight of approximately 150,000 as estimated by gel filtration and approximately 170,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at 4 degrees C for 3 h. At 37 degrees C for 3 h, 25% of enzymatic activity was lost. Approximately 55% of the enzyme activity was lost within 30 min at 50 degrees C, with greater than 70% of the enzyme activity being destroyed within 10 min at temperatures of > or = 65 degrees C. Images PMID:8418046

  3. Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

    PubMed Central

    Yang, Hua; Carney, Paul J.; Mishin, Vasiliy P.; Guo, Zhu; Chang, Jessie C.; Wentworth, David E.; Gubareva, Larisa V.

    2016-01-01

    ABSTRACT During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (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 United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of 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 pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment. PMID:27053557

  4. Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

    SciTech Connect

    Gibson, S.; Jung, C.Y.; Takahashi, M.; Lenard, J.

    1986-10-07

    The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the monomer of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.

  5. Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

    SciTech Connect

    Robach, Jessica G.; Lamb, Robert A.

    2010-10-25

    The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

  6. Rapid differentiation of seasonal and pandemic H1N1 influenza through proteotyping of viral neuraminidase with mass spectrometry.

    PubMed

    Schwahn, Alexander B; Wong, Jason W H; Downard, Kevin M

    2010-06-01

    Signature peptides of the neuraminidase antigen across all common circulating human subtypes of type A and B influenza are identified through the bioinformatic alignment of translated gene sequences. The detection of these peptides within the high-resolution mass spectra of whole antigen, virus, and vaccine digests enables the strains to be rapidly and directly typed and subtyped. Importantly, unique signature peptides for pandemic (H1N1) 2009 influenza are identified and detected that enable pandemic strains to be rapidly and directly differentiated from seasonal type A (H1N1) influenza strains. The detection of these peptides can enable the origins of the neuraminidase gene to be monitored in the case of reassorted strains.

  7. Recent H3N2 Influenza Virus Clinical Isolates Rapidly Acquire Hemagglutinin or Neuraminidase Mutations When Propagated for Antigenic Analyses

    PubMed Central

    Chambers, Benjamin S.; Li, Yang; Hodinka, Richard L.

    2014-01-01

    Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses. PMID:24991002

  8. Neuroblast proliferation on the surface of the adult rat striatal wall after focal ependymal loss by intracerebroventricular injection of neuraminidase.

    PubMed

    Del Carmen Gómez-Roldán, María; Pérez-Martín, Margarita; Capilla-González, Vivian; Cifuentes, Manuel; Pérez, Juan; García-Verdugo, Jose Manuel; Fernández-Llebrez, Pedro

    2008-04-01

    The subventricular zone of the striatal wall of adult rodents is an active neurogenic region for life. Cubic multiciliated ependyma separates the subventricular zone from the cerebrospinal fluid (CSF) and is involved in the control of adult neurogenesis. By injecting neuraminidase from Clostridium perfringens into the right lateral ventricle of the rat, we provoked a partial detachment of the ependyma in the striatal wall. The contralateral ventricle was never affected and was used as the experimental control. Neuraminidase caused widening of the intercellular spaces among some ependymal cells and their subsequent detachment and disintegration in the CSF. Partial ependymal denudation was followed by infiltration of the CSF with macrophages and neutrophils from the local choroid plexus, which ependymal cells never detached after neuraminidase administration. Inflammation extended toward the periventricular parenchyma. The ependymal cells that did not detach and remained in the ventricle wall never proliferated. The lost ependyma was never recovered, and ependymal cells never behaved as neural stem cells. Instead, a scar formed by overlapping astrocytic processes sealed those regions devoid of ependyma. Some ependymal cells at the border of the denudated areas lost contact with the ventricle and became located under the glial layer. Concomitantly with scar formation, some subependymal cells protruded toward the ventricle through the ependymal breaks, proliferated, and formed clusters of rounded ventricular cells that expressed the phenotype of neuroblasts. Ventricular clusters of neuroblasts remained in the ventricle up to 90 days after injection. In the subventricular zone, adult neurogenesis persisted.

  9. Neuraminidase produces dose-dependent decrease of slime production and adherence of slime-forming, coagulase-negative staphylococci.

    PubMed

    Sakarya, Serhan; Oncu, Serkan; Oncu, Selcen; Ozturk, Barcin; Tuncer, Gunay; Sari, Cavide

    2004-01-01

    Slime is one of the important structures of certain bacterial strains involved in nonspecific adherence. This study was conducted to determine the role of neuraminidase on slime formation and adherence of slime-forming coagulase-negative staphylococci to inert surface. Quantitative biofilm and qualitative bacterial adherence assays were performed with increasing concentrations of neuraminidase extracted from Clostridium perfringens-treated bacteria in polystyrene plates and polypropylene tubes. Slime production of slime-forming, coagulase-negative staphylococci was significantly decreased dose dependently at > or =100 mU/mL (p <0.001). Bacterial adherence to smooth surface was impeded at > or =100 mU/mL of neuraminidase treatment and adherence results were comparable with slime production assay results. Sialic acid may be a constituent molecule of slime and involved in bacterial adherence to inert surface. These results represent new insight into the mechanism of slime production and adherence of slime-forming, coagulase-negative staphylococci to inert surface.

  10. Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

    PubMed

    Gillman, Anna; Nykvist, Marie; Muradrasoli, Shaman; Söderström, Hanna; Wille, Michelle; Daggfeldt, Annika; Bröjer, Caroline; Waldenström, Jonas; Olsen, Björn; Järhult, Josef D

    2015-09-01

    Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

  11. Influenza A(H7N9) Virus Acquires Resistance-Related Neuraminidase I222T Substitution When Infected Mallards Are Exposed to Low Levels of Oseltamivir in Water

    PubMed Central

    Nykvist, Marie; Muradrasoli, Shaman; Söderström, Hanna; Wille, Michelle; Daggfeldt, Annika; Bröjer, Caroline; Waldenström, Jonas; Olsen, Björn; Järhult, Josef D.

    2015-01-01

    Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 μg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted. PMID:26077257

  12. Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

    PubMed Central

    Changsom, Don; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Chakritbudsabong, Warunya; Siridechadilok, Bunpote; Prasertsopon, Jarunee; Noisumdaeng, Pirom; Masamae, Wanibtisam; Puthavathana, Pilaipan

    2016-01-01

    Influenza neuraminidase (NA) proteins expressed in TK− cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross

  13. ACE inhibitors

    MedlinePlus

    ... ACE inhibitors There are many different names and brands of ACE inhibitors. Most work as well as ... urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. follows ...

  14. Activation of brain endothelium by pneumococcal neuraminidase NanA promotes bacterial internalization.

    PubMed

    Banerjee, Anirban; Van Sorge, Nina M; Sheen, Tamsin R; Uchiyama, Satoshi; Mitchell, Tim J; Doran, Kelly S

    2010-11-01

    Streptococcus pneumoniae (SPN), the leading cause of meningitis in children and adults worldwide, is associated with an overwhelming host inflammatory response and subsequent brain injury. Here we examine the global response of the blood-brain barrier to SPN infection and the role of neuraminidase A (NanA), an SPN surface anchored protein recently described to promote central nervous system tropism. Microarray analysis of human brain microvascular endothelial cells (hBMEC) during infection with SPN or an isogenic NanA-deficient (ΔnanA) mutant revealed differentially activated genes, including neutrophil chemoattractants IL-8, CXCL-1, CXCL-2. Studies using bacterial mutants, purified recombinant NanA proteins and in vivo neutrophil chemotaxis assays indicated that pneumococcal NanA is necessary and sufficient to activate host chemokine expression and neutrophil recruitment during infection. Chemokine induction was mapped to the NanA N-terminal lectin-binding domain with a limited contribution of the sialidase catalytic activity, and was not dependent on the invasive capability of the organism. Furthermore, pretreatment of hBMEC with recombinant NanA protein significantly increased bacterial invasion, suggesting that NanA-mediated activation of hBMEC is a prerequisite for efficient SPN invasion. These findings were corroborated in an acute murine infection model where we observed less inflammatory infiltrate and decreased chemokine expression following infection with the ΔnanA mutant. © 2010 Blackwell Publishing Ltd.

  15. Inhibition of Influenza A Virus Infection by Fucoidan Targeting Viral Neuraminidase and Cellular EGFR Pathway

    PubMed Central

    Wang, Wei; Wu, Jiandong; Zhang, Xiaoshuang; Hao, Cui; Zhao, Xiaoliang; Jiao, Guangling; Shan, Xindi; Tai, Wenjing; Yu, Guangli

    2017-01-01

    Development of novel anti-influenza A virus (IAV) drugs with high efficiency and low toxicity is critical for preparedness against influenza outbreaks. Herein, we investigated the anti-IAV activities and mechanisms of fucoidan in vitro and in vivo. The results showed that a fucoidan KW derived from brown algae Kjellmaniella crassifolia effectively blocked IAV infection in vitro with low toxicity. KW possessed broad anti-IAV spectrum and low tendency of induction of viral resistance, superior to the anti-IAV drug amantadine. KW was capable of inactivating virus particles before infection and blocked some stages after adsorption. KW could bind to viral neuraminidase (NA) and inhibit the activity of NA to block the release of IAV. KW also interfered with the activation of EGFR, PKCα, NF-κB, and Akt, and inhibited both IAV endocytosis and EGFR internalization in IAV-infected cells, suggesting that KW may also inhibit cellular EGFR pathway. Moreover, intranasal administration of KW markedly improved survival and decreased viral titers in IAV-infected mice. Therefore, fucoidan KW has the potential to be developed into a novel nasal drop or spray for prevention and treatment of influenza in the future. PMID:28094330

  16. Neuraminidase Subtyping of Avian Influenza Viruses with PrimerHunter-Designed Primers and Quadruplicate Primer Pools

    PubMed Central

    Huang, Yanyan; Khan, Mazhar; Măndoiu, Ion I.

    2013-01-01

    We have previously developed a software package called PrimerHunter to design primers for PCR-based virus subtyping. In this study, 9 pairs of primers were designed with PrimerHunter and successfully used to differentiate the 9 neuraminidase (NA) genes of avian influenza viruses (AIVs) in multiple PCR-based assays. Furthermore, primer pools were designed and successfully used to decrease the number of reactions needed for NA subtyping from 9 to 4. The quadruplicate primer-pool method is cost-saving, and was shown to be suitable for the NA subtyping of both cultured AIVs and uncultured AIV swab samples. The primers selected for this study showed excellent sensitivity and specificity in NA subtyping by RT-PCR, SYBR green-based Real-time PCR and Real-time RT-PCR methods. AIV RNA of 2 to 200 copies (varied by NA subtypes) could be detected by these reactions. No unspecific amplification was displayed when detecting RNAs of other avian infectious viruses such as Infectious bronchitis virus, Infectious bursal disease virus and Newcastle disease virus. In summary, this study introduced several sensitive and specific PCR-based assays for NA subtyping of AIVs and also validated again the effectiveness of the PrimerHunter tool for the design of subtyping primers. PMID:24312367

  17. The haemagglutinin gene, but not the neuraminidase gene, of 'Spanish flu' was a recombinant.

    PubMed Central

    Gibbs, M J; Armstrong, J S; Gibbs, A J

    2001-01-01

    Published analyses of the sequences of three genes from the 1918 Spanish influenza virus have cast doubt on the theory that it came from birds immediately before the pandemic. They showed that the virus was of the H1N1 subtype lineage but more closely related to mammal-infecting strains than any known bird-infecting strain. They provided no evidence that the virus originated by gene reassortment nor that the virus was the direct ancestor of the two lineages of H1N1 viruses currently found in mammals; one that mostly infects human beings, the other pigs. The unusual virulence of the virus and why it produced a pandemic have remained unsolved. We have reanalysed the sequences of the three 1918 genes and found conflicting patterns of relatedness in all three. Various tests showed that the patterns in its haemagglutinin (HA) gene were produced by true recombination between two different parental HA H1 subtype genes, but that the conflicting patterns in its neuraminidase and non-structural-nuclear export proteins genes resulted from selection. The recombination event that produced the 1918 HA gene probably coincided with the start of the pandemic, and may have triggered it. PMID:11779383

  18. Structural Characterization of the 1918 Influenza H1N1 Neuraminidase

    SciTech Connect

    Xu, X.; Zhu, X.; Dwek, R.A.; Stevens, J.; Wilson, I.A.

    2009-05-28

    Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 'Spanish flu' (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-{angstrom} and 1.45-{angstrom} resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147 to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.

  19. Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

    PubMed

    Park, Sehee; Il Kim, Jin; Lee, Ilseob; Bae, Joon-Yong; Yoo, Kirim; Nam, Misun; Kim, Juwon; Sook Park, Mee; Song, Ki-Joon; Song, Jin-Won; Kee, Sun-Ho; Park, Man-Seong

    2017-09-07

    It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

  20. Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines

    PubMed Central

    Marcelin, Glendie; Sandbulte, Matthew R.; Webby, Richard J.

    2012-01-01

    SUMMARY Vaccines are instrumental in controlling the burden of influenza virus infection in humans and animals. Antibodies raised against both major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), can contribute to protective immunity. Vaccine-induced HA antibodies have been characterized extensively, and they generally confer protection by blocking the attachment and fusion of a homologous virus onto host cells. Though not as well characterized, some functions of NA antibodies in influenza vaccine-mediated immunity have been recognized for many years. In this review we summarize the case for NA antibodies in influenza vaccine-mediated immunity. In the absence of well-matched HA antibodies, NA antibodies can provide varying degrees of protection against disease. NA proteins of seasonal influenza vaccines have been shown in some instances to elicit serum antibodies with cross-reactivity to avian- and swine-origin influenza strains, in addition to HA drift variants. NA-mediated immunity has been linked to [i] conserved NA epitopes amongst otherwise antigenically distinct strains, partly attributable to the segmented influenza viral genome; [ii] inhibition of NA enzymatic activity; and [iii] the NA content in vaccine formulations. There is potential to enhance the effectiveness of existing and future influenza vaccines by focusing greater attention on the antigenic characteristics and potency of the NA protein. PMID:22438243

  1. Neuraminidase subtyping of avian influenza viruses with PrimerHunter-designed primers and quadruplicate primer pools.

    PubMed

    Huang, Yanyan; Khan, Mazhar I; Khan, Mazhar; Măndoiu, Ion; Măndoiu, Ion I

    2013-01-01

    We have previously developed a software package called PrimerHunter to design primers for PCR-based virus subtyping. In this study, 9 pairs of primers were designed with PrimerHunter and successfully used to differentiate the 9 neuraminidase (NA) genes of avian influenza viruses (AIVs) in multiple PCR-based assays. Furthermore, primer pools were designed and successfully used to decrease the number of reactions needed for NA subtyping from 9 to 4. The quadruplicate primer-pool method is cost-saving, and was shown to be suitable for the NA subtyping of both cultured AIVs and uncultured AIV swab samples. The primers selected for this study showed excellent sensitivity and specificity in NA subtyping by RT-PCR, SYBR green-based Real-time PCR and Real-time RT-PCR methods. AIV RNA of 2 to 200 copies (varied by NA subtypes) could be detected by these reactions. No unspecific amplification was displayed when detecting RNAs of other avian infectious viruses such as Infectious bronchitis virus, Infectious bursal disease virus and Newcastle disease virus. In summary, this study introduced several sensitive and specific PCR-based assays for NA subtyping of AIVs and also validated again the effectiveness of the PrimerHunter tool for the design of subtyping primers.

  2. Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent.

    PubMed

    Baradaran, Ali; Yusoff, Khatijah; Shafee, Norazizah; Rahim, Raha Abdul

    2016-01-01

    The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) with its immunotherapeutic activities and sialic acid binding abilities is a promising cancer adjuvant. The HN was surfaced displayed on Lactococcus lactis and its cancer targeting ability was investigated via attachment to the MDA-MB231 breast cancers. To surface display the HN protein on the bacterial cell wall, HN was fused to N-acetylmuraminidase (AcmA) anchoring motif of L. lactis and expressed in Chinese hamster ovary cells. The expressed recombinant fusion proteins were purified and mixed with a culture of L. lactis and Lactobacillus plantarum. Immunofluorescence assay showed the binding of the recombinant HN-AcmA protein on the surface of the bacterial cells. The bacterial cells carrying the HN-AcmA protein interacted with the MDA-MB231 breast cancer cells. Direct and fluorescent microscopy confirmed that L. lactis and Lb. plantarum surface displaying the recombinant HN were attached to the breast cancer MDA-MB231 cells, providing evidence for the potential ability of HN in targeting to cancer cells.

  3. Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent

    PubMed Central

    Baradaran, Ali; Yusoff, Khatijah; Shafee, Norazizah; Rahim, Raha Abdul

    2016-01-01

    The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) with its immunotherapeutic activities and sialic acid binding abilities is a promising cancer adjuvant. The HN was surfaced displayed on Lactococcus lactis and its cancer targeting ability was investigated via attachment to the MDA-MB231 breast cancers. To surface display the HN protein on the bacterial cell wall, HN was fused to N-acetylmuraminidase (AcmA) anchoring motif of L. lactis and expressed in Chinese hamster ovary cells. The expressed recombinant fusion proteins were purified and mixed with a culture of L. lactis and Lactobacillus plantarum. Immunofluorescence assay showed the binding of the recombinant HN-AcmA protein on the surface of the bacterial cells. The bacterial cells carrying the HN-AcmA protein interacted with the MDA-MB231 breast cancer cells. Direct and fluorescent microscopy confirmed that L. lactis and Lb. plantarum surface displaying the recombinant HN were attached to the breast cancer MDA-MB231 cells, providing evidence for the potential ability of HN in targeting to cancer cells. PMID:26918060

  4. Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase.

    PubMed

    Wu, Zhengliang L; Zhou, Hui; Ethen, Cheryl M; N Reinhold, Vernon

    2016-04-29

    The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and (3)H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme.

  5. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  6. New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability

    PubMed Central

    Maurice, Pascal; Baud, Stéphanie; Bocharova, Olga V.; Bocharov, Eduard V.; Kuznetsov, Andrey S.; Kawecki, Charlotte; Bocquet, Olivier; Romier, Beatrice; Gorisse, Laetitia; Ghirardi, Maxime; Duca, Laurent; Blaise, Sébastien; Martiny, Laurent; Dauchez, Manuel; Efremov, Roman G.; Debelle, Laurent

    2016-01-01

    Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity. PMID:27917893

  7. New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability

    NASA Astrophysics Data System (ADS)

    Maurice, Pascal; Baud, Stéphanie; Bocharova, Olga V.; Bocharov, Eduard V.; Kuznetsov, Andrey S.; Kawecki, Charlotte; Bocquet, Olivier; Romier, Beatrice; Gorisse, Laetitia; Ghirardi, Maxime; Duca, Laurent; Blaise, Sébastien; Martiny, Laurent; Dauchez, Manuel; Efremov, Roman G.; Debelle, Laurent

    2016-12-01

    Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

  8. Development of neuraminidase detection using gold nanoparticles boron-doped diamond electrodes.

    PubMed

    Wahyuni, Wulan T; Ivandini, Tribidasari A; Saepudin, Endang; Einaga, Yasuaki

    2016-03-15

    Gold nanoparticles-modified boron-doped diamond (AuNPs-BDD) electrodes, which were prepared with a self-assembly deposition of AuNPs at amine-terminated boron-doped diamond, were examined for voltammetric detection of neuraminidase (NA). The detection method was performed based on the difference of electrochemical responses of zanamivir at gold surface before and after the reaction with NA in phosphate buffer solution (PBS, pH 5.5). A linear calibration curve for zanamivir in 0.1 M PBS in the absence of NA was achieved in the concentration range of 1 × 10(-6) to 1 × 10(-5) M (R(2) = 0.99) with an estimated limit of detection (LOD) of 2.29 × 10(-6) M. Furthermore, using its reaction with 1.00 × 10(-5) M zanamivir, a linear calibration curve of NA can be obtained in the concentration range of 0-12 mU (R(2) = 0.99) with an estimated LOD of 0.12 mU. High reproducibility was shown with a relative standard deviation (RSD) of 1.14% (n = 30). These performances could be maintained when the detection was performed in mucin matrix. Comparison performed using gold-modified BDD (Au-BDD) electrodes suggested that the good performance of the detection method is due to the stability of the gold particles position at the BDD surface.

  9. 6SLN-lipo PGA specifically catches (coats) human influenza virus and synergizes neuraminidase-targeting drugs for human influenza therapeutic potential.

    PubMed

    Sriwilaijaroen, Nongluk; Suzuki, Katsuhiko; Takashita, Emi; Hiramatsu, Hiroaki; Kanie, Osamu; Suzuki, Yasuo

    2015-10-01

    The purpose of this study was to develop a new compound to overcome influenza epidemics and pandemics as well as drug resistance. We synthesized a new compound carrying: (i) Neu5Acα2-6Galβ1-4GlcNAc (6SLN) for targeting immutable haemagglutinins (HAs) unless switched from human-type receptor preference; (ii) an acyl chain (lipo) for locking the compound with the viral HA via hydrophobic interactions; and (iii) a flexible poly-α-L-glutamic acid (PGA) for enhancing the compound solubility and for coating the viral surface, precluding accessibility of the PGA-coated virus to the negatively charged sialic acid on the host cell surface. 6SLN-lipo PGA appears to subvert binding of pandemic H1 and seasonal H3 HAs to receptors, as assessed by using guinea pig erythrocytes, which is critical for virus entry into host cells for multiplication. It shows high potency with IC50 values in the range of 300-500 nM against multiplication of both influenza pandemic H1N1/2009 and seasonal H3N2/2004 viruses in cell culture. It acts in synergism with either of the two FDA-approved neuraminidase inhibitor (NAI) clinical drugs, zanamivir (Relenza(®)) and oseltamivir carboxylate (active form of Tamiflu(®)), and it has the potential to aid NAI drugs to achieve complete clearance of the virus from the culture. 6SLN-lipo PGA is a new potential candidate drug for influenza control and is an attractive candidate for use in combination with an NAI drug for minimized toxicity, delayed development of resistance, prevention and treatment with the potential for eradication of human influenza. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Optimization and comparative characterization of neuraminidase activities from Pseudomonas aeruginosa with Klebsiella pneumoniae, Hep-2 cell, sheep kidney and rat liver lysosome

    PubMed Central

    Ghazaei, C; Ahmadi, M; Hosseini Jazani, N

    2010-01-01

    Background and Objectives The properties of neuraminidase produced by P. aeruginosa strain PAO1 during growth in a defined medium (BHI) was examined and compared with some neuraminidase features of K. pneumoniae in this investigation. Materials and Methods The enzyme was isolated from concentrated culture supernatants of P. aeruginosa which was used in a sensitive fluorometric assay by using 2′-(4-methylumbelliferyl) α-D-N acetylneuraminic acid as substrate. Results Neuraminidase production in P. aeruginosa PAO1 paralleled bacterial growth in defined medium (BHI) and was maximal in the late logarithmic phase of growth but decreased during the stationary phase, probably owing to protease production or thermal instability. Highest production of P. aeruginosa PAO1 neuraminidase was in BHI culture media. The neuraminidase of P. aeruginosa PAO1 possessed an optimum temperature of activity at 56°C and the activity was maximal at pH 5. Heating the enzyme to 56°C for 45 min., in the presence of bovine serum albumin destroyed 33.1% of it's activity and addition of Ca+2, EDTA and NANA also decreased activity markedly. Conclusion The results revealed that the highest specific activity is for p. aeruginosa PAO1. PMID:22347548

  11. Computational study of interdependence between hemagglutinin and neuraminidase of pandemic 2009 H1N1.

    PubMed

    Hu, Wei

    2015-03-01

    Influenza type A viruses are classified into subtypes based on their two surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA protein facilitates the viral binding and entering a host cell and the NA protein helps the release of viral progeny from the infected cell. The complementary roles of HA and NA entail their collaboration, which has important implications for viral replication and fitness. The HA protein from early strains of pandemic 2009 H1N1 of swine origin preferentially binds to human type receptors with a weak binding to avian type receptors. This virus caused several human deaths in December 2013 in Texas, USA, which motivated us to investigate the changes of genetic features that might contribute to the surged virulence of the virus. Our time series analysis on the strains of this virus collected from 2009 to 2013 implied that the HA binding preference of this virus in USA, Europe, and Asia has been the characteristic of swine H1N1 virus since 2009. However, its characteristic of seasonal human H1N1 and its binding avidity for avian type receptors both were on steady rise and had a clear increase in 2013 with American strains having the sharpest surge. The first change could enhance the viral transmission and replication in humans and the second could increase its ability to cause infection deep in lungs, which might account for the recent human deaths in Texas. In light of HA and NA coadaptation and evolutionary interactions, we also explored the NA activity of this virus to reveal the functional balance between HA and NA during the course of virus evolution. Finally we identified amino acid substitutions in HA and NA of the virus that were critical for the observed evolution.

  12. Structure of the Parainfluenza Virus 5 (PIV5) Hemagglutinin-Neuraminidase (HN) Ectodomain

    PubMed Central

    Welch, Brett D.; Yuan, Ping; Bose, Sayantan; Kors, Christopher A.; Lamb, Robert A.; Jardetzky, Theodore S.

    2013-01-01

    Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F. PMID:23950713

  13. Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

    PubMed Central

    Blanchette, Krystle A.; Shenoy, Anukul T.; Milner, Jeffrey; Gilley, Ryan P.; McClure, Erin; Hinojosa, Cecilia A.; Kumar, Nikhil; Daugherty, Sean C.; Tallon, Luke J.; Ott, Sandra; King, Samantha J.; Ferreira, Daniela M.; Gordon, Stephen B.; Tettelin, Hervé

    2016-01-01

    Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro. Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo. Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. PMID:27481242

  14. Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region.

    PubMed Central

    Markoff, L; Lin, B C; Sveda, M M; Lai, C J

    1984-01-01

    A full-length double-stranded DNA copy of an influenza A virus N2 neuraminidase (NA) gene was cloned into the late region of pSV2330, a hybrid expression vector that includes pBR322 plasmid DNA sequences and the simian virus 40 early region and simian virus 40 late region promoters, splice sequences, and transcription termination sites. The protein encoded by the cloned wild-type NA gene was shown to be present in the cytoplasm of fixed cells and at the surface of "live" or unfixed cells by indirect immunofluorescence with N2 monoclonal antibodies. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]methionine-labeled proteins from wild-type vector-infected cells with heterospecific N2 antibody showed that the product of the cloned NA DNA comigrated with glycosylated NA from influenza virus-infected cells, remained associated with internal membranes of cells fractionated into membrane and cytoplasmic fractions, and could form an immunoprecipitable dimer. NA enzymatic activity was detectable after simian virus 40 lysis of vector-infected cells. These properties of the product of the cloned wild-type gene were compared with those of the polypeptides produced by three deletion mutant NA DNAs that were also cloned into the late region of the pSV2330 vector. These mutants lacked 7 (dlk), 21 (dlI), or all 23 amino acids (dlZ) of the amino (N)-terminal variable hydrophobic region that anchors the mature wild-type NA tetrameric structure in the infected cell or influenza viral membrane. Comparison of the phenotypes of these mutants showed that this region in the NA molecule also includes sequences that control translocation of the nascent polypeptide into membrane organelles for glycosylation. Images PMID:6700587

  15. Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

    PubMed

    Cuevas-Romero, Julieta Sandra; Rivera-Benítez, José Francisco; Hernández-Baumgarten, Eliseo; Hernández-Jaúregui, Pablo; Vega, Marco; Blomström, Anne-Lie; Berg, Mikael; Baule, Claudia

    2016-12-01

    Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Phylogenetic analysis of the N8 neuraminidase gene of influenza A viruses.

    PubMed

    Saito, T; Kawaoka, Y; Webster, R G

    1993-04-01

    Phylogenetic analysis of the N8 neuraminidase (NA) genes from 18 influenza A viruses, representing equine and avian hosts in different geographic locations, revealed three major lineages: (i) currently circulating equine 2 viruses; (ii) avian viruses isolated in the Eurasian region, including A/Equine/Jilin/1/89, a recent avian-like N8 isolate found in horses in China; and (iii) avian viruses isolated in North America. Comparison of mutation rates indicated that avian N8 genes have evolved more slowly than their equine counterparts. That is, in both avian lineages, 72% of the nucleotide changes were silent in the terminal branches of the phylogenetic tree, whereas in equine 2 viruses, 59% of the nucleotide changes were silent. This suggests greater selective pressure on the NA gene from the mammalian immune system, leading to progressive evolution. Alternatively, the slower mutation rate for avian N8 genes could reflect a selective advantage gained from a longer, continuous span of evolution. The shape of the phylogenetic tree, the evolutionary rate, and the calculated date of origin for the N8 equine 2 virus lineage were comparable to findings for the equine 2 virus hemagglutinin (HA) gene (Bean et al., J. Virol. 66, 1129-1138, 1992). This suggests that both viral membrane glycoproteins of equine 2 viruses have evolved together and have been subjected to similar levels of selective pressure. Several amino acid residues were found to differ among the three host-specific lineages, but they may not be involved in host restriction of the NA, as they are shared by EQ/Jilin/1/89 and viruses of avian origin. The present findings complement detailed structural information on the N2 and N9 subtypes and should prove valuable in understanding future X-ray diffraction studies of N8 crystals.

  17. Microbial Neuraminidase Induces a Moderate and Transient Myelin Vacuolation Independent of Complement System Activation.

    PubMed

    Granados-Durán, Pablo; López-Ávalos, María Dolores; Cifuentes, Manuel; Pérez-Martín, Margarita; Fernández-Arjona, María Del Mar; Hughes, Timothy R; Johnson, Krista; Morgan, B Paul; Fernández-Llebrez, Pedro; Grondona, Jesús M

    2017-01-01

    Some central nervous system pathogens express neuraminidase (NA) on their surfaces. In the rat brain, a single intracerebroventricular (ICV) injection of NA induces myelin vacuolation in axonal tracts. Here, we explore the nature, the time course, and the role of the complement system in this damage. The spatiotemporal analysis of myelin vacuolation was performed by optical and electron microscopy. Myelin basic protein-positive area and oligodendrocyte transcription factor (Olig2)-positive cells were quantified in the damaged bundles. Neuronal death in the affected axonal tracts was assessed by Fluoro-Jade B and anti-caspase-3 staining. To evaluate the role of the complement, membrane attack complex (MAC) deposition on damaged bundles was analyzed using anti-C5b9. Rats ICV injected with the anaphylatoxin C5a were studied for myelin damage. In addition, NA-induced vacuolation was studied in rats with different degrees of complement inhibition: normal rats treated with anti-C5-blocking antibody and C6-deficient rats. The stria medullaris, the optic chiasm, and the fimbria were the most consistently damaged axonal tracts. Vacuolation peaked 7 days after NA injection and reverted by day 15. Olig2+ cell number in the damaged tracts was unaltered, and neurodegeneration associated with myelin alterations was not detected. MAC was absent on damaged axonal tracts, as revealed by C5b9 immunostaining. Rats ICV injected with the anaphylatoxin C5a displayed no myelin injury. When the complement system was experimentally or constitutively inhibited, NA-induced myelin vacuolation was similar to that observed in normal rats. Microbial NA induces a moderate and transient myelin vacuolation that is not caused either by neuroinflammation or complement system activation.

  18. Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization.

    PubMed

    Blanchette, Krystle A; Shenoy, Anukul T; Milner, Jeffrey; Gilley, Ryan P; McClure, Erin; Hinojosa, Cecilia A; Kumar, Nikhil; Daugherty, Sean C; Tallon, Luke J; Ott, Sandra; King, Samantha J; Ferreira, Daniela M; Gordon, Stephen B; Tettelin, Hervé; Orihuela, Carlos J

    2016-10-01

    Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Methanolic soluble fractions of lingzhi or reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) extract inhibit neuraminidase activity in Newcastle disease virus (LaSota).

    PubMed

    Shamaki, Bala U; Sandabe, Umar K; Ogbe, Adamu O; Abdulrahman, Fanna I; El-Yuguda, Abdul-Dahiru

    2014-01-01

    The antineuraminidase activity of different organic soluble fractions of Ganoderma lucidum extract was investigated using inhibition of hemagglutination and elution of chicken erythrocytes by Newcastle disease virus (NDV). Fractions of methanol, ethylacetate, and normal butanol (n-butanol) of the G. lucidum were tested against neuraminidase producing NDV as antigen. Different dilutions of the organic soluble fractions inhibited elution of 1% red blood cells by neuraminidase of NDV While the methanolic and n-butanol extracts inhibited neuraminidase activity even at a dilution of 1:16 and that of ethylacetate fraction inhibited even at 1:32 respectively. This finding indicates that G. lucidum has some antineuraminidase activity against NDV and may be exploited in the management of NDV infection.

  20. Change in relative mobility of M protein following neuraminidase treatment in patients with multiple myeloma and monoclonal gammopathy of undetermined significance.

    PubMed

    Kurihara, Yuriko; Iijima, Shiro; Fukushima, Kouhei; Hosaka, Toshiaki; Shiba, Kiyoko

    2004-01-01

    The aim of this study was to clarify the relationship between the relative mobility of M protein in various disease states using cellulose acetate membrane electrophoresis. To examine the carbohydrate chain of the M protein, sera from patients with multiple myeloma (MM), various cancers, and benign disease were treated with neuraminidase. The relative mobility in benign disease and MM patient sera following neuraminidase treatment varied among individuals, while that of IgG M protein in sera from cancer patients was from 0.2 to 0.3. Thus, the relative mobility in cancer patients was narrower than in those with MM or benign disease.However, after neuraminidase treatment, there was no significant difference between relative mobility in cancer patient's sera and those in other disease patients.

  1. Neuraminidase decreases in vitro adherence of slime-forming coagulase-negative staphylococci to biosynthetic ovine collagen vascular graft.

    PubMed

    Onem, Gokhan; Sacar, Mustafa; Sacar, Suzan; Sakarya, Serhan; Turgut, Huseyin; Ozcan, Ali Vefa; Baltalarli, Ahmet

    2006-01-01

    Vascular prosthetic graft infection is a major complication of vascular surgery that starts with adhesion of the microorganism to the graft. Because slime-forming microorganisms are the major causative agents in graft infection, the goals of investigators in this study were (1) to investigate the bacterial adherence of slime-forming and non-slime-forming coagulase-negative staphylococci (CNS), and (2) to determine the role of neuraminidase (NANase) in bacterial adherence to the biosynthetic ovine collagen graft. Human plasma was instilled and incubated at 37 degrees C in preparation for fibrin deposition of grafts. After 48 hours, incubation grafts were drained and inoculated with slime-forming and non-slime-forming CNS in tryptic soy broth in the presence and in the absence of neuraminidase. After 24 hours of incubation at 36 degrees C, grafts were vortexed and cultured for colony count. Bacterial counts were expressed as total colony-forming units per longitudinal centimeter of the graft. Slime-forming CNS had greater affinity to the collagen graft compared with non-slime-forming CNS (P<.05). Adherence of slime-forming CNS was impaired by NANase treatment (P<.001). NANase treatment of patients with non-slime-forming CNS did not change adherence to the graft (P>.05). Results show that slime plays an important role in the pathogenesis of vascular graft infection. Adherence of slime-forming CNS can be decreased through the administration of NANase. This may have implications for the development of neuraminidase-embedded vascular grafts designed to reduce the occurrence of biomaterial-related infection.

  2. Preterm human milk contains a large pool of latent TGF-β, which can be activated by exogenous neuraminidase.

    PubMed

    Namachivayam, Kopperuncholan; Blanco, Cynthia L; Frost, Brandy L; Reeves, Aaron A; Jagadeeswaran, Ramasamy; MohanKumar, Krishnan; Safarulla, Azif; Mandal, Partha; Garzon, Steven A; Raj, J Usha; Maheshwari, Akhil

    2013-06-15

    Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects.

  3. Preterm human milk contains a large pool of latent TGF-β, which can be activated by exogenous neuraminidase

    PubMed Central

    Namachivayam, Kopperuncholan; Blanco, Cynthia L.; Frost, Brandy L.; Reeves, Aaron A.; Jagadeeswaran, Ramasamy; MohanKumar, Krishnan; Safarulla, Azif; Mandal, Partha; Garzon, Steven A.; Raj, J. Usha

    2013-01-01

    Human milk contains substantial amounts of transforming growth factor (TGF)-β, particularly the isoform TGF-β2. We previously showed in preclinical models that enterally administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study we hypothesized that premature infants remain at higher risk of NEC than full-term infants, even when they receive their own mother's milk, because preterm human milk contains less bioactive TGF-β than full-term milk. Our objective was to compare TGF-β bioactivity in preterm vs. full-term milk and identify factors that activate milk-borne TGF-β. Mothers who delivered between 23 0/7 and 31 6/7 wk or at ≥37 wk of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling were measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, TGF-β3, and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Preterm milk contains large quantities of TGF-β, but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects. PMID:23558011

  4. Long time scale GPU dynamics reveal the mechanism of drug resistance of the dual mutant I223R/H275Y neuraminidase from H1N1-2009 influenza virus.

    PubMed

    Woods, Christopher J; Malaisree, Maturos; Pattarapongdilok, Naruwan; Sompornpisut, Pornthep; Hannongbua, Supot; Mulholland, Adrian J

    2012-05-29

    Multidrug resistance of the pandemic H1N1-2009 strain of influenza has been reported due to widespread treatment using the neuraminidase (NA) inhibitors, oseltamivir (Tamiflu), and zanamivir (Relenza). From clinical data, the single I223R (IR(1)) mutant of H1N1-2009 NA reduced efficacy of oseltamivir and zanamivir by 45 and 10 times, (1) respectively. More seriously, the efficacy of these two inhibitors against the double mutant I223R/H275Y (IRHY(2)) was significantly reduced by a factor of 12 374 and 21 times, respectively, compared to the wild-type.(2) This has led to the question of why the efficacy of the NA inhibitors is reduced by the occurrence of these mutations and, specifically, why the efficacy of oseltamivir against the double mutant IRHY was significantly reduced, to the point where oseltamivir has become an ineffective treatment. In this study, 1 μs of molecular dynamics (MD) simulations was performed to answer these questions. The simulations, run using graphical processors (GPUs), were used to investigate the effect of conformational change upon binding of the NA inhibitors oseltamivir and zanamivir in the wild-type and the IR and IRHY mutant strains. These long time scale dynamics simulations demonstrated that the mechanism of resistance of IRHY to oseltamivir was due to the loss of key hydrogen bonds between the inhibitor and residues in the 150-loop. This allowed NA to transition from a closed to an open conformation. Oseltamivir binds weakly with the open conformation of NA due to poor electrostatic interactions between the inhibitor and the active site. The results suggest that the efficacy of oseltamivir is reduced significantly because of conformational changes that lead to the open form of the 150-loop. This suggests that drug resistance could be overcome by increasing hydrogen bond interactions between NA inhibitors and residues in the 150-loop, with the aim of maintaining the closed conformation, or by designing inhibitors that can form

  5. Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus.

    PubMed

    Plant, Ewan P; Ye, Zhiping

    2015-04-01

    We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.

  6. In the Shadow of Hemagglutinin: A Growing Interest in Influenza Viral Neuraminidase and Its Role as a Vaccine Antigen

    PubMed Central

    Wohlbold, Teddy John; Krammer, Florian

    2014-01-01

    Despite the availability of vaccine prophylaxis and antiviral therapeutics, the influenza virus continues to have a significant, annual impact on the morbidity and mortality of human beings, highlighting the continued need for research in the field. Current vaccine strategies predominantly focus on raising a humoral response against hemagglutinin (HA)—the more abundant, immunodominant glycoprotein on the surface of the influenza virus. In fact, anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. Yet, the quest to discern the exact importance of NA-based protection is decades old. Also, while antibodies against the NA glycoprotein fail to prevent infection of the influenza virus, anti-NA immunity has been shown to lessen the severity of disease, decrease viral lung titers in animal models, and reduce viral shedding. Growing evidence is intimating the possible gains of including the NA antigen in vaccine design, such as expanded strain coverage and increased overall immunogenicity of the vaccine. After giving a tour of general influenza virology, this review aims to discuss the influenza A virus neuraminidase while focusing on both the historical and present literature on the use of NA as a possible vaccine antigen. PMID:24960271

  7. An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro

    PubMed Central

    Hsieh, Chung-Fan; Chen, Yu-Li; Lin, Chwan-Fwu; Ho, Jin-Yuan; Huang, Chun-Hsun; Chiu, Cheng-Hsun; Hsieh, Pei-Wen; Horng, Jim-Tong

    2016-01-01

    Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication. PMID:27796330

  8. Chemoenzymatic Syntheses of Sialylated Oligosaccharides Containing C5-Modified Neuraminic Acids for Dual Inhibition of Hemagglutinins and Neuraminidases.

    PubMed

    Birikaki, Lémonia; Pradeau, Stéphanie; Armand, Sylvie; Priem, Bernard; Márquez-Domínguez, Luis; Reyes-Leyva, Julio; Santos-López, Gerardo; Samain, Eric; Driguez, Hugues; Fort, Sébastien

    2015-07-20

    A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them.

  9. Functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 H1N1 influenza pandemic.

    PubMed

    Xu, Rui; Zhu, Xueyong; McBride, Ryan; Nycholat, Corwin M; Yu, Wenli; Paulson, James C; Wilson, Ian A

    2012-09-01

    The 2009 H1N1 influenza pandemic is the first human pandemic in decades and was of swine origin. Although swine are believed to be an intermediate host in the emergence of new human influenza viruses, there is still little known about the host barriers that keep swine influenza viruses from entering the human population. We surveyed swine progenitors and human viruses from the 2009 pandemic and measured the activities of the hemagglutinin (HA) and neuraminidase (NA), which are the two viral surface proteins that interact with host glycan receptors. A functional balance of these two activities (HA binding and NA cleavage) is found in human viruses but not in the swine progenitors. The human 2009 H1N1 pandemic virus exhibited both low HA avidity for glycan receptors as a result of mutations near the receptor binding site and weak NA enzymatic activity. Thus, a functional match between the hemagglutinin and neuraminidase appears to be necessary for efficient transmission between humans and may be an indicator of the pandemic potential of zoonotic viruses.

  10. Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

    PubMed Central

    Krejcova, Ludmila; Hynek, David; Kopel, Pavel; Merlos Rodrigo, Miguel Angel; Adam, Vojtech; Hubalek, Jaromir; Babula, Petr; Trnkova, Libuse; Kizek, Rene

    2013-01-01

    Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids. PMID:23860384

  11. Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

    SciTech Connect

    Yuan, Ping; Paterson, Reay G.; Leser, George P.; Lamb, Robert A.; Jardetzky, Theodore S.

    2012-09-06

    Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN0) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase {beta}-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN{sub 0} and associated reduced virulence.

  12. An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro.

    PubMed

    Hsieh, Chung-Fan; Chen, Yu-Li; Lin, Chwan-Fwu; Ho, Jin-Yuan; Huang, Chun-Hsun; Chiu, Cheng-Hsun; Hsieh, Pei-Wen; Horng, Jim-Tong

    2016-10-31

    Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.

  13. In-Silico screening of Pleconaril and its novel substituted derivatives with Neuraminidase of H1N1 Influenza strain

    PubMed Central

    2012-01-01

    Background Neuraminidase (NA) is a prominent surface antigen of Influenza viruses, which helps in release of viruses from the host cells after replication. Anti influenza drugs such as Oseltamivir target a highly conserved active site of NA, which comprises of 8 functional residues (R118, D151, R152, R224, E276, R292, R371 and Y406) to restrict viral release from host cells, thus inhibiting its ability to cleave sialic acid residues on the cell membrane. Reports on the emergence of Oseltamivir resistant strains of H1N1 Influenza virus necessitated a search for alternative drug candidates. Pleconaril is a novel antiviral drug being developed by Schering-Plough to treat Picornaviridae infections, and is in its late clinical trials stage. Since, Pleconaril was designed to bind the highly conserved hydrophobic binding site on VP1 protein of Picorna viruses, the ability of Pleconaril and its novel substituted derivatives to bind highly conserved hydrophobic active site of H1N1 Neuraminidase, targeting which oseltamivir has been designed was investigated. Result 310 novel substituted variants of Pleconaril were designed using Chemsketch software and docked into the highly conserved active site of NA using arguslab software. 198 out of 310 Pleconaril variants analyzed for docking with NA active site were proven effective, based on their free binding energy. Conclusion Pleconaril variants with F, Cl, Br, CH3, OH and aromatic ring substitutions were shown to be effective alternatives to Oseltamivir as anti influenza drugs. PMID:22340192

  14. Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

    PubMed Central

    Salah, Zuhair W.; Gui, Long; DeVito, Ilaria; Jurgens, Eric M.; Lu, Hong; Yokoyama, Christine C.; Palermo, Laura M.; Lee, Kelly K.

    2012-01-01

    The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect. PMID:22993149

  15. An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

    PubMed

    Zanin, Mark; Duan, Susu; Wong, Sook-San; Kumar, Gyanendra; Baviskar, Pradyumna; Collin, Emily; Russell, Charles; Barman, Subrata; Hause, Benjamin; Webby, Richard

    2017-01-15

    Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

  16. Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice

    USDA-ARS?s Scientific Manuscript database

    Following passage of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 t...

  17. Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens

    USDA-ARS?s Scientific Manuscript database

    The fusion (F) protein of Newcastle disease virus (NDV) plays an important role in viral infection and pathogenicity through mediating membrane fusion between the virion and host cells in the presence of the hemagglutinin-neuraminidase (HN). Previously, we obtained a velogenic NDV genotype VII muta...

  18. Could A Deletion in Neuraminidase Stalk Strengthen Human Tropism of the Novel Avian Influenza Virus H7N9 in China, 2013?

    PubMed Central

    Chen, Liang; Zhu, Feng; Xiong, Chenglong; Zhang, Zhijie; Jiang, Lufang; Chen, Yue; Zhao, Genming; Jiang, Qingwu

    2015-01-01

    Objective. A novel avian influenza A virus (AIV) H7N9 subtype which emerged in China in 2013 caused worldwide concern. Deletion of amino-acids 69 to 73 in the neuraminidase stalk was its most notable characteristic. This study is aimed to discuss the tropism and virulence effects of this deletion. Methods: Neuraminidase gene sequences of N9 subtype were collected from NCBI and GISAID. MEGA6.0, Stata12.0, and UCSF Chimera were employed for sequence aligning, significance testing, and protein tertiary structure homology modeling. Results: A total of 736 sequences were obtained; there were 81 human isolates of the novel AIV H7N9, of which 79 had the deletion. Among all the 654 avian origin sequences, only 43 had the deletion (p < 0.001). Tertiary structure displayed that the deletion obviously changed the spatial direction of neuraminidase. Conclusions: The deletion in neuraminidase stalk could have strengthened human tropism of the novel AIV H7N9, as well as its virulence. PMID:25608590

  19. Functional balance between the hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 HA D222 variants.

    PubMed

    Casalegno, Jean-Sébastien; Ferraris, Olivier; Escuret, Vanessa; Bouscambert, Maude; Bergeron, Corinne; Linès, Laetitia; Excoffier, Thierry; Valette, Martine; Frobert, Emilie; Pillet, Sylvie; Pozzetto, Bruno; Lina, Bruno; Ottmann, Michèle

    2014-01-01

    D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2

  20. An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous challenge.

    PubMed

    Wilson, Jason R; Belser, Jessica A; DaSilva, Juliana; Guo, Zhu; Sun, Xiangjie; Gansebom, Shane; Bai, Yaohui; Stark, Thomas J; Chang, Jessie; Carney, Paul; Levine, Min Z; Barnes, John; Stevens, James; Maines, Taronna R; Tumpey, Terrence M; York, Ian A

    2017-11-01

    The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection. Published by Elsevier Inc.

  1. I223R Mutation in Influenza A(H1N1)pdm09 Neuraminidase Confers Reduced Susceptibility to Oseltamivir and Zanamivir and Enhanced Resistance with H275Y

    PubMed Central

    Abou-Jaoudé, Georges; Scemla, Anne; Ribaud, Patricia; Mercier-Delarue, Séverine; Caro, Valérie; Enouf, Vincent; Simon, François; Molina, Jean-Michel; van der Werf, Sylvie

    2012-01-01

    Background Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. Methods The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0–15) and zanamivir (days 15–25 and 70–80). Results Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6–69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. Conclusions Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines

  2. Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

    NASA Astrophysics Data System (ADS)

    Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

    2010-06-01

    Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

  3. Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes.

    PubMed

    Espínola, Emilio E

    2012-01-01

    Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.

  4. [The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86].

    PubMed

    Arbatskiĭ, N P; Zheltova, A O; Iurtov, D V; Kharitonenkov, I G; Abashev, Iu P; Derevitskaia, V A; Kochetkov, N K

    1990-06-01

    The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.

  5. Progress of small molecular inhibitors in the development of anti-influenza virus agents

    PubMed Central

    Wu, Xiaoai; Wu, Xiuli; Sun, Qizheng; Zhang, Chunhui; Yang, Shengyong; Li, Lin; Jia, Zhiyun

    2017-01-01

    The influenza pandemic is a major threat to human health, and highly aggressive strains such as H1N1, H5N1 and H7N9 have emphasized the need for therapeutic strategies to combat these pathogens. Influenza anti-viral agents, especially active small molecular inhibitors play important roles in controlling pandemics while vaccines are developed. Currently, only a few drugs, which function as influenza neuraminidase (NA) inhibitors and M2 ion channel protein inhibitors, are approved in clinical. However, the acquired resistance against current anti-influenza drugs and the emerging mutations of influenza virus itself remain the major challenging unmet medical needs for influenza treatment. It is highly desirable to identify novel anti-influenza agents. This paper reviews the progress of small molecular inhibitors act as antiviral agents, which include hemagglutinin (HA) inhibitors, RNA-dependent RNA polymerase (RdRp) inhibitors, NA inhibitors and M2 ion channel protein inhibitors etc. Moreover, we also summarize new, recently reported potential targets and discuss strategies for the development of new anti-influenza virus drugs. PMID:28382157

  6. Screening and evaluation of commonly-used anti-influenza Chinese herbal medicines based on anti-neuraminidase activity.

    PubMed

    Han, Xue; Zhang, Ding-Kun; Guo, Yu-Ming; Feng, Wu-Wen; Dong, Qin; Zhang, Cong-En; Zhou, Yong-Feng; Liu, Yan; Wang, Jia-Bo; Zhao, Yan-Ling; Xiao, Xiao-He; Yang, Ming

    2016-10-01

    Anti-influenza Chinese herbal medicines (anti-flu CHMs) have advantages in preventing and treating influenza virus infection. Despite various data on antiviral activities of some anti-flu CHMs have been reported, most of them could not be compared using the standard evaluation methods for antiviral activity. This situation poses an obstacle to a wide application of anti-flu CHMs. Thus, it was necessary to develop an evaluation method to estimate antiviral activities of anti-flu CHMs. In the present study, we searched for anti-flu CHMs, based on clinic usage, to select study objects from commonly-used patented anti-flu Chinese medicines. Then, a neuraminidase-based bioassay, optimized and verified by HPLC method by our research group, was adopted to detect antiviral activities of selected 26 anti-flu CHMs. Finally, eight of these herbs, including Coptidis Rhizoma, Isatidis Folium, Lonicerae Flos, Scutellaria Radix, Cyrtomium Rhizome, Houttuynia Cordata, Gardeniae Fructus, and Chrysanthemi Indici Flos, were shown to have strong antiviral activities with half maximal inhibitory concentration (IC50) values being 2.02 to 6.78 mg·mL(-1) (expressed as raw materials). In contrast, the IC50 value of positive control peramivir was 0.38 mg·mL(-1). Considering the extract yields of CHMs, the active component in these herbs may have a stronger antiviral activity than peramivir, suggesting that these herbs could be further researched for active compounds. Moreover, the proposed neuraminidase-based bioassay was high-throughput and simple and could be used for evaluation and screening of anti-flu CHMs as well as for their quality control.

  7. Structure determination of the 1918 H1N1 neuraminidase from a crystal with lattice-translocation defects

    SciTech Connect

    Zhu, Xueyong; Xu, Xiaojin; Wilson, Ian A.

    2008-08-01

    The structure of the 1918 H1N1 neuraminidase was determined to 1.65 Å from crystals with a lattice-translocation defect using uncorrected, as well as corrected, diffraction data. Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 Å resolution are described. The crystal was indexed in space group C222{sub 1}, with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 Å, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C222{sub 1}, structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects.

  8. Comparison of Serum Hemagglutinin and Neuraminidase Inhibition Antibodies After 2010–2011 Trivalent Inactivated Influenza Vaccination in Healthcare Personnel

    PubMed Central

    Laguio-Vila, Maryrose R.; Thompson, Mark G.; Reynolds, Sue; Spencer, Sarah M.; Gaglani, Manjusha; Naleway, Allison; Ball, Sarah; Bozeman, Sam; Baker, Steven; Martínez-Sobrido, Luis; Levine, Min; Katz, Jackie; Fry, Alicia M.; Treanor, John J.

    2015-01-01

    Background. Most inactivated influenza vaccines contain purified and standardized hemagglutinin (HA) and residual neuraminidase (NA) antigens. Vaccine-associated HA antibody responses (hemagglutination inhibition [HAI]) are well described, but less is known about the immune response to the NA. Methods. Serum of 1349 healthcare personnel (HCP) electing or declining the 2010–2011 trivalent-inactivated influenza vaccine ([IIV3], containing A/California/7/2009 p(H1N1), A/Perth/16/2009 [H3N2], B/Brisbane/60/2008 strains) were tested for NA-inhibiting (NAI) antibody by a modified lectin-based assay using pseudotyped N1 and N2 influenza A viruses with an irrelevant (H5) HA. Neuraminidase-inhibiting and HAI antibody titers were evaluated approximately 30 days after vaccination and end-of-season for those with polymerase chain reaction (PCR)-confirmed influenza infection. Results. In 916 HCP (68%) receiving IIV3, a 2-fold increase in N1 and N2 NAI antibody occurred in 63.7% and 47.3%, respectively. Smaller responses occurred in HCP age >50 years and those without prior 2009–2010 IIV3 nor monovalent A(H1N1)pdm09 influenza vaccinations. Forty-four PCR-confirmed influenza infections were observed, primarily affecting those with lower pre-exposure HAI and NAI antibodies. Higher pre-NAI titers correlated with shorter duration of illness for A(H1N1)pdm09 virus infections. Conclusions. Trivalent-inactivated influenza vaccine is modestly immunogenic for N1 and N2 antigens in HCP. Vaccines eliciting robust NA immune responses may improve efficacy and reduce influenza-associated morbidity. PMID:25884004

  9. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

    SciTech Connect

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2014-10-02

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

  10. Human parainfluenza virus infection of the airway epithelium: viral hemagglutinin-neuraminidase regulates fusion protein activation and modulates infectivity.

    PubMed

    Palermo, Laura M; Porotto, Matteo; Yokoyama, Christine C; Palmer, Samantha G; Mungall, Bruce A; Greengard, Olga; Niewiesk, Stefan; Moscona, Anne

    2009-07-01

    Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN's receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN's F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN's F-triggering activity may have promise in vivo.

  11. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  12. Isolation of novel triple‐reassortant swine H3N2 influenza viruses possessing the hemagglutinin and neuraminidase genes of a seasonal influenza virus in Vietnam in 2010

    PubMed Central

    Ngo, Long Thanh; Hiromoto, Yasuaki; Pham, Vu Phong; Le, Ha Thi Hong; Nguyen, Ha Truc; Le, Vu Tri; Takemae, Nobuhiro; Saito, Takehiko

    2011-01-01

    Please cite this paper as: Ngo et al. (2012) Isolation of novel triple‐reassortant swine H3N2 influenza viruses possessing the hemagglutinin and neuraminidase genes of a seasonal influenza virus in Vietnam in 2010. Influenza and Other Respiratory Viruses 6(1), 6–10. Surveillance of swine influenza viruses (SIVs) in 31 pig farms in northern and southern parts of Vietnam was conducted. Six H3N2 influenza A viruses were isolated from a pig farm in southern Vietnam. They were novel genetic reassortants between a triple–reassortant SIV and a human seasonal H3N2 virus. Their hemagglutinin and neuraminidase genes were derived from a human virus circulating around 2004–2006 and the remaining genes from a triple‐reassortant SIV that originated in North America. This is the first report describing the isolation of a novel triple‐reassortant SIV in Vietnam. PMID:21668659

  13. Identification of single amino acid substitutions (SAAS) in neuraminidase from influenza a virus (H1N1) via mass spectrometry analysis coupled with de novo peptide sequencing.

    PubMed

    Peng, Qisheng; Wang, Zijian; Wu, Donglin; Li, Xiaoou; Liu, Xiaofeng; Sun, Wanchun; Liu, Ning

    2016-08-01

    Amino acid substitutions in the neuraminidase of the influenza virus are the main cause of the emergence of resistance to zanamivir or oseltamivir during seasonal influenza treatment; they are the result of non-synonymous mutations in the viral genome that can be successfully detected by polymer chain reaction (PCR)-based approaches. There is always an urgent need to detect variation in amino acid sequences directly at the protein level. Mass spectrometry coupled with de novo sequencing has been explored as an alternative and straightforward strategy for detecting amino acid substitutions, as well - this approach is the primary focus of the present study. Influenza virus (A/Puerto Rico/8/1934 H1N1) propagated in embryonated chicken eggs was purified by ultracentrifugation, followed by PNGase F treatment. The deglycosylated virion was lysed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel band corresponding to neuraminidase was picked up and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three amino acid substitutions: R346K, S349 N, and S370I/L, in the neuraminidase from the influenza virus (A/Puerto Rico/8/1934 H1N1), which were located in three mutated peptides of the neuraminidase: YGNGVWIGK, TKNHSSR, and PNGWTETDI/LK, respectively. We found that the amino acid substitutions in the proteins of RNA viruses (including influenza A virus) resulting from non-synonymous gene mutations can indeed be directly analyzed via mass spectrometry, and that manual interpretation of the MS/MS data may be beneficial. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  15. Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

    PubMed

    Gattu, Srikanth; Crihfield, Cassandra L; Holland, Lisa A

    2017-01-03

    Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.

  16. Amino Acid 316 of Hemagglutinin and the Neuraminidase Stalk Length Influence Virulence of H9N2 Influenza Virus in Chickens and Mice

    PubMed Central

    Sun, Yipeng; Tan, Yuanyuan; Wei, Kai; Sun, Honglei; Shi, Yi; Pu, Juan; Yang, Hanchun; Gao, George F.; Yin, Yanbo; Feng, Wenhai; Perez, Daniel R.

    2013-01-01

    H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice. PMID:23269805

  17. Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion.

    PubMed

    Bose, Sayantan; Welch, Brett D; Kors, Christopher A; Yuan, Ping; Jardetzky, Theodore S; Lamb, Robert A

    2011-12-01

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  18. Monoclonal antibodies for the detection of desialylation of erythrocyte membranes during haemolytic disease and haemolytic uraemic syndrome caused by the in vivo action of microbial neuraminidase.

    PubMed

    Seitz, R C; Poschmann, A; Hellwege, H H

    1997-09-01

    Especially in childhood, the in vivo action of microbial neuraminidase may cause haemolytic anaemia or life-threatening haemolytic uraemic syndrome. The exposure of the Thomsen-Friedenreich (T) crypto-antigen and T-antigen polyagglutinability of erythrocytes has been described as the first sign of toxic cleavage of N-acetylneuraminic acid (Neu5Ac) from sialoglycoproteins of cell membranes. This phenomenon may, however, be too unspecific to initiate treatment for toxin elimination. The present study investigated the diagnostic effectiveness of a panel of three monoclonal antibodies (mcabs) for the estimation of the clinical significance of neuraminidase action in vivo. Depending on the amount of Neu5Ac released, the mcabs I-C4, II-Q9 and III-Y12 recognized different epitopes on erythrocyte asialoglycophorin. In 1345 patients, the mcab II-09 detected cleavage of Neu5Ac in 32 children who had T-antigen polyagglutinability and mild to moderate haemolytic anaemia. However, only 10 patients, whose erythrocytes were agglutinated by the mcabs III-Y12 or I-C4, developed severe haemolysis, thrombocytopenia, and finally the life-threatening haemolytic uraemic syndrome (p<0.0002). In conclusion, these mcabs provided an early marker of the in vivo action of neuraminidase. Two different degrees of erythrocyte desialylation, as defined by these mcabs, are suggested to reflect the severity of toxin-associated disease.

  19. Rescue of a H3N2 Influenza Virus Containing a Deficient Neuraminidase Protein by a Hemagglutinin with a Low Receptor-Binding Affinity

    PubMed Central

    Richard, Mathilde; Erny, Alexandra; Caré, Bertrand; Traversier, Aurélien; Barthélémy, Mendy; Hay, Alan; Lin, Yi Pu; Ferraris, Olivier; Lina, Bruno

    2012-01-01

    Influenza viruses possess at their surface two glycoproteins, the hemagglutinin and the neuraminidase, of which the antagonistic functions have to be well balanced for the virus to grow efficiently. Ferraris et al. isolated in 2003–2004 viruses lacking both a NA gene and protein (H3NA- viruses) (Ferraris O., 2006, Vaccine, 24(44–46):6656-9). In this study we showed that the hemagglutinins of two of the H3NA- viruses have reduced affinity for SAα2.6Gal receptors, between 49 and 128 times lower than that of the A/Moscow/10/99 (H3N2) virus and no detectable affinity for SAα2.3Gal receptors. We also showed that the low hemagglutinin affinity of the H3NA- viruses compensates for the lack of NA activity and allows the restoration of the growth of an A/Moscow/10/99 virus deficient in neuraminidase. These observations increase our understanding of H3NA- viruses in relation to the balance between the functional activities of the neuraminidase and hemagglutinin. PMID:22563453

  20. Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

    PubMed

    Wohlbold, Teddy John; Chromikova, Veronika; Tan, Gene S; Meade, Philip; Amanat, Fatima; Comella, Phillip; Hirsh, Ariana; Krammer, Florian

    2015-10-28

    Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality. Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or

  1. Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

    PubMed Central

    Xu, Rui; Palmer, Samantha G.; Porotto, Matteo; Palermo, Laura M.; Niewiesk, Stefan; Wilson, Ian A.; Moscona, Anne

    2013-01-01

    ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by

  2. Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site

    PubMed Central

    Diederich, Sandra; Berhane, Yohannes; Embury-Hyatt, Carissa; Hisanaga, Tamiko; Handel, Katherine; Cottam-Birt, Colleen; Ranadheera, Charlene; Kobasa, Darwyn

    2015-01-01

    ABSTRACT Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase

  3. Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9).

    PubMed

    Gaymard, Alexandre; Charles-Dufant, Aymeric; Sabatier, Murielle; Cortay, Jean-Claude; Frobert, Emilie; Picard, Caroline; Casalegno, Jean-Sébastien; Rosa-Calatrava, Manuel; Ferraris, Olivier; Valette, Martine; Ottmann, Michèle; Lina, Bruno; Escuret, Vanessa

    2016-11-01

    While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ± I222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness. Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ± I222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, Km of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined. NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA. NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions

  4. Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus.

    PubMed

    Li, Qing; Sun, Xiaoman; Li, Zhixin; Liu, Yue; Vavricka, Christopher J; Qi, Jianxun; Gao, George F

    2012-11-13

    The recent discovery of the unique genome of influenza virus H17N10 in bats raises considerable doubt about the origin and evolution of influenza A viruses. It also identifies a neuraminidase (NA)-like protein, N10, that is highly divergent from the nine other well-established serotypes of influenza A NA (N1-N9). The structural elucidation and functional characterization of influenza NAs have illustrated the complexity of NA structures, thus raising a key question as to whether N10 has a special structure and function. Here the crystal structure of N10, derived from influenza virus A/little yellow-shouldered bat/Guatemala/153/2009 (H17N10), was solved at a resolution of 2.20 Å. Overall, the structure of N10 was found to be similar to that of the other known influenza NA structures. In vitro enzymatic assays demonstrated that N10 lacks canonical NA activity. A detailed structural analysis revealed dramatic alterations of the conserved active site residues that are unfavorable for the binding and cleavage of terminally linked sialic acid receptors. Furthermore, an unusual 150-loop (residues 147-152) was observed to participate in the intermolecular polar interactions between adjacent N10 molecules of the N10 tetramer. Our study of influenza N10 provides insight into the structure and function of the sialidase superfamily and sheds light on the molecular mechanism of bat influenza virus infection.

  5. Phylogenetic analysis of hemagglutinin and neuraminidase genes of H9N2 viruses isolated from migratory ducks.

    PubMed

    Liu, Jin-Hua; Okazaki, Katsunori; Shi, Wei-Min; Kida, Hiroshi

    2003-12-01

    Genetic analysis indicated that the pandemic influenza strains derived from wild aquatic birds harbor viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) antigenic subtypes. Surveillance studies have shown that H9N2 subtype viruses are worldwide in domestic poultry and could infect mammalian species, including humans. Here, we genetically analyzed the HA and NA genes of five H9N2 viruses isolated from the migratory ducks in Hokkaido, Japan, the flyway of migration from Siberia during 1997-2000. The results showed that HA and NA genes of these viruses belong to the same lineages, respectively. Compared with those of A/quail/Hong Kong/G1/97-like and A/duck/Hong Kong/Y280/97-like viruses, HA and NA of the migratory duck isolates had a close relationship with those of H9N2 viruses isolated from the chicken in Korea, indicating that the Korea H9N2 viruses might be derived from the migratory ducks. The NA genes of the five isolates were located in the same cluster as those of N2 viruses, which had caused a human pandemic in 1968, indicating that the NA genes of the previous pandemic strains are still circulating in waterfowl reservoirs. The present results further emphasize the importance of carrying out molecular epidemiological surveillance of H9N2 viruses in wild ducks to obtain more information for the future human influenza pandemics preparedness.

  6. Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase.

    PubMed

    Ramírez-Salinas, Gema L; García-Machorro, J; Quiliano, Miguel; Zimic, Mirko; Briz, Verónica; Rojas-Hernández, Saul; Correa-Basurto, J

    2015-11-01

    The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50 ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir.

  7. Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

    PubMed Central

    Jones, L V; Compans, R W; Davis, A R; Bos, T J; Nayak, D P

    1985-01-01

    We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process. Images PMID:3016520

  8. Sequence analysis of the neuraminidase genes of avian influenza A viruses isolated from live bird markets in the United States.

    PubMed

    Chander, Yogesh; Jindal, Naresh; Sreevatsan, Srinand; Goyal, Sagar M

    2011-08-01

    Neuraminidase (NA) gene of avian influenza viruses isolated from Live Bird Markets (LBMs) on the east coast of the United States was sequenced and analyzed for mutations associated with antiviral resistance. In total, 189 isolates collected from 1994 to 2005 were used in this study. Full length sequences of the NA gene were obtained from 183 of the 189 isolates. Four different lengths of NA gene were observed; 40 isolates had full length (about 1400 nt), 132 isolates had a deletion of 48 nt, 10 isolates had a deletion of 66 nt, and one isolate had a deletion of 72 nt. Amino acid analysis of the sequence data showed point mutations distributed throughout the gene length. None of these deletions was in the catalytic region and most of the mutations were observed in the flanking regions. None of the isolates had mutations which are known to confer antiviral resistance. These results indicate that though NA gene is prone to mutational changes, much of those changes occur outside the catalytic domain.

  9. Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate

    PubMed Central

    Marathe, Bindumadhav M.; Lévêque, Vincent; Klumpp, Klaus; Webster, Robert G.; Govorkova, Elena A.

    2013-01-01

    The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining Km and Vmax kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories. PMID:23977037

  10. Determination of neuraminidase kinetic constants using whole influenza virus preparations and correction for spectroscopic interference by a fluorogenic substrate.

    PubMed

    Marathe, Bindumadhav M; Lévêque, Vincent; Klumpp, Klaus; Webster, Robert G; Govorkova, Elena A

    2013-01-01

    The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining K(m) and V(max) kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.

  11. Neuraminidase 1 (NEU1) promotes proliferation and migration as a diagnostic and prognostic biomarker of hepatocellular carcinoma

    PubMed Central

    Li, Yixue; Yuan, Shengxian; Zhao, Linghao; Wu, Mengchao; Liu, Lei; Zhou, Weiping

    2016-01-01

    Hepatocellular carcinoma (HCC) is among the most malignant cancers worldwide, lacking biomarkers for subtyping and the reliable prognostication. Herein, we report a novel biomarker, NEU1 (neuraminidase 1), is up-regulated in most samples of HCC. The diagnostic value of NEU1 was evaluated by ROC, and the AUC (area under curve) reached 0.87 and 0.96 in two independent datasets, respectively. The survival differences of HCC patients with high or low expression of NEU1 were statistically significant, and a significant correlation between NEU1 expression and clinical information including stage, differentiation, AFP and embolus were observed. NEU1 expression, at both the mRNA and protein levels, were also higher in the portal vein tumor thrombus than tumor tissues. We also measured the proliferation and migration ability of two HCC cell lines following NEU1 interference and over-expression. Migration and proliferation rate were increased in NEU1 high expression groups. Moreover, gene expression studies identified pathways significantly associated with NEU1 expression. Among them, all the genes involved in spliceosomepathway were up regulated in NEU1-high group. In summary, our work identified NEU1 as a novel biomarker for both diagnosis and prognosis in HCC, and one of the most altered pathway of NEU1 is spliceosome. PMID:27602751

  12. Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

    PubMed Central

    Lee, Youn-Jeong; Sung, Haan-Woo; Choi, Jun-Gu; Lee, Eun-Kyoung; Yoon, Hachung; Kim, Jae-Hong

    2008-01-01

    Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program. PMID:18716451

  13. Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering.

    PubMed

    Bose, Sayantan; Zokarkar, Aarohi; Welch, Brett D; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-09-25

    The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.

  14. Punctuated Evolution of Influenza Virus Neuraminidase (A/H1N1) under Opposing Migration and Vaccination Pressures

    PubMed Central

    Phillips, J. C.

    2014-01-01

    Influenza virus contains two highly variable envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The structure and properties of HA, which is responsible for binding the virus to the cell that is being infected, change significantly when the virus is transmitted from avian or swine species to humans. Here we focus first on the simpler problem of the much smaller human individual evolutionary amino acid mutational changes in NA, which cleaves sialic acid groups and is required for influenza virus replication. Our thermodynamic panorama shows that very small amino acid changes can be monitored very accurately across many historic (1945–2011) Uniprot and NCBI strains using hydropathicity scales to quantify the roughness of water film packages. Quantitative sequential analysis is most effective with the fractal differential hydropathicity scale based on protein self-organized criticality (SOC). Our analysis shows that large-scale vaccination programs have been responsible for a very large convergent reduction in common influenza severity in the last century. Hydropathic analysis is capable of interpreting and even predicting trends of functional changes in mutation prolific viruses directly from amino acid sequences alone. An engineered strain of NA1 is described which could well be significantly less virulent than current circulating strains. PMID:25143953

  15. What adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor?

    PubMed

    Gambaryan, A S; Matrosovich, M N

    2015-07-01

    Wild ducks serve as the primary host for numerous and various influenza type A viruses. Occasionally, viruses from this reservoir can be transferred to other host species and cause outbreaks of influenza in fowl, swine, and horses, as well as result in novel human pandemics. Cellular tropism and range of susceptible host species are determined by interaction between virus and receptor molecules on cells. Here we discuss modern data regarding molecular features underlying interactions of influenza viruses with cellular receptors as well as a role for receptor specificity in interspecies transmission. By analyzing the earliest available pandemic influenza viruses (1918, 1957, 1968, 2009), we found that hemagglutinin reconfigured to recognize 2-6 sialic acid-containing receptors in the human upper airway tract together with altered enzymatic activity of neuraminidase necessary for maintaining functional balance with hemagglutinin are responsible for effective spread of influenza viruses in human populations. Resistance to low pH also contributes to this. Thus, a combination of such parameters makes it possible that influenza viruses give rise to novel pandemics.

  16. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

    PubMed

    Doyle, Tracey M; Li, Changgui; Bucher, Doris J; Hashem, Anwar M; Van Domselaar, Gary; Wang, Junzhi; Farnsworth, Aaron; She, Yi-Min; Cyr, Terry; He, Runtao; Brown, Earl G; Hurt, Aeron C; Li, Xuguang

    2013-11-08

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.

  17. A Conformational Restriction in the Influenza A Virus Neuraminidase Binding Site by R152 Results in a Combinational Effect of I222T and H274Y on Oseltamivir Resistance

    PubMed Central

    Huang, Lan; Cao, Yang; Zhou, Jianfang; Qin, Kun; Zhu, Wenfei; Zhu, Yun; Yang, Lei; Wang, Dayan

    2014-01-01

    The I222K, I222R, and I222T substitutions in neuraminidase (NA) have been found in clinically derived 2009 pandemic influenza A/H1N1 viruses with altered susceptibilities to NA inhibitors (NAIs). The effects of these substitutions, together with the most frequently observed resistance-related substitution, H274Y, on viral fitness and resistance mechanisms were further investigated in this study. Reduced sensitivities to oseltamivir were observed in all three mutants (I222K, I222R, and I222T). Furthermore, the I222K and I222T substitutions had a combinational effect of further increasing resistance in the presence of H274Y, which might result from a conformational restriction in the NA binding site. Of note, by using molecular dynamics simulations, R152, the neighbor of T222, was observed to translate to a position closer to T222, resulting in the narrowing of the binding pocket, which otherwise only subtends the residue substitution of H274Y. Moreover, significantly attenuated NA function and viral growth abilities were found in the I222K+H274Y double mutant, while the I222T+H274Y double mutant exhibited slightly delayed growth but had a peak viral titer similar to that of the wild-type virus in MDCK cells. The relative growth advantage of the I222T mutant versus the I222K mutant and the higher frequency of I222T emerging in N1 subtype influenza viruses raise concerns necessitating close monitoring of the dual substitutions I222T and H274Y. PMID:24366752

  18. Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain).

    PubMed

    Useh, N M; Ajanusi, J O; Esievo, K A N; Nok, A J

    2006-01-01

    A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.

  19. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  20. Design of new inhibitors for H5N1 avian influenza using a molecular dynamics simulation

    NASA Astrophysics Data System (ADS)

    Park, Jin Woo; Jo, Won Ho

    2008-03-01

    Recently, there has been a growing interest in the treatment of H5N1 avian influenza. One of the most widely used antiviral agents is oseltamivir. However, it has been reported that oseltamivir is not as effective against the neuraminidase subtype N1 as it is against subtypes N2 and N9. In our research we addressed this problem by designing new inhibitors and these altered inhibitor's binding affinities were calculated. In this study, we introduced chemical groups to the existing oseltamivir, so to fit into the newly discovered cavity in the subtype N1. When the binding strengths of the oseltamivir and the newly designed inhibitors for N1 were calculated to examine the drug efficiency through a molecular dynamics simulation, then compared with each other, it was found that one of the designed molecules exhibited a strong binding affinity, with more than twice the binding strength than that of oseltamivir. Since the aforementioned designed inhibitor appears to have the possibility for oral activity according to the criteria of human oral bioavailability, we propose that the inhibitor is a promising antiviral drug for H5N1 avian influenza.

  1. Vaccination with Adjuvanted Recombinant Neuraminidase Induces Broad Heterologous, but Not Heterosubtypic, Cross-Protection against Influenza Virus Infection in Mice

    PubMed Central

    Wohlbold, Teddy John; Nachbagauer, Raffael; Xu, Haoming; Tan, Gene S.; Hirsh, Ariana; Brokstad, Karl A.; Cox, Rebecca J.; Palese, Peter

    2015-01-01

    ABSTRACT In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection. PMID:25759506

  2. Avian adeno-associated virus-based expression of Newcastle disease virus hemagglutinin-neuraminidase protein for poultry vaccination.

    PubMed

    Perozo, F; Villegas, P; Estevez, C; Alvarado, I R; Purvis, L B; Saume, E

    2008-06-01

    The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus member of the family Parvoviridae that has been proved to be useful as a viral vector for gene delivery. The use of AAAV for transgenic expression of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein and its ability to induce immunity in chickens were assessed. Proposed advantages of this system include no interference with maternal antibodies, diminished immune response against the vector, and the ability to accommodate large fragments of genetic information. In this work the generation of recombinant AAAV virions expressing the HN protein (rAAAV-HN) was demonstrated by electron microscopy, immunocytochemistry, and western blot analysis. Serological evidence of HN protein expression after in ovo or intramuscular inoculation of the recombinant virus in specific-pathogen-free chickens was obtained. Serum from rAAAV-HN-vaccinated birds showed a systemic immune response evidenced by NDV-specific enzyme-linked immunosorbent assay and hemagglutination inhibition testing. Positive virus neutralization in embryonated chicken eggs and indirect immunofluorescence detection of NDV infected cells by serum from rAAAV-HN vaccinated birds is also reported. A vaccine-challenge experiment in commercial broiler chickens using a Venezuelan virulent viscerotropic strain of NDV was performed. All unvaccinated controls died within 5 days postchallenge. Protection up to 80% was observed in birds vaccinated in ovo and revaccinated at 7 days of age with the rAAAV-HN. The results demonstrate the feasibility of developing and using an AAAV-based gene delivery system for poultry vaccination.

  3. Evolutionary History and Phylodynamics of Influenza A and B Neuraminidase (NA) Genes Inferred from Large-Scale Sequence Analyses

    PubMed Central

    Xu, Jianpeng; Davis, C. Todd; Christman, Mary C.; Rivailler, Pierre; Zhong, Haizhen; Donis, Ruben O.; Lu, Guoqing

    2012-01-01

    Background Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking. Methodology/Principal findings Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The dN/dS ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding. Conclusions/Significance The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance. PMID:22808012

  4. Structure Determination of the 1918 H1N1 Neuraminidase From a Crystal With Lattice-Translocation Defects

    SciTech Connect

    Zhu, X.; Xu, X.; Wilson, I.A.

    2009-05-28

    Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 {angstrom} resolution are described. The crystal was indexed in space group C222{sub 1}, with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 {angstrom}, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C222{sub 1}, structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects.

  5. Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains.

    PubMed

    Yamada, Hiroshi; Moriishi, Eiko; Haredy, Ahmad M; Takenaka, Nobuyuki; Mori, Yasuko; Yamanishi, Koichi; Okamoto, Shigefumi

    2012-12-01

    Dextran sulfate (DS), a negatively charged, sulfated polysaccharide, suppresses the replication of an influenza A virus strain, and this suppression is associated with inhibition of the hemagglutinin (HA)-dependent fusion activity. However, it remains unknown whether the replication of all or just some influenza A virus strains is suppressed by DS, or whether HA is the only target for the replication suppression. In the present study, we found that DS inhibited the replication of some, but not all influenza A virus strains. The suppression in the DS-sensitive strains was dose-dependent and neutralized by diethylaminoethyl-dextran (DD), which has a positive charge. The suppression by DS was observed not only at the initial stage of viral infection, which includes viral attachment and entry, but also at the late stage, which includes virus assembly and release from infected cells. Electron microscopy revealed that the DS induced viral aggregation at the cell surface. The neuraminidase (NA) activity of the strains whose viral replication was inhibited at the late stage was also more suppressed by DS than that of the strains whose replication was not inhibited, and this inhibition of NA activity was also neutralized by adding positively charged DD. Furthermore, we found that replacing the NA gene of a strain in which viral replication was inhibited by DS at the late stage with the NA gene from a strain in which viral replication was not inhibited, eliminated the DS-dependent suppression. These results suggest that the influenza virus NA contributes to the DS-suppressible virus release from infected cells at the late stage, and the suppression may involve the inhibition of NA activity by DS's negative charge.

  6. Enhanced Mammalian Transmissibility of Seasonal Influenza A/H1N1 Viruses Encoding an Oseltamivir-Resistant Neuraminidase

    PubMed Central

    Rahmat, Saad; Pica, Natalie

    2012-01-01

    Between 2007 and 2009, oseltamivir resistance developed among seasonal influenza A/H1N1 (sH1N1) virus isolates at an exponential rate, without a corresponding increase in oseltamivir usage. We hypothesized that the oseltamivir-resistant neuraminidase (NA), in addition to being relatively insusceptible to the antiviral effect of oseltamivir, might confer an additional fitness advantage on these viruses by enhancing their transmission efficiency among humans. Here we demonstrate that an oseltamivir-resistant clinical isolate, an A/Brisbane/59/2007(H1N1)-like virus isolated in New York State in 2008, transmits more efficiently among guinea pigs than does a highly similar, contemporaneous oseltamivir-sensitive isolate. With reverse genetics reassortants and point mutants of the two clinical isolates, we further show that expression of the oseltamivir-resistant NA in the context of viral proteins from the oseltamivir-sensitive virus (a 7:1 reassortant) is sufficient to enhance transmissibility. In the guinea pig model, the NA is the critical determinant of transmission efficiency between oseltamivir-sensitive and -resistant Brisbane/59-like sH1N1 viruses, independent of concurrent drift mutations that occurred in other gene products. Our data suggest that the oseltamivir-resistant NA (specifically, one or both of the companion mutations, H275Y and D354G) may have allowed resistant Brisbane/59-like viruses to outtransmit sensitive isolates. These data provide in vivo evidence of an evolutionary mechanism that would explain the rapidity with which oseltamivir resistance achieved fixation among sH1N1 isolates in the human reservoir. PMID:22532693

  7. Transforming Growth Factor-β: Activation by Neuraminidase and Role in Highly Pathogenic H5N1 Influenza Pathogenesis

    PubMed Central

    Moser, Lindsey A.; O'Brien, Kevin B.; Cline, Troy D.; Jones, Jeremy C.; Tumpey, Terrence M.; Katz, Jacqueline M.; Kelley, Laura A.; Gauldie, Jack; Schultz-Cherry, Stacey

    2010-01-01

    Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus–infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis. PMID:20949074

  8. Infection of influenza virus neuraminidase-vaccinated mice with homologous influenza virus leads to strong protection against heterologous influenza viruses.

    PubMed

    He, Biao; Chang, Haiyan; Liu, Zhihua; Huang, Chaoyang; Liu, Xueying; Zheng, Dan; Fang, Fang; Sun, Bing; Chen, Ze

    2014-12-01

    Vaccination is the best measure to prevent influenza pandemics. Here, we studied the protective effect against heterologous influenza viruses, including A/reassortant/NYMC X-179A (pH1N1), A/Chicken/Henan/12/2004 (H5N1), A/Chicken/Jiangsu/7/2002 (H9N2) and A/Guizhou/54/89×A/PR/8/34 (A/Guizhou-X) (H3N2), in mice first vaccinated with a DNA vaccine of haemagglutinin (HA) or neuraminidase (NA) of A/PR/8/34 (PR8) and then infected with the homologous virus. We showed that PR8 HA or NA vaccination both protected mice against a lethal dose of the homologous virus; PR8 HA or NA DNA vaccination and then PR8 infection in mice offered poor or excellent protection, respectively, against a second, heterologous influenza virus challenge. In addition, before the second heterologous influenza infection, the highest antibody level against nucleoprotein (NP) and matrix (M1 and M2) proteins was found in the PR8 NA-vaccinated and PR8-infected group. The level of induced cellular immunity against NP and M1 showed a trend consistent with that seen in antibody levels. However, PR8 HA+NA vaccination and then PR8 infection resulted in limited protection against heterologous influenza virus challenge. Results of the present study demonstrated that infection of the homologous influenza virus in mice already immunized with a NA vaccine could provide excellent protection against subsequent infection of a heterologous influenza virus. These findings suggested that NA, a major antigen of influenza virus, could be an important candidate antigen for universal influenza vaccines.

  9. A sandwich ELISA for the detection of neuraminidase of avian influenza A(H7N9) virus.

    PubMed

    Yu, Yueyang; Zhang, Xi; Zhao, Baihui; Sun, Ying; Zhang, Xiaoguang; Bai, Tian; Lu, Jian; Li, Zi; Liu, Liqi; Wang, Dayan; Shu, Yuelong; Zhou, Jianfang; Qin, Kun

    2017-09-01

    Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1400 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed. The immunoassay employed mouse monoclonal antibody (mAb) 3C1 to specifically capture the N9 and 3E9 for the detection. Its limit of detection is 6.25ng/ml for N9 protein of A/Anhui/1/2013(H7N9, AH1/2013) and 0.125HAU/50μL for live virus, AH1/2013 and A/Environment/Jiangxi/28/2009 (H11N9), respectively. When applied to test the five clinic throat swabs from H7N9 patients confirmed by nuclear acid testing (NAT) using quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), two samples showed positive result in sandwich ELISA while all were negative using commercial Flu A and H7 subtype rapid antigen tests (RAT). The ELISA using anti-N9 mAbs provided a valuable approach to detect H7N9 virus and quantify the N9 protein. Copyright © 2017. Published by Elsevier B.V.

  10. MiR674 inhibits the neuraminidase-stimulated immune response on dendritic cells via down-regulated Mbnl3.

    PubMed

    Lin, Jian; Chen, Ya T; Xia, Jing; Yang, Qian

    2016-08-02

    Neuraminidase (NA), a structural protein of the H9N2 avian influenza virus (H9N2 AIV), can facilitate viral invasion of the upper airway by cleaving the sialic acid moieties on mucin. Dendritic cells (DCs) are major antigen-presenting cells whose immune functions, such as presenting antigens and activating lymphocytes, can be regulated by microRNAs. Here, we studied the molecular mechanism of miRNA-induced repression of immune responses in mouse DCs. First, we screened for and verified the miRNAs induced by NA. Then, we showed that, consistent with the H9N2 virus treatment, the viral NA up-regulated the expression of miR-155, miR-674, and miR-499 in DCs; however, unlike H9N2 virus treatment, the presence of NA was associated with reduced expression of miR-181b1. Our results suggest that NA significantly increased DC surface markers CD80 and MHCII and enhanced the ability of activating lymphocytes and secreting cytokines compared with HA, NP and M2. Meanwhile, we found that miR-674 and miR-155 over-expression increased all surface markers of DC. Nevertheless, by inhibiting the expression of miR-674 and miR-155, NA lost the ability to promote DC maturation. Furthermore, we predicted and demonstrated that Pgm2l1, Aldh18a1, Camk1d, and Mbnl3 were the target genes of miR-674. Among them, Mbnl3 interference strongly blocked the mature DCs. Collectively, our data shed new light on the roles of and mechanisms involved in the repression of DCs by miRNAs, which may contribute to efforts to develop a prophylaxis for the influenza virus.

  11. MiR674 inhibits the neuraminidase-stimulated immune response on dendritic cells via down-regulated Mbnl3

    PubMed Central

    Lin, Jian; Chen, Ya T.; Xia, Jing; Yang, Qian

    2016-01-01

    Neuraminidase (NA), a structural protein of the H9N2 avian influenza virus (H9N2 AIV), can facilitate viral invasion of the upper airway by cleaving the sialic acid moieties on mucin. Dendritic cells (DCs) are major antigen-presenting cells whose immune functions, such as presenting antigens and activating lymphocytes, can be regulated by microRNAs. Here, we studied the molecular mechanism of miRNA-induced repression of immune responses in mouse DCs. First, we screened for and verified the miRNAs induced by NA. Then, we showed that, consistent with the H9N2 virus treatment, the viral NA up-regulated the expression of miR-155, miR-674, and miR-499 in DCs; however, unlike H9N2 virus treatment, the presence of NA was associated with reduced expression of miR-181b1. Our results suggest that NA significantly increased DC surface markers CD80 and MHCII and enhanced the ability of activating lymphocytes and secreting cytokines compared with HA, NP and M2. Meanwhile, we found that miR-674 and miR-155 over-expression increased all surface markers of DC. Nevertheless, by inhibiting the expression of miR-674 and miR-155, NA lost the ability to promote DC maturation. Furthermore, we predicted and demonstrated that Pgm2l1, Aldh18a1, Camk1d, and Mbnl3 were the target genes of miR-674. Among them, Mbnl3 interference strongly blocked the mature DCs. Collectively, our data shed new light on the roles of and mechanisms involved in the repression of DCs by miRNAs, which may contribute to efforts to develop a prophylaxis for the influenza virus. PMID:27285980

  12. Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments.

    PubMed

    Schrauwen, Eefje J A; Bestebroer, Theo M; Rimmelzwaan, Guus F; Osterhaus, Albert D M E; Fouchier, Ron A M; Herfst, Sander

    2013-01-01

    Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.

  13. Immunological and physiological characteristics of the rapid immune hemolysis of neuraminidase-treated sheep red cells produced by fresh guinea pig serum.

    PubMed

    Lauf, P K

    1975-10-01

    The rapid hemolysis by fresh guinea pig serum known to occur with neuraminidase-treated sheep red cells has been investigated with respect to the immunological and physiological properties of the lytic process. The following observations were made: (a) The susceptibility to hemolysis is directly proportional to the amounts of neuraminic acid enzymatically released from the cell surface. Complement lysis is mediated through binding of an IgM antibody protein to membranes of neuraminidase-treated cells. (b) Hemolysis is relatively temperature-independent above about 28 degrees C but below which a decrease in the hemolysis rate occurs. Arrhenius activation energies above and below the transition temperature were therefore found to be different. (c) Colloid osmotic swelling of neuraminidase-treated high potassium sheep red cells with a chloride ion concentration ratio near unity suspended in high potassium medium could not be prevented by sucrose. Hence, colloid osmotic swelling before lysis must be due to the entrance of sucrose and water since sucrose was the only external solute not at equilibrium. (d) From the rate of swelling and the apparent flux of sucrose under nonsteady state conditions an experimental permeability coefficient (P) for sucrose of 3-10(-8) cm-s-1 was computed. Comparison with a theoretical P of 4-10(-6) cm-s-1 for sucrose freely permeating through a single, hypothetical membrane lesion per cell of 60 A effective diameter indicates a membrane lesion which permits the passage of solutes larger than cations, but clearly constrains the free diffusion of sucrose.

  14. Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

    PubMed Central

    2016-01-01

    Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. PMID:27936604

  15. Investigation of the binding and cleavage characteristics of N1 neuraminidases from avian, seasonal, and pandemic influenza viruses using saturation transfer difference nuclear magnetic resonance

    PubMed Central

    Garcia, Jean-Michel; Lai, Jimmy C C; Haselhorst, Thomas; Choy, Ka Tim; Yen, Hui-Ling; Peiris, Joseph S M; von Itzstein, Mark; Nicholls, John M

    2014-01-01

    Objectives The main function of influenza neuraminidase (NA) involves enzymatic cleavage of sialic acid from the surface of host cells resulting in the release of the newly produced virions from infected cells, as well as aiding the movement of virions through sialylated mucus present in the respiratory tract. However, there has previously been little information on the binding affinity of different forms of sialylated glycan with NA. Our objectives were then to investigate both sialic acid binding and cleavage of neuraminidase at an atomic resolution level. Design Nuclear magnetic resonance (NMR) spectroscopy was used to investigate pH and temperature effects on binding and cleavage as well as to interrogate the selectivity of human-like or avian-like receptors for influenza neuraminidase N1 derived from a range of different influenza virus strains including human seasonal H1N1, H1N1pdm09 and avian H5N1. Results We demonstrated that an acidic pH and physiological temperature are required for efficient NA enzymatic activity; however a change in the pH had a minimum effect on the NA-sialic acid binding affinity. Our data comparing α-2,3- and α-2,6-sialyllactose indicated that the variation in neuraminidase activity on different ligands correlated with a change in binding affinity. Epitope mapping of the sialylglycans interacting with NAs from different viral origin showed different binding profiles suggesting that different binding conformations were adopted. Conclusions The data presented in this study demonstrated that physicochemical conditions (pH in particular) could affect the NA enzymatic activity with minor effect on ligand binding. NA cleavage specificity seemed to be associated with a difference in binding affinity to different ligands, suggesting a relationship between the two events. These findings have implications regarding the replication cycle of influenza infection in the host where different sialidase activities would influence penetration

  16. Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures, Toxin Cell Entry Domains, and Non-Influenza Virus Cell Entry Domains

    PubMed Central

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  17. Using common spatial distributions of atoms to relate functionally divergent influenza virus N10 and N11 protein structures to functionally characterized neuraminidase structures, toxin cell entry domains, and non-influenza virus cell entry domains.

    PubMed

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  18. Synthetic Mucus Nanobarriers for Identification of Glycan-Dependent Primary Influenza A Infection Inhibitors

    PubMed Central

    2016-01-01

    Current drugs against the influenza A virus (IAV) act by inhibiting viral neuraminidase (NA) enzymes responsible for the release of budding virions from sialoglycans on infected cells. Here, we describe an approach focused on a search for inhibitors that reinforce the protective functions of mucosal barriers that trap viruses en route to the target cells. We have generated mimetics of sialo-glycoproteins that insert into the viral envelope to provide a well-defined mucus-like environment encapsulating the virus. By introducing this barrier, which the virus must breach using its NA enzymes to infect a host cell, into a screening platform, we have been able to identify compounds that provide significant protection against IAV infection. This approach may facilitate the discovery of potent new IAV prophylactics among compounds with NA activities too weak to emerge from traditional drug screens. PMID:27800553

  19. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways

    PubMed Central

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-01-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro. PMID:27900002

  20. Effects of water models on binding affinity: evidence from all-atom simulation of binding of tamiflu to A/H5N1 neuraminidase.

    PubMed

    Nguyen, Trang Truc; Viet, Man Hoang; Li, Mai Suan

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔG(bind) of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔG(bind) is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔG(bind) decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM.

  1. Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

    PubMed

    Pushko, Peter; Tretyakova, Irina; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tumpey, Terrence M; Kapczynski, Darrell R

    2017-01-15

    Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. Copyright © 2016. Published by Elsevier Inc.

  2. Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.

    PubMed

    Westgeest, Kim B; Bestebroer, Theo M; Spronken, Monique I J; Gao, Jin; Couzens, Laura; Osterhaus, Albert D M E; Eichelberger, Maryna; Fouchier, Ron A M; de Graaf, Miranda

    2015-06-01

    Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Saccharomyces boulardii expresses neuraminidase activity selective for α2,3-linked sialic acid that decreases Helicobacter pylori adhesion to host cells.

    PubMed

    Sakarya, Serhan; Gunay, Necati

    2014-10-01

    Helicobacter pylori is a major causative agent of gastritis and peptic ulcer disease and is an established risk factor for gastric malignancy. Antibiotic combination therapy can eradicate H. pylori. As these same regimens can evoke adverse effects and resistance, new alternative therapies or adjunctive treatments are needed. A probiotic approach may provide a novel strategy for H. pylori treatment. In the current study, two probiotic bacteria, Lactobacillus acidophilus and Lactobacillus reuteri, and a probiotic yeast, Saccharomyces boulardii, were evaluated for their ability to influence H. pylori viability, adherence to gastric and duodenal cells, as well as the effect of S. boulardii on cell surface expression of sialic acid. Our results indicate that S. boulardii contains neuraminidase activity selective for α(2-3)-linked sialic acid. This neuraminidase activity removes surface α(2-3)-linked sialic acid, the ligand for the sialic acid-binding H. pylori adhesin, which in turn, inhibits H. pylori adherence to duodenal epithelial cells. © 2014 APMIS. Published by John Wiley & Sons Ltd.

  4. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

    PubMed Central

    Elango, N; Coligan, J E; Jambou, R C; Venkatesan, S

    1986-01-01

    The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. Images PMID:3003381

  5. Influenza A H1N1pdm 2009 Virus in Paraguay: Nucleotide Point Mutations in Hemagglutinin and Neuraminidase Genes are not Associated with Drug Resistance

    PubMed Central

    Espínola, Emilio E; Amarilla, Alberto A; Martínez, Magaly; Aquino, Víctor H; Russomando, Graciela

    2014-01-01

    Influenza virus is associated with upper respiratory tract infections. The fourth influenza pandemic was declared in 2009. The aim of this study was to determine the genetic variability of the 2009 H1N1 pandemic virus circulating in Paraguay. Nasal swabs were collected from 181 patients with flu symptoms managed at the Hospital of the Medical School in Asunción, Paraguay, between August and October 2009. Virus detection was carried out by real-time reverse transcription-polymerase chain reaction, followed by sequencing of the hemagglutinin and neuraminidase genes, and phylogenetic analysis. H1N1pdm09 was detected in 14.9% (27/181) of the suspected cases. Analysis of 13 samples showed that these viruses the clustered in a single genetic group. Neither the mutation related to exacerbation of disease (D239G in hemagglutinin) nor that related to antiviral resistance (H275Y in neuraminidase), both detected in neighboring countries, were found. This genetic analysis of H1N1pdm09 will help to understand the spread of the disease. PMID:25328558

  6. Effects of Water Models on Binding Affinity: Evidence from All-Atom Simulation of Binding of Tamiflu to A/H5N1 Neuraminidase

    PubMed Central

    Nguyen, Trang Truc; Viet, Man Hoang

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔGbind of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔGbind is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔGbind decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM. PMID:24672329

  7. Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

    PubMed

    Chen, Li-Gang; Liu, Yuan-Sheng; Zheng, Tang-Hui; Chen, Xu; Li, Ping; Xiao, Chuan-Xing; Ren, Jian-Lin

    2016-11-01

    A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

  8. The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication.

    PubMed

    Takahashi, Tadanobu; Kurebayashi, Yuuki; Ikeya, Kumiko; Mizuno, Takashi; Fukushima, Keijo; Kawamoto, Hiroko; Kawaoka, Yoshihiro; Suzuki, Yasuo; Suzuki, Takashi

    2010-12-09

    The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.

  9. Roles of the Fusion and Hemagglutinin-Neuraminidase Proteins in Replication, Tropism, and Pathogenicity of Avian Paramyxoviruses▿

    PubMed Central

    Kim, Shin-Hee; Subbiah, Madhuri; Samuel, Arthur S.; Collins, Peter L.; Samal, Siba K.

    2011-01-01

    Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of

  10. A Contributing Role for Anti-Neuraminidase Antibodies on Immunity to Pandemic H1N1 2009 Influenza A Virus

    PubMed Central

    Marcelin, Glendie; DuBois, Rebecca; Rubrum, Adam; Russell, Charles J.; McElhaney, Janet E.; Webby, Richard J.

    2011-01-01

    Background Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown. Methods and Results Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera. Conclusions The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies

  11. Detection of Neuraminidase Stalk Motifs Associated with Enhanced N1 Subtype Influenza A Virulence via Raman Spectroscopy

    PubMed Central

    Choi, Joo Young; Martin, Sharon J.H.; Tripp, Ralph A.; Tompkins, S. Mark; Dluhy, Richard A.

    2015-01-01

    Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS).. 5′-thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to

  12. Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate.

    PubMed

    Venereo-Sanchez, Alina; Gilbert, Renald; Simoneau, Melanie; Caron, Antoine; Chahal, Parminder; Chen, Wangxue; Ansorge, Sven; Li, Xuguang; Henry, Olivier; Kamen, Amine

    2016-06-17

    Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization

  13. Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers.

    PubMed

    Desheva, Yulia A; Smolonogina, Tatiana A; Donina, Svetlana A; Rudenko, Larisa G

    2015-04-10

    Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses. In sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2-2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people. Our study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were

  14. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  15. Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

    PubMed

    Huang, K F; Chow, L P; Chiou, S H

    1999-10-05

    A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus. Copyright 1999 Academic Press.

  16. Amino acid substitutions contributing to α2,6-sialic acid linkage binding specificity of human parainfluenza virus type 3 hemagglutinin-neuraminidase.

    PubMed

    Fukushima, Keijo; Takahashi, Tadanobu; Ueyama, Hiroo; Takaguchi, Masahiro; Ito, Seigo; Oishi, Kenta; Minami, Akira; Ishitsubo, Erika; Tokiwa, Hiroaki; Takimoto, Toru; Suzuki, Takashi

    2015-05-08

    Human parainfluenza virus type 3 (hPIV3) recognizes both α2,3- and α2,6-linked sialic acids, whereas human parainfluenza virus type 1 (hPIV1) recognizes only α2,3-linked sialic acids. To identify amino acid residues that confer α2,6-linked sialic acid recognition of hPIV3, amino acid residues in or neighboring the sialic acid binding pocket of the hPIV3 hemagglutinin-neuraminidase (HN) glycoprotein were substituted for the corresponding residues of hPIV1 HN. Hemadsorption assay with sialyl linkage-modified red blood cells indicated that amino acid residues at positions 275, 277, 372, and 426 contribute to α2,6-linked sialic acid recognition of the HN3 glycoprotein. Copyright © 2015. Published by Elsevier B.V.

  17. Molecular characterization of chicken-derived genotype VIId Newcastle disease virus isolates in China during 2005-2012 reveals a new length in hemagglutinin-neuraminidase.

    PubMed

    Zhang, Yuan-Yuan; Shao, Meng-Yu; Yu, Xiao-Hui; Zhao, Jing; Zhang, Guo-Zhong

    2014-01-01

    Newcastle disease (ND) is one of the most important diseases of poultry, and causes severe economic losses in the global poultry industry. Although all Newcastle disease virus (NDV) isolates belong to a single serotype, significant genetic diversity has been described between different NDV isolates. Here, we report the molecular characterization of 23 virulent genotype VIId NDV isolates of class II circulating in China. Phylogenetic construction and analysis revealed the existence of distinctly genomic and amino acid differences that clearly distinguished these isolates from other typical NDV genotypes and vaccine strains. We also report a new 582-amino-acid hemagglutinin-neuraminidase in genotype VII NDV strains. This is believed to be the first study to investigate systematically the most predominant NDV strains, and provides more information on the genetic nature of genotype VIId NDV of class II circulating in China.

  18. Functional Properties and Genetic Relatedness of the Fusion and Hemagglutinin-Neuraminidase Proteins of a Mumps Virus-Like Bat Virus

    PubMed Central

    Krüger, Nadine; Hoffmann, Markus; Drexler, Jan Felix; Müller, Marcel Alexander; Corman, Victor Max; Sauder, Christian; Rubin, Steven; He, Biao; Örvell, Claes; Drosten, Christian

    2015-01-01

    ABSTRACT A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of

  19. Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase.

    PubMed

    Takahashi, Tadanobu; Nidom, Chairul A; Quynh Le, Mai Thi; Suzuki, Takashi; Kawaoka, Yoshihiro

    2012-01-01

    Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.

  20. Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus.

    PubMed

    Krüger, Nadine; Hoffmann, Markus; Drexler, Jan Felix; Müller, Marcel Alexander; Corman, Victor Max; Sauder, Christian; Rubin, Steven; He, Biao; Örvell, Claes; Drosten, Christian; Herrler, Georg

    2015-04-01

    A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests

  1. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

    PubMed

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh; Ideris, Aini

    2016-03-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

  2. Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction.

    PubMed

    Li, Yan; Wu, Tao; Qi, Xian; Ge, Yiyue; Guo, Xiling; Wu, Bin; Yu, Huiyan; Zhu, Yefei; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao; Zhou, Minghao

    2013-12-01

    A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

  3. Neuraminidase produces a decrease of adherence of slime-forming Staphylococcus aureus to gelatin-impregnated polyester fiber graft fabric: an experimental study.

    PubMed

    Sacar, Mustafa; Onem, Gokhan; Baltalarli, Ahmet; Sacar, Suzan; Turgut, Huseyin; Goksin, Ibrahim; Ozcan, Vefa; Sakarya, Serhan

    2007-01-01

    Because slime-forming microorganisms are the major causative agents of graft infections, we aimed to investigate bacterial adherence in slime-forming and nonslime-forming Staphylococcus aureus and to determine the role of neuraminidase (NANase) on adherence to gelatin-impregnated polyester fiber graft fabric. An in vitro model was developed to quantitatively measure bacterial adherence to the surface of the graft. The grafts were divided into two groups - those colonized with slime-forming S. aureus and those colonized with nonslime-forming S. aureus. The grafts were put into sterile tubes and human plasma was instilled and incubated at 37 degrees C to perform fibrin deposition on the grafts. After 48 h of incubation, grafts were drained and inoculated with slime-forming or nonslime-forming S. aureus in triptic soy broth in the presence or absence of NANase. Following 36 h of incubation at 36 degrees C, grafts were vortexed and cultured to perform a colony count. Bacterial counts were expressed as total colony-forming units per square centimeter of graft. Slime-forming S. aureus had greater affinity with the graft compared with nonslime-forming S. aureus (P < 0.05). The adherence of slime-forming S. aureus was impaired by NANase treatment (P < 0.001) but NANase treatment of nonslime-forming S. aureus did not change the adherence to the graft (P > 0.05). These results show that slime plays an important role in the pathogenesis of vascular graft infection. Adherence of slime-forming S. aureus can be decreased by NANase treatment. This may have implications for the development of neuraminidase-embedded vascular grafts to diminish biomaterial-related infections.

  4. In situ molecular identification of the Influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City

    PubMed Central

    2013-01-01

    Background In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City’s hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. Methods In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. Results We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. Conclusions We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City. PMID:23327529

  5. Antiviral activity of chlorogenic acid against influenza A (H1N1/H3N2) virus and its inhibition of neuraminidase

    PubMed Central

    Ding, Yue; Cao, Zeyu; Cao, Liang; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2017-01-01

    Lonicera japonica Thunb, rich in chlorogenic acid (CHA), is used for viral upper respiratory tract infection treatment caused by influenza virus, parainfluenza virus, and respiratory syncytial virus, ect in China. It was reported that CHA reduced serum hepatitis B virus level and death rate of influenza virus-infected mice. However, the underlying mechanisms of CHA against the influenza A virus have not been fully elucidated. Here, the antiviral effects and potential mechanisms of CHA against influenza A virus were investigated. CHA revealed inhibitory against A/PuertoRico/8/1934(H1N1) (EC50 = 44.87 μM), A/Beijing/32/92(H3N2) (EC50 = 62.33 μM), and oseltamivir-resistant strains. Time-course analysis showed CHA inhibited influenza virus during the late stage of infectious cycle. Indirect immunofluorescence assay indicated CHA down-regulated the NP protein expression. The inhibition of neuraminidase activity confirmed CHA blocked release of newly formed virus particles from infected cells. Intravenous injection of 100 mg/kg/d CHA possessed effective antiviral activity in mice, conferring 60% and 50% protection from death against H1N1 and H3N2, reducing virus titres and alleviating inflammation in the lungs effectively. These results demonstrate that CHA acts as a neuraminidase blocker to inhibit influenza A virus both in cellular and animal models. Thus, CHA has potential utility in the treatment of the influenza virus infection. PMID:28393840

  6. Role of R292K mutation in influenza H7N9 neuraminidase toward oseltamivir susceptibility: MD and MM/PB(GB)SA study

    NASA Astrophysics Data System (ADS)

    Phanich, Jiraphorn; Rungrotmongkol, Thanyada; Kungwan, Nawee; Hannongbua, Supot

    2016-10-01

    The H7N9 avian influenza virus is a novel re-assortment from at least four different strains of virus. Neuraminidase, which is a glycoprotein on the surface membrane, has been the target for drug treatment. However, some H7N9 strains that have been isolated from patient after drug treatment have a R292K mutation in neuraminidase. This substitution was found to facilitate drug resistance using protein- and virus- assays, in particular it gave a high resistance to the most commonly used drug, oseltamivir. The aim of this research is to understand the source of oseltamivir resistance using MD simulations and the MM/PB(GB)SA binding free energy approaches. Both methods can predict the reduced susceptibility of oseltamivir in good agreement to the IC 50 binding energy, although MM/GBSA underestimates this prediction compared to the MM/PBSA calculation. Electrostatic interaction is the main contribution for oseltamivir binding in terms of both interaction and solvation. We found that the source of the drug resistance is a decrease in the binding interaction combined with the reduction of the dehydration penalty. The smaller K292 mutated residue has a larger binding pocket cavity compared to the wild-type resulting in the loss of drug carboxylate-K292 hydrogen bonding and an increased accessibility for water molecules around the K292 mutated residue. In addition, oseltamivir does not bind well to the R292K mutant complex as shown by the high degree of fluctuation in ligand RMSD during the simulation and the change in angular distribution of bulky side chain groups.

  7. Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease virus hemagglutinin-neuraminidase is due to a temperature-dependent defect in receptor binding.

    PubMed

    Corey, Elizabeth A; Mirza, Anne M; Levandowsky, Elizabeth; Iorio, Ronald M

    2003-06-01

    The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37 degrees C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.

  8. Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

    PubMed Central

    Seladi-Schulman, Jill; Campbell, Patricia J.; Suppiah, Suganthi; Steel, John; Lowen, Anice C.

    2014-01-01

    Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo. PMID:25383873

  9. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  10. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  11. The Second Receptor Binding Site of the Globular Head of the Newcastle Disease Virus Hemagglutinin-Neuraminidase Activates the Stalk of Multiple Paramyxovirus Receptor Binding Proteins To Trigger Fusion

    PubMed Central

    Salah, Zuhair; DeVito, Ilaria; Talekar, Aparna; Palmer, Samantha G.; Xu, Rui; Wilson, Ian A.

    2012-01-01

    The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN′s globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN′s site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation. PMID:22438532

  12. The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route.

    PubMed

    Fujimoto, Yoshikazu; Ito, Hiroshi; Ono, Etsuro; Kawaoka, Yoshihiro; Ito, Toshihiro

    2016-04-01

    Influenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine. Neuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to

  13. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  14. Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

    PubMed

    Byrn, Randal A; Jones, Steven M; Bennett, Hamilton B; Bral, Chris; Clark, Michael P; Jacobs, Marc D; Kwong, Ann D; Ledeboer, Mark W; Leeman, Joshua R; McNeil, Colleen F; Murcko, Mark A; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W; Zhou, Yi; Charifson, Paul S

    2015-03-01

    VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

  15. Preclinical Activity of VX-787, a First-in-Class, Orally Bioavailable Inhibitor of the Influenza Virus Polymerase PB2 Subunit

    PubMed Central

    Byrn, Randal A.; Jones, Steven M.; Bennett, Hamilton B.; Bral, Chris; Clark, Michael P.; Jacobs, Marc D.; Kwong, Ann D.; Ledeboer, Mark W.; Leeman, Joshua R.; McNeil, Colleen F.; Murcko, Mark A.; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W.; Zhou, Yi

    2014-01-01

    VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m7GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection. PMID:25547360

  16. Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors.

    PubMed

    Maillard, Michel C; Brookfield, Frederick A; Courtney, Stephen M; Eustache, Florence M; Gemkow, Mark J; Handel, Rebecca K; Johnson, Laura C; Johnson, Peter D; Kerry, Mark A; Krieger, Florian; Meniconi, Mirco; Muñoz-Sanjuán, Ignacio; Palfrey, Jordan J; Park, Hyunsun; Schaertl, Sabine; Taylor, Malcolm G; Weddell, Derek; Dominguez, Celia

    2011-10-01

    Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ∼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Iota-Carrageenan Is a Potent Inhibitor of Influenza A Virus Infection

    PubMed Central

    Leibbrandt, Andreas; Meier, Christiane; König-Schuster, Marielle; Weinmüllner, Regina; Kalthoff, Donata; Pflugfelder, Bettina; Graf, Philipp; Frank-Gehrke, Britta; Beer, Martin; Fazekas, Tamas; Unger, Hermann; Prieschl-Grassauer, Eva; Grassauer, Andreas

    2010-01-01

    The 2009 flu pandemic and the appearance of oseltamivir-resistant H1N1 influenza strains highlight the need for treatment alternatives. One such option is the creation of a protective physical barrier in the nasal cavity. In vitro tests demonstrated that iota-carrageenan is a potent inhibitor of influenza A virus infection, most importantly also of pandemic H1N1/2009 in vitro. Consequently, we tested a commercially available nasal spray containing iota-carrageenan in an influenza A mouse infection model. Treatment of mice infected with a lethal dose of influenza A PR8/34 H1N1 virus with iota-carrageenan starting up to 48 hours post infection resulted in a strong protection of mice similar to mice treated with oseltamivir. Since alternative treatment options for influenza are rare, we conclude that the nasal spray containing iota-carrageenan is an alternative to neuraminidase inhibitors and should be tested for prevention and treatment of influenza A in clinical trials in humans. PMID:21179403

  18. CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR

    PubMed Central

    Kunitz, M.

    1947-01-01

    A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496

  19. Therapeutic efficacy of peramivir against H5N1 highly pathogenic avian influenza viruses harboring the neuraminidase H275Y mutation.

    PubMed

    Kobayashi, Masanori; Kodama, Makoto; Noshi, Takeshi; Yoshida, Ryu; Kanazu, Takushi; Nomura, Naoki; Soda, Kosuke; Isoda, Norikazu; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Yamano, Yoshinori; Sato, Akihiko; Kida, Hiroshi

    2017-03-01

    High morbidity and mortality associated with human cases of highly pathogenic avian influenza (HPAI) viruses, including H5N1 influenza virus, have been reported. The purpose of the present study was to evaluate the antiviral effects of peramivir against HPAI viruses. In neuraminidase (NA) inhibition and virus replication inhibition assays, peramivir showed strong inhibitory activity against H5N1, H7N1 and H7N7 HPAI viruses with sub-nanomolar activity in enzyme assays. In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation.

  20. Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

    PubMed

    Woods, Christopher J; Shaw, Katherine E; Mulholland, Adrian J

    2015-01-22

    The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

  1. Proteome Response of Chicken Embryo Fibroblast Cells to Recombinant H5N1 Avian Influenza Viruses with Different Neuraminidase Stalk Lengths

    PubMed Central

    Li, Yongtao; Ming, Fan; Huang, Huimin; Guo, Kelei; Chen, Huanchun; Jin, Meilin; Zhou, Hongbo

    2017-01-01

    The variation on neuraminidase (NA) stalk region of highly pathogenic avian influenza H5N1 virus results in virulence change in animals. In our previous studies, the special NA stalk-motif of H5N1 viruses has been demonstrated to play a significant role in the high virulence and pathogenicity in chickens. However, the molecular mechanisms underlying the pathogenicity of viruses with different NA stalk remain poorly understood. This study presents a comprehensive characterization of the proteome response of chicken cells to recombinant H5N1 virus with stalk-short NA (rNA-wt) and the stalkless NA mutant virus (rSD20). 208 proteins with differential abundance profiles were identified differentially expressed (DE), and these proteins were mainly related to stress response, transcription regulation, transport, metabolic process, cellular component and cytoskeleton. Through Ingenuity Pathways Analysis (IPA), the significant biological functions of DE proteins represented included Post-Translational Modification, Protein Folding, DNA Replication, Recombination and Repair. It was interesting to find that most DE proteins were involved in the TGF-β mediated functional network. Moreover, the specific DE proteins may play important roles in the innate immune responses and H5N1 virus replication. Our data provide important information regarding the comparable host response to H5N1 influenza virus infection with different NA stalk lengths. PMID:28079188

  2. Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens.

    PubMed

    Lv, Jing; Wei, Liangmeng; Yang, Yan; Wang, Bingxiao; Liang, Wei; Gao, Yuwei; Xia, Xianzhu; Gao, Lili; Cai, Yumei; Hou, Peiqiang; Yang, Huili; Wang, Airong; Huang, Rong; Gao, Jing; Chai, Tongjie

    2015-04-18

    Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.

  3. An alpha2,6-sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both sialyltransferase and neuraminidase activity.

    PubMed

    Mine, Toshiki; Katayama, Sakurako; Kajiwara, Hitomi; Tsunashima, Masako; Tsukamoto, Hiroshi; Takakura, Yoshimitsu; Yamamoto, Takeshi

    2010-02-01

    We cloned, expressed, and characterized a novel beta-galactoside alpha2,6-sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56-96% identity to the marine bacterial alpha2,6-sialyltransferases classified into glycosyltransferase family 80. The sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial alpha2,6-sialyltransferases. Although alpha2,6-sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only alpha2,6-sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both alpha2,6-sialyltransferase and alpha2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.

  4. The hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus strain Italien (ndv Italien): comparison with HNs of other strains and expression by a vaccinia recombinant.

    PubMed

    Wemers, C D; de Henau, S; Neyt, C; Espion, D; Letellier, C; Meulemans, G; Burny, A

    1987-01-01

    A cDNA library was constructed with poly(A+) mRNA from cells infected with the virulent Italien NDV strain. A clone that hybridized to the HN gene mRNA was sequenced. A long open reading-frame encodes for a protein of 571 amino acids, with a calculated molecular weight of 61,900, including 13 cysteine residues and six potential glycosylation sites. To define the sequence changes that occurred in the avian paramyxovirus hemagglutinin-neuraminidase (HN) during the evolution of virulence, we have studied the HNs of the virulent Italien NDV strain, the mesovirulent Beaudette strain and the nonvirulent Hitchner strain. The majority of amino acid variations are conservative changes but they cluster at 4 preferential sites in the putative head of HN. The clusters of amino acid substitutions are intimately associated or overlap with regions of HN rich in charged amino acid residues and in cysteines. The latter are conserved not only between HNs from all 3 NDV strains but also between HNs of 4 different paramyxoviruses, NDV, SV 5, Sendai and PI 3. The HN coding sequence was inserted into the genome of vaccinia virus under the control of vaccinia P 7.5 K transcriptional regulatory sequences. Expression of native HN proteins at the surface of recombinant HN vaccinia-infected cells was demonstrated by indirect immunofluorescence with 2 anti-HN monoclonals.

  5. Heterogeneity of rat FSH by chromatofocusing: studies on in-vitro bioactivity of pituitary FSH forms and effect of neuraminidase treatment.

    PubMed

    Blum, W F; Riegelbauer, G; Gupta, D

    1985-04-01

    This study concerned the resolution of rat pituitary FSH utilizing chromatofocusing. Among the 11 components resolved and positively identified, ten had apparent isoelectric points (pI) between 3.1 and 5.1. Approximately 1% of pituitary FSH eluted at pH 9.4. Treatment with varying amounts of neuraminidase followed by refocusing generated FSH components of higher pI values. Treatment with other glycosidases did not alter the elution characteristics in chromatofocusing, while exclusion chromatography established an inverse relationship between apparent molecular weight and pI. Dose-response curves of various FSH components and of the reference preparation in the current radioimmunoassay system were parallel to each other. A study of their in-vitro bioactivity, utilizing granulosa cells which produce a plasminogen activator due to FSH in a dose-dependent manner, provided the following evidence: increased acidity of the components led to an increase of maximum response and an increase of the dose necessary for half-maximum response. Considering the observed alterations in the heterogeneity of FSH with changing physiological states of the animal, it is concluded that qualitative changes of the FSH molecule are perhaps involved in a modulatory role in the biopotencies of the hormone.

  6. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

    PubMed

    Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

    2016-05-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

  7. Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets.

    PubMed

    Smith, Gale E; Sun, Xiangjie; Bai, Yaohui; Liu, Ye V; Massare, Michael J; Pearce, Melissa B; Belser, Jessica A; Maines, Taronna R; Creager, Hannah M; Glenn, Gregory M; Flyer, David; Pushko, Peter; Levine, Min Z; Tumpey, Terrence M

    2017-09-01

    Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses. Published by Elsevier Inc.

  8. Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase.

    PubMed

    Bang, Sunghee; Li, Wei; Ha, Thi Kim Quy; Lee, Changyeol; Oh, Won Keun; Shim, Sang Hee

    2017-05-15

    To determine the compounds responsible for its anti-influenza activities, we isolated the three flavonoids, 6-hydroxyluteolin 7-O-β-d-glucoside (1), nepitrin (2), homoplantaginin (3) from the MeOH extract of Salvia plebeia R.Br. and identified them by comparing the spectroscopic data with that reported in the literature. The contents of the three flavonoids in the whole extract were 108.74 ± 0.95, 46.26 ± 2.19, and 69.35 ± 1.22 mg/g for 6-hydroxyluteolin 7-O-β-d-glucoside, nepitrin, and homoplantaginin, respectively, which demonstrates that they are the major constituents of this plant. The three flavonoids were evaluated for their inhibitory activities against influenza virus H1N1 A/PR/9/34 neuraminidase and H1N1-induced cytopathic effect (CPE) on Madin-Darby canine kidney (MDCK) cells. Our results demonstrated the following arrangement for their anti-influenza activities: nepitrin (2) > 6-hydroxyluteolin 7-O-β-d-glucoside (1) > homoplantaginin (3). The potent inhibitory activities of these flavonoids against influenza suggested their potential to be developed as novel anti-influenza drugs in the future.

  9. Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids.

    PubMed

    Jariwalla, R J; Roomi, M W; Gangapurkar, B; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2007-01-01

    Influenza, one of the oldest and most common infections, poses a serious health problem causing significant morbidity and mortality, and imposing substantial economic costs. The efficacy of current drugs is limited and improved therapies are needed. A unique nutrient mixture (NM), containing ascorbic acid, green tea extract, lysine, proline, N-acetyl cysteine, selenium among other micronutrients, has been shown to exert anti-carcinogenic and anti-atherogenic activity both in vitro and in vivo. Many of the constituents of NM have been shown to have an inhibitory effect on replication of influenza virus and HIV. This prompted us to study the effect of NM on influenza A virus multiplication in infected cells and neuraminidase activity (NA) in virus particles. Addition of NM to Vero or MDCK cells post infection resulted in dose-dependent inhibition of viral nucleoprotein (NP) production in infected cells. NM-mediated inhibition of viral NP was selective and not due to cytotoxicity towards host cells. This antiviral effect was enhanced by pretreatment of virus with the nutrient mixture. Individual components of NM, namely ascorbic acid and green tea extract, also blocked viral NP production, conferring enhanced inhibition when tested in combination. Incubation of cell-free virus with NM resulted in dose-dependent inhibition of associated NA enzyme activity. In conclusion, the nutrient mixture exerts an antiviral effect against influenza A virus by lowering viral protein production in infected cells and diminishing viral enzymatic activity in cell-free particles.

  10. Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.

    PubMed

    Cerriteño-Sánchez, José Luis; Santos-López, Gerardo; Rosas-Murrieta, Nora Hilda; Reyes-Leyva, Julio; Cuevas-Romero, Sandra; Herrera-Camacho, Irma

    2016-04-10

    Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  12. Molecular characterization of the hemagglutinin-neuraminidase gene of porcine rubulavirus isolates associated with neurological disorders in fattening and adult pigs.

    PubMed

    Sánchez-Betancourt, J I; Santos-López, G; Alonso, R; Doporto, J M; Ramírez-Mendoza, H; Mendoza, S; Hernández, J; Reyes-Leyva, J; Trujillo, M E

    2008-10-01

    "Blue eye disease" is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6-PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6-PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6-PAC9 viruses seems to be associated with their increased neurovirulence.

  13. Genetic evolution of the neuraminidase of influenza A (H3N2) viruses from 1968 to 2009 and its correspondence to haemagglutinin evolution

    PubMed Central

    Westgeest, Kim B.; de Graaf, Miranda; Fourment, Mathieu; Bestebroer, Theo M.; van Beek, Ruud; Spronken, Monique I. J.; de Jong, Jan C.; Rimmelzwaan, Guus F.; Russell, Colin A.; Osterhaus, Albert D. M. E.; Smith, Gavin J. D.; Smith, Derek J.

    2012-01-01

    Each year, influenza viruses cause epidemics by evading pre-existing humoral immunity through mutations in the major glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). In 2004, the antigenic evolution of HA of human influenza A (H3N2) viruses was mapped (Smith et al., Science 305, 371–376, 2004) from its introduction in humans in 1968 until 2003. The current study focused on the genetic evolution of NA and compared it with HA using the dataset of Smith and colleagues, updated to the epidemic of the 2009/2010 season. Phylogenetic trees and genetic maps were constructed to visualize the genetic evolution of NA and HA. The results revealed multiple reassortment events over the years. Overall rates of evolutionary change were lower for NA than for HA1 at the nucleotide level. Selection pressures were estimated, revealing an abundance of negatively selected sites and sparse positively selected sites. The differences found between the evolution of NA and HA1 warrant further analysis of the evolution of NA at the phenotypic level, as has been done previously for HA. PMID:22718569

  14. Natural inhibitors of thrombin.

    PubMed

    Huntington, James A

    2014-04-01

    The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

  15. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  16. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM.

  17. Theoretical analysis of the neuraminidase epitope of the Mexican A H1N1 influenza strain, and experimental studies on its interaction with rabbit and human hosts.

    PubMed

    Loyola, Paola Kinara Reyes; Campos-Rodríguez, R; Bello, Martiniano; Rojas-Hernández, S; Zimic, Mirko; Quiliano, Miguel; Briz, Verónica; Muñoz-Fernández, M Angeles; Tolentino-López, Luis; Correa-Basurto, Jose

    2013-05-01

    The neuraminidase (NA) epitope from the Mexican AH1N1 influenza virus was identified by using sequences registered at the GenBank during the peak of a pandemic (from April 2009 to October 2010). First, NA protein sequences were submitted for multiple alignment analysis, and their three-dimensional models (3-D) were then built by using homology modeling. The most common sequence (denominated wild-type) and its mutants were submitted to linear and nonlinear epitope predictors, which included the major histocompatibility complex type II (MHC II) and B-cell peptides. The epitope prediction was in accordance with evolutionary behavior and some protein structural properties. The latter included a low NA mutation rate, NA 3-D surface exposure, and the presence of high hindrance side chain residues. After selecting the epitope, docking studies and molecular dynamics (MD) simulations were used to explore interactions between the epitope and MHC II. Afterward, several experimental assays were performed to validate the theoretical study by using antibodies from humans (infected by pandemic H1N1) and rabbits (epitope vaccination). The results show 119 complete sequences that were grouped into 28 protein sequences according to their identity (one wild-type and 27 representative mutants (1-5 mutations)). The predictors yielded several epitopes, with the best fit being the one located in the C-terminal region. Theoretical methods demonstrated that the selected epitope reached the P4, P6, P7, and P9 pockets of MHC II, whereas the experimental evidence indicates that the epitope is recognized by human antibodies and also by rabbit antibodies immunized with the peptide.

  18. Protection of pigs against pandemic swine origin H1N1 influenza A virus infection by hemagglutinin- or neuraminidase-expressing attenuated pseudorabies virus recombinants.

    PubMed

    Klingbeil, Katharina; Lange, Elke; Blohm, Ulrike; Teifke, Jens P; Mettenleiter, Thomas C; Fuchs, Walter

    2015-03-02

    Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba.

  19. Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

    PubMed

    Henritzi, Dinah; Zhao, Na; Starick, Elke; Simon, Gaelle; Krog, Jesper S; Larsen, Lars Erik; Reid, Scott M; Brown, Ian H; Chiapponi, Chiara; Foni, Emanuela; Wacheck, Silke; Schmid, Peter; Beer, Martin; Hoffmann, Bernd; Harder, Timm C

    2016-11-01

    A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe. © 2016 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  20. Molecular characterization of partial fusion gene and C-terminus extension length of haemagglutinin-neuraminidase gene of recently isolated Newcastle disease virus isolates in Malaysia

    PubMed Central

    2010-01-01

    Background Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene. Results The coding region of eleven NDV isolates fusion (F) gene and carboxyl terminal region of haemagglutinin-neuraminidase (HN) gene including extensions were amplified by reverse transcriptase PCR and directly sequenced. All the isolates have shown to have non-synonymous to synonymous base substitution rate ranging between 0.081 - 0.264 demonstrating presence of negative selection. Analysis based on F gene showed the characterized isolates possess three different types of protease cleavage site motifs; namely 112RRQKRF117, 112RRRKRF117 and 112GRQGRL117 and appear to show maximum identities with isolates in the region such as cockatoo/14698/90 (Indonesia), Ch/2000 (China), local isolate AF2240 indicating the high similarity of isolates circulating in the South East Asian countries. Meanwhile, one of the isolates resembles commonly used lentogenic vaccine strains. On further characterization of the HN gene, Malaysian isolates had C-terminus extensions of 0, 6 and 11 amino acids. Analysis of the phylogenetic tree revealed that the existence of three genetic groups; namely, genotype II, VII and VIII. Conclusions The study concluded that the occurrence of three types of NDV genotypes and presence of varied carboxyl terminus extension lengths among Malaysian isolates incriminated for sporadic cases. PMID:20691110

  1. Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

    PubMed Central

    Brown, Lorena E.; Barr, Ian G.; Gilbertson, Brad; Lowther, Sue; Kachurin, Anatoly; Kachurina, Olga; Klippel, Jessica; Bodle, Jesse; Pearse, Martin; Middleton, Deborah

    2013-01-01

    In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO4 or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed. PMID:23283953

  2. The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface.

    PubMed

    Qian, Guomin; Wang, Song; Chi, Xiaojuan; Li, Hua; Wei, Haitao; Zhu, Xiaomei; Chen, Yuhai; Chen, Ji-Long

    2014-12-01

    Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully underst