Influenza Virus Neuraminidase Research Articles

Reduction in Sympathetic Nerve Activity as a Possible Mechanism for the Hypothermic Effect of Oseltamivir, an Anti‐influenza Virus Drug, in Normal Mice

Oseltamivir, an anti-influenza virus drug, has strong antipyretic effects in mice (Biological and Pharmaceutical Bulletin, 31, 2008, 638) and patients with influenza. In addition, hypothermia has been reported as an adverse event. The prodrug oseltamivir is converted to oseltamivir carboxylate (OC), an active metabolite of influenza virus neuraminidase. In this study, core body temperature was measured in mice, and oseltamivir and OC were administered intracerebroventricularly (i.c.v.) or intraperitoneally (i.p). Low i.c.v. doses of oseltamivir and OC dose-dependently produced hypothermia. Zanamivir (i.c.v.), another neuraminidase inhibitor, did not produce hypothermia. These results suggested that the hypothermic effects of oseltamivir (i.p. and i.c.v.) and OC (i.c.v.) are not due to neuraminidase inhibition. OC (i.p.) did not lower body temperature. Although mecamylamine (i.c.v.) blocked the hypothermic effect of nicotine-administered i.c.v., the hypothermic effects of oseltamivir and OC (i.c.v.) were not blocked by mecamylamine (i.c.v.). The effect of oseltamivir (i.p.) was markedly increased by s.c.-pre-administered mecamylamine and also hexamethonium, a peripherally acting ganglionic blocker, suggesting their potentiating interaction at peripheral sites. The hypothermic effect of nicotine (i.c.v.) was decreased by lower doses of oseltamivir (i.c.v.), suggesting the anti-nicotinic action of oseltamivir. These results suggest that oseltamivir (i.p.) causes hypothermia through depression of sympathetic temperature regulatory mechanisms via inhibition of nicotinic receptor function and through unknown central mechanisms.

Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

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.

The pharmacokinetic and safety profiles of zanamivir after single and repeat intravenous administration in healthy Japanese males

Neuraminidase inhibitors are important options for the treatment of infection by the influenza virus. For the treatment of severe influenza, parenteral administration of a neuraminidase inhibitor may be desirable. This study was conducted to evaluate the pharmacokinetic and safety profiles of intravenous zanamivir, an influenza viral neuraminidase inhibitor, in Japanese subjects to further characterize these profiles particularly following relatively high-doses when compared with inhalation doses and to provide reassurance that there are no marked differences with profiles reported for other ethnically different populations. Single doses of 100, 300, 600mg zanamivir were administered to healthy Japanese men in a randomized, double-blind, ascending dose, placebo-controlled, incomplete three-period cross-over study. In period 3, subjects were given 600mg of zanamivir on day 1, followed by a 60h washout period and then a 5-day course of 600mg zanamivir twice daily. Each subjects received two of three active dosages of zanamivir from 100, 300 and 600mg, and placebo. Adverse events reported in the study were all mild in intensity and resolved without any treatment. The mean AUC0-∞ values after single intravenous administration of 100, 300 and 600mg were 16768, 53462 and 100400ng·h/mL, respectively, demonstrating dose proportionality. No accumulation or time variance was observed after 5days of twice-daily administration of 600mg zanamivir. Urinary concentrations of zanamivir after single doses ranging from 100 to 600mg indicated that over 94% of the zanamivir administered was excreted in urine within 24h. Single and 5-day BID repeat dosing of 600mg were safely administered in Japanese healthy subjects. The pharmacokinetic profile of zanamivir after intravenous administration was consistent with previously reported findings in non-Japanese subjects.

Trapping influenza neuraminidase

A team from The University of British Columbia has used a mechanism-based approach to design irreversible inhibitors of influenza virus neuraminidase. The compounds are being developed by the venture arm of The Centre for Drug Research and Development, a public-private partnership that commercializes discoveries from Canadian researchers.

Influenza virus A(H1N1)pdm09 hemagglutinin polymorphism and associated disease in southern Germany during the 2010/11 influenza season

A novel influenza A virus emerged in early 2009 to cause the first influenza pandemic of the 21st century. Understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. In this study, we investigated the molecular evolution of influenza virus A(H1N1)pdm09 in the 2010/11 influenza season in southern Germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. Phylogenetic analysis revealed co-circulation of different genetic groups. The D222G mutation, which had previously been observed in severe cases, was not detected. Immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. Interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. The H275Y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient.

Nanoparticles from Plant Saponins as Delivery System for Mucosal Influenza Vaccine

This article were studied nanoparticles assembled purified influenza virus hemagglutinin and neuraminidase antigens and low toxicity immunostimulating saponins Gg6 and Ah6, isolated from Kazakhstanian plants <i>G.glabra</i> and <i>A.hippocastanum</i>, in the animal vaccination/challenge experiments as delivery system for mucosal influenza subunit vaccine. Incorporation of influenza virus HA and NA antigens into the structure of nanoparticles significantly increased IgG, IgM and IgA antibody immune responses as well as production of IFN-gamma and IL-2 after single intranasal immunization. Mucosal immunization with nanoparticles possessed HA+NA antigens and saponins Gg6 or Ah6 resulted much better protection of chickens against challenge of highly pathogenic H7N1 avian influenza virus in comparison with immunization of whole virus inactivated vaccine or vaccine mixed with alum hydroxide adjuvant.

Carbosilane glycodendrimers

Glycodendrimers fascinate both carbohydrate chemists and biologists because of their ability to recognize lectins and enhance carbohydrate-protein interactions. These characteristics make glycodendrimers a valuable tool in glycoscience and chemical biology. Many glycodendrimers have been described to date; this tutorial review focuses specifically on carbosilane glycodendrimers. We present methodologies for synthesizing parent carbosilane dendrimers and describe their use in biological assays. We also describe representative functionalizations of parent carbosilane dendrimers at terminal positions which are necessary for chemical ligation with carbohydrate ligands. This is followed by a description of all coupling reactions between carbohydrate and carbosilane dendrimer functionalities used in the synthesis of carbosilane glycodendrimers. The major emphasis of this review is the use of carbosilane glycodendrimers as medical agents against Shiga toxins, dengue viruses, relapsing fever Borrelia, and hemagglutinin and neuraminidase of influenza viruses, as well as on the relationship between dendrimer structure and these biological activities. The last two sections introduce recent attempts to use carbosilane glycodendrimers as new versatile and widely-applicable lectin sensors, and the use of carbosilane glycodendrimers as a novel drug carrier in an active targeting drug delivery system. This review article will be of interest to scientists in the areas of organic chemistry, chemical biology, carbohydrate chemistry, heteroatom chemistry, and organosilicon chemistry.

Active Peptide from Acetes Chinensis with Inhibitory Activity on Neuraminidase of Influenza Virus

Low-valued Acetes Chinensis was chosen to prepare the enzymatic hydrolysate with neuraminidase(NA) inhibitory activity by controlled hydrolysis. A novel NA inhibitory peptide was purified by gel chromatography and reversed phase high performance liquid chromatography. The IC50value of the peptide was only96. 1 μmol / L and it could also significantly inhibit influenza A virus replication in MDCK cells. The amino acid sequence of this peptide was EISYIHAEAYRRGELK which identified by LC-MS / MS and its binding with NA protein was proved by UV spectrum analysis. The accurate sites of NA protein that the peptide bind to were determined using the computer assisted software SYBYL. The largest non-toxic concentration( TC0)was1. 26 mg / mL which assayed by cell cytotoxicity experiments. The hemagglutination essay showed that this pepetide could obviously decrease the agglutination titer influenza virus in a dose-dependent manner which suggested that the antiviral activity of this peptide might be attributed to its action on multiple targets.

In silico study on multidrug resistance conferred by I223R/H275Y double mutant neuraminidase

It was recently reported that an I223R/H275Y double mutant of neuraminidase (NA) creates a multidrug-resistant form of the pandemic influenza A (H1N1) virus. However, a comprehensive understanding of the molecular mechanisms is still lacking. We conducted a systematic in silico study to explore the structural basis underlying this multidrug resistance. By molecular docking analyses and molecular dynamics (MD) simulations, we compared various biochemical and biophysical properties of the wild type, the I223R single mutant and the I223R/H275Y double mutant NA with two inhibitors, zanamivir (ZMR) and oseltamivir (G39). The binding free energy of oseltamivir with all types of NA was substantially lower than its zanamivir counterpart. On the other hand, the binding free energy of each inhibitor with wild type NA was generally higher than that with mutant NAs. MD simulation outcomes exemplify distinct patterns for oseltamivir and zanamivir with all types of NA. In particular, the stronger resistance of the double mutant NA relative to the wild and single mutant types can be ascribed to the overall looser but locally more compact structure of the former. Specifically, as a whole the double mutant NA adapts to the larger gyration radius and greater distance between charged atom groups, which is contrary to the pattern in the local binding site region. The enhanced resistance of all types of NA to oseltamivir rather than zanamivir might be accounted for similarly. We expect these findings to provide significant insights into improving inhibitors for the multidrug-resistant neuraminidase of H1N1 influenza viruses.

DelEnsembleElec: Computing Ensemble-Averaged Electrostatics Using DelPhi

AbstractA new VMD plugin that interfaces with DelPhi to provide ensemble-averaged electrostatic calculations using the Poisson-Boltzmann equation is presented. The general theory and context of this approach are discussed, and examples of the plugin interface and calculations are presented. This new tool is applied to systems of current biological interest, obtaining the ensemble-averaged electrostatic properties of the two major influenza virus glycoproteins, hemagglutinin and neuraminidase, from explicitly solvated all-atom molecular dynamics trajectories. The differences between the ensemble-averaged electrostatics and those obtained from a single structure are examined in detail for these examples, revealing how the plugin can be a powerful tool in facilitating the modeling of electrostatic interactions in biological systems.

Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus.

Photodegradation and inhibition of drug-resistant influenza virus neuraminidase using anthraquinone–sialic acid hybrids

The anthraquinone-sialic acid hybrids designed effectively degraded not only non-drug-resistant neuraminidase but also drug-resistant neuraminidase, which is an important target of anti-influenza therapy. Degradation was achieved using long-wavelength UV radiation in the absence of any additives and under neutral conditions. Moreover, the hybrids efficiently inhibited neuraminidase activities upon photo-irradiation.

A single E105K mutation far from the active site of influenza B virus neuraminidase contributes to reduced susceptibility to multiple neuraminidase-inhibitor drugs

Drugs inhibiting the enzymatic activity of influenza virus neuraminidase (NA) are the cornerstone of therapy for influenza virus infection. The emergence of drug-resistant variants may limit the benefits of antiviral therapy. Here we report the recovery of an influenza B virus with reduced susceptibilities to NA inhibitors from a human patient with no history of antiviral drug treatment. The virus, designated B/Kochi/61/2011, was isolated by inoculating Madin–Darby canine kidney (MDCK) cells with respiratory specimens from the patient. NA inhibition assays demonstrated that the B/Kochi/61/2011 isolate showed a remarkable reduction in susceptibility to peramivir. The isolate also exhibited low to moderately reduced sensitivity to oseltamivir, laninamivir, and zanamivir. A sequence analysis of viruses propagated in MDCK cells revealed that the isolate contained a mutation (E105K) not previously associated with reduced susceptibility to NA inhibitors. However, pyrosequencing analysis showed that the NA E105K mutation was below a detectable level in the original clinical specimens, suggesting that the mutant virus may be preferably selected during propagation in MDCK cells. Analysis of the three-dimensional model of E105 and K105 NAs with peramivir suggested that the E105K mutation at the monomer–monomer interface of the NA tetramer may destabilize the tetrameric form of NA, leading to decreased susceptibility to NA inhibitors. These results have implications for understanding the mechanism of resistance against NA-inhibitor drugs.

The neuraminidase of bat influenza viruses is not a neuraminidase

Influenza A viruses are classified into subtypes according to their two viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA). These viruses are known to infect multiple animal species, including humans, pigs, horses, dogs, sea mammals, chickens, ducks, and other birds. Although humans are sporadically infected with avian influenza virus strains belonging to multiple subtypes, only H1, H2, and H3 HA subtypes and N1 and N2 NA subtypes have been documented to cause pandemics and annual epidemics of influenza in humans. By contrast, all H16 and N9 subtypes of influenza A viruses are found circulating in birds. The recent discovery of influenza A viruses clearly distinct from any known avian influenza A virus subtype in little yellow-shouldered bats from Guatemala has been the first example of an influenza A virus subtype not found in avian species (1). Bat influenza viruses have glycoproteins related to the HA and NA of influenza A virus, which prompted to the classification of these viruses as H17N10 influenza A virus. The crystal structure of the N10 glycoprotein is now solved independently by Li et al. (2) and Zhu et al. (3) in PNAS, and these works provide important clues on the characteristics of these newly discovered influenza viruses.

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(-1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3' sialyllactosamine (3'-SLN) and 6'-SLN sialosides with equilibrium dissociation constant (K(D)) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.

Influenza virus neuraminidase contributes to the dextran sulfate-dependent suppressive replication of some influenza A virus strains

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.

Enhanced Anti-influenza Agents Conjugated with Anti-inflammatory Activity

Influenza therapy with a single targeted compound is often limited in efficacy due to the rapidly developed drug resistance. Moreover, the uncontrolled virus-induced cytokines could cause the high mortality of human infected by H5N1 avian influenza virus. In this study, we explored the novel dual-targeted bifunctional anti-influenza drugs formed by conjugation with anti-inflammatory agents. In particular, the caffeic acid (CA)-bearing zanamivir (ZA) conjugates ZA-7-CA (1) and ZA-7-CA-amide (7) showed simultaneous inhibition of influenza virus neuraminidase and suppression of pro-inflammatory cytokines. These ZA conjugates provided remarkable protection of cells and mice against influenza infections. Intranasal administration of low dosage (<1.2 μmol/kg/day) of ZA conjugates exhibited much greater effect than the combination therapy with ZA and the anti-inflammatory agents in protection of the lethally infected mice by H1N1 or H5N1 influenza viruses.

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

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 reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).

Molecular docking, molecular dynamics simulation, and QSAR model on potent thiazolidine-4-carboxylic acid inhibitors of influenza neuraminidase

In the current work, the relationship between the structure and activity of a series of novel thiazolidine-4-carboxylic acid derivatives as potent influenza virus neuraminidase inhibitors was studied using docking, molecular dynamics (MD) simulations, and QSAR analysis. A 7,000 ps MD simulation in a cubic water box were employed to build 3D structure of the 2HU4 in a water environment. After reaching the equilibrium, the inhibitors were docked into the 2HU4 to realize the binding site of the enzyme. The docking analysis showed that the interaction of the inhibitors with residues Arg371, Arg430, Gly429, Ile427, Lys432, Pro431, Trp403, and Tyr347 plays an important role in the activities of the inhibitors. The docked configurations of the inhibitors with the lowest free energy were used to calculate the most feasible descriptors. The selected descriptors were related to the inhibitory activities using stepwise multiple linear regression, classification and regression trees, and least squares support vector regression techniques. The satisfactory results (R p 2 = 0.883, Q LOO 2 = 0.872, R L25%O 2 = 0.835, RMSELOO = 0.310, and RMSEL25%O = 0.352) demonstrate that CART-LS-SVR models present the relationship between influenza virus neuraminidase inhibitors activity and molecular descriptors clearly. An energetic analysis based on MD calculations, revealed that the potency of the most active compound binding is governed by electrostatic and van der Waals contacts. The results provide a set of useful guidelines for the rational design of novel influenza virus neuraminidase inhibitors.