Avidin-biotin complex-based capture coating platform for universal Influenza virus immobilization and characterization.

Multiple sialic acid (SA) residues conjugated to a linear polyacrylamide backbone are more effective than monomeric SA at inhibiting influenza-induced agglutination of red blood cells. However, "polymeric inhibitors" based on polyacrylamide backbones are cytotoxic. Dendritic polymers offer a nontoxic alternative to polyacrylamide and may provide a variety of potential synthetic inhibitors of influenza virus adhesion due to the wide range of available polymer structures. We evaluated several dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, comb-branched, and dendrigraft polymers, for the ability to inhibit virus hemagglutination (HA) and to block infection of mammalian cells in vitro. Four viruses were tested: influenza A H2N2 (selectively propagated two ways), X-31 influenza A H3N2, and sendai. The most potent of the linear and spheroidal inhibitors were 32-256-fold more effective than monomeric SA at inhibiting HA by the H2N2 influenza virus. Linear-dendron copolymers were 1025-8200-fold more effective against H2N2 influenza, X-31 influenza, and sendai viruses. The most effective were the comb-branched and dendrigraft inhibitors, which showed up to 50000-fold increased activity against these viruses. We were able to demonstrate significant (p < 0.001) dose-dependent reduction of influenza infection in mammalian cells by polymeric inhibitors, the first such demonstration for multivalent SA inhibitors. Effective dendrimer polymers were not cytotoxic to mammalian cells at therapeutic levels. Of additional interest, variation in the inhibitory effect was observed with different viruses, suggesting possible differences due to specific growth conditions of virus. SA-conjugated dendritic polymers may provide a new therapeutic modality for viruses that employ SA as their target receptor.

Cryptantigens: time to uncover the real significance of T-activation.

Should the Neuraminidase-1 locus be considered as part of the major histocompatibility complex?

The infection of pandemic influenza viruses such as swine flu (H1N1) and avian flu viruses to the host cells is related to the following two factors: First, the surface protein such as HA (hemagglutinin) and NA (neuraminidase) of the influenza virus. Second, the specific structure of the oligosaccharide [sialic acid(alpha2-6) galactose(beta1-4)glucose or sialic acid(alpha2-3)galactose(beta1-4)glucose] on the host cell. After recognizing the specific structure of the oligosaccharide on the surface of host cells by the surface protein of the influenza virus, the influenza virus can secrete sialidase and cleave the sialic acid attached on the final position of the specific structure of the oligosaccharide on the surface of host cells. Tamiflu (oseltamivir), known as a remedy of swine flu, has a saccharide analog structure, especially the sialic acid analog. Tamiflu can inhibit the invasion of influenza viruses (swine flu and avian flu viruses) into the host cells by competition with sialic acid on the terminal position of the specific oligosaccharide on the surface of the host cell. Because of the emergence of Tamiflu resistance, the development of new potent anti-influenza inhibitors is needed. The inhibitors with positive-charge groups have potential as antiviral therapeutics, and the strain specificity must also be resolved.

Uptake of Influenza Virus By Platelets Occurs Via Phagocytosis

Objective To observe the expression of influenza virus sialic acid receptors(SAa2, 3-gal and SAa2, 6-gal)and the influence of dexamethasone intervention to its expression in the airway of asthmatic mice. Methods Thirty BALB/c mice were randomly divided into normal group (group N, n=10), asthma group (group A, n=10) and dexamethasone intervention group (group D, n=10). Mice of group A were sensitized and challenged by ovalbumin (OVA) to build asthmatic model. Mice in group D were given dexamethasone (2 mg/kg i. p.) 30 minutes before challenging, other procedures were the same as those of group A. The levels of IL-5 in the brochoalveolar lavage fluid (BALF) of mice in each group were determined by ELISA, the pathological changes of lung tissues were observed by HE staining, the degree of mucus secretion in airways were observed by AB-PAS staining, the expression of Mucin5ac (Muc5ac) in lung tissues were observed by immunohistochemical (IHC) staining, the expression of virus sialic acid receptors were observed by immunofluorescence. Results The counts of eosinophils, the level of IL-5 in BALF, the airway inflammatory pathological scores, the degree of mucus secretion, the expression of Muc5ac protein of group A were higher than those in group N and D (P<0.05). Receptors of SA α 2, 3-gal and SA α 2, 6-gal were expressed in all groups (P<0.05). The average positive rate for SA α 2, 3-gal receptor in group A were higher than that in group N. The average positive rate for SA α 2, 3-gal and SA α 2, 6-gal receptor in group D were higher than those of group N and A (P<0.05). Conclusion All these results indicate that the high expression of influenza virus sialic acid receptors due to asthma and dexamethasone intervention may be the mechanism of the enhanced susceptibility to influenza virus of asthma patients. Key words: Asthma; Mouse; Immunofluorescence; Influenza virus; Sialic acid

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity. Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to alpha2-->6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the alpha2-->3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of alpha2-->3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of alpha2-->6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.

The role of cell‐surface sialic acid in the interaction of 125I‐labeled choriogonadotropin with gonadotropin receptors from plasma membranes of bovine corpus luteum was investigated. Pretreatment of plasma membranes with neuraminidases purified from Clostridium perfringens or Vibrio cholera enhanced the binding activity of 125I‐choriogonadotropin in a concentration‐dependent manner. C. perfringens neuraminidase (100 mU/ml) and V. cholerae neuraminidase (1.0 I.U./ml) maximally stimulated gonadotropin‐binding activity. The neuraminidase stimulation of binding was due to unmasking of gonadotropin‐binding sites and not due to a change in affinity of the receptor for 125I‐choriogonadotropin. The activity of plasma‐ membrane 5′‐nucleotidase, a glycoprotein enzyme, however, was not affected by neuraminidase treatment. The stimulatory effect of C. perfringens enzyme was abolished by co‐incubation of plasma membrane with glycoproteins, fetuin and mucin. Furthermore, the stimulatory effect of neuraminidase was due to an intrinsic property of the enzyme and was accompanied by a parallel dose‐dependent loss of plasma‐membrane‐associated sialic acid residues. Other neuraminidase preparations including that purified from Arthrobacter ureafaciens also enhanced 125I‐choriogonadotropin binding with concomitant hydrolysis of plasma‐membrane sialic acid. By contrast, enzyme purified from influenza virus did not release plasma‐membrane sialic acid and consequently failed to modulate receptor activity. Treatment of plasma membrane with neuraminidase and subsequent fractionation demonstrated that the enzyme hydrolyzed 50–60% sialic acid from glycoprotein and ganglioside components. Preincubation of plasma membrane with wheat‐germ agglutinin partially blocked the hydrolysis of glycoprotein sialic acid as well as stimulation of 125I‐choriogonadotropin‐binding activity. Incubation with other lectins, concanavalin A and peanut agglutinin, neither affected the hydrolysis of glycoprotein/ganglioside sialic acid by neuraminidase nor prevented the stimulatory action of enzyme on 125I‐choriogonadotropin‐binding activity. These studies demonstrate a possible involvement of plasma‐membrane‐associated sialic acid (sialoglycoproteins) in the activity of gonadotropin receptors.

Influenza virus is a respiratory pathogen that can cause disease in humans, with symptoms ranging from mild to life-threatening. The vast majority of influenza virus infections in humans are observed during seasonal epidemics and occasional pandemics. Given the substantial public health burden associated with influenza virus infection, yearly vaccination is recommended for protection against seasonal influenza viruses. Despite vigilant surveillance for new variants and careful selection of seasonal vaccine strains, the efficacy of seasonal vaccines can vary widely from year to year. This often results in lowered protection within the population, regardless of vaccination status. In order to broaden the protection afforded by seasonal influenza vaccines, the National Institute of Allergy and Infectious Diseases (NIAID) has deemed the development of a universal influenza virus vaccine to be a priority in influenza virus vaccine research. This universal vaccine would provide protection against all influenza virus strains, eliminating the need for the yearly reformulations of seasonal influenza vaccines. In addition to universal influenza vaccine efforts, substantial progress has been made in developing novel influenza virus therapeutics that utilize broadly neutralizing antibodies to provide protection against influenza virus infection and to mitigate disease outcomes during infection. In this review, we discuss various approaches toward the goal of improving influenza virus vaccine efficacy through a universal influenza virus vaccine. We also address the novel methods of discovery and utilization of broadly neutralizing antibodies to improve influenza disease outcomes.

Influenza viruses (IVs) are a continual threat to global health. The high mutation rate of the IV genome makes this virus incredibly successful, genetic drift allows for annual epidemics which result in thousands of deaths and millions of hospitalizations. Moreover, the emergence of new strains through genetic shift (e.g., swine-origin influenza A) can cause devastating global outbreaks of infection. Neuraminidase inhibitors (NAIs) are currently used to treat IV infection and act directly on viral proteins to halt IV spread. However, effectivity is limited late in infection and drug resistance can develop. New therapies which target highly conserved features of IV such as antibodies to the stem region of hemagglutinin or the IV RNA polymerase inhibitor: Favipiravir are currently in clinical trials. Compared to NAIs, these treatments have a higher tolerance for resistance and a longer therapeutic window and therefore, may prove more effective. However, clinical and experimental evidence has demonstrated that it is not just viral spread, but also the host inflammatory response and damage to the lung epithelium which dictate the outcome of IV infection. Therapeutic regimens for IV infection should therefore also regulate the host inflammatory response and protect epithelial cells from unnecessary cell death. Anti-inflammatory drugs such as etanercept, statins or cyclooxygenase enzyme 2 inhibitors may temper IV induced inflammation, demonstrating the possibility of repurposing these drugs as single or adjunct therapies for IV infection. IV binds to sialic acid receptors on the host cell surface to initiate infection and productive IV replication is primarily restricted to airway epithelial cells. Accordingly, targeting therapies to the epithelium will directly inhibit IV spread while minimizing off target consequences, such as over activation of immune cells. The neuraminidase mimic Fludase cleaves sialic acid receptors from the epithelium to inhibit IV entry to cells. While type III interferons activate an antiviral gene program in epithelial cells with minimal perturbation to the IV specific immune response. This review discusses the above-mentioned candidate anti-IV therapeutics and others at the preclinical and clinical trial stage.

The Galanthus nivalis agglutinin (GNA)-related lectin family exhibit significant anti-HIV and anti-HSV properties that are closely related to their carbohydrate-binding activities. However, there is still no conclusive evidence that GNA-related lectins possess anti-influenza properties. The hemagglutinin (HA) of influenza virus is a surface protein that is involved in binding host cell sialic acid during the early stages of infection. Herein, we studied the 3D-QSARs (three-dimensional quantitative structure-activity relationships) of lectin- and HA-sialic acid by molecular modeling. The affinities and stabilities of lectin- and HA-sialic acid complexes were also assessed by molecular docking and molecular dynamics simulations. Finally, anti-influenza GNA-related lectins that possess stable conformations and higher binding affinities for sialic acid than HAs of human influenza virus were screened, and a possible mechanism was proposed. Accordingly, our results indicate that some GNA-related lectins, such as Yucca filamentosa lectin and Polygonatum cyrtonema lectin, could act as drugs that prevent influenza virus infection via competitive binding. In conclusion, the GNA-related lectin family may be helpful in the design of novel candidate agents for preventing influenza A infection through the use of competitive combination against sialic acid specific viral infection.

The neuraminidase protein of influenza viruses is a surface glycoprotein that shows 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 the aggregation of the virus by the hemagglutinin protein binding to other viral proteins. Antibodies to the neuraminidase protein can be protective alone in animal challenge studies, but the neuraminidase antibodies appear to provide protection in a different manner than antibodies to the hemagglutinin protein. Neutralizing antibodies to the hemagglutinin protein can directly block virus entry, but protective antibodies to the neuraminidase protein are thought to primarily aggregate virus on the cell surface, effectively reducing the amount of virus released from infected cells. The neuraminidase protein can be divided into nine distinct antigenic subtypes, where there is little cross-protection of antibodies between subtypes. All nine subtypes of neuraminidase protein are commonly found in avian influenza viruses, but only selected subtypes are routinely found in mammalian influenza viruses; for example, only the N1 and N2 subtypes are commonly found in both humans and swine. Even within a subtype, the neuraminidase protein can have a high level of antigenic drift, and vaccination has to specifically be targeted to the circulating strain to give optimal protection. The levels of neuraminidase antibody also appear to be critical for protection, and there is concern that human influenza vaccines do not include enough neuraminidase protein to induce a strong protective antibody response. The neuraminidase protein has also become an important target for antiviral drugs that target sialic acid binding which blocks neuraminidase enzyme activity. Two different antiviral drugs are available and are widely used for the treatment of seasonal influenza in humans, but antiviral resistance appears to be a growing concern for this class of antivirals.

Influenza virus is a major human pathogen that causes annual epidemics and occasional pandemics. Moreover, the virus causes outbreaks in poultry and other animals, such as pigs, requiring costly and laborious countermeasures. Therefore, influenza virus has a substantial impact on health and the global economy. Here, we review entry of this important pathogen into target cells, an essential process by which viral genomes are delivered from extracellular virions to sites of transcription/replication in the cell nucleus. We summarize current knowledge on the interaction of influenza viruses with their receptor, sialic acid, and highlight the ongoing search for additional receptors. We describe receptor-mediated endocytosis and the recently discovered macropinocytosis as alternative virus uptake pathways, and illustrate the subsequent endosomal trafficking of the virus with advanced live microscopy techniques. Release of virus from the endosome and import of the viral ribonucleoproteins into the host cell nucleus are also outlined. Although a focus has been on viral protein function during entry, recent studies have revealed exciting information on cellular factors required for influenza virus entry. We highlight these, and discuss established entry inhibitors targeting viral and host factors, as well as the latest prospects for designing novel 'anti-entry' compounds. New entry inhibitors are of particular importance for current efforts to develop the next generation of anti-influenza drugs - entry is the first essential step of virus replication and is an ideal target to block infection efficiently.

Effect of Viruses on Platelet Aggregation and Platelet Survival in Rabbits