RESUMEN
In this study, we describe the input data and processing steps to find antiviral lead compounds by a virtual screen. Two-dimensional and three-dimensional filters were designed based on the X-ray crystallographic structures of viral neuraminidase co-crystallized with substrate sialic acid, substrate-like DANA, and four inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir). As a result, ligand-receptor interactions were modeled, and those necessary for binding were utilized as screen filters. Prospective virtual screening (VS) was carried out in a virtual chemical library of over half a million small organic substances. Orderly filtered moieties were investigated based on 2D- and 3D-predicted binding fingerprints disregarding the "rule-of-five" for drug likeness, and followed by docking and ADMET profiling. Two-dimensional and three-dimensional screening were supervised after enriching the dataset with known reference drugs and decoys. All 2D, 3D, and 4D procedures were calibrated before execution, and were then validated. Presently, two top-ranked substances underwent successful patent filing. In addition, the study demonstrates how to work around reported VS pitfalls in detail.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Humanos , Inhibidores Enzimáticos/farmacología , Estudios Prospectivos , Zanamivir/farmacología , Antivirales/uso terapéutico , Virus de la Influenza A/metabolismo , Neuraminidasa/metabolismo , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & controlRESUMEN
Influenza A virus (IAV) causes an important respiratory disease in mammals and birds leading to concerns in animal production industry and public health. Usually, antibodies produced in mammals are employed in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated. The aim of this study was to produce egg yolk immunoglobulin (IgY) in laying hens against a highly conserved protein (nucleoprotein- NP) of IAV and to evaluate the application of anti-NP IgY antibodies in virus detection by immunocytochemistry (ICC) and immunohistochemistry (IHC). Three laying hens of the White Leghorn line were inoculated seven times with a recombinant NP protein and their eggs collected seven days after the 3rd, 5th and 7th inoculations. Immunoglobulin Y antibodies were purified from egg yolk through precipitation with ammonium sulfate. The titers and specificity of the purified antibodies were determined by ELISA, western blotting, ICC and IHC. High levels of specific anti-NP antibodies were detected by ELISA after the 5th inoculation, reaching a peak after the 7th inoculation. The mean yield of total protein in yolk after the 7th inoculation was 13.5â¯mg/mL. The use of western blotting and ICC demonstrated that anti-NP IgY binds specifically to NP protein. Moreover, the use of anti-NP IgY antibody in ICC test revealed positive staining of MDCK cells infected with IAV of the three subtypes circulating in swine (H1N1, H1N2, and H3N2). However, no staining was observed in lung tissues through the IHC test. The data obtained showed that anti-NP IgY antibodies bound specifically to influenza virus NP protein, detecting the main virus subtypes circulating in swine, reinforcing their usefulness in the influenza diagnosis.
Asunto(s)
Anticuerpos Antivirales , Inmunoglobulinas , Virus de la Influenza A , Infecciones por Orthomyxoviridae , Enfermedades de los Porcinos , Proteínas del Núcleo Viral , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Pollos/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Infecciones por Orthomyxoviridae/sangre , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/veterinaria , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas del Núcleo Viral/sangre , Proteínas del Núcleo Viral/inmunologíaRESUMEN
Proteins undergo dynamic structural changes to function within the range of physical and chemical conditions of their microenvironments. Changes in these environments affect their activity unless the respective mutations preserve their proper function. Here, we examine the influenza A virus spike protein hemagglutinin (HA), which undergoes a dynamic conformational change that is essential to the viral life cycle and is dependent on endosomal pH. Since the cells of different potential hosts exhibit different levels of pH, the virus can only cross species barriers if HA undergoes mutations that still permit the structural change to occur. This key event occurs after influenza A enters the host cell via the endocytic route, during its intracellular transport inside endosomes. The acidic pH inside these vesicles triggers a major structural transition of HA that induces fusion of the viral envelope and the endosomal membrane, and permits the release of the viral genome. HA experiences specific mutations that alter its pH stability and allow the conformational changes required for fusion in different hosts, despite the differences in the degree of acidification of their endosomes. Experimental and theoretical studies over the past few years have provided detailed insights into the structural aspects of the mutational changes that alter its susceptibility to different pH thresholds. We will illustrate how such mutations modify the protein's structure and consequently its pH stability. These changes make HA an excellent model of the way subtle structural modifications affect a protein's stability and enable it to function in diverse environments.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Virus de la Influenza A/metabolismo , Adaptación Biológica/genética , Adaptación Biológica/fisiología , Animales , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/genética , Estabilidad Proteica , Internalización del VirusRESUMEN
While the effect of the influenza A virus non-structural protein (NS) on cytokine production during viral infection is well known, inconsistent results have been observed with some other influenza A virus backbone studied. In this study, in order to focus on the impact of the avian NS gene segments on viral virulence, the NS genes encoded by different strains of avian influenza A viruses were incorporated into an identical [A/Puerto Rico/8/1934(H1N1), PR8] virus background to generate various NS recombinant viruses. Thus, PR8NS, PR8×[A/Hong Kong/483/97(H5N1) 483NS, PR8×[A/Ck/Korea/150/03(H9N2) 150NS, and PR8×[A/EM/Korea/W149/06(H5N1) W149NS were constructed utilizing reverse genetics. Here, we show the effects of each of these recombinant viruses upon viral pathogenesis and cytokine production during viral replication in vivo. In this regard, we found that infection of mice with the PR8×150NS recombinant virus resulted in the lowest pathogenicity (6.0×10(4)MLD50), yet elicited the highest levels of TNF-α production in bronchoalveolar lavage (BAL) fluid compared to infection with the other recombinant influenza viruses. In contrast, infection with the PR8 virus showed the highest pathogenicity (1.0×10(2)MLD50) as well as relatively high cytokine levels (IL-1α, IL-1ß, IL-17, and eotaxin) in mouse BAL fluid. In addition, the PR8 and PR8×483NS viruses induced severe and extensive inflammation in infected lungs compared with that of PR8×150 NS recombinant virus-infected mice. These results clearly demonstrate that the NS genes of diverse influenza A strains can variable impact pathogenicity, histopathology, and cytokine production in mice even when expressed in an identical genetic background.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Proteínas no Estructurales Virales/metabolismo , Animales , Aves , Pollos , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Subtipo H9N2 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/metabolismo , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Gripe Humana/inmunología , Ratones , Recombinación Genética , Proteínas no Estructurales Virales/genética , VirulenciaRESUMEN
The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal beta-D-galactopyranosyl residues. The sialic acid linkages to galactose are alpha 2,6 and alpha 2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The alpha 2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.
Asunto(s)
Ácido N-Acetilneuramínico/análisis , Ácido N-Acetilneuramínico/metabolismo , Paracoccidioides/química , Paracoccidioides/metabolismo , Aglutininas/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Ferritinas/farmacocinética , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Galactosa/metabolismo , Virus de la Influenza A/metabolismo , Lectinas/metabolismo , Microscopía Electrónica , Neuraminidasa/farmacología , Paracoccidioides/ultraestructura , Unión Proteica , Receptores de Superficie Celular/metabolismoRESUMEN
The present study was conducted to investigate the characteristics of two samples of influenza A/England/42/72 (H3N2) virus, one of them selected by an adsorption-elution technique, to determine the possible existence of virus variants or subpopulations. Based on specificity of virulence-related cell receptor-binding and sialidase activities, this selection technique using human O group erythrocytes revealed the presence of variants within a standard virus sample with diversity for their hemagglutinating and sialidase activities. The standard-like (E1) sample exhibited titers of 4 and 32 HAU (hemagglutinating units in 25 microliters) with human O group and chicken erythrocytes, respectively, while the sample obtained by the adsorption-elution process (E2) exhibited titers of 32 and 4 HAU, respectively, with these same types of erythrocytes. The E2 sample showed higher sialidase activity at pH values between 5.4 and 6.6 with human erythrocytes (128-256 HAU), but the E1 sample did not exhibit significant sialidase activity with either human or chicken erythrocytes. The different pH optima for hemolysis (5.2) and sialidase (5.4-6.6) activities and the higher hemolysis indexes present in samples with sialidase activity inhibited by heating (at 56 degrees C for 30 min) or by treatment with EDTA (dilution in buffer containing 2 mM EDTA, a chelating agent on calcium-dependent sialidase activity) demonstrate the independence of these activities in the selected sample: native E2 (absorbance = 0.18), EDTA-treated native E2 (absorbance = 0.28), heated E2 (absorbance = 0.26), EDTA-treated heated E2 (absorbance = 0.41).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hemaglutininas Virales/fisiología , Virus de la Influenza A/clasificación , Neuraminidasa/fisiología , Animales , Embrión de Pollo , Variación Genética , Pruebas de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/análisis , Hemólisis/fisiología , Calor , Humanos , Concentración de Iones de Hidrógeno , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Neuraminidasa/análisis , Factores de TiempoRESUMEN
The present study was conducted to investigate the characteristics of two samples of influenza A/England/42/72 (H3N2) virus, one of them selected by an adsorption-elution technique, to determine the possible existence of virus variants or subpopulations. Based on specificity of virulence-related cell receptor-binding and sialidase activities, this selection technique using human O group erythrocytes revealed the presence of variants within a standard virus sample with diversity for their hemagglutinating and sialidase activities. The standard-like (E1) sample exhibited titers of 4 and 32 HAU (hemagglutinating units in 25 microliters) with human O group and chicken erythrocytes, respectively, while the sample obtained by the adsorption-elution process (E2) exhibited titers of 32 and 4 HAU, respectively, with these same types of erythrocytes. The E2 sample showed higher sialidase activity at pH values between 5.4 and 6.6 with human erythrocytes (128-256 HAU), but the E1 sample did not exhibit significant sialidase activity with either human or chicken erythrocytes. The different pH optima for hemolysis (5.2) and sialidase (5.4-6.6) activities and the higher hemolysis indexes present in samples with sialidase activity inhibited by heating (at 56 degrees C for 30 min) or by treatment with EDTA (dilution in buffer containing 2 mM EDTA, a chelating agent on calcium-dependent sialidase activity) demonstrate the independence of these activities in the selected sample: native E2 (absorbance = 0.18), EDTA-treated native E2 (absorbance = 0.28), heated E2 (absorbance = 0.26), EDTA-treated heated E2 (absorbance = 0.41).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Humanos , Animales , Embrión de Pollo , Hemaglutininas Virales , Neuraminidasa , Virus de la Influenza A/clasificación , Variación Genética , Pruebas de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales , Hemólisis/fisiología , Calor , Concentración de Iones de Hidrógeno , Neuraminidasa , Factores de Tiempo , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismoRESUMEN
The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SA alpha 2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SA alpha 2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SA alpha 2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SA alpha 2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.