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Neuraminidase inhibitors are a class of antivirals used to treat influenza infections. Screening assays for potential neuraminidase inhibitors would benefit from the development of in vitro procedures that do not require handling viruses. The aim of the study was to develop and validate a procedure for in vitro determination of inhibitory effects on neuraminidase (EC 3.2.1.18), using 2’-4(methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NANA) as a fluorogenic substrate and quinonoid pigments, potential neuraminidase inhibitors, as a case study. Materials and methods: the method is based on neuraminidase cleavage of 4MU-NANA to release fluorescent 4-methylumbelliferone, which is detected at the excitation and emission wavelengths of 360 and 450 nm, respectively. Results: the procedure was validated for specificity, range, accuracy, and precision. It remained linear over the range of 0.31–80 μM of 4-methylumbelliferone. The accuracy for four concentration levels (including the LLOQ) was 87–114%; i.e., the relative error of accuracy evaluation was less than 15%. The intra- and inter-day precision ranged from 1.5 to 10.4% and from 2.3 to 9.6%, respectively. Inhibitory effect evaluation using zanamivir hydrate (0.6–150 nM) demonstrated the accuracy of 89–120% and the precision of 3.1–11.0%. The IC50 values for positive controls (zanamivir hydrate and oseltamivir) were 27 ± 3 and 16 ± 2 nM, respectively. The following solvents may be used: 50% dimethyl sulfoxide, 5% Polysorbate 80, 50% ethanol, 50 and 100% methanol. If a compound is insoluble in the solvents, it is possible to form inclusion complexes with 2-hydroxypropyl-β-cyclodextrin. For bisnaphthazarin, the natural quinonoid pigment used in the study, the IC50 amounted to 273 ± 28 nМ. Conclusion: the procedure demonstrated adequate accuracy and reproducibility and is recommended for screening for potential neuraminidase inhibitors. In order to use the procedure for insoluble substances, the authors suggest forming inclusion complexes with cyclodextrins.

Introduction. The classic hemagglutination inhibition reaction (RTGA) is used to determine the level of antiviral antibodies in human and animal serum specimens. During the performance of RTGA the tested sera must be treated with a receptor-destroying enzyme (RDE) to remove serum glycans that degrade the accuracy of the RTGA results. To optimize the amounts of RDE compounds used, it is necessary to know their real neuraminidase activity. This article describes a simple and economical method for testing the neuraminidase activity of receptor-destroying compounds using standard reagents and laboratory equipment. Aims of investigation. Design of an improved simple and convenient method for evaluating the neuraminidase activity using the flu virus. Material and methods. Here, we propose a convenient method for evaluating the activity of neuraminidase by double-fold dilution procedure with human or animal erythrocytes followed by hemagglutination assay with influenza A virus. Results and discussion. The method is based on the ability of neuraminidase to hydrolyze sialic acid residues on the cell surface of erythrocytes, that deprives red blood cells to be agglutinated with the flu virus, since these sialic glycans provide virus attachment and hemagglutination. Conclusion. The designed method allows the accurate measurement of the receptor-destroying (neuraminidase) activity of RDE compounds and the comparison of the compounds with each other. This test is necessary to optimize the RTGA protocol when monitoring blood sera of animals and humans after influenza infection and/or Acute Respiratory diseases (ARD). The designed method can be included in the guidelines of regulations for the RTGA protocol, which is used in different laboratories to monitor the epidemic process of influenza and ARD infections.

Aim. Study of the inhibitory effect of polyelectrolytes with antiviral effect against influenza neuraminidase. Determination of the type and inhibition constant. The study of the effect of polyelectrolytes (PE) on the process of oxidative phosphorylation in the mitochondria of cells host organism. Materials and methods. Purified influenza virus strains were used: A/VPCH/Weybridge (H7N7), A/Mallard Pennsylvania/10218/84 (H5N2), A/NIBRG-14 (H5N1) with an initial infectious titer of 4,5 lgTCD50/ml. PE solutions of polystyrenesulfonate with a degree of polymerization of 8 (PSS-8) in concentrations of 0,5—4,0 mM and polyallylamine (6 kDa) PAA (6 kDa) in concentrations of 0,5—4,0 μM. To determine the activity of influenza neuraminidase, influenza virus strains were used after the removal of low molecular weight inhibitors of neuraminidase by dialysis against water. The neuraminidase substrate was fetuin at final concentrations of 0,052 to 1,2 μM for PAA (6 kDa) and from 0,052 to 1,2 mM for PSS-8. As quantitative characteristics of respiration and phosphorylation of mitochondria, respiratory coefficients according to Lardi-Velman and Chans-Williams, as well as the ratio of ADP/O were used. Mitochondria were isolated from skeletal muscle. The determination of respiratory coefficients and the ratio of ADP/O) was determined by the polarographic method. Results. A noncompetitive type of inhibition of these PEs was detected in relation to the neuraminidase activity of influenza viruses with inhibition constants KI = 1,6 ± 0.08 μM for PAA (6 kDa) and KI = 1,7 ± 0.085 mM for PSS-8. Respiratory coefficients and the ratio of ADP / 0 were determined in the absence and after addition of PSS-8 and PAA (6 kDa) to mitochondria at concentrations of 20 mM and 10 μM, respectively. A decrease in respiratory coefficients and an ADP/O ratio was observed, indicating an inhibition of the enzymes of the electron-transport chain of mitochondria. At concentrations of less than 20 mM and 10 μM for PSS-8 and PAA (6 kDa), all indicators did not significantly change. Conclusion. The non-competitive mechanism of inhibition of the neuraminidase activity of the influenza PE viruses is explained by the conformational changes in the molecules of the enzyme and/or enzyme-substrate complex and, accordingly, the structural and functional changes in its secondary structure. When going beyond the range of non-toxic concentrations of20 mM for PSS-8 and 10 μM for PAA (6 kDa), inhibition of the mitochondrial respiratory chain enzymes was observed while maintaining the antiviral effect.

It is known that the functioning of many proteins and enzymes depends on the degree of hydration of their surfaces. In our studies, neuraminidase (NA) of influenza virus was selected as a model for surface antigenic viral protein. The Brunauer–Emmett–Teller (BET) model of adsorption was used to calculate the values of water monolayer (am) at different values of water vapor pressure. The obtained BET isotherms allow for concluding that hysteresis takes place manifested by the difference between the monolayer am values for sorption and desorption of water from the surface of the enzyme, which is probably associated with a high degree of cooperation of the hydration shell formed. The maximum binding of water molecules was observed for the vapor pressure p/ps value of 0.65 and was am = 224 water molecules per a molecule of the enzyme. Basing on the calculated surface area of a NA tetramer (S = 256 nm2 ) and the maximum projection area of water molecule, it may be concluded that the entire surface of the enzyme is completely covered with a water monolayer. For said am value the maximum activity of NA was observed, whereas the minimum enzyme activity corresponded to the am value of 98 water molecules per a molecule of the enzyme, which corresponded to the water vapor pressure p/pS value of 0.38. Thus, for the influenza virus NA protein a dependency of the enzymatic activity on the degree of hydration of the surface of the enzyme is demonstrated. The dependence of immunogenicity of influenza virus from the extent of hydration of NA.

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Copyright of News of Kazakhstan Science / Novosti nauki Kazahstana is the property of NCSTE (JSC National Center for State Scientific and Technical Evaluations) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aim. To investigate the mechanisms of variability of the influenza virus.Discussion. Influenza is one of the most significant problems facing the health care system. Human influenza causes a number of social and economic problems: mortality in high-risk groups, increasing in the load on personnel agencies health and reduced manpower. Features of the influenza virus genome enable the pathogen to cause seasonal epidemics each year, despite the preventive measures (vaccination) and using of specific antiviral drugs.Conclusion. Variability of the influenza virus genome is provided by antigenic drift, antigenic shift and recombination. This mechanisms of the variability of influenza virus that allow to remain a highly contagious infectious agent able to infect a large number of host species and cause significant problems as a result of epidemics and pandemics.

The recombinant strain Flu-NS1-124-Omp16 (H5N1) of the influenza virus expressing the brucellar Omp16 gene was constructed on the basis of the technology of reverse genetics for the purpose of developing vector antibrucellosis vaccine. The obtained recombinant strain is a genetically stable construction. This stability is confirmed by the comparative analysis of the nucleotide sequences of the HA, NA, and NS genes of the recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the Omp16 gene of the Brucella abortus (genBank: AAA59360.1). The comparative analysis showed that the nucleotide sequence of the Ns gene of the first and the fifth passage level of the Flu-NS1-124-Omp16 (H5N1) virus corresponded for 100% to the initial part of 12ААS2TC_124Omp16g containing the chimera NS1-124-Omp16 in the composition of DNA (deoxyribonucleic acid) plasmids pHW2000. Total identity with HA and NA genes of the strain А/AstanaRG/6:2/2009 (H5N1) was shown by the comparative analysis of the nucleotide sequences of HA and NA genes of the first and the fifth passage level of the recombinant strain Flu-NS1-124-Omp16 (H5N1). The recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the brucella Omp16 gene maintains the genetic stability during 5 passages in 10-day developing chicken embryos.

Copyright of News of Kazakhstan Science / Novosti nauki Kazahstana is the property of NCSTE (JSC National Center for State Scientific and Technical Evaluations) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In the result of the monitoring for influenza in the Novosibirsk region during the season 2011-2012 85 strains of influenza virus on the culture of MDCK cells were isolated: 79 strains A (H3N2) and 6 strains of influenza virus B (five strains belonged to the Yamagata line and. 1 strain belonged to the Victoria line). During the entire epidemic season 2011-2012 on the territory of the Novosibirsk region no strains of influenza A (H1N1) pdm09 were allocated. For molecular genetic analysis 3 strains of influenza A (H3N2) were selected. We determined full nucleotide sequence of NA and. HA genes encoding respective surface glycoproteins of influenza virus, as well as the comparative analysis of amino acid sequences of these proteins to the strains of influenza A (H3N2), vaccine strain and. the strain A/Victoria/361/2011 earlier seasons, for which diagnostic serum were obtained.

Several variants of models for predicting the IC50 values of inhibitors of influenza virus neuraminidase are presented for both individual strains and for combinations of data for neuraminidases of several strains. They are based on the use of calculated energy contributions to the amount of change in the free energy of enzyme-inhibitor complexes. In contrast to previous works, aimed at the complex modeling, we added a procedure of comparison of the docking variants with one of the neuraminidase inhibitors, for which the structure of the complexes was determined experimentally. The choice of the comparison structure was made according to the similarity of structures evaluated using the Tanimoto metrics and the limit of the RMSD value for a similar part of the structure was no more than 2 Å. Using this limitation and filtering datasets for a particular strain by the Q2 value obtained in the leave-one-out control procedure it is possible to construct equations for predicting the IC50 value with a Q2 value close to the minimum confidence threshold (0.57 in this work). Taking into consideration that in this version of the prediction, a minimum set of energy contributions is used, which does not provide for expensive calculations of entropy contributions, the result obtained supports the correctness of using a generalized model based on the data on the position of known ligands to predict the inhibition of neuraminidase of the influenza virus of various strains.

Introduction: The classic hemagglutination inhibition reaction (RTGA) is used to determine the level of antiviral antibodies in human and animal serum specimens. During the performance of RTGA the tested sera must be treated with a receptor-destroying enzyme (RDE) to remove serum glycans that degrade the accuracy of the RTGA results. To optimize the amounts of RDE compounds used, it is necessary to know their real neuraminidase activity. This article describes a simple and economical method for testing the neuraminidase activity of receptordestroying compounds using standard reagents and laboratory equipment. Aims of investigation. Design of an improved simple and convenient method for evaluating the neuramin1idase activity using the flu virus., Material and Methods: Here, we propose a convenient method for evaluating the activity of neuraminidase by double-fold dilution procedure with human or animal erythrocytes followed by hemagglutination assay with influenza A virus., Results and Discussion: The method is based on the ability of neuraminidase to hydrolyze sialic acid residues on the cell surface of erythrocytes, that deprives red blood cells to be agglutinated with the flu virus, since these sialic glycans provide virus attachment and hemagglutination., Conclusion: The designed method allows the accurate measurement of the receptor-destroying (neuraminidase) activity of RDE compounds and the comparison of the compounds with each other. This test is necessary to optimize the RTGA protocol when monitoring blood sera of animals and humans after influenza infection and/or Acute Respiratory diseases (ARD). The designed method can be included in the guidelines of regulations for the RTGA protocol, which is used in different laboratories to monitor the epidemic process of influenza and ARD infections., Competing Interests: The authors declare no conflict of interest.

In this work the possibility of using neuraminidase for increasing the content of ganglioside GM1 in the mixture of gangliosides used for the sensitization of erythrocytes has been studied. The study has revealed that the treatment of gangliosides with neuraminidase is sufficient for obtaining active hemosensitin; there is no need for the purification of the preparation by gel filtration.

Gangliosides of human placenta were studied, using biochemical methods and specific antibodies. The placenta was found to contain three types of gangliosides with oligosaccharide chains Lac, GgOse4 and nLcOse4.

It has been shown that 4-sphingenine is the main sphingoid in lamprey brain gangliosides. Saturated and monoenoic fatty acids were found to predominate, the main fatty acids in the lamprey brain are presented by stearic (43-49% of total fatty acids) and oleic ones. N-acetylneuraminic acid is the only sialic acid found in gangliosides from the lamprey brain. Other components of ganglioside molecules are glucose, galactose and N-acetylgalactosamine. Two main lamprey brain gangliosides which constitute more than 90% of lipid-bound sialic acid, were found to be trisialogangliosides. It was shown that both gangliosides have the following structure in their molecules: (formula see text). They differ in the position of two other sialic acid residues. Data on lamprey brain gangliosides in the literature are practically absent. The data obtained in the present study confirmed the conclusions on changes in the hydrophobic part of ganglioside molecule in evolution of vertebrates and made it possible to define more exactly the molecular evolution of ganglioside carbohydrate component.

A comparative examination of 226 paired sera from patients with pneumonia was carried out by CFT, HI, neuraminidase activity inhibition (NI) and double immunodiffusion. A correlation of the results of agar gel precipitation and the CFT and HI tests was observed. Convalescent sera contained antibody to influenza virus ribonucleoprotein frequently, less so to its neuraminidase or hemagglutinin. The precipitation test was shown to be highly sensitive, easy to perform, and therefore should be used in examinations of sera from patients with influenza-bacterial pneumonia.

Gangliosides from murine B-lymphomas (MOPC 21 and MOPC 406) and T-lymphoma EL-4 were studied by thin-layer chromatography, immunoprecipitation with specific antisera to gangliosides and by treatment with neuraminidase. It was found that the gangliosides of all three lymphomas differ in their interaction with antisera and neuraminidase although they are similar in their chromatographic behaviour.

The leukemogenic activity of modified leukemic cells was studied in experiments on the 385 BALB/c mice with Rauscher leukemia by transplantation of different amount of treated cells. Immunogenicity of modified leukemic cells was estimated under conditions of three-fold immunization of animals 2 days later by the cells dose equal to 1.10(6) which began 24 h after the leukemia transplantation. The effect of immunotherapy of neuraminidase-modified leukemic cells results in elongation of animal lifetime, presence of less pronounced changes in the peripheral blood and spleen as well as in stimulation of cell immunity factors.

The method of electrophoresis in a single polyacrylamide gel plate was used for comparative study on virion polypeptides mobility in human influenza A and B virus strains. Molecular masses of individual polypeptides and their portion in the virion were determined. No variations in the migration speed of nucleoprotein (NP) and membrane protein (MP) were found in strains belonging to the same genus, but there were differences in the migration speed of these proteins in the genus A and genus B. Significant differences in migration and the content of glycoproteins, particularly of the heavy chain of hemagglutinin (HAI) in the genus A strain having different subtypes of hemagglutinin, including strains A/WS/33 (HONI), A/FM/1/47 (HINI), A/Sing/1/57 (H2N2), A/Port Chalmers/73 (H3N2), as well as in the genus B in strains B/Lee/40, B/Yamagata/1/73, and B/Johanesbourg/56. No differences in the mobility of glycoproteins in strains similar in the antigenic specificity of hemagglutinin and neuraminidase were found. On the basis of the comparative analysis of virion polypeptide electrophoregrams, three new strains on influenza A virus isolated in December, 1977, were identified as belonging to the species A (HINI).

The biological features of the strains of influenza virus isolated in the 1974-1975 epidemic were compared with those of the strains which had circulated earlier. Most of the properties examined (toxicity for mice, sensitivity to gamma-inhibitors, thermostability of hemagglutinin and neuraminidase) in strains isolated in 1974-1975 did not differ from those in strains of previous epidemics. Clear-cut differences between the new and earlier strains were found in the studies of their chromatographic profiles.

A comparative study of the rate of appearance of specific virus antigen in immune competent organs of mice immunized with inactivated influenza vaccine with active neuraminidase or a vaccine devoid of the neuraminidase activity was carried out by means of the immunofluorescence test and the serological tests. A considerable delay in the time (by 16--18 hours) of detection of virus antigen in cells of lymph nodes of mice as well as later (1--2 days) and less intensive (2--4-fold) production of specific virus antibody was demonstrated after immunization of mice with the vaccine without the active enzyme.