Kinetics of Influenza A/BANGKOK/1/1979(Н3N2) Virus Thermal Inactivation in the Presence of Polyallylamine.

Polyelectrolytes currently play an increasingly important role in antivirus therapy. Antiviral activity towards influenza virus, measles virus, herpes simplex virus type 1, and cytomegalovirus was demonstrated for the 6000 Da polyelectrolyte polyallylamine. A nontoxic polyallylamine concentration of 30 µM at which the compound retains its antiviral effect towards measles and influenza viruses but lacks any toxic effect on human cells was previously determined. It is well known, at the same time, that simultaneous virus exposure to physical environmental factors and chemical substances causes a more significant decrease in virus infectivity. Temperature is among these physical factors since thermal exposure causes virus inactivation. Analysis of virus thermal inactivation parameters is of high practical importance when it comes to the development of vaccines against influenza virus and to the study of how virus particles infectivity decreases on various surfaces. In this view, the study of kinetic and thermodynamical characteristics of influenza virus thermal inactivation in the presence of the antiviral preparation polyallylanime is of particular interest. The paper reports that thermal inactivation of influenza virus in the temperature range of 38-60°C in the presence of polyallylamine follows the first-order reaction kinetics. Thermodynamic parameters of influenza virus thermal inactivation evidence that influenza virus surface proteins are involved in the inactivation process as a result of their interaction with polyallylamine. The obtained results show that polyallylamine may be used to accelerate thermal inactivation of the influenza virus.

[Determination of thermodynamic binding parameters and type of interaction between the А/Bangkok/1/1979 (Н3N2) influenza virus hemagglutinin and phosphatidylcholine liposome]. / Opredelenie termodinamicheskikh parametrov sviazyvaniia i kharakter vzaimodeistviia gemaggliutinina virusa grippa A/Bangkok/1/1979(N3N2) s liposomami iz fosfatidilkholina.

The study of interaction between surface viral proteins and model phospholipids is important for learning more details about the mechanisms of viral penetration into cells during infection. In this context, liposomes represent suitable systems for modeling a cell membrane. The binding of hemagglutinin (HA) of influenza virus with phosphatidylcholine liposomes was studied by equilibrium adsorption. It was interesting elucidate changes occurring in the structure of a protein during its translocation from the surface into the interior part of the membrane. In this work, we have studied characteristics of the protein-lipid interaction during HA complex formation with phospholipids including adsorption of HA on a phospholipid bilayer. Using the Scatchard equation and the Gibbs-Helmholtz equation at pH 4.0 and pH 6.0 thermodynamic parameters were determined. The results concluded the hydrophobic type of interaction between viral protein and liposomes. The additional confirmation of hydrophobic protein-lipid interaction presence was determination of HA distribution constants in two-phase systems: dextran-polyethylene glycol (K1) and dextran-polyethylene glycol esterified with palmitic acid (K2). The presence of hydrophobic interaction between HA and the liposome membrane was also confirmed using the quenching method of intrinsic protein fluorescence by a neutral quencher with acrylamide. At pH 4.0, an increase in the Stern-Volmer quenching constant was observed for the HA+liposome from phosphatidylcholine system, which is caused by structural changes in HA upon incorporation into the liposome bilayer. The fluorescence quenching rate constants calculated using the Stern-Volmer equation indicate a static quenching mechanism in which the quencher interacts with fluophors of a stationary protein molecule. The obtained results are interesting for not only studying virus and cell fusion theoretically, but also have practical applications. Using values of the protein-bilayer binding constant and free energy constant, it is possible to select the optimal phospholipid composition of liposomes or virosomes to obtain a stronger complex with various viral proteins. With two-phase systems, it is possible to determine the presence of hydrophobic sites on the viral protein surface, which can be used for evaluation both protein-lipid and protein-protein interaction.

[The study of the antiviral activity of polyelectrolytes with respect to the influenza virus].

It is well known that polyelectrolytes (PE) have a damaging effect on the secondary structure of the proteins and enzymes. It is also known that some PEs exert an immunostimulating action on T- and B-lymphocytes. However, currently, almost nothing is known about the impact of the PE of the viral proteins and viral envelope. Therefore, a detailed study of the mechanisms of the antiviral action of various polyelectrolytes would create the scientific-practical base for the construction of the antiviral drugs on the polyelectrolyte basis. In these works, for the first time the influence of PE polystyrene sulfonate (PSS) with varying degrees of polymerization and polyallylamin (PAA) and with molecular mass 6 and 8 kDa on the infectivity of different strains of the influenza virus was studied. It was shown that the expressed antiviral action PSS with degree of polymerization-8 (ETS-8) and PAA (6 kDa) against the influenza viruses was characterized by a significant reduction in the infectious titer of the virus. It was determined that the span of the nontoxic concentrations for the ETS-8 was 1-40 mM; for PAA (6 kDa), 1-40 µM, with IC50 = 3.8 ± 0.19 mm and 1.8 ± 0.09 µM, respectively. For the first time the impact of the PE on the structural-functional state of the viral membrane was assessed on the basis of monomolecular monolayers used as models of cell membranes.

[Study of surface-active properties antivirials compound 1-boraadamantane for model monomoleculars phosphlipids layers].

The surface-active properties of 1-boraadamantane have been studied using model phospholipid monolayers. Results suggest that the increase in 1-boraadamantane concentrations from 10(-7) to 10(-6) M is accompanied by the increase of the area per phospholipid molecule. This decreases to frequency of lateral diffusion of phospholipids molecules, the potential difference and the angle of the phospholipid monolayer arrangement. These phenomena may lead to impossibility of interaction between the virus and cell membranes.

[The study of structural changes in influenza virus hemagglutinin (HA) after interaction with liposomes by the fluorescence method].

Using the method of self fluorescence quenching influence of phospholipids on conformational state of HA has been studied. Interactions of HA with model phospholipids membranes are accompanied by changes of structure-dynamic protein organization. This method could be used for controlling the structure organization of proteins after receiving by virus the influenzal virusomal vaccine.

[Study of interaction of polystirolsulphonate with polymerization degree of 8 and polyallylamin with bilayer lipids membranes].

Interaction of polystirolsulphonate with polymerization degree of 8 (PSS-8) and polyallylamin PAA (molecular mass 60 kilodaltons) with viruses from bloodline of paramixo- and orthomixoviruses by the example of measles virus, parotitis and flu leads to the decreasing of infective activity. The possible mechanism of viral inhibitive action of these chemical compounds is damaging of interfacial antigenic proteins of paramixo- and orthomixoviruses. In this study it was detected the change of surface tension of bilayer lipid membrane in the presence of PSS-8 and PAA. The change of surface tension leads to disorder in viral proteins adsorption in bilayer lipid membrane. This process could lead to disorder of juncture and self-assembly of virions.

[Influence of polyelectrolytes on measles virus infectivity].

AIM: Mechanism of virus inhibiting action against measles virus of polyelectrolytes (PE) polystyrolsulfonate (PSS) of various polymerization degree and 60 kDa molecular weight polyallylamine (PAA) was studied. MATERIALS AND METHODS: Measles virus Leningrad-16 strain was used for the study. Virus infectious titer reduction kinetics after interaction with PSS with the degree of polymerization of 8 (PSS 8), 31, 77, 170, 360, 430 and PAA were determined by titration method with cytopathogenic effect detection in Vero continuous cell line. Circular dichroism and fluorescence spectra of viral proteins were obtained by using Zenith 200st spectrophotometer (Russian Federation) and Jasco J-810 dichrograph (Japan). RESULTS: A significant decrease of measles virus infectious titers after interaction with PSS with the degree of polymerization of 8 and PAA in concentration of 30 mM was detected. Analysis of circular dichroism spectra and protein fluorescence allowed to determine the mechanism of interaction of the indicated PE with measles virus surface proteins. The secondary structure of viral proteins is damaged by hydrophobic polar frame of these PE, polyanion PSS 8 also interacts with positive charges of protein groups that leads to the formation of loops and tails that disrupt alpha-spirals. CONCLUSION: The studied PE could be considered as potential antiviral preparations, and methods of circular dichroism and protein fluorescence could be used to detect damage of viral protein secondary structure by agents of different kinds.

[A study of the interaction of boraadamantane with liposomes].

Changes in the surface potential of liposomes obtained from dipalmitoyl phospatidylcholine during their interaction with the new antiviral preparation boraadmantane have been studied. It has been concluded that the saturation of the lipid bilayer of liposomes by boraadmantane occurs at concentrations of the preparation above 10 microg/ml.

[New method for measurement of size and concentration of lyposomes].

AIM: To develop new method of determination of size and concentration of lyposomes based on measurement of opacity in dispersed media. MATERIALS AND METHODS: Dispersions of lyposomes from dipalmitoylposphatidylcholine were the object of the study. Opacity spectrum of lyposome suspension was measured with Zenyth 200st spectrophotometer. Mean values of diameter of lyposomes determined by opacity spectrum were compared with the same values measured by electron microscopy (JEM 100-CX, JEOL, Japan) with magnification 58,000 - 100,000. Refraction index was measured with refractometer RPL-3 (Russia). RESULTS: Sizes of lyposomes measured by the new method and by electron microscopy did not differ significantly. Determination of sizes and concentration of lyposomes by the new method did not depend from effect of secondary multiple scattering of light. CONCLUSION: Obtained data allowed to conclude that developed method could be used in practice. Advantages of the new method are usage of common spectrophotometers and photoelectrocolorimeters for deriving of liposomes suspension opacity curve as well as its high validity, which are confirmed by data obtained with electron microscopy.