Anti-Influenza Activity of Cetraria islandica Lichen Extracts in In Vitro Experiments.

The toxicity and antiviral activity of extracts obtained by the methods of aqueous and ethanol extraction of bioactive substances from Cetraria islandica lichen as a raw material were studied. Aqueous and ethanol extracts of lichen were characterized by low toxicity with respect to the passaged MDCK cell culture and exhibited antiviral activity. The ethanol extract showed more potent in vitro antiviral activity against human A/H3N2 and avian A/H5N1 influenza viruses: in a concentration of 50 µg/ml, it suppressed replication of these viruses by 3.5 and 4 log10, respectively, while the aqueous extract inhibited replication of viruses by 2 and 6 log10, respectively, when taken in a concentration of 500 µg/ml that was 10-fold higher than the concentration of the ethanol extract.

Synthesis of esters and amides of 2-aryl-1-hydroxy-4-methyl-1H-imidazole-5-carboxylic acids and study of their antiviral activity against orthopoxviruses.

Smallpox was eradicated >40 years ago but it is not a reason to forget forever about orthopoxviruses pathogenic to humans. Though in 1980 the decision of WHO to cease vaccination against smallpox had seemed logical, it led to the decrease of cross immunity against other infections caused by orthopoxviruses. As a result, in 2022 the multi-country monkeypox outbreak becomes a topic of great concern. In spite of existing FDA-approved drugs for the treatment of such diseases, the search for new small-molecule orthopoxvirus inhibitors continues. In the course of this search a series of novel 2-aryl-1-hydroxyimidazole derivatives containing ester or carboxamide moieties in position 5 of heterocycle has been synthesized and tested for activity against Vaccinia virus in Vero cell culture. Some of the compounds under consideration revealed a selectivity index higher than that of the reference drug Cidofovir. The highest selectivity index SI = 919 was exhibited by ethyl 1-hydroxy-4-methyl-2-[4-(trifluoromethyl)phenyl]-1H-imidazole-5-carboxylate 1f. The most active compound also demonstrated inhibitory activity against the cowpox virus (SI = 20) and the ectromelia virus (SI = 46).

Estimation of Absolute Bioavailability of the Chemical Substance of the Anti-Smallpox Preparation NIOCH-14 in Mice.

We compared absolute bioavailability of the chemical substance of the anti-smallpox preparation NIOCH-14 and chemical compound ST-246 active against orthopoxviruses after oral administration to mice in doses of 10 and 50 µg/g and intravenous administration to mice in a dose of 2 µg/g body weight. The absolute bioavailability of NIOCH-14 is comparable with the absolute bioavailability of ST-246.

Using the Ground Squirrel (Marmota bobak) as an Animal Model to Assess Monkeypox Drug Efficacy.

In experiments to study the sensitivity of ground squirrels (Marmota bobak) to monkeypox virus (MPXV) at intranasal challenge, expressed pox-like clinical symptoms (hyperthermia, lymphadenitis, skin rash all over the body and mucous membranes and others) were observed 7-9 days post-infection. The 50% infective dose (ID50 ) of MPXV for these marmots determined by the presence of clinical signs of the disease was 2.2 log10 PFU. Some diseased marmots (about 40%) died 13-22 days post-infection, and the mortality rate was weakly dependent on MPXV infective dose. Lungs with trachea were primary target organs of marmots challenged intranasally (with ~30 ID50 ). The pathogen got to secondary target organs of the animals mainly via the lymphatic way (with replication in bifurcation lymph nodes). Lungs with trachea, nasal mucosa and skin were the organs where the maximum MPXV amounts accumulated in these animals. Evidences of the pathogen presence and replication were revealed in these and subcutaneously infected marmots in the traditional primary target cells for MPXV (macrophages and respiratory tract epitheliocytes), as well as in some other cells (endotheliocytes, plasmocytes, fibroblasts, reticular and smooth muscle cells). Our use of this animal species to assess the antiviral efficacy of some drugs demonstrated the agreement of the obtained results with those described in scientific literature, which opens up the prospects of using marmots as animal models for monkeypox to develop therapeutic and preventive anti-smallpox drugs.

[EVALUATION OF THE HUMAN SENSITIVITY TO SMALLPOX VIRUS BY THE PRIMARY CULTURES OF THE MONOCYTE-MACROPHAGES].

Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

[THE USE OF THE MODEL MOUSE ICR--VARIOLA VIRUS FOR EVALUATION OF ANTIVIRAL DRUG EFFICACY].

Mice of the ICR outbred population were infected intranasally (i/n) with the variola virus (VARV, strain Ind-3a). Clinical signs of the disease did not appear even at the maximum possible dose of the virus 5.2 lg PFU/head (plaque-forming units per head). In this case, 50% infective dose (ID50) of VARV estimated by the presence or absence of the virus in the lungs three days after infection (p.i.) was equal to 2.7 ± 0.4 lg PFU/head. Taking into account the 10% application of the virus in the lungs during the intranasal infection of the mice, it was adequate to 1.7 lg PFU/lungs. This indicates a high infectivity of the VARV for mice comparable to its infectivity for humans. After the i/n infection of mice with the VARV at a dose 30 ID50/ head the highest concentration of the virus detected in the lungs (4.9 ± 0.0 lg PFU/ml of homogenate) and in nasal cavity tissues (4.8 ± 0.0 lg PFU/ml) were observed. The pathomorphological changes in the respiratory organs of the mice infected with the VARV appeared at 3-5 days p.i., and the VARV reproduction noted in the epithelial cells and macrophages were noticed. When the preparations ST-246 and NIOCH-14 were administered orally at a dose of 60 µg/g of mouse weight up to one day before infection, after 2 hours, 1 and 2 days p.i., the VARV reproduction in the lungs after 3 days p.i. decreased by an order of magnitude. Thus, outbred ICR mice infected with the VARV can be used as a laboratory model of the smallpox when evaluating the therapeutic and prophylactic efficacy of the antismallpox drugs.

The Possibility of Using the ICR Mouse as an Animal Model to Assess Antimonkeypox Drug Efficacy.

As a result of the conducted experimental studies on intranasal challenge of ICR mice, rabbits and miniature pigs (even in the maximum variant) with the doses of 4.0-5.5 lg PFU of monkeypox virus (MPXV), some clinical signs such as purulent conjunctivitis, blepharitis and ruffled fur were found only in mice. The 50% infective dose (C ID50 ) of MPXV for these animals estimated by the presence of external clinical signs was 4.8 lg PFU, and L ID50 estimated by the virus presence in the lungs of mice 7 days post-infection taking into account its 10% application in the animal respiratory tract was 1.4 lg PFU. When studying the dynamics of MPXV propagation in mice challenged intranasally with 25 L ID50 of MPXV, the maximum pathogen accumulation was revealed in nasal cavity, lungs and brain: 5.7 ± 0.1, 5.5 ± 0.1 and 5.3 ± 0.3 lg PFU/ml, respectively. The pathomorphological examination of these animals revealed the presence and replication of the pathogen in the traditional primary target cells for MPXV (mononuclear phagocyte system cells and respiratory tract epitheliocytes) as well as in some other types of cells (endothelial cells, reticular cells, connective tissue cells). Our use of these animals to assess the antiviral efficacy of some drugs demonstrated the agreement of the results (a significant positive effect of NIOCH-14 and ST-246) with those described in scientific literature, which opens up the prospects of using ICR mice as animal models for monkeypox to develop preventive antismallpox drugs.

[Role of the tissue antioxidant status in response to chronic irradiation of mice during early ontogenesis].

The response of the liver and blood erythrocyte lipids to the low intensity chronic γ-irradiation action of mice at the dose of 8 cGy during early ontogenesis immediately and 7 months after irradiation is studied. The maintainance of the changed structural state of the lipid component in liver and especially blood erythrocytes which are characterized by a lower antioxidant status compared with the liver lipids is revealed during a long time after the cessation of irradiation. This confirms the possibility of using blood erythrocyte lipids as a perspective model for the estimation of the effects of the weak impact of unfavorable factors on the organism.

[Evaluation of therapeutic-prophylactic effectiveness of chemical compound NIOC-14 against ectromelia virus in vivo].

AIM: Study pharmacodynamic parameters of anti-viral effectiveness of a chemical compound NIOC-14 in experiments in mice infected with ectromelia virus (EV). MATERIALS AND METHODS: EV (K-1 strain) was obtained from the State Collection of Viral Infections and Rickettsioses Causative Agents of the State Scientific Centre of Virology and Biotechnology "Vector". Outbred ICR mice were intranasally infected with EV at a dose of 10 LD50 per animal (10 x 50% lethal doses/animal) and per orally received NIOC-14 or ST-246 as a positive control. Chemical compound NIOC-14 (7-[N'-(4-trifluoromethylbenzoyl)-hidrazincarbonyl]-tricyclo[3.2.2.0(2,4)]non-8-en-6-carbonic acid) was synthesized in Novosibirsk Institute of Organic Chemistry (NIOC). Anti-pox preparation ST-246, developed by SIGA Technologies Inc. (USA), was synthesized in NIOC using the technique described by the authors. RESULTS: 50% effective doses against EV in vivo were shown not to differ significantly between the preparations NIOC-14 (3.59 µg/g mouse mass) and ST-246 (5.08 µg/g mouse mass). During determination of therapeutic window, administration of NIOC-14 to mice 1 day or 1 hour before EV infection, as well as 1, 2 and 4 days after EV infection and then for 9 days was found to ensure 100% animal survival. Administration of NIOC-14 as well as ST-246 resulted in the decrease relative to control of EV titers in lungs, nasal cavity, brains, liver, spleen, kidneys and pancreas. CONCLUSION: Anti-viral effectiveness of NIOC-14 against EV in vivo was thus comparable by all the studied pharmacodynamic parameters with anti-viral activity of anti-pox-virus preparation ST-246.

[Development of the disease in marmot at the intranasal infection with the monkeypox virus].

In experimental study the sensitivity of the Marmota bobak species to the monkeypox virus (MPXV) with the intranasal (i/n) infection was tested. It was demonstrated that 50% of the infective dose (ID50) of the MPXV on external clinical signs of the disease was 2.2 Ig plaque forming units (PFU). The percentage of the marmot mortality is slightly dependent on the infecting dose of the MPXV, therefore it is not possible to correctly determine the value of 50 % fatal dose (FD50) for these animals. The most pronounced external clinical signs of the disease were obtained in the marmots: pox-like skin rash throughout the surface of the body and mucous membranes, purulent discharge from the nose, lymphadenitis, discoordination, tremor of the extremities, fever, increased aggression, and ruffled fur. In the course of experiments intended to determine the dynamics of the accumulation of the MPXV in various organs, tissues, and blood serum of marmot infected i/n with dose of 3.7 Ig PFU, it was found that the trachea, lungs, and the bifurcation lymph nodes are the primary target organs. The trachea, lungs, nasal mucosa membrane, and skin are the organs with maximal virus replication recorded at 5, 7, 9, and 12 days after the infection. The transfer of the MPXV into the secondary target organs (nasal mucosa membrane, brain, spleen, duodenum, adrenal glands, and skin) was carried out in marmots with lymphogenic and hematogenic ways of the dissemination of the infection.

[A new approach to analysis of participation of oxidative processes in regulation of metabolism in animal tissues].

A new approach to the analysis of the oxidative processes participated in regulation of metabolism in norm and the assessment of the biological consequences under the action of damaging factors of different nature and intensity for animal groups is suggested using the analysis of interrelations between the different parameters of the physicochemical regulatory system of the lipid peroxidation in tissues of the laboratory mice (the phospholipid composition, amounts of the oxidation products in lipids). by changes of the scale and direction of interrelations between the reciprocal parameters of the lipid peroxidation system in norm.

[A comparative study of the antiviral activity of chemical compounds concerning the orthopoxviruses experiments in vivo].

In the experiments using intranasal (i/n) infection of mice with the ectromelia virus (EV) in a dose 10 LD50/head (10 x 50% lethal doselhead) or with the monkaypox virus (MPXV) in a dose 10 ID50/head (10 x 50% infective dose/ head) it was demonstrated that the antiviral efficiency of chemical compounds - the condensed derivatives of pyrrolidin-2,5-dion, as well as their predecessors and the nearest analogues, synthesized in Novosibirsk Institute of Organic Chemistry of the Siberian Branch of the Russian Academy of Sciences (NIOCH SB RAS) was observed. As a positive control we used the antipoxvirus chemical preparation ST-246 available from SIGA Technologies Inc. (USA), synthesized in NIOCH SB RAS by the technique suggested by the authors. It was demonstrated that the compound NIOCH-14 (7-[N'-(4-Trifluoromethylbenzoil)-hydrazidecarbonil]-tricyclo[3.2.2.02,4]non-8-en-6-carbonic acid) possessed comparable with ST-246 antiviral activity concerning EV and MPXV on all indicators used. Therefore, at infection of mice with EV (strain K-1) and peroral administration of NIOCH-14 and ST-246 in a dose 50 mkg/g of mouse weight (12-14 g) within 10 days the survival rate and average life expectancy of mice authentically exceeded the control levels. EV titers in lungs through 6 days after infection in the same groups were lower than in the control. In addition to that, after 7 days of infection of mice with MPXV (strain V79-1-005) and daily peroral administration of NIOCH-14 and ST-246 in a dose 60 mkg/g of mouse weight (9-11 g) authentic decrease in a part of infected animals and MPXV titers in lungs was observed.

[Interrelation between structural state, physicochemical properties and lipid composition in microbial cells].

We performed complex investigations of antioxidant properties and the lipid composition of the five gram-negative bacteria depending on the cultivation season. Structural state, the lipid composition and physicochemical properties of lipids of the three gram-negative bacteria (Renobacter vacuolatum, Flectobacillus major WKM 869, Pseudomonas fluorescens) during their growth and mycelium of four species of the xylotrophic basidiomycetes (Panus tigrinus IBK-131, Fomes fomentarius M71, Laetiporus sulfureus M131, Piptoporus betulinus M60) during the lag phase were also studied. Changes in the composition of the lipid component in the studied bacteria led to considerable structural rearrangements in their membrane system during the growth of bacteria. It is shown that more hydrophilic regions of the lipid component are of paramount importance in maintaining the fluidity of the entire lipid bilayer in the basidiomycete mycelium. The scale and direction of the revealed interrelations between rotational correlation times of probes and the lipid composition depend both on localization of the probe in membrane and the composition and physicochemical properties of the microorganism lipids.

[Peculiarities of antioxidants as radioprotectors under radiation damage of different extent].

Results of the own and literature data about the effectiveness of antioxidants as radioprotectors under the action of ionizing irradiation in animals are generalized dependingon the extent of the radiation damage and the linear energy loss of radiation. The initial antioxidant status of tissues is shown to make a different con- tribution in the manifestation of the radioprotective properties of natural and synthetic antioxidants owing to the non-linearity of the lipid peroxidation processes depending on the radiation dose and dose rate. Thus, a complex approach and consideration of the time factor are necessary when estimating radioprotective effectiveness of antioxidants as radoprotectors under radiation damage of different extent.

Infection of chickens caused by avian influenza virus A/H5N1 delivered by aerosol and other routes.

This study presents results of the study of infectivity of avian influenza virus (AIV) A subtype H5N1 strains isolated from agricultural birds across the territory of the Russian Federation and CIS countries. The results of the susceptibility of chickens to the AIV isolates delivered by the aerosol route and the dissemination of the virus in the organs of infected birds are presented. As was observed, the sensitivity of birds to AIV by the aerosol route of infection is 30 times higher than by intranasal route, 500 times higher than by the oral route and 10000 times higher than by the intragastric route of infection, which is indicative of higher permissivity of respiratory organs to AIV. The highest titres of AIV A subtype H5N1(A/Chicken/Kurgan/05/2005 strain) in aerosol-infected chickens were found in nasal cavity mucosa, lungs, cloaca, serum and kidney, where viable virus accumulation was detected by 18h post-infection (p.i.). The highest virus titres were observed 54h p.i. in lungs, serum and kidney, reaching the value of 8.16 lg EID50 /g(ml) in the lungs. The results showed that birds infected by the aerosol route developed higher titres of virus than those infected by other routes.

[The blood erythrocyte lipid composition of mice exposed to the chronic radiation action at low doses during the early stages of ontogeny].

The comparative analysis of the blood erythrocyte lipid composition of the laboratory mice (males and females) after exposure to the chronic radiation action at the dose of 8 cGy during the early stages of the ontogeny was performed within the early and remote periods after irradiation. This action is shown to cause the marked effect both on the quantitative changes in the lipid composition and the structural state of the blood erythrocyte lipid component as follows from the numerous disturbances of the correlation interrelations between parameters of the lipid composition which are due to the structural state of the erythrocyte membrane. The most substantial changes in the blood erythrocyte lipid composition were detected for males within the remote period after the exposure, which provides evidence in favor of the disorder in the hemopoiesis process. The data obtained allow us to suggest that the erythrocyte membrane viscosity under the chronic low intensity irradiation is supported by the balance maintenance between the relative content of the phospholipid lysoforms and the molar ratio of the [sterols]/[phospholipids].

[Significance of the initial state of the murine liver for the development of consequences after the combined action of the factors of different nature].

The search of the most significant parameters of the liver initial state of mice Balb/c (males) for the development of the biological consequences under a combined action of the Tween-80 (0.3% solution) in the 10% solution of the aqueous acetone and X-rays at the doses of 4 and 5 Gy during one month after exposure was performed by means of the software program for the experimental data analysis. The study has shown both the absence of the same correlation relationships and the existence of two groups of relationships in terms of the coefficient correlation values under the alteration of the X-ray dose. The assumption has been made about different mechanisms underlying regulation of the biochemical process in the murine liver after the combined action of low-toxicity chemical agents at low doses and X-rays irradiation at sublethal doses, whose contribution to the development of effects of different factors depends on the radiation dose.

[Change of the lipid parameters in the murine liver during one month after low intensity X-ray exposure at low doses].

Parameters of the physicochemical regulatory system of lipid peroxidation in the liver of white outbred mice (females) were studied before and during one month after X-ray exposure at the doses less than 1.5 mGy in the autumn and spring-summer seasons. The initial value of parameters is found to exert the most substantial influence on the liver relative mass, the phosphatidylcholine and lysoform relative content in the liver phospholipids of mice. The reliable diminution and the substantial influence of the dose rate dynamics during irradiation are revealed for the molar ratio of [sterols]/[phospholipids], the phosphatidylcholine/phosphatidylethanolamine ratio and the ratio of sums of the more easily oxidizable to the more poorly oxidizable fractions ofphospolipids. The experimental data testify to the complicated nonlinear character of the biological effects of X-irradiation at low doses.

[Surface active properties of isobornylphenols in systems with different degrees of complexity].

Interrelations between the structure of the semi-synthetic phenolic antioxidants -- isobornylphenols and their surface active properties were studied in the chemical (the lecithin aggregation in hexane) and biological (the incubation with the blood erythrocytes) model systems. It has been shown that all studied compounds are able to affect the lecithin aggregation in hexane: the share of the main fraction of the L micelles decreases with increasing the share of particles of greater size. The effect substantially depends on hindered OH group and the presence of the intramolecular hydrogen bond in molecule. The cytotoxic properties of isobornylphenols (the concentration is 100 M) are predominantly due to the molecule structure. The interrelation between the aggregate size of the main fraction of L in the presence of the studied compounds and the discocyte share during mice blood erythrocyte incubation in their presence for 4 h is revealed. Thus, this provides the possibility to assume that the ability of the different biological active substances to affect the lecithin aggregation in non-polar solvent could be used as a model system for the initial assessment of their surface active properties.

[Efficacy of combined administration of ridostin and oseltamivir (tamiflu) for therapy and prophylaxis of experimental infection induced by influenza virus A (H5N1) in mice].

AIM: Study of possibility of treatment-prophylaxis effect increase during combined administration of ridostin and tamiflu in experiments in mice infected with highly pathogenic influenza virus strain A/chicken/Kurgan/05/2005 (H5N1). MATERIALS AND METHODS: Balb/c line mice infected intranasally with influenza virus at 100 and 10 LD50 doses received ridostin and tamiflu as monopreparation or the combined variant before or after the infection. The mice were observed for 16 days, lethality rate, protection coefficient and average life span were evaluated. Virus concentration in lungs was determined by using titration in MDCK cell line. RESULTS: Combined administration ofridostin and tamiflu after the infection increased survivability of the animals when compared with the control group, and reduced influenza virus concentration in lungs. CONCLUSION: Treatment effect during combined administration of ridostin and tamiflu after influenza virus infection increased.