Background and objectives: The use of antagonistic probiotic microorganisms and their byproducts represents a promising approach for the treatment of viral diseases. In the current work, the effect of exopolysaccharides (EPSs) produced by lactic acid bacteria from different genera on the structural and functional characteristics of cells and the development of adenoviral infection in vitro was studied. Materials and Methods: Cytotoxicity of six EPSs of lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The influence of the EPSs on the infectivity of human adenovirus type 5 (HAdV-5) and on the cell cycle under a condition of adenovirus infection was studied using plaque reduction assay and flow cytometric analysis, respectively. Results: It was shown that exopolysaccharides were non-toxic to Madin-Darby bovine kidney cells (MDBK) as they reduced their viability by 3–17%. A change in the distribution of the cell cycle phases in the non-infected cell population treated with EPSs was observed. The analysis demonstrated an increase in the number of cells in the S phase by 47% when using EPSs 15a and a decrease in the number of cells in the G1 phase by 20–27% when treated with the EPSs 15a, 33a, and 19s. The use of EPSs did not led to the normalization of the life cycle of HAdV-5 infected cells to the level of non-infected cells. The EPSs showed low virucidal activity and reduced the HAdV-5 infectivity to 85%. Among the studied exopolysaccharides, anti-adenovirus activity was found for EPS 26a that is produced by Lactobacillus spp. strain. The treatment of cells with the EPS following virus adsorption completely (100%) suppressed the formation and release of HAdV-5 infectious. Conclusions: EPS 26a possessed distinct anti-HAdV-5 activity and the obtained data demonstrate the potential of using exopolysaccharides as anti-adenoviral agents.

Background and objectives: A considerable increase in the levels of adenoviral diseases among both adults and children necessitate the development of effective methods for its prevention and treatment. The synthesis of the new fluorinated 1,2,3-triazoles, and the study of the mechanisms of their action, are promising for the development of efficient antiviral drugs of our time. Materials and Methods: Antiviral activity and cell cytotoxic effect of 2-(3-chlorotetrahydrofuran-2-yl)-4-tosyl-5-(perfluoropropyl)-1,2,3-triazole (G29) were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The influence of the compound on the infectivity of human adenovirus type 5 (HAdV-5) was carried out via the cytomorphology method. The influence of the compound on the cell cycle under a condition of adenovirus infection was studied using flow cytometric analysis of propidium iodide-stained cells. Results: It was found that G29 suppressed HAdV-5 reproduction by 50% in concentrations of 37 μg/mL. Furthermore, the compound reduced the titer of virus obtained de novo, and inhibited HAdV-5 inclusion bodies formation by 84–90%. The use of fluorinated compounds under the conditions of adenovirus infection decreased the number of apoptotic cells by 11% and the number of cells in S phase by 21–42% compared to the profile of infected cells. Conclusions: The fluorinated compound G29 showed moderate activity against HAdV-5 based on several mechanisms. It led to the normalization of the life cycle of cells infected with adenovirus to the level of non-infected cells and caused the obstruction of HAdV-5 reproduction, inducing the formation of non-infectious virus progeny.

Introduction: In the current scenario of antiviral research, lactic acid bacteria (LAB) and their derived polymers or polysaccharides are considered potential candidates in antiviral therapy to prevent or treat viral infections in both human and animals with remarkable efficacy and might have significant contribution in medicine and pharmaceutical industries in future. The aim of this work was to study the anti-herpetic activity of exopolysaccharides produced by Leuconostoc sp. Methodology: The strains of the LAB were isolated from fermented homemade vegetables: apples, tomato juice, and sauerkraut. Exopolysaccharides (EPSs) were isolated from the culture fluid. Cytotoxicity and antiherpetic activity of 7 EPSs (15a, 48a, 33a, 43a, 2t, 19s, and 6s) were studied using MTT assay. The influence of EPSs on the herpes simplex virus 1 type (HSV-1) was determined by the virucidal, adsorption and penetration assays. The impact of the EPSs on the cell cycle under a condition of HSV-1 infection was analyzed using flow cytometric analysis of propidium iodide-stained cells. Results: All EPSs demonstrated the minimal cytotoxicity of cells and their CC₅₀ values were >3.5 mg/ml. It was determined, that only EPSs 2t, 19s, and 6s significantly inhibited HSV-1 reproduction; their EC₅₀ value equal to 0.2 and 0.5 mg/ml, and the selectivity index was in the range of 39 – 52. Moreover, their showed virucidal activity when were added to virus 3 h before adsorption reduced HSV-1 infectivity by 86 – 97%. Our studies revealed that these EPSs were able to prevent the HSV-1 attachment to cells and penetration into cells in a different manner, reducing HSV-1 production by 70 – 99 %. Furthermore, the normalization of the number of cells in all phases of the cell cycle compared with the profile of infected cells and the increasing number of cells in G1 phase to 79% compared with the control values of viral infections were determined after using of EPSs. Conclusion: This study presents the first data indicating an enormous potential of using EPSs the genus Leuconostoc sp. for lower or hinder the spread of diseases caused by herpesviruses. Funding: Publications are based on the research provided by the grant support of the State Fund for Fundamental Research (project F83).

Mixed viral infection is one of the current and unexplored issues of human infectious diseases. A special place in the development of these pathologies is occupied by adeno- and herpes viruses that are able to persist for a long time in a latent condition in the body. There is a huge lack of knowledge about antiviral activities of specific drugs during the mixed infections. The study of know drugs and discovery of new compounds using not only standard mono-infections but also with created mixed infections is a topical and a new direction in antivirus screening. Previously in our department, the models of adeno-herpetic infections in cells of different origins were created and the features of the development of viral infections in these systems were studied. The model of simultaneous adeno-herpetic infection of MDBK cells was used for the analysis of the antiherpetic drug acyclovir (ACV) and for research of new fluorine-containing derivatives of L-phenylalanine (10S-23 and 10S-24, synthesized in Institute of Organic Chemistry of the NAS of Ukraine). Determination of the antiviral activity accessed via real-time PCR and infectious virus yield reduction assay demonstrated the inhibitory effect of these compounds on the late stage of the HSV-1 and HAdV5 reproduction. With the presence of 10S-24 and ACV in the conditions of mono-infection, HSV-1 DNA replication was inhibited on 39 and 100%, respectively. Furthermore, the significant delays of HSV-1 reproductions were observed, the titer of virus obtained de novo reduces on >99%. It was shown that for adenovirus infection of the cells, all compounds reduced the titer of the virus on 34-96%. Use of the ACV under mixed infection led to the 46% loss of the drug activity against HSV-1. The application of 10S-23 and 10S-24 at mixed infection induced a decrease of effectiveness of the compounds relatively herpes simplex virus by 58-73% and 16-57%, respectively. It was shown that compounds have been not effective against HAdV5 under conditions of co-infection of cells. An abnormal action of drugs in co-infected cells indicates the need for further study of the mechanisms and targets of antiviral activity of compounds in such conditions, because using these compounds can be ineffective in medical practice. Acknowledgements This work was supported by a President’s of Ukraine grant for competitive projects F70 of the State Fund for Fundamental Research.

The aim of the study: to investigate the peculiarities of the development of the pathological process in cells in conditions of mixed viral infection and to study the effectiveness of antiviral drugs in this model. Materials and methods of research. A model of simultaneous mixed infection of MDBK cells with human adenovirus serotype 5 (HAdV-5) and herpes simplex virus type 1 (HSV-1) was developed. Mitochondrial activity, ultrastructure and the state of the cell population were studied using MTT assay, transmission electron microscopy and flow cytometry with propidium iodide dye. The intensity of virus reproduction in cells and their infectious titer were studied by the cytomorphology method. The level of the synthesis of the major proteins of associate viruses was analyzed using flow cytometry and the corresponding monoclonal antibodies. Results of the research. Co-infected cells demonstrated a lower rate of development of pathomorphological changes compared to mono-infections, related to the inhibition of the reproduction of associate viruses. It was found that the co-infection of cells with HSV-1 and HAdV-5 results in a decrease in the number of cells with virus-induced intranuclear inclusions of both viruses by up to 40 % and viruses titer by 1.6 lg and 2.6 lg, respectively. Inhibition of synthesis of major capsid protein and glycoproteins of the herpes virus by 83 % and 64 %, respectively, and a less pronounced decrease in the amount of adenovirus hexon protein (by 17 %) were also noted. It is shown that the mitochondrial activity of co-infected cells increases to 64 % in comparison with herpetic mono-infection. An analysis of the influence of co-infection on cell cycle revealed that the number of cells in G1 phase remained unchanged compared with both mono-infections, while the number of apoptotic cells compared with herpes infection was reduced by 24 %. An analysis of the officinal drugs Acyclovir and Ribavirin effectiveness in conditions of mixed infection showed a reduction in their antiviral activity against associate viruses by 1 to 2.3 lg compared to mono-infections. Conclusions. The presence of a specific innovative cellular model of mixed infection with known aspects of the course of associated infections allows it to be used for preclinical study of antiviral activity of compounds and to obtain new data of the role of viral-viral interactions in the development of inefficient application of antiviral agents in medical practice. Key words: mixed viral infection, cytomorphological changes, cell cycle, reproduction of associate viruses, and antiviral activity. For citation: Biliavska LO, Povnitsa OY, Pankivska YB, Zagorodnya SD. Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection. Journal of the National Academy of Medical Sciences of Ukraine. 2019;25(4):476–87

INTRODUCTION: Human adenovirus and herpes simplex virus cause infectious diseases of eyes, respiratory, enteric and urogenital tracts, of central and peripheral nervous system and are capable to persistence in latent form and are activated under the influence of endogenous and exogenous factors. A high spread of infections caused by herpes virus and adenovirus and their mixed infections are the problems of modern medicine. During mixed infections, an absence of viruses’ interaction as well as a mutual influence of viruses may originate. Search of drugs with a relatively high activity against the co-associated viruses by inhibiting their reproduction and transmission is an important task. Activity of antiviral drugs under condition of co-infection is not studied enough. METHODS: Human adenovirus type 5 (HadV-5) and herpes simplex virus type 1 (HSV-1/US) were used as the model viruses. The reproduction of viruses in MDBK cells was studied using the cell-ELISA, flow cytometry and Western-blot. The levels of synthesis of the major proteins of adenoviruses and herpes viruses under conditions of the mixed infection were studied. The created model of mixed infection in the populations of MDBK cells was confirmed by the electron microscopy and immunofluorescence analysis. The cytotoxic activity of the drugs was studied by the MTT-assay. The model of co-infection of MDBK cells was used to evaluate the efficiency of the antiviral agents of the broad action spectrum (ganciclovir, cidofovir and ribavirin) against HAdV-5 and HSV-1/US. The antiviral effects of the drugs were studied using the medical scheme of introduction of preparation (as a component of supporting medium). The analysis of the virus DNA synthesis in infected cells after treatment with the drugs was carried out by the RT-qPCR. The effectiveness of the preparations under mono- and mixed infections was studied by the cytomorphological method. RESULTS: The simultaneous infection of the cells with two viruses resulted in a significant inhibition of the reproduction of herpes virus and in a less pronounced inhibition of the adenovirus reproduction. Use of ribavirin in conditions of mono infection led to the inhibition of DNA replication of adenovirus by 41% and herpes virus by 29%. Ribavirin was ineffective against HАdV-5 and its activity against herpes virus remained unchanged under conditions of co-infection. The analysis of antiviral activity of ganciclovir on the models of mono infections showed the reduction of reproduction of HSV-1/US by 60% and HAdV-5 by 4%. Ganciclovir inhibited the reproduction of herpes virus by 61% during the co-infection. The effectiveness of the drug against adenovirus increased 20%. Under condition of the mixed infection the cidofovir activity against HAdV-5 and HSV-1/US decreased 3-fold and 79-fold, respectively, compared to the mono infections. CONCLUSIONS: The changes of the nature of the pathological processes were found, as a result of the interference of the viruses and the differences of the drugs activities against the co-associated viruses in conditions of the mono and mixed infections of MDBK cells. Both the increase and inhibition of the drugs activities were detected that may lead to the formation of resistant strains of viruses.