Antiviral activity of the exopolysaccharides produced by Leuconostoc sp. against HSV-1

2 Herpes viral infection is the most common human viral infection. According to the WHO data, about 80% of the world's population has the antibodies to herpes viruses. Modern chemotherapy of infectious diseases, caused by the herpes virus, is associated with the use of guanine-containing drugs, which are the modified acyclic nucleosides of acyclovir, penciclovir, cidofovir and others. The main disadvantage of these drugs, despite their high antiviral activity, is their toxicity and formation of drug resistance. This leads to the necessity for comprehensive study and discovery of the alternative and safe remedies for the prevention and treatment of various forms of herpetic lesions. The attention of researchers is focused on the compounds derived from natural sources. A promising approach for the treatment of diseases caused by herpes simplex virus is the use of lactic acid bacteria (LAB) and their metabolites. Our work is related to the study of the anti-herpetic activity of exopolysaccharides produced by Leuconostoc sp.


Introduction:
3 Lactic acid bacteria have shown great potential in the prevention of severe gastrointestinal disorders in human beings and animals. Although the mode of antiviral action of lactic acid bacteria has not been elucidated in details, they have shown significant ability to inhibit viral infections and/or replication either directly or indirectly caused by respiratory, gastroenteric, murine, influenza, herpes and Newcastle disease viruses. However, variations in the antiviral effect have been observed at species level based on the efficiency and biological properties of the test strain.
Exopolysaccharides are known as potentially useful and biologically active polymer substances for medicinal and pharmaceutical uses due to their versatile biological properties. Liu et al. reported that polysaccharide may prevent viral infection through blockage of virus adsorption onto the host cells by interacting either with virus particles or with the host cell. Some study confirmed that strong evidences on interaction of polysaccharide molecule and cell membrane should be occurred in order to confirm the proper blocking of receptor resulting in the adsorption of virus on the cell membrane. Inhibition of virus-cell adsorption onto the host cell is considered to be the first steps in viral infection process. It has been found that sulfated polysaccharides inhibit the virus-cell attachment and display antiviral effect against various types of viruses including hepatitis B virus, human cytomegalovirus, herpes simplex virus and influenza virus.
Hence, in the current scenario of antiviral research, lactic acid bacteria 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. Objects: Сell culture BHK-21 (kidney of Syrian hamster) and herpes simplex virus 1 (HSV-1) were used. The strains of the Leuconostoc sp were isolated from fermented homemade vegetables: apples, tomato juice, and sauerkraut. Exopolysaccharides (EPSs) were isolated from the culture fluid.

Results and discussion
The first stage of the antiviral assay is necessary to determine the concentration of the compounds that is not toxic to the cells.

Cellular toxicity:
Cellular toxicity of EPSs was tested in vitro according to a cell viability MTT-assay. Monolayers of BHK-21 cells in 96-multiwell plates were incubated with the compounds at concentration of 1500 -370 μg/ml for 72 h, then in a medium was added 20 μl of a 5 mg/ml solution of MTT (Sigma, USA). The concentrations of EPSs that inhibit 50% of cell viability compared to control cells (CC 50 ) were measured.
All EPSs at a concentration of 1500 µg/ml exhibited little cytotoxic effect, and ∼ 71 -100% of cells survived (data not shown) and their CC50 values were >3.5 mg/ml.

Cell Cycle Analysis
Suppressіоn of сеll grоwth аnd prоlіferation frеquently rерresents сеll response to comроund cytotoxicity and virus infection. Therefore, the influence of the EPSs on the cell cycle under normal conditions and the conditions of herpetic infection was analyzed. For the purpose cells (1×10 6 ) were harvested by centrifugation at 300 g (2000rpm) for 7 min, resuspended in 96% ice-cold ethanol, washed with PBS, resuspended in 500 µl solution of PBS (Sigma) that contained RNAse (100 µg/ml) and propidium iodide (PI) (50 µg/ml), and incubated at room temperature for 1 h. The cell fluorescence intensity was measured by an flow cytometer (Beckman Coulter Epics LX, USA) with laser wavelength 488 nm. Cell cycle profiles were analyzed with the program Flowing Software, version 2.5. As the intensity of the PI signal is directly associated with DNA content, the number of cells in a certain cell cycle phase and cells containing fragmented DNA (apoptotic cells), as well as cell structure, were estimated.
The effect of the EPSs on BHK-21 cells population is demonstrated on the following histogram and graphics. It was revealed that after 48 hours of growth 38% of BHK-21 cells remained in G1 phase, 10% were in S phase and 16% were in G2/M phase of cell cycle. Under the conditions of the EPSs treatment, the distribution of cells according to the structure and cell cycle phases was similar to control cells but not identical. Thus, depending on the used EPSs concentrations, 33 -47% of cells remained in G1 phase, 8 -13% were in S phase and 14 -18% were in G2/M phase of cell cycle.

Influence of the EPSs on the cell cycle of BHK-21 cells
Results corresponding to the percentage of cells in G1, S and G2/M phases of three independent experiments are presented as mean ± S.D. *Significant difference between control sample and treated cells (P < 0.05).
Virus infection frequently results in the disturbance of key cellular processes within the host cell. The subversion of cell cycle pathways is a well-established mechanism by which viruses create the most suitable environment for their replication. Notably, the induction of S-phase is either mandatory or at least advantageous for lytic replication of a number of viruses. The characteristic changes in DNA synthesis and content induced by HSV-1 infection allow the use of flow cytometry to detect not only an infection but also the potential antiviral activities.

Antiviral Assay
We used a modification of an MTT-assay developed for screening of anti-HSV compounds. 50 µl of virus suspension (a multiplicity of infection (MOI) of 3,2) was added to BHK-21 cells After 2 hour, any unabsorbed virus was aspirated and 200 µl of serial two-fold EPSs-containing medium was added to each well, and incubated at 37˚C and 5% CO 2 for 2 days. The % of protection and the concentrations of the EPSs that inhibit virus reproduction at 50% compared with virus control (IC 50 ) were measured.
Herpes virus reproduction is characterized by a complex sequence of different steps with which antiviral agents might interfere. It is known that a protein bound microbial polysaccharide showed potent ability to reduce the viral titer of HSV-1 and HSV-2. The significant antiviral activity against herpes simplex viruses might be attributed to the following reasons including disintegration of the entire herpes simplex virus particles, solubilization of the virus envelope, modification in chemical composition, protein degradation, or by covering the essential proteins of virus envelop. Some study revealed that protein bound polysaccharide may exert its partial antiviral mode of action against HSV-2 via binding the glycoproteins of HSV-2 to protein bound polysaccharide which may interfere at the initial state of virus replication or release of any virions.
To determine the stage of HSV-1 infection that was inhibited by EPSs, virus and cells were treated with EPSs at various times before and after HSV-1 infections.

Virucidal assay
100 µL of 1.5 mg/ml of the EPSs were mixed with 100 µL of 2X HSV-1 in microfuge tubes and incubated at 37˚C for 3 hours. Then, the BHK-21 cells were infected with 0.05 ml per well of ten-fold serial dilutions of the virus-containing suspension. The virus adsorption was carried out for 2 hours, then 0.15 ml of serum-free medium was added. After 3 days of incubation, virus titer (TCD50/ml) was calculated.

Attachment assay
The BHK cell monolayer was pre-chilled at 4°C for 1 h and then infected with 3,2 MOI HSV-1 in the absence or presence of serial dilutions of the EPSs. After 3 h of incubation at 4°C cell monolayer was washed with PBS three times and overlaid with medium containing 0.06% agarose. The infected cell monolayer was fixed and stained with 1% crystal violet in 20% ethanol. The antiviral activity of the EPSs was determined by the following formula: % Percentage inhibition = (1-number of plaque (tested)/ number of plaque (control))*100%

Viral Penetration Assay
The BHK-21 cell monolayer was pre-chilled at 4°C for 1 h, infected with 3,2 MOI HSV-1 and incubated at 4°C for a further 3 h to allow the attachment of HSV-1. After incubation, 50 µL of 2X concentration of compounds were added. The infected cell monolayer was then incubated at 37°C to maximize the penetration of virus. At 45 min intervals, the infected cell monolayer was treated with PBS at pH 3 for 1 min to inactivate a non-penetrated virus. PBS at pH 11 was then immediately added to neutralize acidic PBS. The neutral PBS was removed and the cell monolayer was overlaid with overlay medium. After a further 48 h of incubation, the cell monolayer was fixed and stained. Plaques were counted as described above.

Conclusions:
Taken together, our results showed that the EPSs 2t, 19s, and 6s, isolated from the genus Leuconostoc sp., in non-toxic concentrations possess the anti-HSV activity, which is realized via multiple mechanisms. The EPS 6s showed significant virucidal activity and reduces the viral adsorption, penetration, and yield of virus particles by 97 -99%. Whereas the EPSs 2t and 19s block the viral adsorption to the cells and inhibits infectivity of viral progeny. Moreover, it was demonstrated that using the EPSs leads to the normalization of a life cycle of cells infected with herpes virus to the level of non-infected cells. Taking into account the promising antiherpetic activity of the EPSs produced by Leuconostoc sp. herein reported further investigation is needed to explore the antiviral mechanism of these compounds in detail.

Funding:
Publications are based on the research provided by the grant support of the State Fund for Fundamental Research (project F83).
A various inhibitory effect of the EPSs was observed when there were added 3 h before adsorption, during virus adsorption and penetration, and after virus adsorption. The EPSs 2t, 19s, and 6s showed virucidal activity when were added to the virus before adsorption reduced HSV-1 infectivity by 86 -97%. Our studies revealed that this 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 %. It was shown that all EPSs reduced the titer of the virus obtained de novo by 23-99%.