**1. Introduction**

Despite successes of vaccination, chemoprevention, and chemotherapy of human respiratory viral infections, these diseases still are one of the leading causes of human infectious disorders. Adenoviruses cause keratoconjunctivitis, as well as respiratory, urogenital and intestinal tract infectious diseases. In addition, adenoviruses may persist in the lymphoid tissue of the tonsils and adenoids for a long time, causing upper respiratory tract infections in adults [1,2].

Various compounds have been found to be e ffective against adenoviruses. Anti-adenoviral activity was shown in vitro for nucleotide analogs [3,4]. Ribavirin, which a ffects many DNA and RNA-containing viruses, was found to be e ffective, partially e ffective, or not e ffective against adenoviral infection. Ganciclovir, which is efficient against cytomegalovirus infection, is effective against adenoviruses both in vitro and in vivo [5]. Cidofovir, which is characterized by a broad spectrum of antiviral activity, is used for the treatment of severe adenoviral infections [6]. Ribavirin, ganciclovir, and cidofovir are more or less successfully used for the treatment of severe human adenoviral pathologies, including hepatitis, cystitis, and pneumonia in immunocompromised organ transplant recipients [7,8]. At the same time, apparent adverse effects and the ability of adenoviruses to form resistant strains limit the use of these drugs [9]. Some chemically distinct compounds, including lipids, acridones, and imidazoquinolinamines, as well as other analogs of nucleosides, also show anti-adenoviral activity. Virus reproduction in cell cultures may be inhibited by high concentrations of green tea catechins [10]. Interferon drugs used to treat respiratory infections are most useful for prevention and cannot be considered as the primary medications for the treatment of severe infection. As adenoviruses have effective mechanisms that suppress the interferon-induced antiviral cascade, they are resistant to the action of interferon and its inducers [11].

Therefore, the development of new antiviral drugs that would be effective against adenoviruses without adverse effects, for a wide range of patients, including long-term users, is very urgent.

Currently, the use of probiotic antagonist microorganisms and their metabolic products represents a promising approach for the treatment of viral diseases [12]. Lactic acid bacteria (LAB), which can be found in the microbiota of the mucous membranes of the nose and mouth, and the gastrointestinal and urinary tracts, have a positive effect on these ecosystems as they stimulate the immune system and increase resistance to infections of bacterial and viral origin. LAB are the sources of multiple biologically active substances that vary in chemical structure and action spectrum [13]. During metabolic processes, lactic acid bacteria produce vital substances, including vitamins, amino acids, enzymes, fatty acids, exopolysaccharides, lactic acid, bacteriocins, and hydrogen peroxide, and significantly improve the intestinal absorption of micro- and macro-elements essential for the organism. It was also shown that exopolysaccharides that are secreted during the cultivation of LAB, in addition to their probiotic activity, can act selectively on pathogenic microbes and viruses without significant disturbances of the microbiocenosis and the development of inflammatory processes that occur during the use of broad-spectrum antibiotics and chemical antiviral agents [14]. As a result, exopolysaccharides can be used as a base for the production of harmless, non-toxic and acellular probiotic products and drugs that have a targeted therapeutic effect.

The present study aimed to investigate the antiviral activity of exopolysaccharides produced by lactic acid bacteria of the genera *Pediococcus*, *Leuconostoc* and *Lactobacillus* against human adenovirus type 5.

#### **2. Materials and Methods**

#### *2.1. Virus and Cell Culture*

In this study MDBK cells were obtained from the tissue culture collection of the Institute of Virology of the Bulgarian Academy of Sciences (Sofia, Bulgaria) and HAdV-5 was obtained from the collection of the Institute of Microbiology of the Budapest University of Medical Sciences (Budapest, Hungary) and propagated in HEp-2 (ATCC CCL-23) cells.

MDBK cells were maintained in sterile plastic falcon ® (Sarstedt AG & Co. KG, Nümbrecht, Germany) in a growth medium composed of 45% DMEM (Sigma-Aldrich, St. Louis, MO, USA), 45% RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) heat inactivated at 56 ◦C with antibiotics penicillin (Sigma-Aldrich, St. Louis, MO, USA) and streptomycin (100 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA).

Cultivation, purification, and preservation of HAdV-5 were performed according to the standard method [15]. The infectious titer of the virus in MDBK cell culture was 7.0 × 10<sup>7</sup> PFU/mL.

#### *2.2. Tested Substances*

Exopolysaccharides (EPSs) produced by lactic acid bacteria of the genera *Pediococcus*, *Leuconostoc* and *Lactobacillus* were extracted, purified and accumulated by the researchers from the Department of Physiology of Industrial Microorganisms at the Zabolotny Institute of Microbiology and Virology National Academy of Sciences of Ukraine (Table 1) [16,17]. Ribavirin (Rib) (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference compound.

**Table 1.** Sources used to extract exopolysaccharides (EPSs) and genera of lactic acid bacteria (LAB) producing respective EPS.


#### *2.3. Cytotoxicity Assays*

MTT-assay was used for the analysis of cell viability [18]. After 24 h of growth in growth medium, monolayers of MDBK cells in 96-multiwell plates were incubated with EPS at the concentrations of 375, 750 and 1500 μg/mL. Control cells were incubated with fresh medium lacking EPS for 72 h. A total of 20 μL of MTT solution 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) was added into wells and cells were incubated at 37 ◦C and 5% CO2 for 3–4 h, then the medium was removed and 150 μL of 96% ethanol was added. The plates were read using a Multiskan FC (Thermo Scientific, Waltham, MA, USA) with a 538-nm test wavelength.

Percentage decrease of cell viability under the condition of EPS action was calculated by the following formula:

$$\% \text{ decrease of cell viability} = 100 - (\text{A/B} \times 100) \tag{1}$$

where A is the mean optical density of the studied samples at a certain concentration, and B is the mean optical density of the control cell samples.

EPS concentration at which cell viability was inhibited by 50% (CC50) was estimated in comparison to the control cells not treated with EPS.

#### *2.4. Cell Cycle Analysis by Flow Cytometry*

MDBK cells infected with adenovirus (treated and not treated with the EPSs) were fixed in 96% ice-cold ethanol for 1 h, resuspended in 500 mL solution of phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) that contained RNAse (100 μg/mL) (AppliChem GmbH, Darmstadt, Germany) and propidium iodide (PI) (50 μg/mL) (Sigma-Aldrich, St. Louis, MO, USA), and incubated at room temperate in a dark place for1h[19]. The cell fluorescence intensity was measured by a flow cytometer (Beckman Coulter Epics LX, Minneapolis, MN, USA) with a laser wavelength of 488 nm. A total of 20,000 cells were sorted per sample. Cell cycle profiles were analyzed with the program Flowing Software, v. 2.5 (Cell Imaging Core, Finland).

## *2.5. Antiviral Assay*

To investigate the best order of EPSs' antiviral effects, three experimental procedures were used as follows:

#### 2.5.1. Pre-Treatment of Cells with EPSs

To MDBK cells were added 100 μL of EPSs at the concentrations of 20, 100 and 500 μg/mL, which were then incubated at 37 ◦C for 24 h. Then, cells were infected with HAdV-5 (the multiplicity of the infection (MOI) of 7 PFU per cell). After 1.5 h, the virus-containing medium was removed, and 200 μL of serum-free medium was added.

#### 2.5.2. Co-Incubation of EPSs and HAdV-5

Adenovirus (MOI 70 PFU/cell) was mixed with an equal volume of EPS at a concentration of 1500 μg/mL and incubated at 37 ◦C for 3 h. The MDBK cell monolayer was infected with 50 μL per well of suspension of EPS-HAdV-5 of the appropriate dilution. Following the virus adsorption that was carried out at 37 ◦C for 1.5 h, the virus-containing material was removed, and 200 μL of serum-free medium was added.

#### 2.5.3. EPSs Were Added Post-Infection

The MDBK cells were first infected with HAdV-5 at 50 μL/well (MOI 3.5 PFU/cell), and the virus was adsorbed at 37 ◦C for 1.5 h. Then, the virus was removed, and the growth medium with the appropriate EPSs concentration was added (20, 100 and 500 μg/mL).

Plates with cells were kept at 5% CO2 at 37 ◦C until a visible cytopathic effect of adenovirus appeared (3–5 days), and the virus material was selected for further investigation of the virus titer. Non-treated cells and cells treated with HAdV-5 served as controls.

#### *2.6. Virus Titration*

The titer of the virus was determined by the plaque method, based on the formation of necrotic sites in the infected cells due to the reproduction of the virus. Cells were grown in a 24-well plate (TPP, Trasadingen, Switzerland) to form a 100% monolayer and were infected with serial ten-fold diluted lysates of cells previously infected with adenovirus (untreated or treated with different concentrations of EPSs) at 0.3 mL per well and incubated for 1.5 h at 37 ◦C at 5% CO2. After adsorption of the virus, the medium was removed, and the cells were covered with 1% methylcellulose (Sigma-Aldrich, St. Louis, MO, USA), DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) and 2% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated at 37 ◦C at 5% CO2 for 5 days. Next, the cover was removed and 200 μL of 0.2% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in 20% ethanol was added to the cell monolayer [20,21]. The titer of the virus was determined by the highest dilution of the virus in which the virus-induced plaques were formed according to the formula:

$$\text{Virus titer PFU/mL} = \text{A/(B} \times \text{C)}\tag{2}$$

where A is the number of plaques, B is the dilution of the virus, and C is the volume of the inoculum. The antiviral activity of EPSs was determined using the following formula:

$$1\% \text{ inhibition} = (1 - \text{Virus timer (experiment)}) \text{virus timer (control)}) \times 100\% \tag{3}$$

A decrease in the infectious titer of the virus by 2 lg or more (or 99% or more), compared with the control, indicates significant activity of the compound against the virus, 97–99% demonstrates a moderate effect and less than 97% shows a relative activity [22].

#### *2.7. Statistical Analysis*

Statistical data processing was performed according to standard approaches for calculating statistical errors (standard deviation) using Microsoft Excel 2010. The results were expressed as mean ± S.D. for three independent experiments. The Student's *t*-test (unpaired, two-sided *t*-test) was used

to evaluate the difference between the test sample and control. A *p* value of <0.05 was considered statistically significant.
