**3. Results**

#### *3.1. The E*ff*ect of Lactic Acid Bacteria Exopolysaccarides on Viability and Mitotic Activity of MDBK Cells*

The issue of directed inhibition of the reproduction of infectious disease pathogens requires a search for medicines that are characterized by low toxicity and a broad spectrum of antiviral activity. As viruses are intracellular parasites, selective suppression of virus reproduction without adverse effect on the viability of host cells is one of the requirements for antiviral inhibitors.

MTT analysis is one of the most simple and available methods for the estimation of cytotoxicity. The technique enables the identification of the negative impact of EPSs on the viability of cells and the functional activity of mitochondria (Figure 1).

**Figure 1.** Cytotoxicity of exopolysaccharides in MDBK cells. Serial three-fold dilutions of EPSs in DMEM-RPMI medium were added onto the monolayer of MDBK in a 96-well plate for 72 h at 37 ◦C. Cell viability effect was determined by MTT assay. Values represent the mean ± S.D. for three independent experiments.

It was found that EPSs at a concentration of 375–1500 μg/mL were not toxic for MDBK cells, as they suppressed their viability only by 3–17%, whereas the CC50 value was significantly higher than 1500 μg/mL. The CC50 value for referent compound Ribavirin was 400 μg/mL.

As toxic compounds often cause the termination of cell growth and proliferation, the effect of EPSs on the MDBK cell populations was studied. Cells were fixed and dyed with fluorochrome propidium iodide and analyzed using flow cytometry. The approach allowed the estimation of the distribution of cells by their structure and cell cycle phases, as well as the identification of apoptotic cells, as the propidium iodide signal is directly proportional to DNA content.

As can be seen from cell cycle profiles (Figure 2A) based on the distribution of cells according to the structure and cell cycle phase, the histograms of EPS-treated and control MDBK cells are similar but not identical. It was revealed that after 48 h of growth, 50% of control MDBK cells remained in the G1 phase, 16% were in the S phase, and 13% were in the G2/M phases (Figure 2B).

**Figure 2.** Influence of the EPSs on the cell cycle of the MDBK cells. (**A**) Cell cycle profiles of the control cells and EPS-treated cells (concentration of exopolysaccharides and ribavirin are 1500 and 125 μg/mL, respectively). (**B**) Cell cycle features after treatment with the EPSs were measured by flow cytometry following cell fixation and propidium iodide staining. Cell cycle profiles were analyzed with the Flowing Software, version 2.5. 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 test sample and control cells (*p* < 0.05).

Under the conditions of the compound treatment, the distribution of cells in G1 and G2/M phases was similar to control cells. However, compared to control cells, EPSs 48a and 19s decreased the number of cells in the G1 phase up to 16%, whereas EPSs 6a, 48a, and 6s increased the number of cells in the G2/M phase by 11–42%.

#### *3.2. Anti-Adenoviral Activity of EPSs*

The anti-HAdV-5 activity of EPSs (in non-toxic concentrations) was confirmed by measuring virus yield synthesized de novo using a plaque reduction assay.

Analysis of the antiviral activity of EPSs added to MDBK cells 24 h before adenovirus infection demonstrated that only EPSs 48a, 26a and 6s showed low antiviral activity, reducing virus reproduction by 23–67% (Figure 3), indicating the inefficiency of EPS use according to the suggested treatment approach.

**Figure 3.** Effect of EPSs on infectivity of adenoviral offspring: the results of the analysis of the EPS-mediated antiviral effect for pre-treatment of cells with EPSs. Results are presented as percentage of infectious virus titer reduction and are the mean of three independent experiments.

It was found that EPS showed weak virucidal activity decreasing the infectious titer of HAdV-5 by 3–85%, whereas incubation of the virus with EPS 6a increased HAdV-5 reproduction by 15% (Figure 4).

**Figure 4.** Influence of EPSs on the formation of infectious progeny of adenovirus. The results of the analysis of the EPS-mediated antiviral effect for co-incubation of EPSs (concentration 1500 μg/mL) and HAdV-5. Results are presented as percentage of infectious virus titer reduction and are the mean of three independent experiments.

Using EPSs at late stages of adenovirus reproduction and immediately after virus adsorption, it was found that only EPS 26a shows apparent anti-HAdV-5 activity. At the concentrations of 20 and 100 μg/mL, the EPS absolutely blocked the synthesis of viral progeny (Figure 5). Other EPSs used in the analyzed concentrations reduced the infectious titer of the virus by 18–93%. The use of Ribavirin at concentrations of 32–125 μg/mL after infection of the cells with adenovirus resulted in a decrease in HAdV-5 reproduction by 96.3–99.8% (data not shown).

**Figure 5.** Effect of EPSs on adenovirus reproduction: the results of the analysis of the antiviral activity of EPSs added after infection. Results are presented as percentage of infectious virus titer reduction and are the mean of three independent experiments.

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 [23]. Notably, the induction of the S-phase is either mandatory or at least advantageous for lytic replication of a number of viruses. Adenoviral infection has been reported to have e ffects on the cell cycle. It is well-known that adenoviral E1 gene products interact with pRb (retinoblastoma protein), causing the release of E2F transcription factor, which potentiates transition from the G1 to the S phase, in which productivity is greatest. HAdV infection of a range of epithelial cell lines, including a primary cell line, causes G2 phase synchronization and cell cycle arrest [24,25]. This synchronization in the G2 phase may be a significant factor contributing to the cell-size increase [25]. Therefore, the influence of EPSs on the cell cycle under the conditions of adenovirus infection was analyzed using flow cytometric analysis.

Significant changes in the cell cycle of cells infected with HAdV-5 compared to non-infected cells were revealed. In particular, the number of cells in the G1 phase decreased by 67%, whereas the number of cells in the S phase is doubled (Figure 6). The reduction of the cell population in G1 phases demonstrated the suppression of the transition of cells through the mitotic phase. As infected cells enter the S phase and the G2/M phase is blocked, cells produce viral DNA, late viral proteins and virions. Further, these cells are destroyed and detach from the monolayer.

**Figure 6.** Influence of the EPSs on the cell cycle of the MDBK in the presence of adenovirus infection. The cells' fluorescence intensity was measured by a flow cytometer (Beckman Coulter Epics LX, MN, USA) with laser wavelength 488 nm. Cell cycle profiles were analyzed with the program Flowing Software, version 2.5. Results corresponding to the percentage of cells in the G1, S, and G2/M phases of three independent experiments are presented as mean ± S.D. \* Significant di fference between test sample and control of infected cells (*p* < 0.05). \*\* Significant di fference between a control of infected cells and control cells *(p* < 0.05).

It was found that after the treatment of cells infected with adenovirus using exopolysaccharides, the distribution of cells in cell cycle phases was similar to the distribution of cells infected with adenovirus, demonstrating the ine fficiency of the exopolysaccharide treatment (data not shown). A decrease in the number of cells in the S phase by 5–33% compared with the distribution of infected cells was observed. However, there was no increase in the number of cells in the G1 phase, indicating continued blocking of the mitotic phase of the cell cycle due to viral infection. Only the use of EPS 48a at a concentration of 100 and 20 μg/mL resulted in a significant increase in the number of cells in the G1 phase (1.5–2 times) compared with infected cells, and a decrease in the number of cells in the synthetic phase by 18–29%, indicating normalization of the cell cycle of infected cells to the level of uninfected cells (Figure 6).
