Antiviral activity of lipopolysaccharides of Pseudomonas chlororaphis subsp. Aureofaciens

C virus is completely inhibited.

The problem of viral infections control is one of the more relevant in modern medicine.Against acquired immunodeficiency syndrome (AIDS), cytomegalovirus infection, hemorrhagic fevers (one of which is deadly Ebola fever), there are still no effective prevention tools.Influenza and other acute respiratory viral diseases occupy a leading place in the structure of human infectious diseases, quickly and widely distributed and cause significant morbidity and mortality worldwide.As pointed out by WHO experts, there is the tendency to constant growth of diseases [1].
Causative agents of these diseases often acquire partial or complete resistance to the treatment.Modern chemotherapy has a wide arsenal of antiviral agentsabnormal nucleosides, adamantane and thiosemicarbazone derivatives, synthetic amino acids, virucidal preparations, numerous interferons and interferonogens [2].However, traditional methods of treatment using these antiviral preparations are often ineffective.The problem of lack of highly effective substance for prevention and treatment of viral infections is relevant not only due to their morbidity and possible serious complications, but due to provoking many others, including cancer.Therefore, the search for new alternative means of influence on viruses and viral infections is an urgent task of modern medicine and biotechnology.
Reports of bacterial lipopolysaccharides (LPS) or endotoxins ability to prevent the development of some experimental viral infections are present in the literature for long.It has been suggested that the antiviral effect of bacterial LPS can be caused by stimulation of endogenous interferon biosynthesis [3].This assumption is confirmed by many experiments.So, it is shown [4] that Escherichia coli and Salmonella typhimurium LPS increased -interferon secretion by cell culture of human skin fibroblasts, which in turn ensues protection against vesicular stomatitis virus.Data on the protective effect of bacterial endotoxins concern only some experimental viral infections, and most studied LPS were derived from the strains of pathogenic or opportunistic Gramnegative bacteria, which are characterized by significant toxicity.Unlike the works cited [5], it was previously shown by us that LPS of saprophytic bacteria of the genus Pseudomonas, which are widely inhabiting soil, water, plant rhizosphere, including P. chlororaphis subsp.aureofaciens, and used for biological crop protection from pests and pathogens are highly active antiviral agents [6].During research in 2010-2012, they consistently demonstrated efficiency against the tobacco mosaic virus (TMV) in the plant of Solanaceae family.At LPS concentrations of 1-10 mg/ml the inhibition of virus infectivity was 98-100%, 0.1 mg/ml -57-69%, 0.01 mg/ml -43-44%.At a concentration of 1 mg/ml the reduction in infectivity of the virus at various plants-inductors -datura and tobacco -was from 10.2 to 46%.The activity of both LPS was similar.It is interestingly, that LPS derived from microorganisms belonging to other families and species (Rahnella aquatilis, Ralstonia solanacearum), were not active against the TMV, and sometimes even stimulated the formation of necrosis.
Therefore, the purpose of this study was to investigate the ability of lipopolysaccharides of Pseudomonas chlororaphis subsp.aureofaciens two strains to inhibit in vitro the reproduction of human viruses (influenza and herpes), bovine diarrhea virus, which is used as a model of hepatitis C virus, and also to inhibit the production of hepatitis C virus in the model system of cells transfected with the cDNA of this virus.

Materials and Methods
The objects of research were lipopolysaccharides (LPS 1 and LPS 2) of two strains of Pseudomonas chlororaphis subsp.аureofaciens: UCM B-306 and UCM B-111 respectively, kindly provided to us for research by prof.Kiprianova O. A. Cultures cultivation and LPS preparation were performed as described previously [5].
LPS ability to inhibit the development of viral infection was tested on three types of cell cultures: MDCK, Vero and MDVK.There are subinoculated cultures of dog kidney cells, African green monkey and bull respectively.As a test objects there were used: 1) influenza virus -the strain A/FM/1/47 (H1N1)infectious titer of allantois culture -8,0 lg TCD 50 , hemagglutinin titer -1: 512 HAunits; 2) herpes simplex virus type 2 (HSV-2) strain VN, infectious titer by CPE in cell culture was 7.0 lg TCD 50 .Prior to experimental studies virus was stored at -70 C; 3) bovine viral diarrhea virus (VBVD), which is a surrogate test model of hepatitis C virus (HCV) [7], virus infectious titer after ten passages in MDBK cell culture was 6-7 lg TCD 50 .
To determine the cytotoxic concentrations of LPS preparations, at least ten holes in plastic plots with cell culture for each preparation dilution (from 1.55 to 100.0 μg/ ml) in culture medium were used.Plates with cell culture were incubated at 37 C with supply of 5% CO 2 for 5 days.Test and control cultures were revised every day to detect the presence or absence of cytopathic effect (CPE) on cells.CPE degree was determined by the change in the morphology of cells (cell rounding and shrinkage, degenerated cells abruption from holes surface) by 4-plus system from "1+" to "4+".The maximum non-toxic concentration (MNTC) of LPS preparation that does not cause degeneration of cells was determined.
For anti-flu LPS preparations activity determination in vitro, daily inoculated MDCK cell culture with continuous monolayer was used.Cells were grown in plates on the medium RPMI-1640 with 10% of fetal serum (Nunclon, Surface, Denmark) at 37 C in thermostat with CO 2 supply.To increase the sensitivity of cells to infection with influenza virus the treatment with trypsin was carried out.Trypsin mother solution (Trypsin, TPCK) was prepared by adding of 10 ml of nutrient medium DMEM (Sigma) to 20 mg of enzyme.Cells were washed three times with this solution in a final concentration of 2 μl/ml by 50 μl per well.Growth medium was poured out, investigated LPS preparations in different concentrations were added to the cells and flu virus at a dose of 100 TCD 50 was introduced.Cultures were incubated in thermostat with CO 2 supply for 3 days, daily monitoring using a microscope.After 48-72 hour of incubation, the culture fluid was collected and the influenza virus infectious titer was determined in cell culture by titration.
To the study of LPS preparations antiherpetic activity, the herpes simplex virus type 2 (HSV-2) strain VN, infectious titer of 7.0 lg TCD 50 , was used.CPE of HSV in cells morphologically manifested in symplasts or rounded cells formation in combination with the proliferation and giant multi-cells appearance.The study was performed on daily cell cultures Vero with their continuous monolayer.Cells were grown in plates on the medium RPMI-1640 with 10% of fetal serum (Nunclon, Surface, Denmark) at 37 C in thermostat with CO 2 supply.Growth medium was poured out; investigated LPS preparation in different concentrations was added on the monolayer.After 1 hour of contact, herpes virus was added at a dose of 100 TCD 50 .Cultures were incubated in thermostat with CO 2 supply for 5 days, daily monitoring with a microscope and noting the virus reproduction by HSV cytopathic action on Vero cells compared with control cultures where the monolayer is not subjected to any influence.
As a surrogate hepatitis C virus (HCV) the bovine viral diarrhea virus (VBVD) was used.Antiviral activity was studied in culture MDBK, in which various concentrations of LPS and VDVB at a dose of 100 TCD 50 were added.Cultures were incubated in thermostat to specific CPЕ in control of the virus, and then the infectious titer in the culture medium was determined.
In all experiments, the virus infectious titer reduction under the action of preparation at 1.0 lg TCD 50 (ED 90 ) or 2.0 lg TCD 50 (ED 99 ) compared with the virus control was considered the indicator of inhibitory action.
Transfection was performed using a standard protocol with transfection reagent Turbofect (Thermo Scientific).The density of cells on the day of transformation was 510 4 (for the inoculated cells) and 510 5 (for suspension cells) in 1 ml of nutrient media.To obtain HCV-producing cell cultures, HCV RNA was isolated from patients with hepatitis C. Further cDNA was obtained on the matrix of HCV RNA via reverse transcription. 1 μg of cDNA was diluted in 100 μl of serum-free medium RPMI-1640, shaking the solution, and 2 ml of transfection reagent (Turbofect) were added.After pipetting or shaking in Vortex, specimens were incubated for 15-20 min at room temperature.Then 100 ml of transfection reagent containing cDNA were added dropwise to each well with cell culture.Plates were incubated at 37 C in thermostat with CO 2 .Cultures of transfected HCV cDNA were incubated at 36.6 C in thermostat with 5% of CO 2 supply.Virus testing was performed by PCR at the second passage.All transfected cultures produced hepatitis C virus as on the 7 th and on the 9 th day of culture Jurkat-cDNA cultivation.
All experiments were performed at least in 3-5 replicates.Statistical analysis of experimental series results was performed by standard methods using Student t-test for the 5% significance level.

Results and Discussion
Lipopolysaccharides of investigated strains of Pseudomonas chlororaphis subsp.aureofaciens UCM B-306 and UCM B-111, which are components of gaupsin biopreparation, were isolated and described earlier [8].Based on fatty acids and monosaccharides composition, including the presence of heptose, 2-keto-3-desoxyoctanoic acid and 3-hydroxy fatty acid, it is demonstrated that LPS of P. chlororaphis for the first time isolated by us contain all characteristic for these biopolymers components.
The influence of P. chlororaphis subsp.aureofaciens UCM B-306 and UCM B-111 LPS was studied on three types of viruses: influenza, herpes type 2 and bovine diarrhea viruses, which was used as hepatitis C virus model.They belong to different families: Orthomyhoviridae, Herpesviridae and Flaviviridae, respectively.These viruses were selected for studies concerning LPS influence on them, as they cause a series of life-threatening human diseases: influenza H1N1, which, since 1947, occasionally causes a flu epidemics; herpes simplex type 2, which infects the body of almost 90% of our planet population and capable of longterm persistence in the body, causing chronic and latent forms of infection with periodic exacerbations.Some herpes viruses are able to spread transplacentally and cause intrauterine and neonatal pathology; and hepatitis C virus, which causes dangerous liver disease capable in some cases to be complicated by cirrhosis and carcinoma.Therefore, finding of preparations able to retard the development of diseases caused by viruses described above represents the theoretical and practical interest.
To determine the cytotoxicity of investigated preparations and their effects on viruses reproduction, LPS were tested at concentrations ranging from 1.55 to 100.0 μg/ml.
The results of experiments to determine the MNTC show that both LPS on three types of cultures: MDCK, Vero and MDVK were nontoxic and did not cause degeneration of cells even at a concentration of 100.0 μg/ml.LPS minimal concentration that can inhibit the reproduction of the virus by 1.0-2.0lg TCD 50 , was equal to influenza, herpes and bovine diarrhea viruses (Fig. 1-3).The reduction of viruses' infectious titer at least by 2.0 lg TCD 50 influenced by LPS was observed even at a concentration of 1.55 μg/ml for both LPS.In fact, LPS preparations inhibited influenza virus reproduction by 5.0-6.0 lg TСD 50 , VBVD (surrogate HCV)by 4.0-5.0lg TСD 50 , herpes virus type 2 -by 3 lg TСD 50 .
According to the MNTC/ED 99 ratio, indices of selectivity of P. chlororaphis subsp.aureofaciens UCM B-306 and UCM B-111 LPS preparations against the influenza virus A/FM/1/47 (H1N1), herpes simplex type 2 and bovine diarrhea accounted for at least 64.Therefore, investigated LPS are effective inhibitors of investigated viruses reproduction.
The purpose of the following studies was to perform anti-virus LPS screening on the model of cell culture Jurkat producing human hepatitis C virus.As a result of suspension cultures Jurkat transfection with cDNA of HCV from a patient using Turbofect we received producing cell cultures transfected with cDNA of HCV that provide stable production of HCV with viral loading of 2.9•10 3 , 1.6•10 5 , 1.1•10 5 , 7.6•10 4 , 1.0•10 5 genome/equivalents.
In the model of transfected human hepatitis C virus with viral loading of 2.9•10 3 genome/ equivalents the effect of LPS was determined.Into producing culture Jurkat HCV the LPS preparations in dilutions of 1:1000 were added.After 5 days in each sample the HCV RNA loading was determined.The study shows that the introduction of LPS 1 and 2 fully inhibits the HCV production, i.e. in producing cultures Jurkat HCV the viral loading was not found, while in the control cultures Jurkat HCV the viral loading was 3.83•10 2 genome/equivalents.Thus, it is revealed that LPS of P. chlororaphis subsp.aureofaciens UCM B-306 (LPS 1) and UCM B-111 (LPS 2) are highly active antiviral agents.They inhibit the reproduction of influenza viruses A/FM/1/47 (H1N1), herpes simplex type 2, and bovine viral diarrhea that is surrogate hepatitis C virus.Although the mechanism of LPS antiviral activity until today is not deciphered, we can assume that there are two such mechanisms.Since the majority of LPS biological activity types is due to lipid A, the most conservative part of the molecule, as evidenced by almost the same fatty acid composition [8], we can assume that lipid A is responsible for the antiviral effect that in P. chlororaphis subsp.aureofaciens UCM B-306 and UCM B-111 was similar.We cannot exclude the impact of the O-specific polysaccharide (OPS), the most variable part of LPS molecule.Although earlier [9] we found that there are significant differences in the OPS structure of two studied strains of P. chlororaphis subsp.aureofaciens: one OPS represented by linear trisaccharide while another by branched tetrasaccharide, they have one common characteristic: contain the residues of 2-amino-2-deoxy-galacturonic acid (GalNAcA).This fact is very important because it is known that receptors of some viruses are galactose-specific.