key: cord-0854290-ty1w9x0c
authors: Sánchez-León, E.; Bello-Morales, R.; López-Guerrero, J.A.; Poveda, A.; Jiménez-Barbero, J.; Gironès, N.; Abrusci, C.
title: ISOLATION AND CHARACTERIZATION OF AN EXOPOLYMER PRODUCED BY BACILLUS LICHENIFORMIS: IN VITRO ANTIVIRAL ACTIVITY AGAINST ENVELOPED VIRUSES
date: 2020-07-08
journal: Carbohydr Polym
DOI: 10.1016/j.carbpol.2020.116737
sha: b9ff2c5643a691578f7f711810230fc4e10351be
doc_id: 854290
cord_uid: ty1w9x0c

The exopolymer (EPSp) produced by the strain B. licheniformis IDN-EC was isolated and characterized using different techniques (MALDI-TOF, NMR, ATR-FTIR, TGA, DSC, SEM). The results showed that the low molecular weight EPSp contained a long polyglutamic acid and an extracellular teichoic acid polysaccharide. The latter was composed of poly(glycerol phosphate) and was substituted at the 2-position of the glycerol residues with a αGal and αGlcNH(2). The αGal O-6 position was also found to be substituted by a phosphate group. The antiviral capability of this EPSp was also tested on both enveloped (herpesviruses HSV, PRV and vesicular stomatitis VSV) and non-enveloped (MVM) viruses. The EPSp was efficient at inhibiting viral entry for the herpesviruses and VSV but was not effective against non-enveloped viruses. The in vivo assay of the EPSp in mice showed no signs of toxicity which could allow for its application in the healthcare sector.

The rapid appearance of microorganisms that can cause zoonotic diseases can cause severe health problems as, due to their sudden emergence, there typically are no vaccines available to counteract them. In the last two decades, two novel zoonotic viruses have emerged causing fatal epidemics in humans: the severe acute respiratory syndrome coronavirus (SARS-CoV), and the Middle East (MERS-CoV), which appeared in 2002 and 2012 respectively. Most recently, the SARS-CoV-2 (Gorbalenya et al., 2020) has been the causal agent for the coronavirus pandemic J o u r n a l P r e -p r o o f which has posed a serious threat to global health and economy. In the cases where the mechanisms of action of the pathogen are not well known, these can lead to the collapse of whole countries' health systems. It is therefore necessary to research and implement antiviral compounds that have effects on a broader spectrum in order to prevent and combat possible pandemics. This type of antiviral treatments can effectively and rapidly reduce infection rates (Harrison, 2020) These antiviral compounds can be obtained through biotechnological processes undertaken by microorganisms that can adapt to different environments. This is the case of bacteria that can produce and secrete extracellular polymeric substances (EPS) with a highly heterogeneous composition (More, Yadav, Yan, Tyagi, & Surampalli, 2014) . These compounds play a role in the protection against desiccation, predation by protozoans and viruses and in the survival in nutrient-starved environments (Panosyan, Di Donato, Poli, & Nicolaus, 2018) . These polymers'

attributes have led to their use in a wide range of applications in different industrial sectors (Ates, 2015; Donot, Fontana, Baccou, & Schorr-Galindo, 2012) .Their antimicrobial properties have been the focus of past research (Yu, Shen, Song, & Xie, 2018) and, in particular, several studies have reported the antiviral effect on several viruses. These include herpes simplex type 1 (HSV-1) (Gugliandolo et al., 2015; Marino-Merlo et al., 2017) , herpes simplex type 2 (HSV-2) (Arena et al., 2006) , encephalomyocarditis virus (EMCV) (Yim et al., 2004) , influenza virus (Zheng, Chen, Cheng, Wang, & Chu, 2006) , infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) (Nácher-Vázquez et al., 2015) .

The lack of efficient drugs to treat fast emerging pandemics makes it imperative to speed up the research for antiviral agents that are effective against future viral threats. This study reports the finding of a novel EPSp composed of Poly-γ-glutamic acid and an extracellular teichoic acid polysaccharide (Birch, Van Calsteren, Pérez, & Svensson, 2019) . This EPSp was isolated and characterized from B. licheniformis and it was found to exhibit a drastic inhibitory activity in cell cultures against multiple human and animal viruses. The EPSp was applied to four enveloped viruses (HSV-1 and HSV-2 which infect humans and PRV and VSV which infects animals); and a non-enveloped virus (MVM) that infects animals. In particular, the EPS was most effective J o u r n a l P r e -p r o o f when used against enveloped viruses as it significantly reduced the viral yield. This EPSp has been proven to be non-toxic in mice and, given its potent antiviral capability in vitro, it is proposed as a good candidate for further studies in cell cultures with other enveloped viruses and potentially in animal models, in order to establish its efficacy in vivo against enveloped viral infection.

The anti-HSV gD LP2 antibody was sourced from Alexa555, conjugated secondary anti-mouse and anti-rabbit antibodies were sourced from Molecular Probes (Eugene, OR, USA), and Mowiol was obtained from Calbiochem (Merck Chemicals, Germany). The rest of reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The indigenous bacterial strain, Bacillus licheniformis IDN-EC, had been isolated from films based on Poly(Butylene Adipate-co-Terephthalate) and its blend with Poly (Lactic Acid) (Morro, Catalina, Sanchez-León, & Abrusci, 2019).

Bacillus licheniformis IDN-EC was inoculated from the stock culture in trypticase soya agar medium (TSA) and incubated at 45 ºC for 24 h. After that, the strains were transferred into flasks of 100 ml filled with 20 ml of minimal growth medium (MGM), prepared as described by Abrusci et al (Abrusci et al., 2011) .: g/L: K2HPO4 0.5, KH2PO4 0.04, NaCl 0.1, CaCl2 2H2O 0.002, (NH4) 2SO4 0.2, MgSO4 7H2O 0.02, FeSO4 0.001, and glucose as a carbon source at a concentration of 10 g/L, pH adjusted to 7.0. The flasks were incubated in a rotary shaker incubator (Biogen) at 45ºC and 110 rpm for 24 h. After the first incubation, 10 ml of this broth (2.5 × 10 7 cells/ml concentration) was inoculated into flasks of 1000 ml filled with 100ml of MGM with glucose supplementation. The flasks were incubated at 45ºC and 110 rpm for 48 h, when the stationary phase was reached. Three independent assays were performed. J o u r n a l P r e -p r o o f

The biodegrading bacteria were evaluated by indirect impedance measurements. The aerobic biodegradation of glucose compound by B. licheniformis was performed at 45°C. The bioassays were carried out in bioreactors of 7 ml, filled with 1.5 mL of bacterial suspension prepared as described above. These containers were introduced into disposable cylindrical cells of 20 mL filled with 1.5 mL of 2 g/L KOH aqueous solution and provided by four stainless steel electrodes to measure impedance on a Bac-Trac 4300 apparatus (SY-LAB Geräte GmbH, Neupurkerdorf, Austria). The method has a typical error in the measurements of 1-2%. The experimental device and procedure have been previously described in the literature (San Miguel, Peinado, Catalina, & Abrusci, 2009 ). The device monitors the relative change (each 20 min) in the initial impedance value of KOH solution, which is converted in concentration of carbon dioxide by a calibration curve of impedance variation versus concentration of CO2. The percentage of biodegradation of glucose was calculated as a percentage of the ratio between the cumulative amount of CO2 produced in the biodegradation at time, t, and the theoretical amount of carbon dioxide assuming that all the carbon of the glucose structures introduced in the bioreactor are transformed into CO2. The cell growth number was evaluated by different dilution plating incubated at 45ºC for 48 h with TSA agar medium. A Thermo Orion pH Meter (model, 2 Star) was used to determine the pH values during a fermentation period of 48 h.

The cultures obtained from the strain B. licheniformis IDN-EC were centrifuged at 13,154 × g for 30 min at 4ºC (Duppont -RC5). The EPS was precipitated with cold ethanol (three times the volume) and left overnight. The precipitate was collected by centrifugation at 13,154 × g for 30 min at 4ºC and dissolved in Milli-Q water. Then the crude EPS was dialyzed at 4ºC with Milli-Q water for 48 h. The dialyzed contents were then freeze dried by lyophilization for 48 h and the dry weight of the powdered EPS was determined.

The further purification of crude EPS (10 mL, 10 mg/mL) was subjected to a DEAE-52 anion exchange column (2.6×30 cm) and eluted with deionized water, 0.05 and 0.3 M NaCl at 1 mL/min flow rate.

MALDI-TOF mass spectra were recorded on an Ultraflex III TOF/TOF mass spectrometer (BrukerDaltonics) equipped with a Nd:YAG laser (355 nm). Mass spectra were recorded in positive reflector (range 1-10 KDa) and lineal (range 1-20 KDa) modes, using a matrix of 10mg/mL 2,5-dihydroxibenzoic acid (DHB) in methanol/water (90/10).

To determine the monosaccharide composition, the EPSp was hydrolyzed with trifluoroacetic acid (TFA) 0,5M at 120°C for 2 h. The samples were treated before and after the process with N2.

The monosaccharide content of EPSp was analyzed by HPLC using a 920LC Varian apparatus equipped with a PL-EDS 2100 Ice detector. A sugar SP0810 (Shodex) column as a stationary phase was used with an isocratic mobile phase of water as a solvent and a flow rate of 0,5mL/min. The column temperature was maintained at 30°C. The samples injection volume was 50 μL. The monosaccharide such as glucose, arabinose, rhamnose, xylose, mannose, galactose, fructose and sorbose were used as standards. The concentration of glucuronic acid was determined by HPLC/MSMS using an Agilent Technologies 1100 series -6410B (TQ). An ACE Excel 3 C18-Amide column as a stationary phase was used with a mobile phase of 0, 1% formic acid in water.

Flow rate of 0,2ml/min. The temperature for analysis was set at 40 °C.

For NMR sample preparation, ca 4 mg of the EPSp sample were dissolved in 0.5 ml of deuterated water D2O. NMR spectra were acquired using either a Bruker AVIII-600 spectrometer equipped with a 5mm PATXI 1H/D-13C/15N XYZ-GRD probe (for 1 H and 13 C experiments) or a 5 mm QXI 1H XYZ-GRD probe (for 1 H and 31 P experiments), or in a Bruker AVIII-800 equipped with pulse sequence from the Bruker library. An exponential gradient list of 24 values was created by using the standard AU program dosy. Experiments were acquired using 8 scans, δ/2 of 1.8 ms, diffusion time ∆ of 500 ms, and eddy current delay value of 5 ms.

Attenuated Total Reflectance/FT-Infrared Spectroscopy (ATR/FTIR).

The structural-functional groups of the EPSp were detected using Attenuated Total

Reflectance/FT-Infrared Spectroscopy (ATR-FTIR). IR spectra were obtained using a Perkin

Elmer BX-FTIR spectrometer coupled with an ATR accessory, MIRacleTM-ATR from PIKE

Technologies and interferograms were obtained from 32 scans at a 4 cm -1 with a resolution from 400 to 4000 cm -1 .

Thermogravimetric analysis (TGA) of the polymer was done using a TGA Q-500 (Perkin-Elmer).

The heating rate for the dynamic conditions was 10°C/min, and the nitrogen flow was maintained constant at 60 mL/min.

Scanning electronic microscopy micrographs were obtained using a Philips XL 30 scanning electron microscope operating in conventional high-vacuum mode at an accelerating voltage of 25 kV. Previously, EPSp was coated with a 3 nm thick gold/palladium layer.

Vero, HOG, MeWo and Hela cell lines were propagated in DMEM supplemented with 10% FBS, penicillin (50 U/mL) and streptomycin (50 μg/mL) at 37°C in an atmosphere of 5% CO2, (Bello-Morales et al., 2012). The Jurkat cell line was cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mMHepes, and 100 mg/mL each of penicillin and streptomycin as described (Alonso, Mazzeo, Mérida, & Izquierdo, 2007) .

In this study, HSV-1 K26GFP (Desai & Person, 1998) (Montgomery, Warner, Lum, & Spear, 1996) , was used to monitor the viral entry.

To Viral titer was quantified by an endpoint dilution assay determining the 50% tissue culture infective dose (TCID50) in Vero cells. Each experiment was conducted thrice.

To investigate dose dependent viral infections, the recombinant HSV-1 (KOS) gL86 was used which expresses beta-galactosidase upon entry into cells (Yakoub et al., 2014) . Vero cells plated in 96-well tissue culture dishes were infected at a m.o.i. of 10 with HSV-1 gL86 treated or mocktreated with two-fold serial dilutions of EPSp at concentrations of 6.25, 12.5, 25 and 50 µg/ml. This was prepared following the method described in Section 2.12 and adjusted to obtain the desired concentration. After 6 h p.i., the beta-galactosidase activity was analyzed at 410 nm in a microplate reader.

The effect of the EPSp on different HSV-1 infected cell lines was also analyzed. Several adherent and non-adherent cell lines were chosen: human HOG, Hela, Jurkat and Mewo cells. These were prepared and evaluated following the methodology described in the Section 2.12.

The effect of the EPSp on different viruses was investigated in Section 2.11. The cells were prepared as described in Section 2.12 except for PRV which had an increased base dosage of 10 µg/ml. For dosage comparison purposes, HSV-2 and PRV infected cells were treated with an J o u r n a l P r e -p r o o f additional 2-fold dose. For MVM and VSV, there were additional 5-fold and 4-fold dosages respectively.

Cells grown on glass coverslips were fixed in 4% paraformaldehyde for 20 min and rinsed with PBS. Then cells were permeabilized with 0.2% Triton X-100, rinsed and incubated for 30 min with 3% bovine serum albumin in PBS with 10% human serum (only for herpes, to block the HSV-1-induced IgG Fc receptors). For double and triple-labeled immunofluorescence analysis, cells were incubated for 1 h at room temperature with the appropriate primary antibodies, rinsed several times and incubated at room temperature for 30 min with the relevant fluorescent secondary antibodies. Herpes antibodies were incubated in the presence of 10% human serum.

Controls to assess labeling specificity included incubations with control primary antibodies or omission of the primary antibodies. After thorough washing, coverslips were mounted in Mowiol.

Images were obtained using an LSM510 META system (Carl Zeiss) coupled to an inverted Axiovert 200 microscope. Processing of confocal images was made by FIJI-ImageJ software.

To perform FACS analysis, cells were dissociated by incubation for 1 minute in 0.05% trypsin/0.1% EDTA (Invitrogen) at room temperature and washed and fixed in 4% paraformaldehyde for 15 minutes. Then, cells were rinsed and resuspended in PBS. Cells were analyzed using a FACSCalibur Flow Cytometer (BD Biosciences).

To dose of EPSp, mice were allowed free access to food and water and monitored daily for morbidity, mortality and behavioral changes. At day 14, mice were sacrificed by CO2 and exsanguinated by cardiac puncture to obtain whole blood, in order to analyze their blood profiles and counts. Several parameters were analyzed to monitor renal, hepatic and immunologic basic profiles. For the biochemical analyses, urea, total protein, alanine aminotransferase (ALT) and bilirubin levels were studied. For hematologic analysis, the percentage of lymphocytes and segmented neutrophils was measured, as well as the WBCs count. The body weight gain from the day 0 (inoculation) to the day 14 (sacrifice) was also quantified, to exclude a weight loss or failure to gain weight that would be indicative of toxicity.

Student's t-test was used to determine differences between groups. All data are represented as mean ± standard deviation.

This study was carried out in strict accordance with the European Commission legislation for the protection of animals used for scientific purposes (directives 86/609/EEC and 2010/63/EU). Mice were maintained under specific pathogen-free conditions at the CBMSO (CSIC-UAM) animal facility. The protocol for the treatment of the animals was accepted by the "Comité de Ética de la Investigación" of the Universidad Autónoma of Madrid, Spain and approved by the "Consejería General del Medio Ambiente y Ordenación del Territorio de la Comunidad de Madrid" (PROEX 148/15). Animals had unlimited access to food and water, and at the conclusion of the studies they were euthanized in a CO2 chamber, with every effort made to minimize their suffering, followed by exsanguination by cardiac puncture to obtain whole blood.

J o u r n a l P r e -p r o o f 3. Results

The growth of B. licheniformis IDN-EC, medium biodegradation, pH values and exopolymer production (EPS) at 45ºC, are shown in Fig. 1a . Cellular growth peaked (9.35 log cfu / ml) after 30 h. In this moment, the strain completely biodegraded the glucose as a carbon source. The maximum production of EPS, 60 mg/ L, occurred after 42 h. During the process no acute pH descent was detected, as the acidification of the medium was very low (from pH 7 to just 6.6).

The results of the obtained fraction from the purified exopolymer was named as EPSp. This was found to be water soluble and colorless. The mass spectrum of the polymer indicated that it had an approximate molecular weight of 5kD (Fig. 1b) . HLPC analysis of EPSp, showed that this was formed by α -D-galactose and α -D-glucosamine with a molar ratio of 3:1. It had lack of glucuronic acid. This protocol allowed identifying the two constituent sugar residues. The signals for the major component showed a typical Gal pattern: The 1D-selective TOCSY experiments from the anomeric proton demonstrated the complete H1-H2-H3-H4 spin system, which is stopped at H4

due to the small H4-H5 coupling. This behavior is typical for Gal moieties. Moreover, the 13 C chemical shift for C-6 indicated that this OH position was substituted. As observed in the 1 H-31 P HMBC and HSQCMBC-TOCSY experiments, phosphorylation of Gal sugar moiety at position 6 was confirmed due to the TOCSY correlation of this signal with Gal-H5 position.

The minor component showed a drastically different coupling pattern in the 1D-selective TOCSY, with typical glucose-type couplings: large vicinal 1 H-1 H J values. In this case, the position C2 in the HSQC showed the typical chemical shift of a carbon attached to a nitrogen atom, instead of oxygen. Since no methyl signals for a putative acetyl group were evidenced in the regular 1 H or in the HSQC or in the HMBC spectra, the minor component was identified as an αGlucosamine residue (αGlcN). Indeed, in the HSQC spectra small signals that could belong to methyl groups J o u r n a l P r e -p r o o f were identified. However, they only displayed 15% of the intensity of that expected for a methyl acetate with respect to those belonging to the H1C1 or H2C2 signals of the minor component in the same spectrum. Given that the intensities of the methyl groups are usually magnified thanks to its fast motion features, the presence of N-acetylation should be basically negligible.

In addition to these monosaccharide moieties, NMR signals for glycerol esters were identified with different substitution patterns. From HSQC-edited and HSQC-TOCSY experiments, three CH and four CH2 glycerol signals were identified. The substitution was deduced from the analysis of the 13 C chemical shifts, which allowed differentiating one unsubstituted signal for each group. With this information it is proposed that the composition of the major products of the sample is the following.

-A long polyglutamic acid with the largest molecular weight. The FT-IR spectroscopic analysis was applied to disclose the polar bonds and the different atom vibrations of the molecules. The results showed (Fig. 3a) the presence of the characteristic bands for poly glutamic acid (γ-PGA) (Mohanraj et al., 2019) . The IR spectra of the EPSp of B.

licheniformis IDN-EC exhibited a broad peak at around 3270 cm -1 (range 3600-3200 cm -1 ) for O-H stretching vibration and the peak at 2924 cm -1 was associated to an amine group (Prado-

Fernández, Rodríguez-Vázquez, Tojo, & Andrade, 2003) . The peak at 1582 cm -1 was associated to the deformation vibration of an amide group (Sardari et al., 2017) . The band at 1399 cm -1 was assigned to the group C=O whereas the peak at 1052 cm -1 was associated to the C-N group. On the other hand, polyphosphate groups were assigned to the presence of characteristic bands at 1219cm -1 , (range 1200-900)cm -1 ), (Grunert et al., 2018) (Fig. 3c) . A three-dimensional structure was observed with structural units in the form of thin scales of different sizes intertwined with fine fibers, resulting in a natural scaffold structure.

J o u r n a l P r e -p r o o f c) Scanning electron micrographs

The antiviral activity of the exopolymer was assessed using the procedures described in Section 2.12. The effects of the EPSp on the HSV-1 K2GFP infection of Vero cells are shown in Fig. 4 .

The immunofluorescence assays showed an almost complete disappearance of the GFP signal in cells infected with EPSp-treated virus at 24 h p.i. with a 5µg/ml dose (Fig. 4a) . To quantify the effect of exopolymer on viral yield, progeny virus was titrated to determine the TCID50/ml. After 24 h p.i., viral yield in Vero cells infected with EPSp-treated virus decreased around 5 orders of magnitude compared to cells infected with mock-treated virus, to become practically undetectable (Fig. 4b) .

To investigate whether the decrease in viral yield was due to a decrease in viral entry, Vero cells were infected with the recombinant HSV-1 (KOS) gL86 and treated or mock-treated with twofold serial dilutions of EPSp as described in Materials and Methods (Viral entry section). After 6 h p.i., the beta-galactosidase activity finding a significant (p<0.05) dose dependent decrease of absorbance in cells treated with EPSp ( Fig. 4c) , compared to the control mock-treated cells. 

Experiments performed with the rest of cell lines, HOG (Fig. 5a) , Mewo (Fig. 5b) , Hela (Fig. 5c) and Jurkat (Fig. 5d) , showed similar results: immunofluorescence assays showed a drastic decrease of GFP signal in cells infected with EPSp-treated virus at 24 h p.i. and, in addition, viral progeny decreased around 4-5 orders of magnitude (depending on the infectivity of the cell lines)

in cells infected with EPSp-treated virus compared to the control.

J o u r n a l P r e -p r o o f (Fig. 6b) . Thus, infection with PRV-GFP XGF-N treated with EPSp at 10 µg/ml did not caused any observable effect compared to mock-treated control and, to inhibit infection, a dose of EPSp at 20 µg/ml was needed (Fig. 6b) . Similarly, quantification of viral production with HSV-2and PRV-GFP XGF-N, both treated with same doses EPSp (20 µg/ml) showed a decrease of about 4 and 2 orders of magnitude respectively (Figs. 6c and 6d).

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Twenty male Balb/c mice were mock-inoculated or inoculated with different single doses of the EPSp Section 2.11. Several parameters were analyzed. For the biochemical analysis, levels of urea (Fig. 9a) , total protein (Fig. 9b) , alanine aminotransferase (ALT) (Fig. 9c) and bilirubin (Fig.   9d) were measured. For hematologic analysis, the percentage of lymphocytes (Fig. 9e) , the white blood cells (WBCs) count (Fig. 9f ) and segmented neutrophils (Fig. 9g) were evaluated. For 14 days there were no significant changes in any of the parameters between the control and experimental groups. No toxic signs were observed such as hypothermia, weakness, diarrhea or ataxia. There were also no signs of acute pain, distress or weight loss.

J o u r n a l P r e -p r o o f 

The aim of this work was to isolate and characterize a natural exopolymer produced by B.

licheniformis IDN-EC strain and to evaluate its role as a potential antiviral agent.

During the polymer production process (Fig. 1a) , essential factors like the carbon and nitrogen sources, aeration, agitation and medium pH were controlled. These factors can have an important effect on the quantity and quality of the polymer (Kongklom, Luo, Shi, & Pechyen, 2015) . The characterization assays conducted on the produced polymer (EPSp) (figs. 2 and 3a) identified two components: Poly-γ-glutamic acid (γ-PGA) and an extracellular teichoic acid (EC-TA).

γ-PGA is an anionic polymer due to the presence of carboxylic groups that can interact electrostatically with positive charges (Pereira et al., 2017) . These have been found to be secreted by different species of the Bacillus genus, such as B. licheniformis and B. subtilis (Bajaj & Singhal, 2011) and have been described to have antiviral properties but this is only for high molecular weights (Lee et al., 2013) .

The teichoic acid polysaccharide was composed of polyphosphate and was substituted at the 2position of glycerol residues with a αGal, αGlcNH2. In addition to this, the αGal O-6 position was substituted by a phosphate group. This polysaccharide can be identified as a teichoic acid due to the presence of the polyphosphate compound. A teichoic acid is a polysaccharide that is only produced by gram-positive bacteria. An unusual trait of this teichoic acid is that it is extracellular (EC-TA) since this type of TA has only been described in a limited number of species (Xiao & Zheng, 2016) (Jabbouri & Sadovskaya, 2010) . As well as being extracellular, this TA was also J o u r n a l P r e -p r o o f found to be atypical due to the nature of its charges (Weidenmaier & Peschel, 2008) . These only contain negatively charged phosphate groups and positive charges are absent, this has only been previously described for Bacillus subtilis. In addition to this, the substitution by phosphate groups in the O-6 position of the αGal would increase the electrostatic properties of the EC-TA. It has been found with other compounds that when the αGal O-6 position is substituted with negative charges such as sulphates, there is an increase in the antiviral capability of these (Ghosh et al., 2009 ). The significant negative overall charge of the two polymer components of the EPSp (the γ-PGA and the EC-TA) would have a synergy effect thus increasing the antiviral effect of the polymer.

The molecular weight of the EPSp, (5kDa) ( fig. 1b) is similar to the pentosan polysulphates (3 kDa), and dextran sulphates (5 kDa) (Mbemba, Chams, Gluckman, Klatzmann, & Gattegno, 1992) . These compounds with a high number of negative charges have previously been described as highly effective in inhibiting the replication of enveloped viruses and ineffective against nonenveloped viruses (Wang, Wang, & Guan, 2012) . As well as a low molecular weight, the morphology of the polymer can ease contact. The natural scaffold of the B. licheniformis IDN-EC EPSp presented a non-uniform morphology ( fig. 3c ). This is especially relevant as a nonuniform surface could promote viral contact, in a similar way to cell adhesion. This would in turn favor the polymer-virus interaction and significantly increase the efficiency of antiviral activity (De Colli et al., 2012) ..

The antiviral capability of this EPSp was tested by infecting Vero cells with HSV-1 K26-GFP and varying the dose and cell type. HSV-1 was pretreated with the EPSp prior to cell infection.

The titration results and the immunofluorescence images confirmed that the EPSp has a virucidal dose-dependent effect in the viral entry as the EPS was more effective with larger doses (Fig. 4 ) (Schnitzler, Schneider, Stintzing, Carle, & Reichling, 2008) . The effectiveness against different cell lines also suggested that the EPS could inhibit the first step of viral infection by preventing entry through both endocytosis and fusion (Figs. 4 and 5).

The antiviral capability of this EPSp was also tested on both enveloped (herpesviruses and VSV) and non-enveloped (MVM) viruses. The obtained differences in inhibition efficiency of the EPSp among the herpesviruses (HSV-1 (5 µg/ml ) >HSV-2 ( 10 µg/ml ) >PRV (20 µg/ml) (figs. 4 and 6) and VSV, (20 µg/ml) ( fig. 7 ) could be due to the different capabilities of the viral glycoproteins to establish unspecific electrostatic bonds. HSV-1 has been found to be most effective in establishing this type of bonds with the cells that it infects (Ho, Jeng, Hu, & Chang, 2000) . The

EPSp did not present any antiviral effects against MVM (Fig. 8 ). This suggests that the antiviral properties might be a universal phenomenon against enveloped viruses which would make the EPSp a potential antiviral treatment against other enveloped viruses such as SARS-CoV-2.

Furthermore, the in vivo assay of the EPSp in mice showed no signs of toxicity (Fig. 9) .

This study demonstrates that the exopolymer produced by B. licheniformis IDN-EC was composed of Poly-γ-glutamic acid and a teichoic acid. The negative charge content increased the electrostatic properties of the EPSp. This meant that the EPSp was highly effective as an antiviral treatment against a group of human and animal enveloped viruses. This further suggests that the novel EPSp could be a good candidate for further studies in cell cultures with other enveloped viruses. The in vivo results also imply that there is a potential application in the pharmaceutical industry as a prophylactic therapeutic biomolecule. Future tests in animal models would establish its efficacy in vivo against enveloped viral infection. 

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Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

Efficacy of an aqueous Pelargonium sidoides extract against herpesvirus

1,3-Poly(glycerol phosphate) chains in the cell wall of Streptomyces rutgersensis var. castelarense VKM Ac-238

Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function

The antiviral activities and mechanisms of marine polysaccharides: An overview

Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions

Overview of microalgal extracellular polymeric substances (EPS) and their applications

Comprehensive Analysis of Herpes Simplex Virus 1 (HSV-1) Entry Mediated by Zebrafish 3-O-Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection

Antiviral effects of sulfated exopolysaccharide from the marine microalga Gyrodinium impudicum strain KG03

Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review

Oral administration of exopolysaccharide from Aphanothece halophytica (Chroococcales) significantly inhibits influenza virus (H1N1)-induced pneumonia in mice

Acknowledgments UAM (P. Ref. 905089). We are grateful to the technical staff of the "Servicio Interdepartamental de Investigación (SIdI) de la UAM". We are grateful to the technical staff of the CBMSO, for their support.