A Novel Eukaryotic Cell Model to Study Antiviral Activity

8/8/2019 A Novel Eukaryotic Cell Model to Study Antiviral Activity

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A novel eukaryotic cell culture model to study antiviral activity of

potential probiotic bacteria

Tanja Boti a, Trine Dan Klingberg b, Hana Weingartl c, Avrelija Cenci a,d,

a University of Maribor, Faculty of Agriculture, Vrbanska c.30, 2000 Maribor, Sloveniab The Royal Veterinary and Agricultural University, Department of Food Science, Rolighedsvej 30, 1958 Frederiksberg C, Denmark

c Canadian Food Inspection Agency, National Centre for Foreign Animal Disease, 1015, Arlington Street, Winnipeg, Manitoba, Canada R3E 3M4d University of Maribor, Medical Faculty, Slomskov trg 15, 2000 Maribor, Slovenia

Received 13 March 2006; received in revised form 8 August 2006; accepted 26 October 2006

Abstract

As shown in many intervention studies, probiotic bacteria can have a beneficial effect on rotavirus and HIV-induced diarrhoea. In spite of that

fact, antiviral effects of probiotic bacteria have not been systematically studied yet. Non-tumorigenic porcine intestinal epithelial cells (IPEC-J2)

and alveolar macrophages (3D4/2) were treated in different experimental designs with probiotic and other lactic bacteria and their metabolic

products. Vesicular stomatitis virus (VSV) was used in the study as a model virus. Cell survival and viral inhibition were determined by antiviral

assay and confirmed by immunofluorescence. Pre-incubation of cell monolayers with probiotic bacteria reduced viral infectivity up to 60%. All

bacteria used prevented VSV binding to the cell monolayers by direct binding of VSV to their surface. Probiotic and other lactic bacteria

prevented viral infection also by establishment of the antiviral state in pre-treated cell monolayers. Probiotic and other lactic bacteria secreted

antiviral substances during their growth, as the infectivity of the virus was diminished by 68% when bacterial supernatants were tested. It was

shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model. Possible mechanisms of antiviral

activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) cross-talk with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect.

2007 Elsevier B.V. All rights reserved.

Keywords: Antiviral activity; Probiotic bacteria; Lactobacillus; Bifidobacteria; VSV; Cell model

1. Introduction

The most common cause of gastroenteritis in people of all ages

is still enteric caliciviruses (SRSVs) and rotaviruses followed by

other enteric viruses like hepatitis A and E virus and human

cytomegalovirus (CMV) (Untermann, 1998; Koopmans et al.,2002; Worm et al., 2002; Emerson and Purcell, 2003; Koopmans

et al., 2003; Lopman et al., 2004). Rotaviruses are a significant

cause of infant morbidity and mortality, particularly in developing

countries (Majamaa et al., 1995). In the host, intestinal epithelia

represent the first barrier against food-borne pathogens, including

viruses, followed by the response of the mucosal immune system.

The gut barrier was for a long time seen primarily as a physical

blockade to pathogen entry until its active role in shaping the

immune response to luminal pathogens was deciphered (Berin

et al., 1999; Acheson and Luccioli, 2004). Concomitantly, recent

experimental and clinical studies demonstrate a dependency on

healthy host

microbe interaction to cope with pathogen chal-lenges, and consequently attribute the gut microbiota as an active

role in barrier function (Isolauri, 2003; Kidd, 2003; Servin, 2004).

Probiotic bacteria, as a part of gut microflora, are reported to

promote the host defense and to modulate immune system (Cross,

2002; Clancy, 2003). Among them the genera Lactobacillus sp.

and Bifidobacterium sp., are shown to stimulate systemic, cell-

mediated immunity (TH1) and are nowadays widely used in

probiotic therapies (Cross, 2002; Clancy, 2003). Theyhave several

potential benefits to the host, including the potential to boost the

antiviral activity (Kaila et al., 1992, 1995; Cross, 2002; Kidd,

2003). The scientific basis of probiotic use has been established

International Journal of Food Microbiology 115 (2007) 227 234

www.elsevier.com/locate/ijfoodmicro

Corresponding author. Department of Microbiology, Biochemistry and

Biotechnology, University of Maribor, Faculty of Agriculture, Vrbanska c. 30,

2000 Maribor, Slovenia. Tel.: +386 2 25 05 800; fax: +386 2 229 60 71.

E-mail address: [email protected] (A. Cenci).

0168-1605/$ - see front matter 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2006.10.044
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only recently, based mostly on clinical studies (Saavedra, 2001).

Benefits of probiotic bacteria have been demonstrated for patients

with rota-virus- and HIV-associated diarrhoea (Cunningham-

Rundles et al., 2000; Rolfe, 2000; Rosenfeldt et al., 2002;

Goossens et al., 2003). The mechanisms by which they fight

infections are suggested to include exclusion of pathogens by

means of competition for attachment and stimulation of host-cellimmune defenses (reviewed by Isolauri, 2003).

An increased interest in potency of probiotic bacteria as viral

inhibitors arose recently in treatment of HIV-associated diseases

and infections (Cunningham-Rundles et al., 2000; Chang et al.,

2003). However, the probioticsvirushost interactions have

never been studied in detail. Moreover, no biological study of

probioticvirushost interactions in cell models exists so far.

Therefore, our study is theinvestigation of thepotentialantiviral

activityof probiotic bacteriain established cell model, consisting of

a pig small-intestinal epithelial cell line (IPEC-J2) (Rhoads et al.,

1994) and a pig alveolar macrophage derived cell line 3D4/2

(Weingartl et al., 2002). Although vesicular stomatitis virus (VSV)is not considered to be a classical intestinal pathogen, it is a well

developed model for studies as it has no tissue restriction.

2. Methods

2.1. Cells

The IPEC-J2 cell line (Intestinal Pig Epithelial Cell Jejenum)

was a generous gift from Prof. Anthony Blikslager (North

Carolina State University, USA). The 3D4/2 pig alveolar

macrophage derived cell line was obtained as described

previously (Weingartl et al., 2002).

Cells were grown in Dulbecco's modified Eagle's medium(DMEM) (Sigma-Aldrich, Grand Island, USA), supplemented

with 10% foetal calf serum (Cambrex, Verviers, Belgium), L-

glutamine (2 mmol/l, Sigma, St. Louis, USA), penicillin (100 U/

ml, Sigma, St. Louis, USA) and streptomycin (1 mg/ml, Fluka,

Buchs, Switzerland) at 37 C in 5% CO2 atmosphere in tissue

culture flasks until confluency. Cell culture medium was

regularly changed. To perform biological assays, the cells

were separately seeded in 96 well plates at concentration 1106

viable cells ml 1 (6105 cells/well), as determined by 0.4%

trypan blue (Sigma-Aldrich, St. Louis, USA) viability staining,

and incubated for 24 h at 37 C in atmosphere of 5% CO 2 to

reach the monolayer. Just before use, the monolayers werewashed twice with 100 l DMEM without supplements.

2.2. Bacterial strains and growth conditions

Experiments were carried out by the use of two Bifidobacter-

ium spp.:B. breve DSM 20091 (Bb) andB. longum Q 46 (Bl)and

five Lactobacillus spp.: L. paracasei A14 (Lpa), L. paracasei

paracasei F19 (Lpp), L. paracasei/rhamnosus Q 85 (Lpr),

L. plantarum M1.1 (Lpl) and L. reuteri DSM 12246 (Lr), that

showed good adhesive properties to intestinal epithelial cells

(unpublished results) since this property is one of the main criteria

that bacteria should have to be called probiotic. All bacteria were

from the culture collection of the Dept. of Food Science, KVL,

Denmark. They were maintained at 40 C in 20% (v/v) glycerol

(Merck, Damstadt, Germany). Prior to experiment performance,

bacteria were propagated in 15 ml MRS broth (Oxoid,

Basingstoke, England) for 24 h at 37 C, under anaerobic

conditions in Anaerogen system (Oxoid, Basingstoke, England).

The number of viable bacteria in 1 ml of bacterial culture was

determined by measuring optical density at 620 nm. Thenumber was determined by extrapolation from internal

laboratory standard curve, obtained by optical density measure-

ments of bacterial cultures at same wavelength versus Colony

Forming Units (CFU) of bacteria per ml determined by plating

bacteria on De Man, Ragosa, Sharp agar (MRS agar, Fluka,

Buchs, Switzerland). Bacterial cultures were then centrifuged at

2400 rpm (Centric 3000R, Tehtnica, Slovenia) for 10 min and

bacteria were washed twice to remove excess MRS and re-

suspended in DMEM without supplements. The final bacterial

suspension contained 1 108 bacterial cells ml 1, as determined

from the standard curve.

2.3. Virus

Vesicular stomatitis virus (VSV) Indiana strain was used in

the experiments. Virus passages were performed in IPEC-J2

monolayers. Supernatant containing the virus was collected from

the flasks when cytopathic effect (CPE; cell destruction) was

observed (2448 h at 37 C) by light microscopy and clarified by

centrifugation at 2400 rpm. Virus was storedat 70 C until used.

For the antiviral assay, virus with 1.5 tissue culture infective dose

50% units per ml (TCID50 ml-1) was used (100 l/well).

2.4. TCID50 determination

TCID50 represents the dose that gives rise to cytopathic

effect (CPE or in our case cell lysis) in 50% of inoculated

cultures. The endpoint is taken as the highest dilution of the

virus, which produces cell lyses in 50% of the infected cells.

Calculation of the TCID50 was done by the ReedMuench

method usually used for calculation of LD50 (Krah, 1991).

2.5. Antiviral assay

Different experimental procedures to assay potential antiviral

activity of viable probiotic bacteria were used.

2.5.1. Pre-treatment of cells with bacteria

Washed monolayers of IPEC-J2 or 3D4/2 cells where first

incubated with viable probiotic bacteria (100 l, 108 CFU ml 1)

for 90 min or 24 h at 37 C in the atmosphere of 5% CO2. After

incubation the non-bound bacteria were washed off with

DMEM without supplements and monolayers where challenged

immediately with VSV (100 l of 1.5 TCID50 ml-1) or after 24 h

of further incubation with bacteria. In each variant, the results

were collected 24 h and 48 h after the virus infection.

2.5.2. Co-incubation of bacteria and VSV (competition assay)

Each strain of viable probiotic bacteria (100 l, 108 CFU ml 1)

and VSV (100 l of 1.5 TCID50 ml 1) were simultaneously

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added to pre-washed monolayers of IPEC-J2 or 3D4/2 cells in

P96 plate. Plates were then further incubated at 37 C, 5% CO2 for

24 h or 48 h.

A dose dependence between the titer of the virus, number of

bacteria and antiviral effect was assessed by 1) preparing 10-

fold dilutions of VSV in DMEM without supplements, starting

from 7.3 TCID50 ml 1 while bacterial number was unchanged(108 CFU ml 1) and 2) preparing 10-fold dilutions of each of

the probiotic bacteria in DMEM without supplements, starting

from 108 CFU ml 1 while the VSV titer remained unchanged

(7.3 TCID50 ml 1).

2.5.3. Virus adsorption to the bacteria

Viable probiotic bacteria (108 CFU ml 1) were first co-

incubated with VSV (1.5 TCID50 ml 1) in DMEM for 60 min at

37 C and 5% CO2. Bacteria were removed by centrifugation at

12,000 rpm (Micro242, Tehtnica, Slovenia) for 3 min and the

pellet was prepared for immunofluorescence. Residual viral

infectivity in supernatants was assayed on cell monolayers after

24 h incubation for comparing the TCID50 to the inoculum titer.

VSV alone was treated in the same way as the control.

2.5.4. Antiviral effect of bacterial supernatants

Bacterial culture supernatants were obtained from growingbacterial cultures in MRS broth under anaerobic conditions for

16 h at 37 C. Bacteria were removed by centrifugation at

2400 rpm for 10 min. Supernatants were collected and pH value

was adjusted to 7.0 with the addition of NaOH (1 mol/l). Serial

two-fold dilution of supernatants in DMEM without supple-

ments were added on the monolayer of IPEC-J2 or 3D4/2 as

incubation medium (100 l per well) followed by immediate

VSV challenge. CPE was determined after additional 24 h

incubation at 37 C in an atmosphere of 5% CO2. Pure MRS

broth (pH 7.0) was used as a negative control in the assay.

Fig. 1. Antiviral activity of viable probiotic bacteria on VSV infected IPEC-J2 (A, C, E) and 3D4/2 (B, D) cells. Antiviral effect was observed when probiotic strains

were applied onto cell monolayers for 90 min. After removal of non-bound bacteria, cells were further incubated for 0 (A, B) or 24 h (C, D) before VSV infection.

Results were collected after 24 hfor bothcell lines (A, B, C, D) or after 48 h for IPEC-J2 only (E), post-infection. Results are expressed as percentage (means standarddeviation) of cell survival, as compared to the control cells, not treated with probiotic bacteria or virus (100% survival rate).

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2.6. Immunofluorescence

In the competition assay, monolayers of IPEC-J2 cells were

treated exactly as previously described. After the incubation

period, cell monolayerswere washed with phosphate buffer saline

(PBS) and air-dried. Wells were filled with cold methanol

acetone mixture (1:2 ratio) and fixed for 15 min at 20 C. The

mixture was removed, wells air-dried and a polyclonal antibody

against VSV Indiana strain (whole serum developed in rabbit,

kindly provided by Dr. Alfonso Clavijo, NCFIAD, Canada) was

added to each well (3040 l, 1:100 diluted in sterile PBS) except

to the control wells. Plates were incubated for 30 min at 37 C.

Following five subsequent washes in PBSTween (0.5% Tween

in PBS), cell monolayers were further incubated for 1 h at 37 Cwith fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG

(Sigma, Aldrich, USA) in PBS. After being washed five times in

PBSTween and once with PBS, each well was examined, using

an inverted fluorescence microscope (Nikon, LTD, Japan).

To examine the immunofluorescence of virus adsorbed to

bacteria, washed and re-suspended pellet in PBS was spotted

onto a slide and air-dried. After fixation with cold methanol

acetone mixture (1:2 ratio) for 15 min at 20 C, slides were air-

dried and prepared for immunofluorescence exactly as de-

scribed for the competition assay.

3. Results

3.1. Pre-treatment of cell monolayers with viable probiotic and

other lactic bacteria reduces the virus infectivity

Pre-treatment period of 90 min on IPEC-J2 (Fig. 1A) and

3D4/2 (Fig. 1B) cell lines with viable probiotic and other lactic

Fig. 2. Antiviral activity of viable probiotic bacteria on VSV infected IPEC-J2

cells. Antiviral effect was observed when probiotic strains were applied onto cell

monolayers together with VSV. Results were collected after 48 h after VSV

challenge and expressed as percentage (means standard deviation) of cell

survival, as compared to the control cells, not treated with probiotic bacteria or

virus (100% survival rate).

Fig. 3. Confluent monolayers of IPEC-J2 cells infected with VSV in the presence and absence of probiotic bacteria used, were fixed and processed for

immunofluorescence. Monolayers were infected (7.3 TCDI50) with VSVonly (A) and simultaneously with probiotic bacteria and VSV (Bl+VSVon B, Lpa+VSVon

C). Negative control (D). Reduction of positive signal for anti-VSV antibodies observed comparing A with B, C confirmed successful competition of probiotic bacteriawith VSV for attachment to the cell surface (magnification 200).



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bacteria followed by immediate challenge with VSV resulted in

low inhibition ( 30% cell survival) of VSV activity in the case

of two used bacterial strain (Bl and Lpa). No significant

difference in reduction of the virus infectivity was observed

between the two cell lines. The same low level of inhibition of

VSV infectivity was observed after 24 h pre-treatment with

probiotic strains, followed by 24 h incubation prior to

determining the protective effect (Fig. 1C and D). Significant

protective effect, up to 60%, was however observed in theIPEC-J2 cells incubated for 24 h with the probiotic bacteria

prior to challenge with VSV, and followed by subsequent

incubation for 48 h. No such effect was observed for the 3D4/2

cells (achieved protection was lower than 10%).

3.2. Reduction of the virus infectivity by co-incubation of VSV

with viable probiotic and other lactic bacteria in cell

monolayers (competition experiment)

To determine whether probiotic and other lactic acid bacteria

compete with the virus for attachment to the cells, bacteria and

VSV were incubated together with the IPEC-J2 cell line for 48 h.

As shown in Fig. 2, all tested strains were able to reduce the virus

infectivity up to 60% within 48 h incubation period (Fig. 2). As

confirmed by qualitative immunofluorescence, a reduction of

signal, indicating the presence of VSV in cell monolayers was

observed for all tested bacterial strains (Fig. 3); as shown for Bl

(Fig. 3B) and Lpa (Fig. 3C). The reduction of the virus

infectivity by probiotic bacteria was not dependent on the virus

titer, since no significant change in the cells survival rate

occurred due to virus dilutions (Fig. 2). As shown in Table 1, thereduction of the virus infectivity was strongly affected by the

number of probiotic bacteria applied to the cells. The Lpa and

Lpl appeared to have the best antiviral effect, since they

protected the cells already at a concentration of 105 CFU ml 1.

3.3. Adsorption of the VSV to probiotic and other lactic acid

bacteria reduces the virus infectivity

It was further analysed whether the inhibition of VSV

infectivity was due to the adsorption of the virus to probiotic

bacteria. After 1 h pre-incubation of viable probiotic bacteria

with VSV, residual virus infectivity was assayed on IPEC-J2

and 3D4/2 monolayers. Virus alone, treated under the sameexperimental conditions as with bacteria, was used as a control.

As shown in Fig. 4, VSV adsorbed with the high affinity to all

Table 1

Effect of number of viable probiotic bacteria on the reduction of the virus

infectivity in IPEC-J2 cells (results are presented as cell survival a (%)standard

deviationb)

Bacteria Bacterial number (CFU/ml)

105 106 107 108

Bb 10 8 21 4 30 6 32 5Bl 0 0 0 0 28 3 40 3

Lpa 16 13 15 2 28 7 44 5

Lpp 36 15 45 7 44 6 56 2

Lpr 0 0 20 2 23 0.5 31 12

Lpl 35 10 47 8 36 4 50 4

Lr 0 0 33 5 47 4 51 1

a A dose dependence between the strength of the virus, number of bacteria and

antiviral effect was determined by preparing 10-fold dilutions of each of the

probiotic bacteria in DMEM without supplements; starting from 108 CFU ml 1

while the VSV titer remained unchanged (7.3 TCID50 ml 1). Each probiotic

strain (100 l, 108 CFU ml 1) and VSV (100 l of 7.3 TCID50 ml 1) were

simultaneously added to pre-washed monolayers of IPEC-J2 in P96 plate. Plates

were then further incubated at 37 C, 5% CO2 for 24 h or 48 h. Cell survival was

determined by measuring optical density at 595 nm after staining

destainingwith crystal violet.

b Mean (standard deviation) for four wells (w =4) treated with each dilution in

two separate experiments.

Fig. 4. Cell survival rate of VSV infected IPEC-J2 (A) and 3D4/2 (B) cells. Residual viral infectivity was measured after co-incubation of probiotic strains with VSV

for 60 min. Supernatants obtained after centrifugation were applied onto cell monolayers. Results were collected after 24 h post-infection and expressed as percentage

(means standard deviation) of cell survival, as compared to the control cells, not treated with probiotic bacteria. The virus itself was treated under the same conditions,except that bacteria were not added, and used as a control in the experiment (100% survival rate).

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probiotic strains used in the experiment, that resulted in up to

70% cell survival rate in the IPEC-J2 cell line (Fig. 4A),

whereas lower inhibition of the virus infectivity ( 50% cell

survival) was observed for most of the bacteria tested in 3D4/2

cells (Fig. 4B). Differences in cell survival rate are most

probably due to the diversity in cell surface glycans that are

used for the attachment of virus. The effect of the bacteria wasnot dependent on virus dilutions, as cell survival rate remained

in the same range between the highest and the lowest virus

dilution (Fig. 5). Positive signal for anti-VSVantibody that was

observed in direct immunofluorescence of bacterial pellets,

confirmed the virus adsorption to the probiotic and other lactic

bacteria (results not shown).

3.4. Metabolic products of probiotic and other lactic bacteria

have an antiviral activity

In order to analyse whether metabolic products of probiotic

and other lactic bacteria have an antiviral activity, overnightculture supernatants of the bacteria were used to treat the IPEC-

J2 or 3D4/2 monolayers in two-fold serial dilution, followed by

24 h delayed VSV challenge. pH of the supernatants was raised

to 7.0, in order to eliminate the pH effect on the virus infectivity.

As shown in Table 2, metabolic products of overnight

supernatants of Bl, Lpl and Lr had the highest antiviral effect

( 65% cell survival). The inhibition of virus infectivity

exceeded 60% only in the first dilution (1:2) in higher dilutions;

the concentration of the antiviral substances in the culture

supernatants was probably too low to establish the antiviral

protection in the IPEC-J2 cells. Almost no reduction of virus

infectivity was observed in supernatants of Bb, Lpa and Lpr

indicating that production of potential antiviral metabolites isstrain specific.

4. Discussion

A novel cell culture model to study viruslactic bacteria

host interactions was established. The cell model consisted of

porcine intestinal epithelial cells (IPEC-J2) or porcine alveolar

macrophage cell line (3D4/2), representing different cell types

present in the gut, and vesicular stomatitis virus (VSV)

Indiana strain, as a model virus due to no tissue restriction.

In this model, we have demonstrated decrease of viral

infectivity in IPEC-J2 and 3D4/2 cells by short term (90 min

time needed for the bacteria passage through the gut and theiradherence) pre-incubation of cells with probiotic bacteria prior

to virus challenge, long term (24, 48 h) pre-treatment of cell

monolayers with probiotic and other lactic bacteria, and by pre-

incubation of virus with the bacteria prior to cell inoculation.

These results indicate indirectly, that several mechanisms

may be employed in the antiviral effect of the probiotic and

lactic bacteria. The first possible mechanism may be interfer-

ence with virus attachment or entry into the cells, perhaps by

steric hindrance (short term pre-incubation).

Secondly, it appears that the probiotic and other lactic

bacteria are capable nonspecifically or perhaps specifically of

trapping the VSV, as demonstrated by drop in virus titers afterco-incubation of selected bacterial strains with VSV and IPEC-

J2, as well as drop in virus titers after pre-incubation of the

virus with bacteria prior to the exposure of the cells to the

inoculum. Positive signal in immunofluorescence for VSV in

the residual bacterial pellet confirmed binding of the virus to

the bacteria. Tao (2004) showed that two Lactobacillus strains

were capable of specifically trapping HIV virions by binding

the mannose sugar rich dome of their attachment glycopro-

tein gp120. (The HIV attachment proteins are protein spikes

with sugardome at the end, which prevent HIV from being

recognised by the human immune response). It is therefore

possible that a similar mechanism may also work in other

bacteriavirus systems.

Fig. 5. Cell survival rate of VSV infected IPEC-J2 (A) and 3D4/2 (B) cells.

Residual viral infectivity was measured after co-incubation of probiotic strains

with 10-fold dilutions of VSV, starting with 7.3 TCID50 for 60 min. Supernatants

obtained after centrifugation were applied onto cell monolayers. Results were

collected after 24 h post-infectionand expressedas percentage (means standard

deviation) of cell survival, as compared to the control cells, not treated with

probiotic bacteria. The virus itself was treated under the same conditions, except

that bacteria were not added, and used as a control in the experiment (100%survival rate).

Table 2

Antiviral activity of probiotic bacteria metabolic products (results are presented

as cell survival a (%) standard deviationb)

Bacteria Dilution of the samples

1:2 1:4 1:8 1:16 1:32 1:64 1:128

Bb 21 5 12 1 9 2 0 0 0 0

Bl 67 2 4 1 2 0.5 0 0 0 0Lpa 25 4 15 3 15 4 15 3 0 0 0

Lpr 12 3 0 0 0 0 0 0

Lpl 62 7 6 3 1 0.03 0 0 0 0

Lr 65 5 6 3 5 1 0 0 0 0

a Results are expressed as percentage of cell survival (means standard

deviation) of IPEC-J2 as compared to the control (non-treated cells). The effect

of MRS was eliminated by deduction of the values of the control wells titrated

with pure MRS (pH=7.0).b Mean (standard deviation) for four wells (w= 4) treated with each dilution in

two separate experiments.

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A Novel Eukaryotic Cell Model to Study Antiviral Activity

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