Apoptosis as pathogenic mechanism of infection with ... · plates (50.000 cells per well). Briefly, using MEM supplemented with 5% fetal calf serum, serial ten-fold dilutions from

Apoptosis as pathogenic mechanism of infection with vesicularstomatitis virus. Evidence in primary bovine fibroblast cultures

A. LÓPEZ-HERRERA1*, J. RUIZ-SÁENZ 2, Y.P. GÓEZ3, W. ZAPATA3, P.A. VELILLA3, A.E. ARANGO3, AND

S. URCUQUI-INCHIMA3

1. Grupo BIOGEM, Universidad Nacional de Colombia, Sede Medellín.2. Grupo de Microbiología y Epidemiología, Universidad Nacional de Colombia, Sede Bogotá.3. Grupo Inmunovirología-Biogénesis. Calle 62 No. 52-59, Laboratorio 532, Sede de Investigación Universitaria, Universidad

de Antioquia, Medellín, Colombia.

Key words: vesicular stomatitis virus, apoptosis, resistance, bovine fibroblasts (Source: NLM)

ABSTRACT: To determine whether fibroblasts from Blanco Orejinegro cattle, exhibit any level of resis-tance to infection against vesicular stomatitis virus (VSV) serotypes Indiana (VSV-I) or New Jersey (VSV-NJ), 30 fibroblast cultures were phenotyped to evaluate their resistance/susceptibility. Thirty three % of BlancoOrejinegro fibroblast cultures were classified as very resistant, 50% as resistant, and 17% as susceptible toVSV-I infection, whereas 20% were classified as very resistant, 50% as resistant and 30% as susceptible toVSV-NJ infection. Therefore, there appears to be a large variation in phenotypic polymorphism among thefibroblasts to infection by VSV. To elucidate the mechanisms responsible for this diversity, we searched for apossible relationship between resistance/susceptibility and production of factors with antiviral activity; how-ever fibroblasts did not secrete factors with antiviral activity. We examined also whether apoptosis whereinduced by infection and its correlation with the polymorphism of resistance/susceptibility to VSV. Usingmorphological analyses, hypoploidy measurements, and level of phosphatidyl serine expression, high levelsof apoptosis were measured in VSV infected fibroblasts. However, no correlation exists between apoptosisand the category of resistance/susceptibility to infection, indicating that apoptosis is a pathogenic mechanismof VSV.

BIOCELL2009, 33(2): 121-132

ISSN 0327 - 9545PRINTED IN ARGENTINA

Introduction

In Colombia, there are seven local cattle breeds thatare part of the country’s natural resources. Among them,the “Blanco Orejinegro” cattle occupies a very impor-tant place within the coffee-producing regions. Thisbreed has not been submitted to selection and harbourslarge genetic diversity; in addition it is slowly becom-ing extinct (Arboleda, 1980; Bedoya et al., 2001). There

are reports indicating that in vivo, Blanco Orejinegrocattle are resistant to infestation by the larva of Derma-tobia hominis (Buitrago and Gutierrez, 1999). We haverecently shown that primary fibroblast cultures ofBlanco Orejinegro infected with foot-and-mouth dis-ease virus (FMDV) subtypes A24 or 01 present broadphenotypic polymorphism (López-Herrera et al., 2005).A statistically significant correlation exists between theantiviral activity of the media from fibroblast infectedwith FMDV and the phenotype of resistance/suscepti-bility. That is, the supernatants of fibroblast with highantiviral activity are resistant to both FMDV subtypes,whereas those with low antiviral activity are susceptibleto infection.

*Address correspondence to: A. López-Herrera. GrupoBIOGEM, Universidad Nacional de Colombia, Sede Medellín,COLOMBIA.E-mail: [email protected]: November 5, 2008. Accepted: June 1, 2009.

A. LÓPEZ-HERRERA et al.122

In Colombia both serotypes of vesicular stomatitisvirus (VSV), Indiana (VSV-I) and New Jersey (VSV-NJ)are endemic. VSV is an enveloped virus, member of theRhabdoviridae family, genus Vesiculovirus. In its naturalhosts (horses, cattle and pigs) VSV causes clinical singsthat are indistinguishable from those produced by FMDV,producing vesicular lesions in the oral cavity, extendingto the teats and to the coronary band of the hoofs. Inhumans, a few sporadic cases of VSV have been described(de Mattos et al., 2001). The viral genome is composedof a single strand RNA negative of about 11 Kb that codesfor 5 proteins (reviewed in Lichty et al., 2004): nucleo-capsid, phosphoprotein, matrix, envelope glycoprotein,and RNA-dependent RNA polymerase.

It has been shown that for a few members of thePicornaviridae and Rhabdoviridae families to whichFMDV and VSV belong respectively, resistance mecha-nisms are induced by interferon (IFN) type-I that acti-vates the 2´-5´oligoadenylate synthetase (2-5)-RNAseL, and the double stranded RNA-dependent protein ki-nase. Both pathways lead to inhibition of viral as wellas cell protein synthesis, and to apoptosis of the infectedcells (Biron and Sen, 2001; Chinsangaram et al., 2001).

Apoptosis is a genetically regulated and coordinatedcell death mechanism that is essential for the control ofembryonic development and cell homeostasis. It is acti-vated via a variety of stimuli and is characterized by sev-eral morphological changes and biochemical processes,including chromatin condensation, intranucleosomalcleavages by activation of endonucleases and endogenousproteases, and finally cell fragmentation into apoptoticbodies, thereby avoiding inflammatory responses(O’Brien, 1998).

Apoptosis has been also described as a means ofcounteracting certain infections by inhibiting replica-tion of pathogenic microorganisms. Velilla et al. (2005)recently demonstrated that monocytes of the peripheralblood of a group of people repeatedly exposed to hu-man immunodeficiency virus type-1 (HIV-1) butuninfected, have a higher propensity to apoptosis in-duced by infection with HIV-1 in vitro compared toapoptosis induced by HIV-1 infection in monocytes ofa population at low risk of infection. The authors sug-gest that such monocytes from exposed non infectedsubjects, when infected in vitro with HIV-1 initiateapoptosis to avoid replication and dissemination of thevirus. In the case of Influenza A virus, it was demon-strated that apoptosis of monocytes and macrophagesis a mechanism used to limit replication of the virus(Fesq et al., 1994; Ohyama et al., 2003); the authorsproposed that during infection, apoptosis is induced to

block early events of the replicative influenza A viruscycle. Nevertheless, Nunoi et al. (2005) suggest that ininfluenza A virus infection, apoptosis in organs such asthe brain and liver corresponds to a mechanism of viralpathogenesis. Similarly, it has been described that in-fection of mice by cytomegalovirus can damage the brainby induction of apoptosis in cells of the central nervoussystem (Reuter, 2005). On the other hand, apoptosisinduced by reovirus in several tissues of 2-day old miceis associated with the expression of the viral proteinsigma 1S (Hoyt et al., 2005), that determines the extentof apoptosis in the heart and in the central nervous sys-tem, converting apoptosis into a pathogenic mechanismand a virulence factor.

In the present study, we demonstrate that apoptosisis induced in fibroblast of Blanco Orejinegro cattle in-fected by VSV-I or VSV-NJ. Our results also show thatthere is a phenotypic polymorphism in resistance/sus-ceptibility of such fibroblast infected with VSV that isnot associated either with the expression of antiviralactivity factors or with apoptosis induced by the infec-tion. Apoptosis as observed in Blanco Orejinegro fi-broblast is a pathogenic mechanism of infection trig-gered by VSV, and not a resistance mechanism.

Materials and Methods

Cell Cultures

All cells used in this study were grown or incu-bated at 37ºC in 5% CO

2 for the times indicated.

Primary Fibroblast Cultures

A group of 30 Blanco Orejinegro fibroblast samplesfrom the cell bank of the Immunovirology-BiogenesisGroup were cultivated in RPMI-1640 (Sigma®) growthmedium supplemented with 1% penicillin-streptomy-cin, 1% vitamins, 1% L-glutamine (Sigma®) and 10%fetal calf serum (Gibco®). Phenotypification of resis-tance/susceptibility and apoptosis after infection withVSV-I or -NJ was carried out in vitro using these cells,and the culture media were recovered to quantify anti-viral activity.

Baby hamster kidney (BHK) and African green monkeykidney (Vero) cells

Kidney cells from hamsters (BHK) and from Afri-can green monkeys (Vero) were from the bank of the

123APOPTOSIS AS PATHOGENIC MECHANISM OF VSV

Immunovirology-Biogenesis Group and were cultivatedin minimum essential medium (MEM) (Sigma®) supple-mented as above for the RPMI medium. BHK cellsserved to titer the virus and as susceptibility control indetermining the index of resistance/susceptibility of thefibroblast to infection with either VSV serotype. Thesupernatants of the infected BHK and fibroblast cellcultures served to assay the antiviral activity. Vero cellswere used to quantify the antiviral activity, since thesecells are sensitive to antiviral activity (including to IFN),but are incapable of producing the last one (Emery andMorgan, 1979).

VSV stocks

The VSV-I and -NJ serotypes were from theImmunovirology-Biogenesis Group. Virus stocks wereproduced by infecting BHK monolayers in 75 cm2 cul-ture flasks with 2 ml of VSV-I or -NJ containing titersof 8.8 or 8.5 log

(10) of 50% tissue culture infectious dose/

ml (TCID(50)

/ml) respectively, incubating the cells for 1h to allow adhesion and penetration of the virus, andthen adding medium to 15 ml. The culture media fromthe infected cells were collected 24 h post infection whencytopathic effects were detected in 80% of the cells us-ing an inverted microscope, the media was collected,centrifuged at 220 xG for 5 min at 4ºC, and the super-natants aliquoted (300 μl) and kept at -70ºC. They wereused for phenotypification of resistance/susceptibilityof fibroblasts, to quantify the antiviral activity and todetermine induction of apoptosis.

Virus titer -TCID(50)

/ml

The titer of the VSV stocks was determined by theTCID

(50)/ml method on BHK cells cultivated in 96-well

plates (50.000 cells per well). Briefly, using MEMsupplemented with 5% fetal calf serum, serial ten-folddilutions from 10-1 to 10-12 of VSV-I or -NJ were pre-pared. 100 μl of the virus dilution were added to eachwell containing a monolayer of BHK cells (7 wells perdilution, and 12 wells with uninfected cells as cell con-trols). The plates were incubated for 24 h. The viruswas then inactivated by addition of 100 μl phosphatebuffer-saline (PBS) containing 10% formaldehyde andexposed to UV light for 1 h. Finally, the number of wellspresenting cytopathic effects for each dilution was quan-tified, and compared to the wells with control cells. Thevirus titer (TCID

(50)/ml) was determined by calculating

the virus dilution that produced a cytopathic effect in50% of the infected wells (Leennette, 1995).

Plaque forming units (PFU)

To quantify antiviral activity, the PFU produced bythe VSV-NJ stock were determined (Leennette, 1995).Briefly, starting from virus stock, ten-fold dilutions from10-1 to 10-7 of the virus were prepared in MEM supple-mented with 5% fetal calf serum, and used to infect Veromonolayers in 24-well plates, 3 wells per dilution and200 μl/well; 3 wells also contained uninfected cells.After 1 h, the virus inoculum was removed, 1 ml/wellof a semisolid medium (MEM with 5% fetal calf serumand 0.4% agarose) was added and the plates incubatedfor 24 h. The virus was inactivated with 500 μl PBScontaining 10% formaldehyde and exposed to UV lightfor 1 h. The monolayers were coloured with crystal vio-let and the cytopathic effect determined by the numberof plaques formed per well.

Phenotypification of resistance/susceptibility in BlancoOreginegro Fibroblast

The resistance/susceptibility of each fibroblast cul-ture to infection by VSV-I or -NJ was determined bycomparing the TCID

(50)/ml of each one of the 30 samples

with the TCID(50)

/ml of the control susceptible BHKcells. Plates of 96 wells were seeded with 5 x 104 cells/well of each fibroblast culture (two wells per sample)in 100 μl of RPMI growth medium. After 24 h, eachwell was infected with 100 μl of ten-fold dilutions ofthe virus from 10-2 to 10-7, 14 wells per dilution, andone plate for each VSV serotype. Each plate included12 wells with uninfected cells as control. The cells wereincubated 24 h and after that the virus was inactivatedby the addition of 100 μl of PBS containing 10% form-aldehyde, exposed to UV light for 1 h, and stained withcrystal violet. Destruction of the monolayer was evalu-ated considering each well with cytopathic effects aspositive. The TCID

(50)/ml titer was determined by the

method of Spearman-Karber (Leennette, 1995). In par-allel, the titer was also evaluated in the BHK cells serv-ing as controls, using the same viral dilutions as for thefibroblast samples.

The resistance/susceptibility index was calculatedcomparing the TCID

(50)/ml value obtained with BHK

cells with the TCID(50)

/ml value of each fibroblast sampleinfected with either VSV serotype, and is expressed asthe log of the TCID

(50)/ml in BHK over the TCID

(50)/ml

of the fibroblast, such that:

R/S index = log10

TCID(50)

/ ml BHK

TCID(50)

/ ml Fibroblast


Based on the results thus obtained, the infected fi-broblast samples were grouped in three categories: (1)all the samples with an resistance/susceptibility index<1 that require an equal concentration or a 10-fold ex-cess of virus over BHK cells to reach a 50% level ofcytopathic effect are classified as susceptible, (2) allsamples in which the resistance/susceptibility index liesbetween 1.01 and 3 that require an 11- to a 1000-foldvirus excess over BHK cells to reach the 50% level ofcytopathic effect are classified as resistant, and (3) andall samples with an resistance/susceptibility index > 3.01that require more than a 1000-fold excess virus overBHK cells to reach the 50% level of cytopathic effectare classified as very resistant.

Induction and quantification of antiviral activity

To determine whether synthesis and secretion intothe supernatant of factors with antiviral activity are in-duced in Blanco Orejinegro fibroblast infected with VSV-I or -NJ, 2 x 105 cells/well were seeded in 24-well platescontaining 1 ml of RPMI medium. After 24 h, the me-dium was removed and the cells infected with 10 TCID

(50)/

ml of VSV-I or -NJ in 100 μl of RPMI medium contain-ing 5% of fetal calf serum (12 wells for each virussample). After 1 h, to allow adhesion and penetration ofthe virus into the cells, 900 μl of growth medium wereadded to each well. The supernatants of each well wererecovered 12, 24, 36 or 48 h post infection (the mediumfrom 3 wells for each point time) and placed at –70ºC.The supernatants collected at various time points werecombined to obtain a pool of supernatants for each fi-broblast sample containing the total antiviral activity pro-duced up to 48 h post infection. The virus contained inthe supernatants was inactivated either by decreasing thepH to 2 for 12 h at 4ºC which does not destroy the acid-resistant IFN, or by heating to 65ºC for 30 min whichdoes not destroy thermo-resistant RNases. Supernatantsfrom BHK cells infected with VSV-I or -NJ and treatedin conditions identical to the fibroblast samples and servedas positive controls. Such BHK-infected cells producelarge amounts of factors with antiviral activity, as for in-stance IFN, but are insensitive to IFN because they con-tain a genetic defect in the expression of the IFN recep-tor (Kramer et al., 1983).

To quantify the antiviral activity, a biological stan-dard as established by Ahmed et al. (2003) was used.The wells of 24-well plates were seeded with 1.5 x 105

Vero cells per well. After 24 h, the monolayers weretreated with 2-fold dilutions (1:2 to 1:128) of the poolsof supernatants obtained from the VSV infected fibro-

blast and incubated for 24 h (3 wells per dilution; 3 wellswithout supernatant served as controls). Each well wasthen infected with 15 PFU of VSV-NJ in 100 μl ofgrowth medium for 1 h; the medium was replaced by 1ml of MEM supplemented with 5% fetal calf serum and0.4% of agarose. After 24 h, the virus was inactivatedwith PBS containing 10% formaldehyde, the mixturesexposed to ultraviolet light for 1 h and the cells stainedwith crystal violet. The antiviral activity is expressed inInternational Units (IU) where 1 IU of antiviral activityis the maximum dilution of supernatant capable of de-creasing the PFU by 50% compared to control infectedcells not treated with the supernatants.

Apoptosis in Blanco Orejinegro fibroblast induced byinfection with VSV-I or –NJ

Apoptosis induced by infection of each of the 30fibroblats samples with VSV-I or -NJ (1 molecule ofvirus/cell; MOI = 1) was evaluated based on morpho-logical analyses, hypoploidy and the expression of phos-phatidyl serine induced by infection. Spontaneousapoptosis (control) was estimated in uninfected fibro-blast treated as the infected samples.

Morphological analyses

To evaluate the morphology and the viability ofthe cells 24 or 48 h post infection with VSV byepifluorescence microscopy, 105 cells in 25 μl of PBSwere stained with 1 μl of ethidium bromide (100 μg/ml;Sigma®) and 1 μl of acridine orange (100 μg/ml; Sigma®).The parameters of apoptotic morphology were charac-terized by loss of nuclear lobules, condensation of chro-matin and loss of cell volume. Moreover, based on thedifference of ethidium bromide and acridine orange pen-etration into living or dead cells, it was possible to distin-guish between normal cells in which chromatin is brightgreen, early apoptotic cells with highly condensed orbright green fragmented chromatin, late apoptotic cellswith highly condensed or orange-colored fragmentedchromatin, and necrotic cells with orange-coloured chro-matin (Vélez-Pardo et al., 2002). A total of 200 cells perfibroblast sample were counted per well 24 and 48 h postinfection, and for each serotype.

Hypoploidy

For each fibroblast culture, 106 cells were infectedwith VSV-I or -NJ at a MOI = 1 and incubated for 24 or48 h. The cells were mechanically detached from the


plate and then fixed with 70% ethanol for 30 min at4ºC, washed twice with PBS containing 1 mM KCl, re-suspended in 500 μl of 100 μg/ml of propidium iodide,and incubated for 30 min in the dark. The number ofhypoploidal cells was determined by flow cytometry(Coulter EPICS, XL) using acquisition of 10.000 cells,the data was analyzed by CellQuest program. Cells thatcontained a low level of DNA compared to the normalDNA content of uninfected fibroblast were consideredhypoploid (Mitchels et al., 2003).

Expression of phosphatidyl serine

Phosphatidyl serine is expressed on the outer cellmembranes during the initial phases of apoptosis. Toquantify these residues, the commercial TACSTM

Annexin V-FitC kit (TREVIGEN®) was used. In 16-wellplates, 5 x 105 fibroblast were seeded, infected with VSV-I or -NJ at a MOI = 1, and incubated for 24 or 48 h.They were then mechanically detached, washed with 1ml PBS, centrifuged at 1500 rpm for 5 min at 4ºC, re-suspended in 100 μl of a solution containing 89 μl ofbinding buffer (solution included in the kit), 10 μl ofpropidium iodide at 50 μg/ml and 1 μl of Annexin, andincubated for 30 min at room temperature in the dark.Finally, 400 μl of PBS were added and measurementsperformed by flow cytometry using acquisition of10.000 cells, the data were analyzed by CellQuest pro-gram. Uninfected fibroblasts were used to evaluate spon-taneous apoptosis.

Statistical analyses

The showed data for resistance/susceptibility phe-notypes, antiviral activity and apoptosis quantification

are the result of the average of three repetitions of eachexperiment. To evaluate the statistics normal distribu-tion of the data, the statistics test of Kollmogorov-Smirnov, Shapiro Wilks and Bartlett’s were performed.Since the data did not present a normal distribution, nonparametric analyses such as the Mann-Whitney methodwere performed with a confidence level of 95%. Thestatistical analyses were carried out using the GraphPadPrism® version 3.02.

Results

Fibroblast samples infected with VSV-I or -NJ presentdifferent resistance/susceptibility phenotypes

The titers of the VSV cultivated in BHK cells were7.3 log

(10)TCID

(50)/ml for both serotypes. In the fibro-

blast samples it varied from 1.36 to 6.66 log(10)

TCID(50)

/ml for VSV-I, and between 2.15 and 6.93 log

(10)TCID

(50)/

ml for VSV-NJ. The resistance/susceptibility index thatresulted from the comparison between the TCID

(50)/ml

in fibroblasts and BHK cells (see Materials and Meth-ods) varied between 0.7 and 6 for VSV-I, and 0.43 and5.21 for VSV-NJ. These results indicate that there ex-ists polymorphism in the fibroblast samples with re-spect to resistance infection with VSV-I that it slightlygreater than with VSV-NJ.

Based on the results of the resistance/susceptibil-ity index, the fibroblasts were grouped in three catego-ries of resistance/susceptibility: very resistant, resistantand susceptible (see Materials and Methods). As dem-onstrated in Table 1, the fibroblasts samples present aconsiderable polymorphism with respect to infection byeither VSV serotype, but with different patterns of veryresistant, resistant and susceptible to the two serotypes:Of the samples infected with VSV-I, 33% were veryresistant, 50% were resistant, and only 17% were sus-ceptible, indicating that 83% of the samples show resis-tance to infection. In comparison, 70% showed resis-tance to VSV-NJ (20% were very resistant and 50%resistant) and there were 30% susceptible samples. Con-sequently, the fibroblasts behave differently to infec-tion by the two VSV serotypes.

Quantification of the antiviral activity present in thesupernatants of Blanco Orejinegro fibroblast infectedwith VSV

We have previously shown that the supernatants ofBlanco Orejinegro fibroblast infected with FMDV pro-

TABLE I.

Phenotypification of the resistance/susceptibility of 30primary fibroblast cultures to VSV-I or VSV-NJ infec-tion. To classified each fibroblast culture in a categoryof resistance/susceptibility, the challenge was repeatedthrice with each virus serotype.

Phenotype VSV-I (%) VSV-NJ (%)Very resistant 33 20Resistant 50 50Susceptible 17 30Total 100 100


duce factors with antiviral activity that protect Veromonolayers from infection with VSV (López-Herreraet al., 2005). Consequently, we investigated whether thefibroblast samples infected with VSV also secrete fac-tors with antiviral activity capable of protecting Vero

cells from infection with VSV-NJ. To quantify the anti-viral activity present in the supernatants fibroblast orBHK cells, the virus remaining in the medium was in-activated by lowering the pH or by a heat treatment (seeMaterials and Methods). Quantification was carried out

FIGURE 1. Morphological assay of spontaneous versus induced apoptosis measured after 24 and 48

h in VSV-I or VSV-NJ infected Blanco Orejinegro Fibroblasts. Apoptotic morphology spontaneous (Spo)

or induced by infection (Ind-I: induced by VSV-I; Ind-NJ: induced by VSV-NJ) was visualized by epifluorescence

microscopy. A and B: Average of spontaneous and induced morphological apoptosis after 24 and 48 h re-

spectively. C and D: spontaneous versus induced morphological apoptosis induced by VSV-I in very resis-

tant (VR), resistant (R) and susceptible (S) fibroblasts 24 and 48 h post infection respectively. E and F: Same

situation that C an D, but infected with VSV-NJ. The horizontal lines in each box represent average, the lower

and upper borders of the boxes are the 25 and 75 percentiles respectively, and the short horizontal bars

correspond to the minimum and maximum values. P: level of statistical significance.


in Vero cells treated with the supernatants recoveredfrom the fibroblast or the BHK cells infected with VSV-I or VSV-NJ. Vero cells were not protected from infec-tion with VSV-NJ by the supernatants from VSV-in-fected fibroblasts, although they were protected frominfection by up to a 1:64 fold dilution of the superna-tants from VSV-infected BHK cells. Hence, after infec-tion, BHK cells produce 64 IU/ml of antiviral activity,a phenomenon not observed with infected fibroblast(data not shown).

VSV Infection of fibroblast induces apoptosis

Some degree of spontaneous apoptosis was ob-served in uninfected fibroblast samples maintained invitro and, except for one case, was always much lowerthan the VSV induced apoptosis in fibroblast infectedfor 24 or 48 h.

Effect of VSV infection on the morphology of fibroblastsamples

The level of apoptotic morphology of fibroblastsinfected with each VSV serotype was higher than inuninfected samples. After 24 h, the average percentageof apoptosis in uninfected cells was 6%, compared to37% and 36% in cells infected with VSV-I and -NJ re-spectively (Fig. 1A). After 48 h, the values were 5% inuninfected cells, and 38% in cells infected with eachVSV serotype (Fig. 1B). The difference between spon-taneous apoptosis and apoptosis induced by VSV is sta-tistically highly significant (p < 0.0001) after 24 and 48h. These results show that VSV can induce cell death infibroblasts irrespective of the virus serotype or the du-ration of infection.

The effect of infection on the morphological modi-fications of the fibroblast of the three categories veryresistant, resistant and susceptible were evaluated. Atboth time points considered, there was a large differ-ence in the percentage of induced apoptotic cells be-tween the categories compared to uninfected cells inwhich spontaneous apoptosis was very low (Fig. 1C,D). Nevertheless, the major difference between sponta-neous and induced apoptosis was observed in the resis-tant category with p = 0.001. These results show thatthere is a statistically significant difference betweenspontaneous apoptosis and apoptosis induced by infec-tion for all three categories of infected fibroblasts.

When spontaneous apoptosis was analyzed andcompared between the three categories, a higher inci-dence of apoptosis was visible with the very resistant

fibroblast. However, this difference was statistically sig-nificant at 48 but not at 24 h post infection (p = 0.0163versus p = 0.0751 respectively; results not shown).

Similar results were obtained when comparing thecategories of resistance/susceptibility of fibroblasts in-fected with VSV-NJ, except that at 24 h post infection,cells classified as resistant presented a large decreasein average apoptosis compared to cells infected withVSV-I (28% and 52% for serotype VSV-NJ and sero-type VSV-I; Fig. 1E, C).

When the spontaneous apoptotic morphology be-tween the three categories of resistance/susceptibility toinfection by VSV-NJ was compared, the fibroblasts witha susceptible phenotype tended to present a higher levelof spontaneous apoptosis at 24 h post infection, whereasat 48 h post infection the behavior of the cells was verysimilar between the three categories. Induced apoptoticmorphology was compared between the different catego-ries of resistance/susceptibility to VSV-NJ infection infibroblasts belonging to the three categories. Although aslight tendency to show a higher level of apoptosis post-infection was noted in the very resistant category, therewere no statistically significant differences in inducedapoptosis between the three categories, neither at 24 norat 48 h post infection (data not shown).

Induction of hypoploidy in VSV infected fibroblasts

An evaluation of late apoptotic events based on thecontent of double-stranded DNA in uninfected fibro-blasts (spontaneous) and in infected fibroblasts (in-duced) with each VSV serotype, showed that the per-centage of apoptotic cells is larger in infected than inuninfected fibroblasts at both time points (Fig. 2A, B).After 24 h, VSV-I infected fibroblasts presented a higherlevel of hypoploidy than samples infected with VSV-NJ (55% and 42% of apoptotic cells, respectively).However, at 48 h post infection there was no differencebetween the two serotypes (81.2 and 81.7% respec-tively). Therefore, VSV infection of Blanco Orejinegrofibroblasts induced apoptosis, whereas in the uninfectedcontrol cells, cell death was very low, at 24 and at 48 hpost infection (1.9 and 4.0%, respectively). The resultsof hypoploidy observed in infected VSV is statisticallyhighly significant with each serotype (p < 0.0001) com-pared to spontaneous apoptosis in the same sample,where it is not significant.

It was also noted that hypoploidy in very resistant,resistant and susceptible classified fibroblasts and in-fected with VSV-I is higher than in uninfected cells (Fig.2C, D) at 24 and at 48 h post infection. As can be seen


in Figure 2C there is a strong induction of apoptosis incell cultures infected with VSV-I in the three catego-ries. The percentage of apoptotic cells was about 50%,although the level of spontaneous apoptosis was insig-nificant in these cultures. Surprisingly, after 48 h of in-fection the evolution of cell death was very different

between the three categories (Fig. 2D). In susceptiblecell cultures infected with VSV-I, apoptosis reached92%, whereas in resistant cell cultures cell death was87%. Intriguingly, in the case of the very resistant cellcultures, there was little variation between 24 and 48 hpost infection (from 55% to 62% respectively). These

FIGURE 2. Percentage of hypoploid cells with features of apoptosis measured in Blanco Orejinegro

Fibroblasts infected with VSV-I or VSV-NJ for 24 and 48 h. Number of hypoploid cells determined by flow

cytometry. A and B: general average of spontaneous hypoploidy versus hypoploidy induced by infection with

VSV-I or VSV-NJ in the 30 fibroblasts samples for 24 and 48 h. C and D: spontaneous hypoploidy versus

induced hypoploidy produced by infection with VSV-I in very resistant (VR), resistant (R) and susceptible (S)

fibroblasts, 24 and 48 h post infection respectively. E and F: Same situation that C an D, but infected with

VSV-NJ. Other indications like in Figure 1.


results demonstrate that infection with VSV-I inducehigh levels of apoptosis in Blanco Orejinegro fibroblasts.

As opposed to the results of cell death induced byVSV-I, in VSV-NJ infected fibroblasts each of the threeresistance categories presents a large variation in theresponse to infection. After 24 h, the percentage of

apoptotic cells in the very resistant cultures was 27%,in the resistant cultures it was 37%, and in the suscep-tible cultures it was 55%. It should be noted that a slightincrease in spontaneous apoptosis was observed inuninfected cultures; nevertheless, when comparing thespontaneous to the induced apoptosis in each of the three

FIGURE 3. Expression of phosphatidyl serine on the outer surface of cell membranes with character-

istics of early apoptosis in VSV-I or VSV-NJ infected Blanco Orejinegro Fibroblasts for 24 and 48 h.

Phosphatidyl serine residues expressed on the outer cell membrane quantified by flow cytometry. A and B:

Comparison of the general average of spontaneous and induced expression of phosphatidyl serine by infec-

tion of the 30 fibroblasts samples with the two VSV serotypes for 24 or 48 h. C and D: spontaneous versus

induced expression of phosphatidyl serine by VSV-I in very resistant (VR), resistant (R) and susceptible (S)

fibroblasts, after 24 and 48 h respectively. E and F: Same situation that C an D, but infected with VSV-NJ.

Other indications like in Figure 1.


categories, the difference was statistically significant(Fig. 2E). After 48 h, there was an increase in inducedapoptosis in the cell cultures of categories resistant andsusceptible as also observed with VSV-I; yet, in the veryresistant cultures there was a slight decrease in cell death(Fig. 2F). Taken together, these results suggest that VSV-NJ infected cells are poorer inducers of apoptosis thanVSV-I infected fibroblasts cultures.

Expression of phosphatidyl serine in VSV infected fi-broblasts

First, the average level of expression of phosphati-dyl serine in the 30 uninfected fibroblasts cultures orinfected with either VSV serotype was evaluated, butwithout taking into consideration the category to whichthe samples belong. In uninfected cells, a low level ofphosphatidyl serine was detected, but upon infectionwith either serotype, this level increased dramatically24 and 48 h post infection (Fig. 3A, B). After 24 h, bothserotypes induced a similar level of apoptosis, and at48 h post infection there was an important increase incell death. The percentage of cells expressing phosphati-dyl serine after infection with either serotype was sig-nificantly higher that spontaneous expression (p =0.0001) at both time points considered.

Upon evaluating the percentage of cells expressingphosphatidyl serine in each category of resistance/sus-ceptibility and infected with VSV-I, there was an in-crease in apoptotic cells as compared to spontaneousapoptosis in uninfected cells. After 24 h, the frequencyof induced apoptosis varied between 20 and 32%, butthe difference between spontaneous and inducedapoptosis was statistically significant (Fig. 3C). After48 h, there was a large increase in induced apoptosis inthe case of the very resistant cultures (70%), whereasthe increase in the two other categories was less impor-tant (40% and 45% for the resistant and susceptible cat-egories respectively; Fig. 3D). Similar results were ob-served in fibroblasts samples infected with VSV-NJ for24 or 48 h (Fig. 3E, F). Thus, the annexin assay con-firms the results obtained by the morphological andhypoploid assays, i.e.: VSV is a potent inducer ofapoptosis in Blanco Orejinegro fibroblasts.

Taken together, the results obtained using the threemethods to evaluate induction of apoptosis in BlancoOrejinegro fibroblasts samples infected with the twoVSV serotypes suggest that VSV is a strong inducerof apoptosis, independent of the resistance/suscepti-bility level of the fibroblasts. This indicates that theapoptosis observed in infected fibroblasts is an inte-

gral part of a pathogenic effect of virus infection, sincespontaneous apoptosis in these cultures is low andapoptosis induced by either VSV serotype is indepen-dent of the resistance/susceptibility category of theBlanco Orejinegro fibroblasts.

Discussion

VSV is an endemic virus in Colombia that producesa disease whose clinical symptoms are similar to thoseproduced by infection with FMDV. The presence of VSVis one of the main barriers to exportation of bovinesand pigs and their by-products to those countries whichare either free of the disease or in which the disease isunder a tight sanitary control.

The Blanco Orejinegro cattle breed is a natural re-source of Colombia that is on its way to extinction, andpopular ideas exist about its resistance to VSV. Our re-sults show that Blanco Orejinegro fibroblasts is poly-morphic for resistance/susceptibility to infection byVSV-I and -NJ, and support the results reported byLopez-Herrera et al. (2002) about resistance/suscepti-bility to VSV, and polymorphism in resistance/suscep-tibility to VFA (Lopez-Herrera, 2005).

VSV serotypes I and NJ are recognized VSV sero-types that produce a disease with similar clinical charac-teristics in sick animals. Even though no significant dif-ference has been described for these two serotypes exceptto antibody response, the resistance/susceptibility poly-morphism observed in fibroblasts due to infection isgreater with VSV-I than VSV-NJ. Likewise, for VSV-I, alarger percentage of fibroblasts are in the very resistantor resistant category (83.3%) than for VSV-NJ (70%).

FMDV infected fibroblasts secrete factors withantiviral activity into the medium capable of inhibitingreplication of VSV-NJ in Vero cells (López-Herrera etal., 2005). Nevertheless, neither of the two VSV sero-types induced antiviral activity in Blanco Orejinegrofibroblasts. Surprisingly however, in the supernatantsrecovered from BHK cells infected with VSV, antiviralactivity was detected up to a 1:64 dilution.

The matrix protein of VSV is reportedly (Ahmedet al., 2003) implicated in inhibition of host gene ex-pression triggered by an antiviral response such as IFNtype 1. It is also believed to inhibit the function of thethree cell RNA polymerases, preventing protein syn-thesis. The absence of factors with antiviral activity inthe supernatants of the examined fibroblast could beassociated with inhibition of gene expression in VSVinfected fibroblast via matrix protein. It should be re-


called that bovine fibroblasts are natural target cells ofVSV, whereas BHK cells serve as model for researchpurposes.

Our results also indicate that both VSV serotypesinduce apoptosis in fibroblasts, a phenomenon that hasbeen associated with pathogenesis in cells infected bythe virus (Nunoi et al., 2005; Reuter, 2005). The changein the nuclear morphology of the infected cells observedby epifluoresence microscopy is an indication ofapoptotic cells. This phenomenon can also be associ-ated with necrosis of the cell. However, our results alsoindicate that infected fibroblast present a low level ofDNA as compared to uninfected ones. In addition, alarge difference in phosphatidyl serine expression isobserved in infected versus uninfected. These observa-tions confirm the link that exists between the presenceof VSV and apoptosis, and could form a basis to ex-plain the formation of vesicular lesions in vivo.Apoptosis induced by VSV is a mechanism that leadsto the destruction of tissues and death of infected mice,as described by Sur et al. (2003). Nevertheless the dam-age caused could result from a combination of mecha-nisms directly or indirectly associated with viral infec-tion. Recent studies have shown that the matrix proteinand other viral components are responsible for the in-duction of apoptosis in infected cells (Kopecky et al.,2001). It was also shown that viral matrix protein caninhibit synthesis of cell proteins by blocking the threecell RNA polymerases, also leading to apoptosis(Ahmed and Lyles, 1998; Ahmed et al., 2003; Kopeckyet al., 2003).

Apoptosis has also been reported to be a resis-tance mechanism against virus infection (Fesq et al.,1994; Velilla et al., 2005). However, our results showno significant differences in induction of apoptosis byVSV between the three categories very resistant, re-sistant and susceptible of Blanco Orejinegro fibro-blasts. For this reason, the polymorphism in resistance/susceptibility described here suggests the presence ofanother mechanism implicated in resistance of BlancoOrejinegro fibroblasts to infection by VSV. In a previ-ous study (López-Herrera et al., 2005), we proposedthat resistance of Blanco Orejinegro fibroblasts toFMDV infection is associated with a low level of ex-pression of integrin α

v-β

3, the natural receptor for this

virus. Likewise, the samples classified as very resis-tant or resistant could present low levels of the recep-tor as opposed to those classified as susceptible. Lowexpression of the receptor for VSV could explain theresistance to this virus described here, but this recep-tor has not been characterized yet.

In conclusion, our results demonstrate that thereexists a considerable polymorphism in the resistance/susceptibility of Blanco Orejinegro fibroblasts to infec-tion by VSV and confirm that a native Colombian breed,which is on its way to extinction, is resistant, at least invitro, to infection by viral agents that produce vesiculardiseases. In addition, we demonstrated that natural re-sistance to infection of Blanco Orejinegro fibroblastsby VSV-I and VSV-NJ serotypes is not due to the pro-duction of factors with antiviral activity, and we alsoshowed that apoptosis induced by VSV infection in fi-broblasts is a mechanism of viral pathogenesis.

Acknowledgements

Dr Anne-Lise Haenni and Dr Jorge Ossa are ac-knowledge for their help in the revision and correctionof the English version of the manuscript.

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Apoptosis as pathogenic mechanism of infection with ... · plates (50.000 cells per well). Briefly, using MEM supplemented with 5% fetal calf serum, serial ten-fold dilutions from