inhibitory effect of african swine fever virus on lectin-dependent swine lymphocyte proliferation

~'eterinary Immunology and Immunopathology, 26 ( 1990 ) 71-80 71 Elsevier Science Publishers B.V., Amsterdam

Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation

Silvia Gonz~ilez, Carmen Mendoza, Jos6 M. S~inchez-Vizcaino and Fernando Alonso*

Departamento de Sanidad Animal Instituto Nacional de Investlgaciones Agrarias, Embajadores 68, 28012-Madrid, Spain

(Accepted 9 January 1990)

ABSTRACT

Gonz~tlez, S., Mendoza, C., S~nchez-Vizcaino, J.M. and Alonso, F., 1990. Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation. Vet. Immunol. Immuno- pathol., 26:71-80.

The incubation of swine peripheral blood mononuclear cells (PBMC) with African swine fever (ASF) virus preparations strongly inhibited the proliferative response of lymphocytes to PHA and other lectins. The inhibition, which persisted after inactivation of the virus by UV radiation, was dependent upon the dose and the time that virus preparations were present in cultures. When virus preparations were fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble, whereas no activity was found in the sedimented viral fraction. However, the preincubation during 4 days of this sedimented fraction with swine PBMC, before the addition of the mitogen, restored the inhibitory activity. The results obtained suggest that the inhibition is mediated by one or more soluble factors released by swine PBMC after coincubation with ASF virus in a time dependent process. These factors show a molecular weight between 40 and 80 kDa by gel filtration chromatography. The inhibitory activity described in the present paper is an indication of inhibition of lymphocyte function produced by ASF virus which can help to understand how this virus escapes from the host immune system.

I N T R O D U C T I O N

Inhibition of immune function by virus infection is a common feature described for numerous viruses (Soontiens and Van der Veen, 1973; McChesney and Oldstone, 1987). The inhibition can result from the direct infection of immune competent cells or by an indirect action in which viral infection induces the release of soluble mediators. Other reports have shown that some virus particles can inhibit the proliferative response to lectins by mechanisms non-dependent of active infection (Wainberg and Israel, 1980 ).

*To whom correspondence should be addressed.

0165-2427/90/$03.50 © 1990 - - Elsevier Science Publishers B.V.

72 S. GONZALEZ ET AL.

African swine fever (ASF) virus is an icosahedral deoxivirus classically classified in the Iridoviridae family (Matthews, 1982), which specifically infects swine mononuclear phagocytes and produces an infectious disease in pigs, causing in its acute form a mortality close to 100%. However, pigs infected with attenuated virus develop viremia for long periods of t ime (Hess, 1971; McVicar, 1984; Pan and Hess, 1984 ). The mechanisms by which virus persist in these animals are still unknown.

In the present work we describe and partially characterize a strong inhibitory activity induced by ASF virus on the proliferative response of swine lymphocytes to phytohemagglutinin (PHA) and other lectins. Several isolates of ASF, differing in virulence, geographical origin and cells in which the virus was grown, consistently produced inhibition. The inhibitory activity studied appears to be mediated through a soluble factor released to the medium by the mononuclear cultured cells after incubation with ASF virus.

M A T E R I A L S A N D M E T H O D S

lll'rUS Most of the experiments of this study were performed using the Spain-70

(ETo) strain of ASF virus, adapted and grown in MS (monkey stable) cells after 15 passages (E7oMSIs). In addition, 10 other ASF virus isolates, including spanish and foreign ASF isolates, were used. ASF-viruses were grown in MS or swine buffy coat cells (Ruiz Gonzalvo et al., 1986). Foot-and-mouth disease virus of serotype C (C-$8) was grown in BHK cells (Sobrino et al., 1983 ). Infected cells were detached from the growing surface, centrifuged and sonicated after they were resuspended in RPMI medium supplemented with 20% of fetal calf serum. Sonicates were centrifuged ( 1000 Xg for 15 min) to remove cellular debris and the resulting supernatants were stored at - 7 0 ° C until use in inhibition assays. Virus titrations were performed in MS or swine buffy coat cells as it has been described (Ruiz Gonzalvo et al., 1986 ). Titers were calculated and expressed as TCIDs0/ml (Reed and Muench, 1938). In some experiments, virus was inactivated by UV radiation for 20 min at a distance of 10 cm, using a Sylvania G 15T8 UV lamp. Elimination of infectivity was tested by the inoculation of the irradiated virus onto fresh monolayers of MS cells and assessment of viral infection by direct immunofluorescence after 10 days.

Cells Mononuclear cells were isolated following a modification of the method

described by Haslett et al. ( 1985 ), adapted for swine cells. Six ml of 6% dex- tran (Pharmacia, Mw 500 000) was added to 50 ml polypropylene tubes (Nunc, Denmark) containing 25 ml of fresh blood anticoagulated with 5 mM EDTA (final concentration ) and the volume was made up to 50 ml with 0.9%

EFFECT OF ASF VIRUS ON SWINE LYMPHOCYTE PROLIFERATION 7 3

saline, mixed throughly and allowed to stand for erythrocyte sedimentation to occur. The leukocyte rich plasma was taken and centrifuged at 300 X g for l0 min, the pellet was resuspended in 2 ml of RPMI medium, transferred to a 11 ml polystyrene tube and underlayed with 2 ml 45% Percoll in RPMI medium. The tubes were centrifuged at 300xg for 15 min and the mononuclear cells remaining at the interface were aspirated, washed twice in RPMI and resuspended in this medium supplemented with 20% of autologous serum for further use.

Lectin drive lymphocyte proliferation These experiments were carried out in flat bottom 96-well microtiter plates

using a final volume of 0.25 ml per well. 2× 105 mononuclear cells, resuspended in RPMI medium containing 20% of autologous serum, were placed in each well. Cells were stimulated with phytohemagglutinin (PHA) ranging from 5 to 200/tg/ml or other lectins and after 72 h incubations at 37°C in 5% CO2, 1 /tCi of [3H]thymidine was added to each well. Six h later, cultures were harvested using a Skatron cell harvester (Norway) and the radioactivity incorporated in the filters was determined. The lymphocyte stimulation index was calculated as the ratio of the amount of radioactivity incorporated in the presence of stimulus to that in the absence of stimulus. Each test was performed with three replicate samples.

When virus was coincubated with mononuclear cells to test its inhibitory action on lectin driven proliferation, unless otherwise indicated, a constant amount of the virus preparations was added to each well at the beginning of the incubation, maintaining the total volume constant. In some experiments at the end of the incubation period, tests of cell viability by trypan blue exclusion were done in order to discard a direct cytotoxic effect of the virus.

RESULTS

Effect of ASF virus on lectin driven proliferation assays The coincubation of ASF virus with swine peripheral blood mononuclear

cells (PBMC) inhibited by more than 90% the proliferative response that different doses of PHA induced in cells incubated in the absence of virus (Fig. 1 ). Most of the experiments were carried out with PHA stimulated PBMC; however, the inhibition produced by the virus on the proliferative response of these cells to concanavalin A or pokeweed mitogen was similar (data not shown). Cell viability tested by trypan blue exclusion at the end of the incubation period was higher than 90%, and no difference was found between cells incubated with or without the virus.

The effect was dependent upon the dilution of the virus preparation; however, its inhibitory effect was independent of the PHA concentration. On the

74 S. GONZ.~LEZ ET AL.

t o o

o

& 5 6o F ¢~ . -

f_,. " ; 4 o IE

3 6 12 2 5 5 0 I 0 0 2 0 0

~g /m l PH-A

Fig. l. Effect of the virus on lymphocyte proliferation. 2 X 105 PBMC were cultured during 3 days with the PHA concentrations shown in the figure in the presence ( • ) or absence ( O ) of an AS F virus preparation at a MOI of 0.3. The [3H]thymidine incorporated in the cells was determined 72 h later as described in Materials and Methods. Results represent the mean +_ S.E. of eight experiments done in triplicate. The stimulation index of maximal response was 220 +_ 90.

other hand, the inhibitory activity remained when virus whose infectivity had been abolished by UV radiation was used (Figs. 1 and 2 ).

Low speed supernatants from homogenates of non-infected MS cells, which had been sonicated and treated in the same way as the virus preparations, also inhibited the proliferative response to PHA. However, the inhibition, which was lower than 40% when the non-infected MS cell preparation was undi- luted, dropped rapidly to background levels with dilution. In addition, a virus preparation obtained from BHK cells infected with foot-and-mouth disease virus, which was treated in the same way as the ASF virus infected cells, did not affect the response of swine PBMC to PHA (Fig. 2 ).

Timing of the inhibitory reaction When the virus preparation was added to the cells at 24, 48 and 72 h after

the cultures were initiated in the presence of PHA, the determination of the proliferative response made at the 72 h showed that the inhibitory activity was dependent on the time that the virus preparation was in the culture (Fig. 3). In other experiments the PHA and the virus preparation were added to the cells at the beginning of the culture and the proliferation was determined by a 6 h pulse of [3H]thymidine at 24, 48 and 72 h. Again the proliferative response to PHA was increasingly inhibited up to almost 100% depending on the time the virus preparation was present in the cultures (Fig. 4 ).

EFFECT OF ASF VIRUS ON SWINE LYMPHOCYTE PROLIFERATION 75

._.E

1 0 0

8 0

6 0

4 0

2 0

-- i

I 5 2 0 8 0 3 2 0 1280

I /Virus di ]utJon

Fig. 2. Effect of dilution and UV treatment of virus on the inhibitory activity. 2 × 105 PBMC cultured during 3 days with 25 pg/ml of PHA in the presence of various dilutions of an ASF virus preparation, which had been UV inactivated ( • ) or not ( O ), or in the presence of a non- infected MS cell preparation, treated in the same way as the infected MS cells ( • ). The effect of virus preparations from BHK cells infected with foot-and-mouth disease virus on PHA stimulated swine PBMC was also determined (m). Thymidine uptake was measured as described in Materials and Methods. The values represent the mean of triplicates from a representative experiment.

Partial characterization and localization of the inhibitory activity The resistance of the inhibitory activity to UV treatment suggested that it

was independent of viral infectivity. Accordingly, some experiments were un- dertaken to localize this activity in the fractions resulting from the ultracentrifugation of the virus preparation and to determine whether it was the result of the direct action of virus particles or was mediated by soluble factors. The virus preparation, consisting of a low speed supernatant of sonicated infected MS cells, was centrifuged at 100 000 × g for 60 rain and the inhibitory activity of the resulting fractions determined on PHA stimulated swine PBMC. Al- most 100% of the inhibitory activity was found in the supernatant indepen- dently of the UV treatment, whereas the pellet, where most of the viral particles were present, showed low inhibitory activity (Table 1 ). However, when this pellet was coincubated with PBMC during 4 days before the addition of PHA, the inhibitory activity, determined with respect to controls which were incubated and stimulated with PHA in the same conditions, reached the level showed by the unfractionated virus preparation. Under these conditions, the UV radiation of the pellet had only a slight effect on the inhibition (Table 1 ).

In order to further characterize the inhibitory activity, the supernatant obtained from the ultracentrifugation of the virus was chromatographied in Se- phacryl S-300 and the fractions obtained were tested for their activity. Two peaks of inhibitory activity were obtained. The main peak, consisting in sev-

76 S. G O N Z A L E Z E T A L

I 0 0

8 0

l_ c

~ 6 0

E

o

2 0

H o u r s

I ~ l PHA t00 pg/rnl

~ P H A 12 pg/ml

0 2 4 4 8

o f t e r v i rus o d d i t i o n

:,'::

:'i

7 2

Fig. 3. Effect o f the addit ion o f virus on lymphocyte proliferation at different times. 2 × 105 PBMC were cultured in the presence o f P H A ( 12 or 1 0 0 / l g / m l ) and at the indicated t imes an ASF virus preparation (MOI: 0.3 ) was added to them. Thymidine uptake was determined 72 h after initiating the cultures as described in Materials and Methods. Results represent the mean +_ S.E. o f four experiments.

I 0 0

~ ~eo 0 ~._ .~ 60

~ 40 E

2O

[ ~ ] PHA

PHA÷ virul

3 6 12 25 50 100200 3 6 3

[-

-N .:,~

12 25 50 I00 200 6 12 25 50 leO 200

pglml PHA

Fig. 4. Proliferation o f swine lymphocytes determined at different t imes after the addit ion of the virus. 2 × 105 PBMC were cultured with the PHA concentration indicated in the Fig. and in the presence or absence o f a preparation o f ASF virus. Thymidine uptake was determined at 24 (a ) , 48 (b ) and 72 h (c ) . St imulation indexes o f maximal responses were 54, 328 and 452, respectively.

EFFECT OF ASF VIRUS ON SWINE L Y M P H O C Y T E P R O L I F E R A T I O N

TABLE 1

Localization of the inhibitory activity

77

A B C D Whole virus Supernatant Pellet Preincubated preparation pellet

No treated 99 99 8 97 U.V. treated 98 97 6 76

2 × 10 s PBMC were stimulated with 25 / lg /ml of PHA and cultured during 3 days with the whole virus preparation or the fractions resulting from its centrifugation at 100 000 × g, which were treated or not with an inactivating dose of U V radiation.

Values of column D were obtained after the preincubation with the cells during 4 days of the same pellet used in column C before the addition of PHA. Thymidine uptake was determined 72 h later in regular conditions. Results represent percentage of inhibition of the proliferation shown by cells treated in the same conditions but in the absence of virus.

I 0 0

80

6 0

4 0

20

w . . . . . . . . . . . . . .

5

. Q

IO 15 20 25 5 0 35 4.0

F r o c t i o n s

04

O.3 A O

o

GI

o.2 0

O.I

Fig. 5. Inhibitory activity on swine lymphocytes proliferation ( • ) and OD28 o (O) shown by the fractions obtained from the Sephacryl S-300 chromatography o f the 100 000 X g supernatant o f an ASF virus preparation.

eral fractions and showing an activity close to 100% had a Mw between 60 and 80 kDa. The second peak showed a less consistent activity, ranging between 40% and 80% in different experiments, and had a Mw around 40 kDa (Fig. 5 )

Comparative study of different ASF virus isolates In addition to the EToMS15 isolate, used in most of the experiments re-

ported in this study, 10 other ASF isolates of ASF virus available in our lab-

78 S. GONZ:~,LEZ ET AL.

T A B L E 2

Inhibitor3' effect of different ASF-virus isolates

M . O . I . A ~ B C D E F G H I J

5 99 95 99 96 99 - - 99 - -

3 99 95 99 96 99 - - 97 - - 1 98 93 97 94 98 - - 91 - - 0.9 . . . . . 99 - - 97 -

0 . 6 87 92 96 91 97 - - 9 6 - -

0 . 4 . . . . . 97 - - 96 -

0 . 3 83 92 95 83 9 4 - - 63 - -

0 . 2 . . . . . 95 - - 91 -

0.1 65 - - - 82 92 98 4 9 76 93

0 . 0 8 38 - - - 70 - - 26 - -

0 . 0 7 . . . . . . 98 - - 92

0 . 0 5 . . . . . 85 - - 68 -

0 . 0 4 39 - - - 60 . . . . .

0 . 0 3 - - - 96 - - 83

T h e A S F virus isolates, at the multiplicit ies of infection shown in the Table, were tested for their inhibitor3' activity on pig mononuclear cells st imulated with 25 # g / m l o f P H A , Results represent the percent of inhibition with respect to the proliferation obtained in the absence of v i r u s .

I S p a n i s h i s o l a t e s : A, P o n t e v e d r a ( 1 9 7 0 ), 4 p a s s a g e s in p i g leukocytes: virulent. B, P o n t e v e d r a ( 1 9 7 0 ) ,

7 p a s s a g e s i n p i g leukocytes: virulent. C, Pontevedra ( 1 9 7 0 ), 48 p a s s a g e s i n M S ce l l s : a v i r u l e n t . D ,

P o n t e v e d r a ( 1 9 7 0 ) . 89 p a s s a g e s in V E R O ce l l s ; a v i r u l e n t . E, Z a r a g o z a ( 1 9 8 3 ) , 3 p a s s a g e s in p i g

l e u k o c y t e s : v i r u l e n t . F, C o r d o b a ( 1 9 6 8 ), 5 p a s s a g e s in p i g leukocytes; virulent, G , M u r c i a ( 1 9 8 2 ), 8

p a s s a g e s in p i g l e u k o c y t e s : v i r u l e n t .

Foreign isolates: H , P o r t u g a l ( 1 9 6 3 ), 7 p a s s a g e s in p i g l e u k o c y t e s ; v i r u l e n t . I. H a i t i ( 1 9 8 0 ) , 5 p a s -

s a g e s in p i g leukocytes: virulent. J, Cameroon ( 1 9 8 2 ), 3 p a s s a g e s in p i g l e u k o c y t e s : v i r u l e n t .

oratory were tested at different multiplicities of infection for their inhibitory activity on the response of pig mononuclear cells stimulated with 25 #g/ml of PHA. These ASF isolates were used in order to determine some possible re- lationships between inhibitory activity and virulence, geographical sources or cells where the virus had been grown. As shown in Table 2, no special rela- tionships could be established. However, a clear dose-dependent effect was observed.

D I S C U S S I O N

Virus preparations, consisting of low speed supernatants from sonicated ASF virus infected cultures, strongly inhibit the proliferative response of swine lymphocytes to lectins. The kinetics of the reaction show that the magnitude of the inhibition depends on the time that the virus preparation is present in the cultures and the fact that the effect was not reversible by an excess of PHA suggests that it is not mediated by the competition for a receptor. Many viruses have been described to cause immunosuppression in their hosts (Mc-

EFFECT OF ASF VIRUS ON SWINE LYMPHOCYTE PROLIFERATION 79

Chesney et al., 1987; Soontiens et al., 1973). Basically the mechanisms described for immunosuppression can be the result of the direct infection of immune competent cells, can be induced by soluble mediators produced during infection or can be mediated by non-specific interactions. As ASF virus infects macrophages and blood monocytes, its cytotoxic effect on these cells was first considered as the main cause of inhibition. However, the resistance of the inhibitory activity to UV treatment suggested a mechanism independent of virus infectivity. This was confirmed when, after the virus preparation was fractionated by ultracentrifugation, the inhibitory activity resulted to be soluble. The sedimented viral fraction, which did not inhibit the PHA- dependent lymphocyte proliferation under regular conditions, did inhibit it when it was preincubated with mononuclear cells during 4 days, before the addition of the mitogen. Furthermore, in this case, the previous UV treatment of the pellet had only a slight effect on its inhibitory activity. All these data suggest that the virus, through a mechanism non-dependent of infection, induces the release of one or more soluble inhibitors from the cells with which it is coincubated. These inhibitory factors of lymphocyte proliferation, which by gel filtration chromatography showed a molecular weight between 40 000 and 80 000 are usually present in supernatants from infected cells. The pro- duction of suppressor molecules from macrophages induced by viral infec- tions has been reported for other viruses such as cytomegalovirus (Rodgers et al., 1985 ) and Dengue virus (Gulati et al, 1983 ) and the inhibition of lymphocyte proliferation by a mechanism non-dependent of active infection has been described by Wainberg and Israel (1980).

Previous reports have described the effect of ASF virus on swine lymphocyte response to mitogens (Wardley, 1982; Knudsen and Genovesi, 1987). The results reported here differ from those of Wardley in several important points. Wardley reported the inhibition of lymphocyte proliferation by avirulent but not by virulent viruses. Although most of the experiments described in this report have been carried out using the E7oMSt5 isolate, other isolates tested, including virulent and avirulent viruses as well as viruses grown in swine leukocytes have consistently shown inhibitory activity. There is not a clear explanation for this difference except the use of different isolates. Knudsen and Genovesi (1987) did not find inhibition of the proliferation produced by several mitogens on normal pig lymphocytes when they were tested in the presence of a moderately virulent ASF virus. However, it is not possible to compare their data since they did not report the conditions of the assay nor the treatment of the viral antigen.

The immunology involved in ASF is still poorly understood. When pigs are infected with virulent virus they die within a few days without the chance to build a protective immune response. Pigs infected with attenuated viruses usually survive but become viremic for long periods of time (Hess, 1971; McVicar, 1984; Pan and Hess, 1984). Factors determining the evolution of

80 S. GONZ.~LEZ ET AL.

the disease and the pers is tance o f virus in the b lood o f infected animals have to be explained. A l though o ther au thor s have not found i m p o r t a n t i m m u n o - suppress ion in ASF infected pigs (De Boer, 1967; Hess, 1981 ), the inh ib i t ion found can be an ind ica t ion o f l y m p h o c y t e ma l func t i on in vivo.

ACKNOWLEDGMENTS

We thank Dr. Ru iz G o n z a l v o for p rov id ing some o f the virus isolates and for useful d iscuss ion and Jav ie r Tomi l lo for d rawing the figures.

REFERENCES

De Boer, C.J., 1967. Studies to determine neutralizing antibody in sera from animals recovered from African swine fever and laboratory animals inoculated with African swine fever virus with adjuvants. Arch. Gesamte Virusforscb., 20: 164-179.

Gulati, L. Chaturvedi, U.C. and Mathur, A., 1983. Dengue virus-induced cytotoxic factor induces macrophages to produce a cytotoxin. Immunology, 49:121-130.

Haslett, C., Guthrie, L.A., Kopaniak, M.M., Johnston, R.B. and Henson, P.M., 1985. Modula- tion of multiple neutrophil functions by preparative methods or trace concentrations of bac- terial lipopolysaccharide. Am. J. Pathol., 119: 101-110.

Hess, W.R., 1971. African swine fever virus. Virol. Monogr., 9: 1-33. Hess, W.R., 1981. African swine fever: a reassessment. Adv. Vet. Sci. Comp. Med., 25: 39-69. Knudsen, R.C. and Genovesi, E.V., 1987. In vivo and in vitro effects of moderately virulent

African swine fever virus on mitogenesis of pig lymphocytes. Vet. Immunol. Immuno- pathol., 15: 323-336.

Matthews, R.E.T., 1982. Classification and nomenclature of viruses. Intervirology, 17: 1-199. McChesney, M.B. and Oldstone, M.B.A., 1987, Viruses perturb lymphocyte functions. Selected

principles characterizing virus-induced immunosuppression. Ann. Rev. Immunol., 5: 279- 304.

McVicar, J.W., 1984. Quantitative aspects of the transmission of African swine fever. Am. J. Vet. Res., 45: 1535-1541.

Pan, I.C. and Hess, W.R., 1984. Virulence in African swine fever: its measurement and impli- cation. Am. J. Vet. Res., 45:361-366.

Reed, I.J. and Muench, R.H., 1938. A simple method to estimating fifty percent end points. Am. J. Hyg., 27: 493-497.

Rodgers, B.C., Scott, D.M., Mundin, J. and Sissons, J.G.P., 1985. Monocyte derived inhibitor of interleukin 1 induced by human cytomegalovirus. J. Virol., 55: 527-532.

Ruiz Gonzalvo, F., Carnero, M.E., Caballero, C. and Martinez, J., 1986. Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro. Am. J. Vet. Res., 47: 1249-1252.

Sobrino, F., D~ivila, M., Ortin, J. and Domingo, E., 1983. Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture. Virology, 128:310-318.

Soontiens, F.J.C.J. and Van der Veen, J., 1973. Evidence for a macropbage-rnediated effect of poliovirus on the lymphocyte response to phytohemagglutinin. J. Immunol., 111: 1411-1419.

Wainberg, M.A, and Israel, E., 1980. Viral inhibition of lymphocyte mitogenesis. I. Evidence for the nonspecificity of the effect. J. Immunol., 124: 64-70.

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inhibitory effect of african swine fever virus on lectin-dependent swine lymphocyte proliferation

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