Research in Veterinary Science 1991, 51, 317-321

Inhibition of natural killer activity in porcine mononuclear cells by African swine fever virus C. MENDOZA, S. P. VIDEGAIN, F. ALONSO, Departamento de SanidadAnimal, CIT-INIA, Embajadores 68, 28012-Madrid, Spain

The coincubation at 37°C for 24 hours of swine peripheral blood mononuclear cells with African swine fever virus inhibited in part the natural killer activity shown by cells incubated without the virus. This inhibition depended on the dose of the virus and on the time that cells were incubated with it. When the virus preparation was fractionated by ultracentrifugation, most of the inhibitory activity was found in the sedimented fraction, where viral particles were present; however, the loss of inhibitory activity in respect to the whole virus preparation indicated that some inhibitory activity was present in the supernatant fraction, probably as factors released by infected cells. Most of the inhibitory activity shown by the sedimented fraction was lost when the virus was inactivated by ultraviolet radiation, indicating an active role of virus infectivity in the inhibition.

BESIDES specific effector mechanisms, depending on antibodies or mediated by cytotoxic T lymphocytes, non-specific mechanisms play an important role against viral diseases, especially in primary infections when immunological memory is absent. Among these early non-specific mechanisms, natural killer (NK) cell activity is capable of lysing virus infected cells without prior exposure to antigen (Welsh 1981, Biron and Welsh 1982, Bishop et al 1983). In African swine fever (ASF), an infectious disease produced in pigs by an icosahedral ONA virus classified as an iridovirus, where infection with virulent isolates causes a mortality close to 100 per cent within a few days (Hess 1971), the activity of nonspecific effector mechanisms, such as NK, could be effective in destroying virus infected cells at early stages of infection and avoiding virus multiplication. In previous studies performed 'in vivo' a depression of the NK activity has been reported in pigs infected with ASF virus, between three and six days after infection. However no clear relationship was found between NK activity and recovery from infection (Norley and Wardley 1983). In this study the effect of ASF virus on the NK activity of porcine peripheral blood mononuclear

cells (PBMC)has been investigated in vitro to find out if the failure of this mechanism to stop viral infection is the result of some activity of the virus. The coincubation of ASF virus with effector ceils before the NK assay consistently reduced this activity by an active process depending on time and the dose of virus. Materials and methods Cells

Mononuclear cells were isolated from venous blood anticoagulated with 5 mM EDTAby centrifugation on Percoll discontinuous gradients following the technique previously described (Gonzdlez et al 1990). Large White pigs weighing between 30 and 40 kg were used as donors of blood. Virus

The avirulent ASF virus was the Spain 70 strain (E70) adapted and grown in monkey stable (MS) cells after 15 passages (E70MS15). The virulent ASV virus was obtained at day 4 after infection from the spleen of a pig which had been inoculated intramuscularly four days before, with at least 105 TeIDS0 units of a highly virulent AS; isolate Spain 75 (E75). Spleen was homogenised and centrifuged (500 g, 30 minutes). The supernatant was kept at - 70°C for further use as a virUs source. In some experiments this virus preparation was ultracentrifuged at 100,000 g to obtain the sedimented fraction, which was washed in RPMI medium by a second ultracentrifugation before it was used. Virus titrafions were performed in MS or swine buffy-coat cells as described by Ruiz-Gonzalvo et al (1986). Titres were calculated and expressed in TCIDS0 m1-1 (Reed and Muench 1938). For virus inactivation by ultraviolet radiation, it was placed under a Sylvania G15T8 uv lamp for 10 minutes at a distance of 15 cm. Elimination of infectivity was tested by the inoculation of the irradiated virus onto fresh cultures of buffy-coat cells and assessment of

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C. Mendoza, S. P. Videgain, F. Alonso 70-

viral infection by direct immunofluorescence after 10 days.

60NK assay

Cells from the human myeloid leukaemia lines K-562 or Molt-4 were used as targets. These cells were maintained in RPMI 1640 medium containing 2 mM Lglutamine and 5 per cent fetal calf serum. 2 x 106 target cells in 0.2 ml of RPMI-1640 containing 20 mM HEPES and 10 per cent fetal calf serum (assay medium) were labelled at 37°C for one hour with 100/zCi of Na251CrO4. Labelled cells were washed three times with assay medium and adjusted to 2 x 105 cells m1-1. Tests were run in triplicate in 3 m l polystyrene tubes with varying amounts of effector cells added to test tubes in 100/zl of assay medium. Labelled target cells (2 x 104) were added to each tube and the final volume adjusted to 300/zl with assay medium. After 20 hours of incubation at 37°C, 1 ml of phosphate buffered saline (PBS) was added to all the samples which were then centrifuged. A 1 ml sample of supernatant was harvested from each sample and counted in a gamma counter. The percentage specific release was calculated from the formula:

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Time (h) FIG 1 : Specific lysis produced by swine PBMCcells on molt-4 ( ~*) or K-562 (0) target cells. 2 x 1 0 4 labelled target cells were incubated during the times shown in the figure with 106 freshly obtained swine PBMC and the specific release of 51Cr was determined. Results represent four independent experiments

test cpm % Specific SlCr release = spontaneous cpm x 100. total cpm spontaneous cpm In the experiments in which the effect of the virus was tested, effector cells were incubated for 24 hours with the virus preparations at 2 x 106 cells m1-1 in assay medium and, unless otherwise indicated, at a multiplicity of infection (MOI) of 0"4. At'the end of this incubation cell viability was higher than 90 per cent by trypan blue exclusion. Before the NK assay, effector cells were washed and adjusted to 20 x 106 cells m l - 1. Results

Conditions o f the assay Previous experiments to establish the optimal conditions of the assay showed the convenience of using cells of the line K-562 as targets in a 20-hour assay (Fig 1). The optimal effector to target cell ratio was I00:1. In these conditions spontaneous release was usually lower than 15 per cent of total release.

Effect o f the virus on the NK activity The incubation of effector cells with ASF virus for 24 hours consistently resulted in a decrease of the NK activity shown by these cells compared with the

activity shown by cells incubated without the virus. The inhibitory effect of the virus was maintained at the different effector to target cell ratios tested. The virulent isolate induced a stronger inhibition than the avirulent; however, a homogenate of MS cells treated and incubated with effector cells in the same conditions as infected cells did not change their NK activity (Fig 2).

Timing o f the inhibition The inhibition of the NK activity increased with the time that the effector cells were incubated with the virus preparation in relation to cells incubated without the virus (Fig 3). Incubations longer than 24 hours suppressed most of the activity in cells incubated either in the presence or in the absence of the virus (data not shown).

Effect o f dose and different treatments o f the virus preparation on NK activity Swine mononuclear cells were tested for their NK activity after being incubated for 24 hours with the virulent ASFvirus isolate E75 treated in different ways and at different multiplicities of infection (Fig 4). The whole virus preparation was used without treatment

ASFV inhibition of natural killer activity

319

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FIG 2: Swine PBMCwere incubated for 24 hours in the absence of virus ( * ) or in the presence of the avirulent ASF virus isolate E7OMS 15 ( + ) or the virulent E75 (©) at a multiplicity of infection of O- 4. As controls, PBMC were incubated with a preparation of uninfected MS cells treated in the same way as the virus preparations (D). Then these cells were washed and assayed for their NK activity for 20 hours against 2 x 104 K-562 cells labelled with 51Cr at the effect©r/target ratios shown. Results represent the mean 4- SD of three independent experiments

or was ultracentrifuged twice at 100,000 g for 60 minutes to obtain and wash the sedimented fraction, where more than 90 per cent of viral particles were present as determined by testing its infectivity. The unfractionated virus preparation produced the strongest inhibitory effect, whereas the sedimented fraction produced a lower but still consistent effect depending on the dose. Nevertheless, when this sedimented fraction was inactivated by ultraviolet radiation its inhibitory effect was lost for the virus concentrations used in regular conditions (MOI: 0"4). In addition, a virus preparation obtained from PK-15 cells infected with Aujeszky's disease virus, which was treated in the same way as the ASF virus infected cells, did not affect the NK activity of mononuclear cells.

Discussion In the present study the inhibitory effect of ASF virus on the NK activity of porcine mononuclear cells is reported. The study was done by the coincubation

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FIG 3: Swine PBMC were incubated at 37°C for the times shown with an ASF virus preparation of isolate E75 (MOI 0 " 4 ) (©) or without virus ( * ) and then assayed for their NK activity against 2 x 1 0 4 51Cr labelled K-562 ceils at an effect©r/target ratio = 100. Results represent three independent experiments

of ASF virus with the effector cells for 24 hours before the assay in which K-562 or Molt-4 cells labelled with 51Cr were used as targets.

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Multiplicity of infection FIG 4: Swine PBMC were incubated at 37°C for 24 hours with different virus preparations of the ASF virulent isolate E75 at the MOI shown and then tested for their NK activity in the conditions described in Fig 3. The dotted line represents the NK activity shown by PBMC incubated in the absence of virus. A representative experiment run in triplicate is shown. Virus preparations: © Whole virus preparation, obtained as described in Materials and methods; + sedimented fraction, obtained by ultracentrifugation at 100,000 g for 60 minutes of the whole preparation; [] the same sedimented fraction after inactivation by ultraviolet radiation; * a preparation of Aujeszky's disease virus grown in PK-15 cells

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C. M e n d o z a , S. P. Videgain, F. A l o n s o

NK activity has been shown to be an effective mechanism for controlling cancer and infectious diseases (Welsh 1981, Biron and Welsh 1982, H e r b e r m a n 1985). However, there has been considerable controversy and confusion related to the characterisation of NK effector cells, which have been defined rather as effectors o f a function than as a cell type (Ortaldo and H e r b e r m a n 1984, Reynolds and O r t a l d o 1987). In pigs, where NK activity in peripheral blood cells has been well established (Kim et al 1980, Martin and Wardley 1984), the characterisation of effector cells is even more confusing than in humans and rodents, since porcine NK cells do not belong to the large granular lymphocyte population (Yang et al 1987, Pinto and Ferguson 1988). Accordingly, in the present study most of the experiments were performed with peripheral blood mononuclear cells, without further purification. However, some experiments done with non-adherent mononuclear cells did not show a significant change in the activity (data not shown). Since the main goal o f this study was the analysis of an inhibitory effect upon the porcine NK effector population, to see this inhibition it was necessary to use a target cell line providing the highest possible sensitivity to porcine NI( effector ceils. I n this respect the K-562 line provided the best conditions a m o n g the target cells tested. These cells also permitted the prolongation of the assay to 20 hours without increasing in excess the spontaneous release and, as they are not infected by ASF virus, a possible direct effect of virus infection on the susceptibility to NK lysis can be discarded. These results are mainly in agreement with those from most workers (Martin and Wardley 1984, Yang et al 1987, Pinto and Ferguson 1988). Norley and Wardley (1983) reported a depression in the NK activity in cells from pigs infected with ASF virus between three and six days after infection. The results obtained here are consistent with those of these authors; however, this study was performed in vitro. In this case, the work in vitro allows comparison o f the effect on the NK response o f the same cells treated or not with the virus. Besides this, it allows the use of virulefit viruses, avoiding the death of pigs and the bad conditions affecting coagulation and cell fragility that usually occurs in the blood obtained from pigs infected with these viruses in the first critical days of viraemia. The requirement of virus infectivity and the time dependence showed by the inhibition suggest that it is produced by a direct effect of the virus. Since the main target cell of the virus is the macrophage and infection of lymphocytes has not been clearly established (Casal et a11984, Minguez et a11988), it seems more probable that the effect is produced by factors released by infected macrophages during the 24-hour incubation.

The difference between the inhibitory effects produced by the whole virus preparation and the washed and sedimented virus also support this, showing that part of the inhibitory activity is in the supernatant. In this regard, in vitro inhibition of NK activity has been observed with peptides derived from HIV-1 and feline leukaemia virus (Sirianni et al 1990). Moreover, the present authors have recently reported a similar inhibitory effect mediated by factors released by ASF virus infected cells on the proliferative reponse o f lymphocytes to mitogens (Gonzalez et al 1990). Although it needs to be studied using a wider collection of ASF isolates, some results of this report indicated that virulent strains of the virus produced higher inhibitions of NK activity than avirulent strains. However, Norley and Wardley (1983) did not find any relation in vivo between NK activity and recovery, which somehow reflects the complexity of this disease. Independently, this study shows clearly that ASF virus inhibits the NK activity o f porcine mononuclear cells.

Acknowledgements We thank Dr Javier Dominguez for helpful discussion. This work was supported by the Instituto Nacional de Investigaciones Agrarias grant number 8170.

References BIRON, C. A. & WELSH, R. M. (1982) Activation and role of natural killer cells in virus infection. Medical Microbiology and Immunology 170, 155-172 BISHOP, G. A., GLORIOSO, J. C. & SCHWARTZ, S. A. (1983) Relationship between expression of herpes simplex virus glycoproteins and susceptibility of target cells to human natural killer activity. Journal of Experimental Medicine 157, 1544-1561 CASAL, I., ENJUANES, L. & VINUELA, E. (1984) Porcine leukocyte cellular subsets sensitiveto African swine fever virus in vitro. Journal of Virology 52, 37-46 GONZALEZ, S., MENDOZA, C., SA.NCHEZ-V1ZCAINO,J. M. & ALONSO, F. (1990) Inhibitory effect of African swine fever virus on lectin-dependent swine lymphocyte proliferation. Veterinary Immunology and Immunopathology 26, 71-80 HERBERMAN, R. B. (1985) NK cells and other natural effector cells. Mechanisms of Cytotoxicityby NKcells. Eds R. B. Herberman and D. M. Callewert. Orlando, AcademicPress. pp 1-17 HESS, W. R. (1971) African swinefever virus. VirologyMonographs 9, 1-33 KIM, Y. B., HUH, N. D., KOREN, H. S. & AMOS, D. B. (1980) Natural killing (NK)and antibody dependent cellular cytotoxicity (ADCC)in specific pathogen free (SPF) miniature swine and germ free piglets. 1. Comparison of NK and ADCC. Journal of Immunology 125,755-762 MARTIN, S. & WARDLEY, R. C. (1984) Natural cytotoxicity detected in swine using Aujeszky's disease virus infected targets. Research in VeterinaryScience 37, 211-218 MiNGUEZ, I., RUEDA, A., DOMiNGUEZ, J. & SA.NCHEZVIZCAINO (1988) Double labellingimmunohistologicalstudy of African swine fever virus-infected spleen and lymph nodes. Veterinary Pathology 25, 193-198

ASFV i n h i b i t i o n o f n a t u r a l killer a c t i v i t y NORLEY, S. G. & WARDLEY, R. C. (1983) Investigation of porcine natural-killer cell activity with reference to African swine fever virus infection. Immunology 49, 593-597 ORTALDO, J. R. & HERBERMAN, R. B. (1984) Heterogeneity of natural killer cells. Annual Review of Immunology 2, 359-394 PINTO, A. & FERGUSON, F. (1988) Characteristics of Yorkshire swine natural killer cells. Veterinary Immunology and Immunopathology 20, 15-29 REED, I. J. & MUENCH, R. H. (1938) A simple method to estimating fifty per cent end points. American Journal of Hygiene 27, 493-497 REYNOLDS, C. W. & ORTALDO, J. R. (1987) Natural killer activity: the definition of a function rather than a cell type. Immunology Today 8, 172-174 RUIZ GONZALVO, F., CARNERO, M. E., CABALLERO, C. & MARTfNEZ, J. (1986) Inhibition of African swine fever infection

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in the presence of immune sera in vivo and in vitro. American Journal of Veterinary Research 47, 1249-1252 SIRIANNI, M. C., TAGLIAFERRI, F. & AIUTI, F. (1990) Pathogenesis of the natural killer cell deficiency in AIDS. Immunology Today 11, 81-82 WELSH, R. M. (1981) Natural cell-mediated immunity during viral infections. Current Topics in Microbiology 92, 83-106 YANG, W. C., SCHULZ, R. D. & SPANO, J. S. (1987) Isolation and characterization of porcine natural killer (NK) cells. Veterinary Immunology and lmmunopathology 14, 345-356

Received October 11, 1990 Accepted July 8, 1991