Veterinary Immunology and Immunopathology, 30 (1992) 247-260
247
Elsevier Science Publishers B.V., Amsterdam
Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells S. Chinsakchai and T.W. Molitor Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA (Accepted 9 October 1990)
ABSTRACT Chinsakchai, S. and Molitor, T.W., 1992. Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol., 30: 247260. The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine 1I-,-2and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressiveeffects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph. ABBREVIATIONS CM, complete medium; Con A, ConcanavalinA; IL-2, interleukin-2;MOI, multiplicityof infection; NK, natural killer; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PI, post-infection; PRV, Pseudorabies virus.
INTRODUCTION Pseudorabies virus, Aujeszky's disease virus or porcine herpesvirus type 1 c a u s e s a n e c o n o m i c a l l y i m p o r t a n t d i s e a s e o f s w i n e m a n i f e s t e d as r e s p i r a t o r y
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$03.50
248
S. CHINSAKCHAI AND T.W. MOLITOR
disease, nervous signs and reproductive failure (reviewed in Baskerville et al., 1973; Lee and Wilson, 1979; Gustafson, 1986; Wittmann and Rhiza, 1989). Mortality and morbidity can be high in young pigs, whereas adults may have only clinical signs of respiratory disease and mild depression. The most common mode of transmission is by the nasopharyngeal route. Virus first replicates in nasopharyngeal mucosa, then invades the central nervous system via cranial nerves and disseminates throughout the body via the lymphatic system (McFerran and Dow, 1965; Sabo et al., 1969). Wittmann et al. (1980) suggested that virus spread to other organs through the lymphatic and hematogenic system by infected white blood cells. They detected trace amounts of virus from the peripheral blood mononuclear cells (PBMC) of non-vaccinated, infected pigs. Virus replication was also demonstrated in cultured PBMC collected from vaccinated and non-vaccinated infected pigs. However, detection of virus from PBMC was inconsistent and varied depending upon individual pigs, days post-infection (PI), methods of detection, and the number of white blood cells used in each assay. Donaldson et al. (1983) were able to isolate virus from the PBMC of only three of l0 pigs that were infected with the NIA-strain of Pseudorabies virus (PRV) at Day 5 after infection. Because isolation of PRV from white blood cells was variable, the hypothesis that PRV spreads systemically via PBMC is not convincing. Various facets of PRV infection in swine suggest that PRV is immunosuppressive. PRV-infected pigs showed a reduced humoral immune response to a Bordetella bronchiseptica and hog cholera vaccine (Lee et al., 1986; Lai et al., 1987) and also showed severe clinical signs to secondary infection with Pasteurella multocida and hog cholera, respectively (Fuentes and Pijoan, 1987; Lai et al., 1987). PRV infects most lymphoid tissues of swine as demonstrated by lymphoid necrosis and eosinophilic intranuclear inclusion bodies (Lee et al., 1986). In vitro, PRV replicated and caused functional impairment of swine alveolar macrophages (E1-Awar and Hahn, 1987; Iglesias et al., 1989a,b). Thus, cells of the macrophage lineage appear to be highly susceptible to PRV infection. The objectives of the present studies were to investigate the in vitro replication and potential immunosuppressive effects of PRV in swine PBMC. MATERIALS AND METHODS
Virus
The field isolate S-62 (USDA) strain (Mare et al., 1976) was propagated in Vero cells (ATCC, Rockville, MD). Infected cultures were frozen and thawed three times to release cell-associated virus. Supernatants were partial clarified by centrifugation at 5000 × g, for 10 min (4 °C ) prior to being harvested, aliquoted and stored at - 70 ° C. This will be referred to as virus stock.
REPLICATION AND IMMUNOSUPPRESSIVE EFFECTS OF PSEUDORABIES VIRUS
249
Virus titers were calculated by TCIDso end point (Reed and Meunch, 1938 ). UV-inactivated virus was prepared as described by Hatfield and Mims (1989). To prepare UV-PRV, virus stock was kept at 4°C and exposed to a 15 W UV lamp at a distance of 15 cm for 30 min with regular agitation. By this method, virus was completely inactivated as revealed by TCIDso end point titration. Mock infected controls were prepared in parallel using non-infected Vero cells by the same procedures. Peripheral blood mononuclear cells All blood samples were collected from 1-4-month-old, mixed breed pigs, which were seronegative for PRV by serum neutralization (Banks and Cartwright, 1983 ). PBMC were separated from heparinized blood on Ficoll-Hypaque gradients (Boyum, 1968). Cells were washed three times with phosphate-buffered saline (PBS) and resuspended in complete medium (CM). CM consisted of RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100/zg/ml streptomycin, 1 mM Lglutamine, 12 mM NaHCO3, 14 mM HEPES, and 5 × 10- 5 M 2-mercaptoethanol. Peripheral blood lymphocytes were enriched by removing monocytes via adherence to plastic tissue culture dishes (three cycles of adherence at 37 °C, in a 5% COz incubator for 3 h each). The number of monocytes before (10-15%) and after ( < 1%) panning was determined by morphology with Diff-Quick staining solution (American Scientific Products, McGawPark) and by phagocytosis of fluorescent latex beads (0.79/tm, Pandex, Mundelein, IL). Adherent cells were harvested from tissue culture dishes and were tested and found to be more than 95% monocytes. These cells were resuspended in CM at 1 × 106/ml and seeded into 24-well plates (CoStar, Cambridge, MA). Infection of cells " Lymphocyte and monocyte cultures were infected with the S-62 strain of PRV at 1,0.1, 0.01 and 0.001 multiplicity of infection (MOI). To infect lymphocytes, virus at the selected concentration were inoculated into cell suspensions. After 3 h of adsorption at 37°C, cells were washed with PBS by centrifugation at 4 00× g for 10 min to remove unadsorbed virus. Washing was repeated an additional two times. Infected lymphocytes were then plated into 24-well plates at a concentration of 1 × 106 cells/ml. To infect monocytes, virus was added to adherent monocyte cultures in 24-well plates. Nonadsorbed virus was removed after 3 h PI by washing again with PBS. Fresh CM was then added to the infected monocytes. The infected cultures were collected at 0, 24, 48 and 72 h PI (time 0 was considered after the adsorption period of 3 h) and evaluated for cell viability, virus progeny titer and viral DNA synthesis.
250
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Cell viability Control and infected cells were collected and washed with PBS and then mixed with an equal a m o u n t of one-part-per-million solution of ethidium bromide and acridine orange (Parks et al., 1979 ). Cells were observed with a fluorescent microscope. Viable and dead cells were counted from at least 200 cells from each sample and calculated for percent viability.
Detection of virus replication Infected cultures were frozen and thawed three times before titration on the indicator cell line, Vero. Viral DNA synthesis was detected by slot blot hybridization. Briefly, cells were lysed with an equal a m o u n t of sample buffer (20 m M Tris-HC1, pH 7.4, 20 m M EDTA, 1.0% SDS). Samples were treated with proteinase K ( 2 0 0 / l g / m l ) followed by RNase ( 5 0 / l g / m l ) with phenolchloroform-isoamyl extraction and concentration by ethanol precipitation following each enzyme treatment. The pellets were resuspended in 100/~1 TEl buffer followed by an equal volume of 0.6 N NaOH, then the mixtures were incubated for 30 min at 60 ° C to denature the DNA. Two hundred microliters of 2 M a m m o n i u m acetate were added to each sample and these samples were transferred to a Hybond-N m e m b r a n e (Amersham, Arlington Heights, IL) using a minifold II manifold (Schleicher and Schuell, Keena, N H ) . 3zp-Radionucleotide probes were prepared using the Riboprobe system vector, plasmid Sp64 according to the specification of the manufacturer (Promega Biotec, Madison, WI). The Bam HI fragment 7 of PRV was cloned into the plasmid Sp64 and served as the template for RNA probe synthesis. The membrane was hybridized with the RNA probe under high stringency conditions (50% formamide, 5 X SSC, 42 °C) followed by autoradiography. The specific activity of the radionucleotide probe ranged from 1 X 107 to 1 × 108 counts per minute ( C P M ) / / l g RNA. Non-infected monocyte and lymphocyte cultures and the Bam HI fragment 7 of PRV at various concentrations were included as controls on each membrane.
Lymphocyte proliferation and IL-2 assay PBMC were assayed for their response to the T cell mitogen, Concanavalin A (Con A), in the presence of virus stock, UV-inactivated virus and compared with equivalent mock infected controls. PBMC ( 100 ~tl) at a concentration of 2 × 106 cells/ml were plated into 96-well tissue culture plates (Costar, Cambridge, MA), followed by 100/tl of test virus preparations or mock infected controls over a range of MOI or dilutions (MOI 1 to 0.0001; dilution 1 : 5 to 1 : 50 000). Con A (50/11) at a concentration 12.5/~g/ml in CM or CM alone was added to each set of cultures (final concentration of Con A, 5/~g/ ml). All cultures were performed in triplicate and incubated at 37 ° C, 5% CO2 in a humidified chamber for 48 h. Cultures were labeled with 1 ~tCi/well of 3H-thymidine (Amersham, Arlington Heights, IL, sp. act., 52 C i / m m o l ) for
REPLICATION AND IMMUNOSUPPRESS1VE EFFECTS OF PSEUDORABIES VIRUS
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18 h. Cells were harvested onto glass fiber filter paper by a cell harvester (Skatron, Sterling, VA). The incorporation of 3H-thymidine into PBMC was measured in a scintillation counter. Results were expressed as the mean CPM of triplicate cultures.
Interleukin-2 assay Interleukin-2 (IL-2) synthesis was performed in parallel with lymphocyte proliferation. PBMC at a concentration identical to that described for lymphocyte proliferation were cultured in 24-well plates with the virus stock inoculum ranging from 1 to 0.0001 MOI, and 5/~g of Con A for 48 h, 37°C, 5% CO2. Supernatants were collected and frozen at - 7 0 ° C . Since residual PRV may infect and kill the IL-2 indicator cell line, supernatants from infected and non-infected cultures were treated with swine anti-PRV serum ( 1 : 40 dilution) for 1 h at 37°C prior to evaluating for IL-2. Supernatants were also tested for the presence of infectious virus by inoculation onto Vero cells. Such conditions completely neutralized virus in the supernatants. IL-2 was evaluated by the use of an IL-2-dependent cell line, CTL-FD (Charley et al., 1985 ), a gift from Dr. Keith Kelly (Department of Animal Science, Urbana Champaign, IL). CTL-FD were washed three times with PBS to remove residual IL-2 and were resuspended in CM at 2.5 X 105 cells/ml. Cells were cultured with 100/tl of serial dilutions of the supernatants for 48 h in 96-well tissue culture plates. The cultures were labeled with 1/tCi/well of 3H-thymidine for 18 h. Cells were harvested onto glass fiber filter paper for liquid scintillation counting. Results were expressed as the mean CPM of triplicate cultures. Standard h u m a n recombinant IL-2 (R&D Systems, Minneapolis, M N ) were included in every assay. Natural killer cell activity The chromium-released microcytotoxicity technique was performed using a modification of the m e t h o d of Yang et al. (1987 ). Briefly, a myeloid leukemia cell line, K562 cells (ATCC, Rockville, M D ) were labeled with 51CrNa2CrO4 (Du Pont, Wilmington, DE, sp. act., 518.82 m C i / m g , 1 0 0 / t C i / 5 × 10 6 cells) for 2 h at 37°C, 5% CO2. After labeling, cells were washed once and further incubated for 30 min in CM to reduce spontaneous release. Cells were washed and resuspended in CM at a concentration of 2.5 × 105 cells/ml and used as target cells. PBMC infected with 1, 0.1 and 0.01 MOI of PRV stock for 24 h or non-infected PBMC were used as effector cells. Target cells ( 100/zl) were cultured with 100/tl of effector cells in 96-well tissue culture plates. The NK assay was performed in triplicate at an E: T ratio of 100: 1 and incubated for 18 h at 37°C. Supernatants ( 100/~1) from each well were collected after centrifugation for 10 min at 400 g and radioactivity was measured by a g a m m a counter. The percentage specific lysis was calculated by the following formula.
252
s. CHINSAKCHAI AND T.W. MOLITOR
Percent specific lysis =
test C P M - s p o n t a n e o u s CPM max C P M - spontaneous c P M X 1O0
Maximum CPM was determined by lysing target cells with 100/tl 10% Triton X 100. Spontaneous CPM was 5~Cr released from target cell cultures without effector cells.
Statistical analysis The effect of PRV on lymphocyte proliferation and IL-2 synthesis was analyzed by analysis of variance and multiple comparison test (Tukey's method). The effect of PRV on cell viability at different HPI was determined using an analysis of variance following the factorial experimental design and reconstitution of lymphocyte blastogenesis experiments was analyzed by paired t-test. A statistical package (Statistix version 3.0, NH analytical software, St. Paul, MN ) was used for computing the statistical analyses. RESULTS
The replication of PR V in peripheral blood monocytes and lymphocytes To evaluate whether PBMC were permissive to PRV infection, adherent and non-adherent populations of cells were infected with the S-62 strain of PRV. Virus replicated in both monocytes and lymphocytes (Fig. 1 ). The yield of virus progeny from infected monocyte cultures (Fig. 1A) reached peak
6
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Fig. 1. PRV replication in (A) swine peripheral blood monocytes and (B) lymphocytes cultures. Cultures were infected with MOI of 1.0, 0.1,0.01 and 0.001 and assayed for progeny virus at 24 h intervals PI. Virus progeny titer are expressed as log 10 TCIDso/ml. ( O ) 1 MOI, ( • ) 0.1MOI, ( A ) 0.01 MOI, ( A ) 0.001MOI.
REPLICATION AND IMMUNOSUPPRESSIVE EFFECTS OF PSEUDORABIES VIRUS
LO
Mgb
2 53
PRV
DNA
0
24
50 ng 5.0 ng
72
0.5 ng
null A
B
C
Fig. 2. PRV DNA synthesis determined by slot blot hybridization. Cells were infected at an MOI of 0.1. Infected cultures were collected at either 0, 24, 48 or 72 h PI. Uninfected cells (null) were included as controls. DNA was extracted and transferred to nylon membranes. Membranes were hybridized with 32p-radiolabeled PRV RNA probe. Lane A, lymphocytes; Lane B, monocytes; Lane C, DNA standards containing the Bam HI fragment 7 of PRV genome representing 50, 5 and 0.5 ng. titers o f 107-107.4 T C I D s o / m l at 2 4 - 4 8 h PI, whereas only 104-104.7 T C I D s o / ml were found in infected lymphocyte cultures (Fig. 1B). These titers were found for all four M O I o f inoculated virus. After reaching peak valves, virus titers remained constant up to 72 h PI. Viral D N A synthesis in monocytes and lymphocytes inoculated with 0.1 M O I o f P R V was evaluated from 0 - 7 2 h PI in 24 h intervals by slot blot hybridization with a 32p-radiolabeled R N A probe (Fig. 2). As shown, P R V D N A synthesis from infected m o n o c y t e s cultures increased with time and was higher than that o f infected lymphocyte cultures. There was a P R V specific signal from PRV-infected lymphocytes; however there was only a slight increase in signal over the period o f time examined. There was no cross-hybridization o f the probe with D N A isolated from non-infected cells.
Effect of virus replication on cell viability and immune functions Infection o f m o n o c y t e s by P R V was followed by a dramatic reduction in cell viability; all infected cells from three MOIs o f inoculated virus were completely dead at 72 h PI (Fig. 3A). Although lymphocyte viability declined with culturing ( P < 0.05 ) especially after 24 h, there was no effect o f P R V at any of the M O I on lymphocyte viability when c o m p a r e d with non-infected cells (Fig. 3B). The ability of P B M C to respond to a non-specific mitogenic stimuli, Con A, in the presence o f virus was determined by 3H-thymidine incorporation. P R V caused a reduction o f incorporation which was M O I dependent (Fig. 4A). There was a significant reduction in the responses at an M O I o f 1 to 0.001 ( P < 0.05 ) c o m p a r e d with the controls. To determine whether this im-
254
S. CHINSAKCHAI AND T.W. MOLITOR
12° I 100
80
60
40
20 >i-
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247
Elsevier Science Publishers B.V., Amsterdam
Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells S. Chinsakchai and T.W. Molitor Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA (Accepted 9 October 1990)
ABSTRACT Chinsakchai, S. and Molitor, T.W., 1992. Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol., 30: 247260. The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine 1I-,-2and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressiveeffects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph. ABBREVIATIONS CM, complete medium; Con A, ConcanavalinA; IL-2, interleukin-2;MOI, multiplicityof infection; NK, natural killer; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PI, post-infection; PRV, Pseudorabies virus.
INTRODUCTION Pseudorabies virus, Aujeszky's disease virus or porcine herpesvirus type 1 c a u s e s a n e c o n o m i c a l l y i m p o r t a n t d i s e a s e o f s w i n e m a n i f e s t e d as r e s p i r a t o r y
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$03.50
248
S. CHINSAKCHAI AND T.W. MOLITOR
disease, nervous signs and reproductive failure (reviewed in Baskerville et al., 1973; Lee and Wilson, 1979; Gustafson, 1986; Wittmann and Rhiza, 1989). Mortality and morbidity can be high in young pigs, whereas adults may have only clinical signs of respiratory disease and mild depression. The most common mode of transmission is by the nasopharyngeal route. Virus first replicates in nasopharyngeal mucosa, then invades the central nervous system via cranial nerves and disseminates throughout the body via the lymphatic system (McFerran and Dow, 1965; Sabo et al., 1969). Wittmann et al. (1980) suggested that virus spread to other organs through the lymphatic and hematogenic system by infected white blood cells. They detected trace amounts of virus from the peripheral blood mononuclear cells (PBMC) of non-vaccinated, infected pigs. Virus replication was also demonstrated in cultured PBMC collected from vaccinated and non-vaccinated infected pigs. However, detection of virus from PBMC was inconsistent and varied depending upon individual pigs, days post-infection (PI), methods of detection, and the number of white blood cells used in each assay. Donaldson et al. (1983) were able to isolate virus from the PBMC of only three of l0 pigs that were infected with the NIA-strain of Pseudorabies virus (PRV) at Day 5 after infection. Because isolation of PRV from white blood cells was variable, the hypothesis that PRV spreads systemically via PBMC is not convincing. Various facets of PRV infection in swine suggest that PRV is immunosuppressive. PRV-infected pigs showed a reduced humoral immune response to a Bordetella bronchiseptica and hog cholera vaccine (Lee et al., 1986; Lai et al., 1987) and also showed severe clinical signs to secondary infection with Pasteurella multocida and hog cholera, respectively (Fuentes and Pijoan, 1987; Lai et al., 1987). PRV infects most lymphoid tissues of swine as demonstrated by lymphoid necrosis and eosinophilic intranuclear inclusion bodies (Lee et al., 1986). In vitro, PRV replicated and caused functional impairment of swine alveolar macrophages (E1-Awar and Hahn, 1987; Iglesias et al., 1989a,b). Thus, cells of the macrophage lineage appear to be highly susceptible to PRV infection. The objectives of the present studies were to investigate the in vitro replication and potential immunosuppressive effects of PRV in swine PBMC. MATERIALS AND METHODS
Virus
The field isolate S-62 (USDA) strain (Mare et al., 1976) was propagated in Vero cells (ATCC, Rockville, MD). Infected cultures were frozen and thawed three times to release cell-associated virus. Supernatants were partial clarified by centrifugation at 5000 × g, for 10 min (4 °C ) prior to being harvested, aliquoted and stored at - 70 ° C. This will be referred to as virus stock.
REPLICATION AND IMMUNOSUPPRESSIVE EFFECTS OF PSEUDORABIES VIRUS
249
Virus titers were calculated by TCIDso end point (Reed and Meunch, 1938 ). UV-inactivated virus was prepared as described by Hatfield and Mims (1989). To prepare UV-PRV, virus stock was kept at 4°C and exposed to a 15 W UV lamp at a distance of 15 cm for 30 min with regular agitation. By this method, virus was completely inactivated as revealed by TCIDso end point titration. Mock infected controls were prepared in parallel using non-infected Vero cells by the same procedures. Peripheral blood mononuclear cells All blood samples were collected from 1-4-month-old, mixed breed pigs, which were seronegative for PRV by serum neutralization (Banks and Cartwright, 1983 ). PBMC were separated from heparinized blood on Ficoll-Hypaque gradients (Boyum, 1968). Cells were washed three times with phosphate-buffered saline (PBS) and resuspended in complete medium (CM). CM consisted of RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100/zg/ml streptomycin, 1 mM Lglutamine, 12 mM NaHCO3, 14 mM HEPES, and 5 × 10- 5 M 2-mercaptoethanol. Peripheral blood lymphocytes were enriched by removing monocytes via adherence to plastic tissue culture dishes (three cycles of adherence at 37 °C, in a 5% COz incubator for 3 h each). The number of monocytes before (10-15%) and after ( < 1%) panning was determined by morphology with Diff-Quick staining solution (American Scientific Products, McGawPark) and by phagocytosis of fluorescent latex beads (0.79/tm, Pandex, Mundelein, IL). Adherent cells were harvested from tissue culture dishes and were tested and found to be more than 95% monocytes. These cells were resuspended in CM at 1 × 106/ml and seeded into 24-well plates (CoStar, Cambridge, MA). Infection of cells " Lymphocyte and monocyte cultures were infected with the S-62 strain of PRV at 1,0.1, 0.01 and 0.001 multiplicity of infection (MOI). To infect lymphocytes, virus at the selected concentration were inoculated into cell suspensions. After 3 h of adsorption at 37°C, cells were washed with PBS by centrifugation at 4 00× g for 10 min to remove unadsorbed virus. Washing was repeated an additional two times. Infected lymphocytes were then plated into 24-well plates at a concentration of 1 × 106 cells/ml. To infect monocytes, virus was added to adherent monocyte cultures in 24-well plates. Nonadsorbed virus was removed after 3 h PI by washing again with PBS. Fresh CM was then added to the infected monocytes. The infected cultures were collected at 0, 24, 48 and 72 h PI (time 0 was considered after the adsorption period of 3 h) and evaluated for cell viability, virus progeny titer and viral DNA synthesis.
250
s. CHINSAKCHAI AND T.W. MOLITOR
Cell viability Control and infected cells were collected and washed with PBS and then mixed with an equal a m o u n t of one-part-per-million solution of ethidium bromide and acridine orange (Parks et al., 1979 ). Cells were observed with a fluorescent microscope. Viable and dead cells were counted from at least 200 cells from each sample and calculated for percent viability.
Detection of virus replication Infected cultures were frozen and thawed three times before titration on the indicator cell line, Vero. Viral DNA synthesis was detected by slot blot hybridization. Briefly, cells were lysed with an equal a m o u n t of sample buffer (20 m M Tris-HC1, pH 7.4, 20 m M EDTA, 1.0% SDS). Samples were treated with proteinase K ( 2 0 0 / l g / m l ) followed by RNase ( 5 0 / l g / m l ) with phenolchloroform-isoamyl extraction and concentration by ethanol precipitation following each enzyme treatment. The pellets were resuspended in 100/~1 TEl buffer followed by an equal volume of 0.6 N NaOH, then the mixtures were incubated for 30 min at 60 ° C to denature the DNA. Two hundred microliters of 2 M a m m o n i u m acetate were added to each sample and these samples were transferred to a Hybond-N m e m b r a n e (Amersham, Arlington Heights, IL) using a minifold II manifold (Schleicher and Schuell, Keena, N H ) . 3zp-Radionucleotide probes were prepared using the Riboprobe system vector, plasmid Sp64 according to the specification of the manufacturer (Promega Biotec, Madison, WI). The Bam HI fragment 7 of PRV was cloned into the plasmid Sp64 and served as the template for RNA probe synthesis. The membrane was hybridized with the RNA probe under high stringency conditions (50% formamide, 5 X SSC, 42 °C) followed by autoradiography. The specific activity of the radionucleotide probe ranged from 1 X 107 to 1 × 108 counts per minute ( C P M ) / / l g RNA. Non-infected monocyte and lymphocyte cultures and the Bam HI fragment 7 of PRV at various concentrations were included as controls on each membrane.
Lymphocyte proliferation and IL-2 assay PBMC were assayed for their response to the T cell mitogen, Concanavalin A (Con A), in the presence of virus stock, UV-inactivated virus and compared with equivalent mock infected controls. PBMC ( 100 ~tl) at a concentration of 2 × 106 cells/ml were plated into 96-well tissue culture plates (Costar, Cambridge, MA), followed by 100/tl of test virus preparations or mock infected controls over a range of MOI or dilutions (MOI 1 to 0.0001; dilution 1 : 5 to 1 : 50 000). Con A (50/11) at a concentration 12.5/~g/ml in CM or CM alone was added to each set of cultures (final concentration of Con A, 5/~g/ ml). All cultures were performed in triplicate and incubated at 37 ° C, 5% CO2 in a humidified chamber for 48 h. Cultures were labeled with 1 ~tCi/well of 3H-thymidine (Amersham, Arlington Heights, IL, sp. act., 52 C i / m m o l ) for
REPLICATION AND IMMUNOSUPPRESS1VE EFFECTS OF PSEUDORABIES VIRUS
251
18 h. Cells were harvested onto glass fiber filter paper by a cell harvester (Skatron, Sterling, VA). The incorporation of 3H-thymidine into PBMC was measured in a scintillation counter. Results were expressed as the mean CPM of triplicate cultures.
Interleukin-2 assay Interleukin-2 (IL-2) synthesis was performed in parallel with lymphocyte proliferation. PBMC at a concentration identical to that described for lymphocyte proliferation were cultured in 24-well plates with the virus stock inoculum ranging from 1 to 0.0001 MOI, and 5/~g of Con A for 48 h, 37°C, 5% CO2. Supernatants were collected and frozen at - 7 0 ° C . Since residual PRV may infect and kill the IL-2 indicator cell line, supernatants from infected and non-infected cultures were treated with swine anti-PRV serum ( 1 : 40 dilution) for 1 h at 37°C prior to evaluating for IL-2. Supernatants were also tested for the presence of infectious virus by inoculation onto Vero cells. Such conditions completely neutralized virus in the supernatants. IL-2 was evaluated by the use of an IL-2-dependent cell line, CTL-FD (Charley et al., 1985 ), a gift from Dr. Keith Kelly (Department of Animal Science, Urbana Champaign, IL). CTL-FD were washed three times with PBS to remove residual IL-2 and were resuspended in CM at 2.5 X 105 cells/ml. Cells were cultured with 100/tl of serial dilutions of the supernatants for 48 h in 96-well tissue culture plates. The cultures were labeled with 1/tCi/well of 3H-thymidine for 18 h. Cells were harvested onto glass fiber filter paper for liquid scintillation counting. Results were expressed as the mean CPM of triplicate cultures. Standard h u m a n recombinant IL-2 (R&D Systems, Minneapolis, M N ) were included in every assay. Natural killer cell activity The chromium-released microcytotoxicity technique was performed using a modification of the m e t h o d of Yang et al. (1987 ). Briefly, a myeloid leukemia cell line, K562 cells (ATCC, Rockville, M D ) were labeled with 51CrNa2CrO4 (Du Pont, Wilmington, DE, sp. act., 518.82 m C i / m g , 1 0 0 / t C i / 5 × 10 6 cells) for 2 h at 37°C, 5% CO2. After labeling, cells were washed once and further incubated for 30 min in CM to reduce spontaneous release. Cells were washed and resuspended in CM at a concentration of 2.5 × 105 cells/ml and used as target cells. PBMC infected with 1, 0.1 and 0.01 MOI of PRV stock for 24 h or non-infected PBMC were used as effector cells. Target cells ( 100/zl) were cultured with 100/tl of effector cells in 96-well tissue culture plates. The NK assay was performed in triplicate at an E: T ratio of 100: 1 and incubated for 18 h at 37°C. Supernatants ( 100/~1) from each well were collected after centrifugation for 10 min at 400 g and radioactivity was measured by a g a m m a counter. The percentage specific lysis was calculated by the following formula.
252
s. CHINSAKCHAI AND T.W. MOLITOR
Percent specific lysis =
test C P M - s p o n t a n e o u s CPM max C P M - spontaneous c P M X 1O0
Maximum CPM was determined by lysing target cells with 100/tl 10% Triton X 100. Spontaneous CPM was 5~Cr released from target cell cultures without effector cells.
Statistical analysis The effect of PRV on lymphocyte proliferation and IL-2 synthesis was analyzed by analysis of variance and multiple comparison test (Tukey's method). The effect of PRV on cell viability at different HPI was determined using an analysis of variance following the factorial experimental design and reconstitution of lymphocyte blastogenesis experiments was analyzed by paired t-test. A statistical package (Statistix version 3.0, NH analytical software, St. Paul, MN ) was used for computing the statistical analyses. RESULTS
The replication of PR V in peripheral blood monocytes and lymphocytes To evaluate whether PBMC were permissive to PRV infection, adherent and non-adherent populations of cells were infected with the S-62 strain of PRV. Virus replicated in both monocytes and lymphocytes (Fig. 1 ). The yield of virus progeny from infected monocyte cultures (Fig. 1A) reached peak
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Fig. 1. PRV replication in (A) swine peripheral blood monocytes and (B) lymphocytes cultures. Cultures were infected with MOI of 1.0, 0.1,0.01 and 0.001 and assayed for progeny virus at 24 h intervals PI. Virus progeny titer are expressed as log 10 TCIDso/ml. ( O ) 1 MOI, ( • ) 0.1MOI, ( A ) 0.01 MOI, ( A ) 0.001MOI.
REPLICATION AND IMMUNOSUPPRESSIVE EFFECTS OF PSEUDORABIES VIRUS
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Fig. 2. PRV DNA synthesis determined by slot blot hybridization. Cells were infected at an MOI of 0.1. Infected cultures were collected at either 0, 24, 48 or 72 h PI. Uninfected cells (null) were included as controls. DNA was extracted and transferred to nylon membranes. Membranes were hybridized with 32p-radiolabeled PRV RNA probe. Lane A, lymphocytes; Lane B, monocytes; Lane C, DNA standards containing the Bam HI fragment 7 of PRV genome representing 50, 5 and 0.5 ng. titers o f 107-107.4 T C I D s o / m l at 2 4 - 4 8 h PI, whereas only 104-104.7 T C I D s o / ml were found in infected lymphocyte cultures (Fig. 1B). These titers were found for all four M O I o f inoculated virus. After reaching peak valves, virus titers remained constant up to 72 h PI. Viral D N A synthesis in monocytes and lymphocytes inoculated with 0.1 M O I o f P R V was evaluated from 0 - 7 2 h PI in 24 h intervals by slot blot hybridization with a 32p-radiolabeled R N A probe (Fig. 2). As shown, P R V D N A synthesis from infected m o n o c y t e s cultures increased with time and was higher than that o f infected lymphocyte cultures. There was a P R V specific signal from PRV-infected lymphocytes; however there was only a slight increase in signal over the period o f time examined. There was no cross-hybridization o f the probe with D N A isolated from non-infected cells.
Effect of virus replication on cell viability and immune functions Infection o f m o n o c y t e s by P R V was followed by a dramatic reduction in cell viability; all infected cells from three MOIs o f inoculated virus were completely dead at 72 h PI (Fig. 3A). Although lymphocyte viability declined with culturing ( P < 0.05 ) especially after 24 h, there was no effect o f P R V at any of the M O I on lymphocyte viability when c o m p a r e d with non-infected cells (Fig. 3B). The ability of P B M C to respond to a non-specific mitogenic stimuli, Con A, in the presence o f virus was determined by 3H-thymidine incorporation. P R V caused a reduction o f incorporation which was M O I dependent (Fig. 4A). There was a significant reduction in the responses at an M O I o f 1 to 0.001 ( P < 0.05 ) c o m p a r e d with the controls. To determine whether this im-
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S. CHINSAKCHAI AND T.W. MOLITOR
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