Effect of porcine circovirus infection on porcine alveolar macrophage function

Veterinary immunology and Veterinary

Immunology and lmmunopathology 49 (1996) 295-306

immunopathology

Effect of porcine circovirus infection on porcine alveolar macrophage function F. McNeilly

*,

G.M. Allan, J.C. Foster, B.M. Adair, M.S. McNulty

Department ofAgriculture for Northern Ireland, Veterinary Sciences Division, Stoney Road, Belfast, UK Accepted

26 April 1995

Abstract The effect of porcine circovirns (PCV) infection of porcine alveolar macrophage cultures on some of the functional properties of these cells are reported. PCV infection of alveolar macrophages did not affect their ability to phagocytose and kill complement-coated yeast cells or the expression of Fc or complement receptors. A transient increase in major histocompatibility complex (MHC) class I expression in PCV-infected cells was observed 4 days after infection and a decrease in the number of cells expressing MHC class II antigens was observed 8 days after infection. Infection of alveolar macrophages with PCV also resulted in a transient decrease in their ability to act as accessory cells in mitogen-induced lymphocyte proliferation of monocyte-depleted porcine peripheral blood mononuclear cells.

1. Introduction Porcine circovirus (PCV) was originally described as a picornavirus-like viral contaminant of continuous pig kidney cell lines (T&her et al., 1974). Subsequently, Tischer et al. (1982) demonstrated that PCV was a non-enveloped, small (17 nm) virus, containing a single stranded, circular DNA genome. Recently, it has been proposed that PCV, chicken anaemia virus (CAV) and psittacine beak and feather disease virus (PBFDV) should be classified in a new virus family called the Circoviridae (Studdert, 1993). Immune dysfunction following infection with CAV has been reported (Adair et al., 1991) and, although this virus is not known to replicate in mature macrophages,

’ Corresponding

author.

0165-2427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0165-2427(95)05476-6

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macrophage dysfunction following infection of chicks has also been reported (McConnell et al., 1993). PBFDV is also thought to be immunosuppressive, and this immunosuppression has been associated with replication of the virus in macrophages (Latimer et al., 1990). The replication of PCV in porcine macrophage cultures has recently been reported (Allan et al., 1994) and PCV antigen has been demonstrated in macrophage-like cells in young pigs following experimental infections (Allan et al., 1995). This communication reports an investigation into the effects of PCV infection of cultured porcine alveolar macrophages on some macrophage functions.

2. Materials and methods 2.1. Virus A pool of PCV was prepared using a PCV-persistently infected continuous pig kidney cell line (PK/15/W) using procedures described previously (Allan et al., 1994). The titre of this pool was calculated to be 107.00TCIDS0 per 0.1 ml. A PCV-negative control inoculum was prepared in a similar manner from a PCV-free continuous pig kidney cell line (PK/lS/H). 2.2. Experimental

animals

Eight 3-4 week old pigs, from the same litter, were used as donor animals for all experiments. These animals were obtained from a minimal disease, breeder-finisher unit known to be infected with PCV. All the pigs had maternal antibody titres to PCV in excess of l/1000, as determined by indirect immunofluorescent (IIF) staining (Allan et al., 1994). 2.3. Porcine alveolar macrophages Porcine alveolar macrophages @MS) were collected and processed using the procedures described previously (Allan et al., 19941, except that purified AM preparations were cultured in teflon flasks (Nalgene Nalge Company, Rochester, USA). Cells were seeded in 250 ml volumes at a concentration of 2 X 10’ cells ml-’ and maintained for 72 h at 37°C in 7% CO,, prior to inoculation as described below. Separate AM cultures were inoculated with 2 ml of PCV or control inoculum and incubated at 37°C in a 7% CO, atmosphere in a humidified chamber. The cultures were monitored at selected time intervals for viability by trypan blue dye exclusion and for PCV replication by IIF (Allan et al., 1994). 2.4. Phagocytosis

and microbicidal

activity

The method used to assess AM phagocytic and microbial activity was essentially that of Warr and Jakab (1979) with minor modifications. Briefly, Candida krusei cells were washed three times and resuspended in RPM1 1640 medium containing glutamine and without serum or antibiotics (RPMI-NSA) before counting. The cells were then pelleted

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by centrifugation for 5 min at 350 X g and coated with guinea pig complement (Sera Lab Ltd., Crawley Down, Sussex, UK) by resuspension in RPMI-NSA containing 12.5 X 50% haemolytic complement units ml-‘, at a ratio of 1 ml per 10’ cells. The suspension was incubated at 37°C for 30 min and again centrifuged and resuspended at lo7 cells ml-’ in RPMI-NSA containing 5% fresh guinea pig serum. Aliquots (10 ml) of the PCV and control-inoculated cultures were removed from the teflon flasks at 2, 4 and 8 days after inoculation and distributed in 1 ml volumes into four-well plastic multidishes (Nunclon Gibco BRL, Paisley, UK) containing sterile round glass coverslips and the AMs allowed to adhere for 2 h. Complement-coated yeast cells were added to washed PCV-infected or control AM cultures at 20 yeast cells per AM and incubated for 30 min at 37°C. The cultures were then washed three times with warm RPMI-NSA to remove excess yeast cells. Fresh medium was added and cultures were incubated for a further 2.5 h at 37°C in 5% CO,. The medium was then removed and replaced with a 0.1 mg ml-’ acridine orange (Sigma-Aldrich Co Ltd, Poole, UK) solution, in 0.01 M phosphate buffered saline pH 7.2 (PBS), for 2 min at room temperature. After washing with PBS, the cultures were examined under incident UV illumination. The percentages of AM with ingested yeast cells along with the percentage containing killed yeast cells (stained red) were counted in ten microscopic fields. 2.5. Fc and complement

receptor assays

The effect of PCV infection of AM on Fc receptor (FcR) and complement receptor (CR) expression was determined by rosetting procedures using antibody- or complement-opsonised sheep erythrocytes (SRBC) as described previously (Adair and McNulty, 1992). For demonstration of FcR expression, a 1% suspension of SRBC opsonised with a sub-agglutinating dilution (l/500) of a 7s IgG rabbit anti-SRBC antiserum (Diamedix Corporation, Miami, FL) was added to AM cultures previously seeded as described above, and the cultures incubated at 37°C for 30 min. Coverslips were removed from wells and excess SRBC removed by rinsing twice in PBS. For demonstration of CRs, a 1% suspension of SRBCs opsonised with a sub-agglutinating dilution (l/100) of a 19s IgM rabbit anti-SRBC antiserum (Diamedix Corporation) and complement (fresh mouse serum used as complement source) were incubated with AMs and the procedure completed as described above. Counting of rosettes was carried out following fixation in methanol for 1 min at room temperature and staining with Diff Quik (Baxter Health Care Ltd., Runcorn, UK). Coverslips were then mounted in Per-mount (Fisher Scientific, Fair Lawn, NJ) and examined using a X 40 oil immersion objective. Macrophages were considered positive for receptor expression if three or more SRBCs were attached to a cell. The percentage of rosetted macrophages in ten microscopic fields was determined. 2.6. Expression

of major histocompatibility

complex (MHC) class I and II antigens

Monoclonal antibodies (Mabs) specific for porcine MHC class I (PT85 A) and class II (H42 A) were obtained from VMRD Inc., Washington, USA and used at a l/100 dilution. The Mabs were diluted in PBS, supplemented with 10% heat-inactivated pooled rabbit serum (PBSR).

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PCV-infected and control preparations of porcine macrophages were removed from the teflon flasks at 2, 4 and 8 days after inoculation, the cell concentrations adjusted to lo7 cells ml-’ and aliquoted in 100 ~1 volumes into each of eight wells of a U-bottomed 96-multiwell plate. The cells were then pelleted by rapid acceleration to 2000 X g on a centrifuge followed by immediate braking to a standstill. Duplicate PCV-infected and control wells were resuspended in 25 ~1 volumes of each of the above Mabs. In addition, separate control wells containing cell pellets were resuspended in 25 ~1 volumes of PBSA and a Mab prepared against CAV (4Bl) (McNulty et al., 1990). These mixtures were incubated at 4°C for 30 min. Cells were again pelleted as described above and washed twice in PBSA by centrifugation and suspension. After the final wash the cell pellets were resuspended in 25 ~1 of a l/500 dilution, in PBSR, of a FITC-labelled, rabbit anti-mouse Ig (FITCRAM) (Nordic Immunological Laboratories, Tilburg, Netherlands). The mixtures were then incubated and washed as before and the resulting pellets resuspended in 750 ,ul of PBS containing 1% paraformaldehyde (Sigma, UK). All preparations were stored at 4°C prior to analysis using a fluorescent activated cell sorter (FACS). FACS analysis was carried out using a Becton Dickinson FACS Vantage analyser (Becton Dickinson UK Ltd., Oxford, UK). Samples were gated using the control preparations and the analyser programmed to record 10’ events per sample. Resulting data were analysed and assessed using the Lysis II software programme. 2.7. Depletion

of monocytes from porcine peripheral

blood mononuclear

cells

Depletion of monocytes from porcine peripheral blood mononuclear cells (PBMNCS) was performed using a sequential two-step procedure. A 50 ml volume of heparinised blood was collected from a donor pig and PBMNC separated on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the method described by Adair et al. (1992) and the cells resuspended in RPM1 1640 medium containing glutamine and supplemented with 10% foetal bovine serum (FBS), 1% non-essential amino acids, 1 mM sodium pyruvate, 5 x 1O-5 M 2-mercaptoethanol and 100 pg ml-’ gentamicin (complete medium). Cell viability was determined by trypan blue dye exclusion, the cell concentration was adjusted to 2 X lo6 viable cells ml-’ in complete medium, and 40 ml added to a 75 cm* plastic cell culture flask and incubated at 37°C for 2 h. Non-adherent cells were removed by pipette and recovered by centrifugation at 2000 X g for 10 min. The cell population was resuspended in complete medium and a cell count performed as described above. This cell population was further depleted of monocytes using a negative selection procedure with antibody-coated magnetic beads (Dynal (UK) Ltd, Bromborough, Wirral, UK). A Mab designated GM1 (VMRD), specific for porcine monocytes/macrophages, was diluted in a 2 ml volume of the cell preparation described above, to a final concentration of l/100. The mixture was incubated at 4°C for 30 min, washed three times by centrifugation and suspension, the cells resuspended in 2 ml of complete medium and goat anti-mouse Ig-coated magnetic beads (Catalogue number 110.06; Dynal (UK) Ltd, Bromborough, Wirral, UK) added to a final ratio of 40 beads to one target cell (monocyte/macrophage). The preparation was incubated, with agitation, for 30 min at 4°C after which the beads-target cell complexes were removed using

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2YY

a magnetic particle concentrator (Dynal-MPC 6, Dynal, Oslo, Norway). The remaining depleted cell preparation was collected by centrifugation as described above. The success of monocyte depletion was determined by comparing the lymphocyte stimulation indices of the depleted cell population with equivalent non-depleted preparations following stimulation with concanavalin A (Con A) and pokeweed mitogen (PWM). Briefly, 2 X lo5 cells of the depleted and non-depleted preparations were dispensed, in a 200 ~1 volume, into a 96-multiwell plate. Con A or PWM were then added to triplicate wells of depleted and non-depleted samples to a final concentration of 1.00 pg ml-’ and the mixtures incubated at 37°C for 24 h. A 25 ~1 volume of tritiated (methyl-3H) thymidine (40-60 Ci mmol-’ ; Amersham International, UK) was added to each well for the final 6 h of the incubation period. The contents of each well were then harvested using an automated cell harvester (LKB, Wallac (UK) Ltd, Milton Keynes, UK) onto glass fibre filter mats, and air dried for 1 h. The filter mats were placed into plastic bags, 10 ml of scintillation fluid added to each and the radioactivity in each sample was counted in a liquid scintillation counter (Beta Plate Flat Bet Counter, LKB Wallac (UK) Ltd, Milton Keynes, UK). The mean counts per minute (cpm) in the triplicate wells containing the mitogen-stimulated non-depleted cells were divided by the mean cpm in the triplicate wells containing mitogen-stimulated depleted cells and the results expressed as a percentage of the total response. 2.8. The effect of PCV infection of alveolar macrophages on their accessory function in the lymphocyte proliferation response of porcine peripheral blood mononuclear cells PCV-infected and control macrophages were removed from the stock cultures at 2 and 4 days after inoculation and their ability to reconstitute the mitogen-induced proliferation response of monocyte-depleted PBMNCs determined. PBMNC preparations were obtained, at the same time points chosen for harvesting macrophage cultures, from donor pigs from the same litter. Volumes (50 ml) of whole blood were collected from each of three pigs. Each sample was processed separately for recovery of PBMNCs and depletion of monocytes using the procedure described above. In addition, aliquots of non-depleted PBMNCs from each pig were used as controls. Monocyte-depleted and non-depleted preparations were then aliquoted, in quadruplicates, into 96-multiwell plates. PCV-infected and control macrophage preparations were then added to depletedcells to a final concentration of 5% and the preparations processed for Con A and PWM induced lymphocyte proliferation as described above. Macrophages alone, with and without the addition of mitogens were also cultured. Statistical analysis was carried out using a Student’s f-test.

3. Results 3.1. Alveolar macrophages Alveolar macrophage cultures were successfully maintained in suspension in teflon flasks. Following inoculation with PCV, virus antigen was initially demonstrated as

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pin-like, intracytoplasmic inclusions in 100% of the cells examined by IIF. A steady increase in intracytoplasmic accumulation of virus antigen, with time, was noted and eventual appearance of nuclear staining of PCV antigen in a small proportion of cells by 5 to 6 days after inoculation. No PCV antigen was detected in cells inoculated with the PK/lS/H control inoculum. Cell viability in the alveolar macrophage cultures was examined at regular intervals throughout the course of the experiment. Viability of between 95 and 97% was maintained in both the PCV-inoculated and control cultures for the duration of the experiment. 3.2. Phagocytosis

and microbicidal

activity

Infection of alveolar macrophages with PCV had no adverse effect on their ability to phagocytose and kill Candida krusei cells, when compared with controls. 3.3. Fc and complement

receptor assays

Infection of alveolar macrophages with PCV had no adverse effect on expression Fc and complement receptors on these cells, when compared to controls.

of

3.4. Expression of MHC class I and I1 No substantial differences in the number of cells expressing class I or II antigens, nor the intensity of labelling of these expressed antigens, were detected between the PCV-infected and control preparations processed 2 days after inoculation. An increase in the intensity of labelling of MHC class I antigen was detected in PCV-infected alveolar macrophages, compared with controls (mean f SD: PCV-infected, 64.35 & 56.39; controls, 40.02 + 37.64) in the samples tested 4 days after inoculation (Fig. 1). However, no differences in the numbers of cells expressing MHC class I antigen were seen in PCV-infected preparations, compared with controls, at this time. No differences were observed in class II expression between PCV-infected and control preparations at this time. At day 8 after inoculation, no substantial differences were detected in either the number of cells expressing class I antigens nor the intensity of labelling of these antigens. However, at this time a reduction in the number of cells expressing class II antigens was observed in PCV-infected preparations, compared with controls (PCV-infected, 37.08%; controls, 51.68%) (Fig. 2). 3.5. Depletion

of monocytes from porcine peripheral

blood mononuclear

cells

The two-step monocyte-depletion procedures used resulted in a marked reduction in the Con A induced lymphocyte proliferation response when compared with non-depleted preparations (16.5% of mean cpm obtained in equivalent non-depleted preparation). The results obtained with PWM-induced lymphocyte proliferation of these depleted preparations were less substantial, with only a reduction to 50.7% of the mean cpm obtained in an equivalent non-depleted preparation.

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Fig. 1. Results of FACS analysis of (A) PK/lS/H-inoculated (control) and (B) PCV-inoculated porcine alveolar macrophages for expression of MHC class I antigens at 4 days after inoculation. Note the increase in the expression of MHC class 1 antigens in PCV-inoculated preparation compared with controls.

3.6. The effect of PCV infection of alveolar macrophages on their ability to function as accessory cells in lymphocyte proliferation of monocyte-depleted peripheral blood mononuclear cells

The results of monocyte depletion of PBMNCs obtained from the donor pigs used in this experiment were similar to the results of the preliminary studies reported above. The cpm for depleted and non-depleted preparations, following Con A and PWM stimulation of the three individual pigs used at day 2 are presented in Table 1. Following depletion of PBMNCs taken on day 2, Con A induced stimulation resulted in mean cpm for all preparations of between 11.5 and 29.8% of the response in equivalent non-depleted preparations. Similarly, following PWM-induced stimulation of these preparations, mean

302

F. McNeilly et al. / Veterinary Immunology and Immunopathology 49 (1996) 295-306 c)ll:tE4Fe3l\FLI+wLliwi*~

A

Fig. 2. Results of FACS analysis of (A) PK/lS/H-inoculated (control) and (B) PCV-inoculated porcine alveolar macrophages for expression of MHC class II antigens at 8 days after inoculation. Note the decrease in the number of cells expressing these antigens in PCV-inoculated cells compared with controls.

Table 1 Results of concanavalin A and pokeweed mitogen induced lymphocyte proliferation monocyte-depleted peripheral blood mononuclear cells from three separate pigs Pig

1 2 3

Concanavalin

A

on non-depleted

and

Pokeweek mitogen

Non-depleted

Monocyte-depleted

Non-depleted

Monocyte-depleted

16424&2290 21774*2383 2259Ot2336

3316 f866 (20.1%) 2519f932(11.5%) 6571 zt473 (29.8%)

19661+_ 870 14545+2572 1946lf 975

6013 f 804 (30.5%) 4428 + 1217 (30.4%) 7486% 1419 (38.1%)

Values are mean counts per minute&standard deviation. response compared with equivalent non-depleted control.

Values in parentheses

are the percentage

of total

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Table 2 Results of concanavalin A and pokeweed mitogen induced lymphocyte proliferation following monocyte depletion of porcine peripheral blood mononuclear cells from three separate pigs and addition of accessory alveolar macrophages, 2 days after inoculation of accessory cells with PCV or PK/lS/H Pig

Concanavalin PK/lS/H

1 2 3

Pokeweed mitogen

A a

24393+_1706 27828 f 6003 45153 + 2625

PCV

PK/lS/H

PCV

17698 k 382 (72.0%) 15334* 1390 (55.1%) 33736 + 1301(74.0%)

23050+ 870 15284+3394 27778+2108

16863 + 3446 (73.1%) 13303 f 1084 (87.0%) 244665 1464 (88.0%)

a lnoculum used to infect accessory alveolar macrophages (PK/lS/H, control; PCV, porcine circovirus). Values are mean counts per minute&standard deviation. Values in parentheses are the percentage of total response compared with equivalent control.

cpm ranged

from 30.4 to 38.4% of the response in equivalent non-depleted preparations. The use of PCV-infected alveolar macrophage accessory cells in the restoration of the proliferation response in all three pigs tested at 2 days resulted in a significant (P < 0.05) reduction in the mitogen-induced lymphocyte proliferation response when compared with the controls. These results are presented in Table 2. Restoration of the mitogen-induced proliferation response in Con A stimulated monocyte-depleted cultures, following the addition of PCV-infected alveolar macrophages, ranged from 55.1 to 74.0% of the proliferation response obtained following stimulation of monocyte-depleted preparations containing PK/lS/H inoculated macrophages (controls). Similarly, following PWM stimulation the restoration of the proliferation response with PCV-infected preparations ranged from 73.1 to 88.0% of responses obtained in controls. At 4 days after infection a significant (P < 0.05) reduction in Con A induced lymphocyte proliferation was recorded, following addition of PCV-infected alveolar macrophages to the monocyte-depleted preparation from one pig, compared with the response obtained in controls (56%). However, no significant differences were noted in the mitogen-induced proliferation response in the remaining samples tested at this time point. Con A induced proliferation responses in monocyte-depleted PBMNCs containing PCV-infected macrophages in the remaining two pigs were 97.4% and 123% of equivalent controls. PWM-induced responses in the three preparations at this time point were 95.6%, 97.4%, and 100.1% of equivalent controls. Mean cpm for mitogen stimulated macrophages alone were always less than 200.

4. Discussion

The studies reported in this paper provide new information on the effect of PCV infection of alveolar macrophages on some of the immune functions of these cells. Studies on human macrophage functions following infection with human immunodeficiency virus (Nottet et al., 1993) have indicated that culture of macrophages by adherence to plastic can effect the functions being measured. In the present study,

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porcine alveolar macrophages were cultured in teflon flasks in which the cells grew in suspension. This avoided the problems of adherence of the cells to plastic or glass. In addition, samples could be taken at regular intervals from the same source without the need to dislodge cells by scraping or treatment with proteolytic enzymes. Such manipulations can, in themselves, stimulate the cells under test or inhibit specific functions Wnkelless, 1977). The results of this study indicate that PCV infection of alveolar macrophages in vitro has no effect on their expression of Fc and complement receptors or their ability to phagocytose and/or kill complement coated Can&da krusei cells. In separate studies on the effect of virus infection of porcine macrophages on Fc expression, phagocytosis and killing ability, Martins et al. (1988) reported that infection of alveolar macrophages with African swine fever virus had no effect on Fc expression, although infection did modulate antibody-mediated phagocytosis. The virulence of the African swine fever virus used did not correlate with the ability to alter macrophage function. lnglesias et al. (1989) reported a significant reduction in phagocytosis of yeast cells and opsonised SRBC by Aujeszkys disease virus infected alveolar macrophages compared to uninfected controls and demonstrated that this effect was virus strain dependant. The PCV inoculum used in this study was derived from a persistently infected continuous pig kidney cell line. It is possible that the multiple passage history of this cell line may have attenuated the PCV inoculum. This could explain the inability of this virus to effect phagocytosis, Fc and complement expression. Juarrero et al. (1992), compared infection with several strains of African swine fever virus, on MHC class I and II expression on porcine macrophages and noted decreased and increased MHC class I antigen expression at 8 and 12 h post infection dependent on the virus strain. Higher levels of MHC class II expression were detected in virus-infected macrophages at 8 h after infection but not in macrophages tested at 12 or 24 h after infection. These changes in MHC class I and II expression bore no relationship to virulence of the virus strains used. The authors concluded that loss, or down-regulation, of class I and II expression could be attributed to virus mediated membrane turnover coincident with virus infection of the cells and movement of the virus to the replication centres in the cytoplasm. Increased MHC class I and II expression has been shown to correlate to increased interferon production in pigs infected with hog cholera virus (Torlone et al., 1965). It is possible that aberrant expression of class I and II antigens observed with these viruses and PCV could compromise production of immunoregulatory cytokines, compromising immune function and perhaps favour viral persistence through escape from immune recognition. The ability of AMs to act as accessory cells to reconstitute the mitogen-induced lymphocyte proliferation response of monocyte-depleted PBMNCs has been reported (Adair et al., 1992). In this study, a transient decrease in the ability of PCV-infected AMs to reconstitute the proliferation response of monocyte/macrophage depleted PBMNCs when compared to controls was observed. The significance of these reductions and their transient nature are difficult to explain. However, PCV infection of AMs apparently affects, in some way, their ability to interact with B- and T-cells and stimulate proliferation. This may

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reduce the capability of these, and other cells of the same lineage, to perform their pivotal roles. In conclusion, the results reported in this study show that infection of porcine alveolar macrophage cultures with PCV has a transient effect on some, but not all, of the macrophage functions tested. On the basis of these results, this virus would appear to be capable of interfering with immune responsiveness in the lung, and perhaps other sites of replication of PCV. It is possible that transplacental infection of porcine foetuses occurs with PCV (Hines and Luckert, 1994; Allan et al., 1995). If this is so, then PCV replication in infected foetuses, occurring in cells of the reticuloendothelial system, could effect immune cell function in the foetus. The reason why the dysfunctions reported here should be transient and selective are unclear, however, they could be related to attenuation of virulence of the PCV isolate used following multiple passage in cell cultures.

Acknowledgement We are grateful to I. Walker for excellent

technical

assistance.

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McNuIty, MS., Ma&e, D.P., Pollock, D., McNair, J., Todd, D., Mawhinney, K., Connor, T.J. and McNeilly, F., 1990. Production and preliminary characterisation of monoclonal antibodies to chicken anaemia agent. Avian Dis., 34: 352-358. Nottet, H.S.L.M., de Graaf, L., de Voss, N.M., Bakker, L.J., van Strijp, J.A.G., Visser, M.R. and Verhoef, J., 1993. Phagocytic function of monocyte-derived macrophages is not affected by human immunodeficiency virus type 1 infection. J. Infect. Dis., 168: 84-91. Studdert, J.S., 1993. Circoviridae: new viruses from pigs parrots and chickens. Aust. Vet. J., 70: 121-122. Tischer, I., Peters, D. and Tochtermann, G., 1974. Characterisation of papovavirusand picomavirus-like particles in permanent pig kidney cell lines. Zentralbl. Bakteriol. Hyg. I. Abt. Orig. A., 226: 153-167. Tischer, I., Gelderblom, H., Vettermann, W. and Koch, M.A., 1982. A very small porcine virus with circular single stranded DNA. Nature, 295: 64-66. Torlone, V., Titoli, F. and Gialletti, L., 1965. Circulating interferon production in pigs infected with hog cholera. Life Sci., 4: 1707-1710. Unkelless, J.C., 1977. The presence of two Fc receptors on mouse macrophages. Evidence from a variant cell line and differential trypsin sensitivity. J. Exp. Med., 145: 931-936. Warr, G.A. and Jakab, G.J., 1979. Alterations in lung macrophage antimicrobial activity associated with viral pneumonias. Infect. Immun., 26: 492-497.