1 Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK
2 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Box 6100, MS 39762-6100, USA
Correspondence
adb44{at}cam.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: CIMR, Wellcome Trust/MRC building, Hills Road, Cambridge CB2 2XY, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
According to paradigm, MDV infection occurs by inhalation of cell-free MDV in feather dander. At 46 days post-infection (p.i.), an obligate cytolytic and cell-associated productive infection of B and T lymphocytes is established (Calnek et al., 1984a
, b
). From 7 to 14 days p.i., MDV establishes life-long latency in activated CD4+ T-helper (Th) lymphocytes (Schat et al., 1991
). In genetically susceptible chickens, MDV neoplastically transforms activated CD4+ Th lymphocytes and gross lymphomas form (Burgess & Davison, 2002
). Recently, MDV has been demonstrated also to lytically infect
T lymphocytes (Burgess & Davison, 2002
). MDV is thus insidious, infecting and neoplastically transforming the acquired immune cells evolved to destroy it.
Although phagocytes are postulated to transport MDV from the lungs to primary lymphoid organs, i.e. sites of productive lymphoid infection (Calnek, 2001), this has never been demonstrated. Little work has investigated the question of MDV macrophage infection. Although MDV infects non-lymphoid cells in vitro [chicken embryo fibroblasts (CEF), chick kidney cells and feather follicle epithelium] (Payne, 1985
), neither in vitro macrophage infection with MDV nor in vitro MDV isolation from macrophages taken from infected chickens has been possible (Haffer & Sevoian, 1979
; Haffer et al., 1979
; von Bülow & Klasen, 1983
).
Regardless, in vivo lymphocyte infection alone does not account for the total number of MDV-infected splenocytes during the cytolytic stage of Marek's disease (MD) (Baigent & Davison, 1996; Baigent et al., 1998
). Furthermore, although administration of the T lymphocyte immunosuppressive drug cyclosporin after the establishment of MDV latency results in reappearance of cytolytic infection in primary lymphoid organs, many of the MDV antigen-positive cells are not B or T lymphocytes (Buscaglia et al., 1988
). We hypothesize that macrophages are likely to be MDV-infected in vivo.
In addition to their suspected role as MDV transporters, macrophages are critical to innate immunity. Specifically for MD, macrophages probably restrict initial MDV replication in vivo (Lee et al., 1978a, b
; Haffer & Sevoian, 1979
; Lee, 1979
; Powell et al., 1983
; Gupta et al., 1989
; Schat & Xing, 2000
; Xing & Schat, 2000a
, b
; Djeraba et al., 2000
). In this work, our two specific aims were to identify whether (i) MDV infects macrophages in vivo and (ii) if quantitative differences in macrophage infection in vivo correlate with MDV virulence. We demonstrate for the first time, directly ex vivo, that MDV infects macrophages. Our work has two fundamental implications for understanding MDV pathogenesis. Firstly, MDV evolved to perturb innate, in addition to acquired, immunity. Secondly, and as hypothesized previously by others (Calnek, 2001
), macrophages are excellent candidate cells for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chickens, MDV infection and spleen leukocyte isolation.
Because the spleen is a site of primary MDV infection, all work was done using splenic leukocytes. Specific-pathogen-free, 2-week-old, Houghton Poultry Research Station Rhode-Island Red (HPRS-RIR) chickens bred and maintained at the Institute for Animal Health were infected by intraperitoneal injection of 103 p.f.u. MDV in medium, as described (Barrow & Venugopal, 1999). Chickens infected with the different MDVs, and uninfected control chickens (injected with medium excluding MDV), were housed in separate rooms. At different days p.i. (see below), spleens were removed post-mortem and leukocyte suspensions prepared by density gradient centrifugation (specific gravity 1·077, 500 g, 4 °C, 30 min; FicollPaque, Amersham Pharmacia Biotech). All cells were maintained in PBS with 0·5 % BSA and 0·1 % sodium azide. Experiments followed UK Home Office guidelines.
Antibodies.
All antibodies used in this study are described in Table 1. All monoclonal antibody (mAb) dilutions were determined previously, or were established experimentally, as described (Burgess & Davison, 2002
). mAb BD1 is of isotype IgG2a and recognizes a complex of three antigenically related early phosphoproteins (of 24, 38 and 41 kDa) termed the pp38 complex (Li et al., 1994
). mAb HB3 identifies the glycoprotein B (gB) antigen from all three MDV serotypes and is of isotype IgG2b (Ross et al., 1997
). All other mAbs used were isotype IgG1. CT4 and CT8 identify the chicken T cell antigens CD4 and CD8, respectively (Chan et al., 1988
). mAb TCR1 recognizes the chicken
T cell receptor (Sowder et al., 1988
). mAb AV20 recognizes the chicken pan B cell antigen, chB6 (Rothwell et al., 1996
). mAb KULO1 identifies chicken macrophages and monocytes (Mast et al., 1998
; Mast & Goddeeris, 1999
; Van Immerseel et al., 2002a
, b
). Rabbit polyclonal antibody against the MDV ICP4 homologue was a gift from R. Morgan (University of Delaware, Newark, DE, USA). RSVG- mAbs 29 and 30 were isotype-matched controls for IgG2a and IgG1, respectively, and have been described before (Baigent & Davison, 1996
; Baigent et al., 1998
). RSVG- mAb 11 recognizes the F protein from respiratory syncytial virus and was used as an IgG2b-matched control (Taylor et al., 1984
). Fluorescent-labelled secondary antibodies goat anti-mouse IgG1-FITC, goat anti-mouse IgG1-TRITC, goat anti-mouse IgG2a-FITC, goat anti-mouse IgG2a-Texas red and goat anti-mouse IgG2a-PE were purchased from Southern Biotechnologies. Goat anti-mouse IgG2b-FITC was purchased from Caltag Laboratories. Goat anti-rabbit IgG-Alexa 468 and goat anti-rabbit IgG-Alexa 568 were purchased from Molecular Probes.
|
Confocal microscopy.
Immunostaining of cells for confocal microscopy was done as described for flow cytometric analyses, except that incubations with mAbs recognizing MDV and leukocyte antigens were done separately for 1 h. MDV antigen expression was examined in B lymphocytes and macrophages. The secondary antibodies used in combination were: (i) for pp38 expression, IgG1-FITC+IgG2a-Texas red; (ii) for gB expression, IgG1-TRITC+IgG2b-FITC; and for ICP4 expression, IgG1-FITC+IgG-Alexa 568. Propidium iodide (PI) (10 µg ml-1; Sigma) was used to stain DNA to identify the nucleus. PI was used in combination with anti-MDV ICP4 and IgG-Alexa 488.
To detect ICP4 and pp38 co-expression, leukocytes were incubated with anti-MDV ICP4 and BD1 concurrently, washed and then incubated with IgG-Alexa 568+IgG2a-FITC. As a confocal microscopy control, gB expression was analysed in C12/130-infected CEF that had been removed using PBS and 1 mM EDTA from infected flasks and transferred to microtitre plates for immunostaining. Unpermeabilized and permeabilized CEF were incubated with HB3, washed and then incubated with IgG2b-FITC. Following immunostaining, both CEF and leukocytes were incubated (30 min at room temperature) on poly L-lysine (Sigma)-coated glass coverslips (Merck). The coverslips were then mounted with Vectashield (Vector Laboratories) and examined by laser scanning confocal microscopy (Leica). Transmitted light images were collected with a transmitted light detector on the confocal microscope.
Ex vivo analysis of leukocyte death.
Surface antigen expression and death were analysed in leukcoytes isolated from HPRS-16- and C12/130-infected, and mock-infected, chickens by flow cytometry using an adaptation of the technique described by Nicoletti et al. (1991). The technique was used as described (Burgess & Davison, 2002
) with the following changes: (i) BD1 was conjugated to long-armed biotin (BD1-biotin; Sigma); (ii) leukocytes were incubated concurrently with BD1-biotin and either AV20 or KULO1, washed and then incubated with IgG1-FITC+allophycocyanin (APC)-streptavidin conjugate (Caltag Laboratories)+PI (10 µg ml-1).
Flow cytometric analysis was performed after time-delay calibration (according to the manufacturer's instructions) using APC-conjugated beads and the FACScalibur. At least 500 positive events were collected for the following: (i) pp38+AV20+ and pp38+KULO1+ leukocytes from HPRS-16- and C12/130-infected spleens; (ii) pp38-AV20+ and pp38-KULO1+ leukocytes from HPRS-16- and C12/130-infected spleens; and (iii) pp38-AV20+ and pp38-KULO1+ leukocytes from uninfected spleens. All flow cytometry data were analysed using the WinMDI 2.8 analysis package (http://facs.scripps.edu/software.html).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pp38 antigen was detected in splenocytes from either HPRS-16- or C12/130-infected chickens at 4 and 6 days p.i., but was never detected in splenocytes isolated from mock-infected control chickens (data from 4 days p.i.; Fig. 1). At 4 days p.i., there were 16·8-fold more pp38+ splenocytes in C12/130 (15·1±4·3 %) compared with HPRS-16 (0·9±0·3 %) infected chickens (P<0·05) (Table 2
). At 6 days p.i., two of five C12/130-infected chickens did not express pp38, i.e. these chickens were latently infected. The mean numbers of pp38+ splenocytes from C12/130-infected chickens were 10·7±0·7 % compared with 6·3±2·8 % from HPRS-16-infected chickens at 6 days p.i. (Table 2
).
|
|
According to the generally accepted paradigm for MDV pathogenesis (Calnek, 2001), the initial B lymphocytolytic infection results in T lymphocyte activation. These T lymphoblasts may then become cytolytically or latently MDV infected, although cytolytic infection is, to a lesser degree, compared with B lymphocytes. Following this paradigm, we found pp38+CT4+ and pp38+CT8+ lymphocytes in both HPRS-16- and C12/130-infected chickens at 4 and 6 days p.i. (Fig. 1
and Table 2
). There were differences between the proportions of pp38+CT4+ and pp38+CT8+ lymphocytes at 6 days p.i.; pp38+CT8+ lymphocytes were more frequent (P<0·05) in C12/130 (26·6±2·7 %) compared with HPRS-16-infected chickens (16·6±3·9 %).
Recently, T lymphocytes in MDV lymphomas have been demonstrated to be cytolytically MDV infected (Burgess & Davison, 2002
). Here we show pp38+TCR1+ splenocytes during the cytolytic phase of MD at 4 and 6 days p.i. (Fig. 1
and Table 2
). Notably, pp38+TCR1+ lymphocytes were less frequent (P<0·05) in HPRS-16 (2·3±2·3 % at 4 days p.i.; 2·2±1·1 % at 6 days p.i.) compared with C12/130-infected chickens (8·8±1·0 % at 4 days p.i.; 12·3±1·9 % at 6 days p.i.).
Our most notable and novel finding was pp38+KULO1+ splenocytes in both HPRS-16- and C12/130-infected chickens at 4 and 6 days p.i. (Fig. 1 and Table 2
). Again, pp38+KULO1+ splenocytes were less frequent (P<0·05) in HPRS-16 (3·0±3·0 % at 4 days p.i.; 2·4±1·0 % at 6 days p.i.) compared with C12/130-infected chickens (9·6±1·2 % at 4 days p.i.; 22·6±1·6 % at 6 days p.i.).
Collectively, our data emphasize that C12/130 MDV causes a greater cytolytic infection; 16·8-fold more pp38+ splenocytes were detected at 4 days p.i. compared with HPRS-16-infected chickens. Significantly, our data shows for the first time that macrophages, in addition to B, CD4+, CD8+ and TCR+ lymphocytes, express the early MDV antigen, pp38, indicative of cytolytic infection in vivo (Burgess & Davison, 2002
; Reddy et al., 2002
). The slightly increased proportion of pp38+TCR1+ and pp38+KULO1+ cells at 4 days p.i. is consistent with the increased virulence of C12/130. However, MDV infection of other leukocyte subpopulations was also much higher in C12/130-infected chickens compared with HPRS-16-infected chickens (total pp38+ cells: 15·1±4·3 % in C12/130 compared with 0·9±0·3 % in HPRS-16-infected chickens). Thus, the increased number of pp38+TCR1+ and pp38+KULO1+ splenocytes reflects the pattern of infection seen in other infected cell phenotypes.
At 6 days p.i., there were specific differences in the proportion of pp38+CT8+, pp38+TCR1+ and pp38+KULO1+ cells between C12/130- and HPRS-16-infected chickens. However, the proportions of AV20+ cells were much lower in splenocytes from C12/130-infected chickens at 6 days p.i. (P<0·05; data not shown), compared with the other leukocyte subsets analysed; presumably because of a much earlier loss of B lymphocytes from cytolytic infection and cell death [Fig. 1, compare the proportion of pp38+AV20+ cells (upper right quadrant) to pp38-AV20+ cells (lower right quadrant) from the same bird between HPRS-16- and C12/130-infected chickens]. Loss of B lymphocytes from earlier cytolytic infection would be expected to skew the proportion of pp38+ cells towards other cell types.
MDV-infected B lymphocytes and macrophages express proteins from all three herpesvirus kinetic classes
To investigate further the possibility that the pp38+ macrophages we measured by flow cytometry were MDV infected and not pp38+ because they had phagocytosed MDV antigens from other infected lymphocytes, we measured the expression of representative MDV antigens from the three herpesvirus kinetic classes: ICP4 (immediate early), pp38 (early) and gB (late). We compared directly the presence and distribution of these MDV proteins in macrophages with that in B lymphocytes because these cells are primarily lytically infected by MDV. ICP4 localizes to the nucleus of MDV-infected cells (Knipe et al., 1987; Xing et al., 1999
) and is definitively diagnostic of herpesvirus infection (Roizman, 1996
); cells that have only phagocytosed infected cells will not have nuclear ICP4 (Aderem & Underhill, 1999
).
Splenocytes from uninfected chickens were ICP4-, pp38- and gB- (Fig. 2B, panel i). ICP4 localized to the nucleus of splenocytes from C12/130 MDV-infected chickens (Fig. 2A
, panels iiii). However, a novel, unexpected finding was that ICP4 was also diffusely present within the cytoplasm of some MDV-infected cells (Fig. 2A
, panel ii). This pattern was not an artefact because neither the nucleus nor the cytoplasm of leukocytes from uninfected control chickens was ICP4+ (Fig. 2A
, panel iv).
|
Typical of MDV lytically infected cells (Ross et al., 1997; Burgess & Davison, 2002
; Reddy et al., 2002
), pp38 was strongly expressed and strictly distributed throughout the cytoplasm of both AV20+ and KULO1+ leukocytes from C12/130-infected chickens (Fig. 3
, panels i, ii and v). This widespread distribution suggests the pp38+ immunostaining was not due to phagocytosis of MDV-infected cells. Indeed, these same pp38+KULO1+ cells showed no evidence of having phagocytosed any cells or cellular debris at all by transmitted light confocal microscopy (Fig. 3
, panels iii and iv).
|
|
Macrophages and B lymphocytes expressing pp38 show increased cell death
The only known consequences after any herpesvirus infection of a cell are: lytic infection and death, latent infection, or (more rarely) neoplastic transformation (Roizman, 1996). Lytically MDV-infected macrophages would, by default, die. In contrast, macrophages that phagocytose cell debris do not die; they continue to remove dying cells and remain alive to process the phagocytosed antigens for presentation to acquired immune cells. We compared the proportions of dying pp38+ B lymphocytes and macrophages from HPRS-16- and C12/130-infected chickens at 6 days p.i. by flow cytometry. Since the DNA profiles of cells in G1 or S/G2M phases could be distinguished clearly (Fig. 5
A, panel i), cells that had fragmented DNA profiles because of cell death were therefore evident as subG1 events (Fig. 5A
).
|
Our results indicate that B lymphocytes and macrophages expressing pp38 undergo a cytolytic infection but pp38- macrophages or pp38- B lymphocytes from the same host or those from uninfected chickens do not. pp38+ B lymphocytes have similar levels of death when compared with pp38+ macrophages from the same MDV-infected chickens. However, pp38+ B lymphocytes and pp38+ macrophages from C12/130-infected chickens have higher rates of cell death compared with pp38+ B lymphocytes and pp38+ macrophages from HPRS-16-infected chickens. Our results emphasize further the greater cell death after infection with C12/130 compared with HPRS-16.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MDV expresses ICP4, pp38 and gB, representing the immediate-early, early and late temporal classes of herpesvirus gene expression, respectively. To identify MDV infection definitively, we demonstrate expression of MDV ICP4 in the nucleus of KULO1+ cells (Knipe et al., 1987; Roizman, 1996
). Furthermore, ICP4 will not localize to the nucleus of cells that have phagocytosed dead or dying MDV-infected cells. Such phagocytosis would result in localized degradation of the engulfed cell in phagolysosomes and not ICP4 expression in the nucleus (Aderem & Underhill, 1999
).
The MDV lytic-cycle antigens pp38 and gB are expressed in the cytoplasm and not the nucleus of MDV-infected cells (Ross et al., 1989, 1997
; Reddy et al., 2002
). In our work, the distributions of pp38 and gB were very similar between both AV20+ and KULO1+ splenocytes. pp38 was expressed throughout the cytoplasm of AV20+ and KULO1+ splenocytes but was not present in discrete phagocytic vacuoles. Any MDV antigens present in macrophages as a result of phagocytosis of dead/dying MDV-infected cells will lose their antigenicity (and therefore mAb reactivity) due to rapid degradation in phagolysosomes (Aderem & Underhill, 1999
). Furthermore, the widespread ICP4 and pp38 cytoplasmic staining we observed would not be expected if these proteins were localized in phagolysosomes. Indeed, transmitted light microscopy showed that the MDV antigen+KULO1+ cells had not phagocytosed any dead or dying cells at all. The representative MDV proteins from all three herpesvirus kinetic classes shared highly similar subcellular distributions between B lymphocytes and macrophages, fulfilling the current antigenic criteria for MDV infection specifically (Ross et al., 1989
, 1997
; Burgess & Davison, 2002
; Reddy et al., 2002
) and herpesvirus infection in general (Roizman, 1996
).
In addition to our primary results, we make two interesting observations about the MDV antigens gB and ICP4. Firstly, we demonstrate in B lymphocytes and macrophages that ICP4 localizes both to the nucleus and to the cytoplasm. This phenomenon is not unique to MDV; it also occurs after herpes simplex virus type 1 (HSV-1) infection (Knipe et al., 1987; Zhu & Schaffer, 1995
). MDV ICP4 has a number of spliced polyadenylated sense transcripts, which are not fully characterized yet (R. Morgan, personnel communication). The anti-MDV ICP4 we used recognizes amino acids 5341415 of ICP4. Although identifying the 140 kDa native form of IPC4, anti-MDV ICP4 also identifies an 80 kDa antigen (probable ICP4 isoform) in MDV-infected, but not uninfected, cells (R. Morgan, personnel communication). Either isoform may be in the nucleus or cytoplasm and be functionally significant and this issue deserves further analysis.
Secondly, cell-surface gB expression, by both B lymphocytes and macrophages, was lower, or non-existent, compared with MDV-infected CEF. Notably, human herpesvirus type 6-infected lymphocytes display similar phenomena (Cirone et al., 1994; Torrisi et al., 1999
). In MDV infection, downregulation of gB is reported in response to interferon-
and nitric oxide (Xing & Schat, 2000b
; Djeraba et al., 2000
) as well as an undefined latency maintaining factor (Buscaglia & Calnek, 1988
; Volpini et al., 1995
, 1996
). These factors have been reported during the cytolytic stage of infection in MDV-infected chickens and could be responsible for the unexpected gB surface staining of B lymphocytes and macrophages isolated ex vivo.
All herpesvirus infections result in cell death (Roizman, 1996). Lytically MDV-infected macrophages would, by default, die. In contrast, macrophages that phagocytose cell debris do not die; they continue to remove debris and remain alive to process phagocytosed antigens for expression to acquired immune cells. The numbers of cells in each cell population with subdiploid DNA, resulting from cell death (most likely as a result of cytolytic infection), were analysed. pp38+AV20+ and pp38+KULO1+ leukocytes die, whereas pp38-AV20+ and pp38-KULO1+ leukocytes from the same infected individual do not. These results support our suggestion that pp38+ macrophages are lytically infected by MDV and undergo cell death.
To examine whether the MDV cytolytic infection of macrophages resulted in a productive infection, we have carried out electron microscopic examination of splenocytes from infected birds. Despite repeated efforts, after isolating KULO1+ splenocytes from HPRS-16- and C12/130-infected chickens by cell-sorting and in frozen spleen sections from MDV-infected chickens, we have been unable to identify any MDV particles in KULO1+ splenocytes by electron microscopy (EM) (data not shown). The chicken macrophage antigen recognized by mAb KULO1 is labile under EM fixative conditions (4 % paraformaldehyde±0·05 % gluteraldehyde), making it impossible to definitively identify macrophages in EM sections of spleen from MDV-infected chickens. Despite this, we have MACS-sorted macrophages to 95 % purity using mAb KULO1. Of these sorted cells,
10 % are pp38+ by flow cytometry. Because the cells were sorted before EM fixation, the KULO1+ macrophages can be clearly identified from contaminating cells via the electron-dense MACS beads and still virus particles cannot be found in these purified fractions, despite EM evidence that these cells do not contain phagocytosed lymphocytes.
MDV infection is cell-associated and there is no in vitro model for macrophage infection using conventional co-culture methods (Haffer et al., 1979; von Bülow & Klasen, 1983
). It is because of these early co-culture studies that MDV is considered not to infect macrophages. Despite this, we have also tried our own in vitro co-culture experiments using MDV-infected lymphocytes on either bone marrow-derived chicken macrophages or macrophage cell lines and cannot detect any antigen expression, leading us to believe that MDV macrophage infection clearly requires in vivo conditions. On the contrary, cells are rapidly phagocytosed and degraded and no nuclear ICP4 expression can be detected in vitro, unlike the nuclear ICP4 expression we find in macrophages isolated ex vivo from MDV-infected chickens, consistent with previous studies (von Bülow & Klasen, 1983
). We have also cell-sorted KULO1+ macrophages from MDV-infected chickens and co-cultured them with permissive fibroblasts and chick kidney cells. In the instances where we have obtained infectious foci from cell-sorted KULO1+ and AV20+ cells (used as a positive control), the infectious foci are infrequent (less than one or two foci per T25 flask) from 1x106 sorted KULO1+ or AV20+ cells. These cell-sorted populations are not 100 % pure and the small number of infectious foci we do see could also be critically attributed to the small fraction of cells that often contaminate even MoFlo cell-sorted populations (routinely
98 % pure), leaving a possibility that the low number of foci obtained arose possibly from the
1-2 % contaminants of 1x106 sorted macrophages and B cells (
20 000 contaminating cells).
It is possible that MDV infection of macrophages may result in an abortive infection, with no virus particle production, as occurs when HSV-1 infects macrophages (Morahan et al., 1989; Tenney & Morahan, 1991
; Wu et al., 1993
). In view of these results, further research should define whether MDV induces either an abortive or a productive infection of macrophages. Nevertheless, our ex vivo data showing the nuclear localization of ICP4, widespread cytoplasmic pp38 and gB localization, which is not localized to discrete phagosomes and highly similar distribution to MDV-infected B lymphocytes and death of pp38+ but not pp38-KULO1+ cells (from the same infected bird), demonstrate infection of macrophages by MDV clearly. Our results are the first data, which support previous suggestions (Calnek, 2001
), that macrophages are the proposed carrier cells' responsible for the early transport of MDV to lymphoid organs.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baigent, S. J. & Davison, T. F. (1996). A flow cytometric method for identifying Marek's disease virus pp38 expression in lymphocyte subpopulations. Avian Pathol 25, 255267.
Baigent, S. J., Ross, L. J. & Davison, T. F. (1998). Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes. J Gen Virol 79, 27952802.[Abstract]
Barrow, A. & Venugopal, K. (1999). Molecular characteristics of very virulent European MDV isolates. Acta Virol 43, 9093.[Medline]
Burgess, S. C. & Davison, T. F. (2002). Identification of the neoplastically transformed cells in Marek's disease herpesvirus-induced lymphomas: recognition by the monoclonal antibody AV37. J Virol 76, 72767292.
Buscaglia, C. & Calnek, B. W. (1988). Maintenance of Marek's disease herpesvirus latency in vitro by a factor found in conditioned medium. J Gen Virol 69, 28092818.[Abstract]
Buscaglia, C., Calnek, B. W. & Schat, K. A. (1988). Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus. J Gen Virol 69, 10671077.[Abstract]
Calnek, B. W. (2001). Pathogenesis of Marek's disease virus infection. Curr Top Microbiol Immunol 255, 2555.[Medline]
Calnek, B. W., Schat, K. A., Ross, L. J., Shek, W. R. & Chen, C. L. (1984a). Further characterization of Marek's disease virus-infected lymphocytes. I. In vivo infection. Int J Cancer 33, 389398.[Medline]
Calnek, B. W., Schat, K. A., Ross, L. J. & Chen, C. L. (1984b). Further characterization of Marek's disease virus-infected lymphocytes. II. In vitro infection. Int J Cancer 33, 399406.[Medline]
Chan, M. M., Chen, C. L., Ager, L. L. & Cooper, M. D. (1988). Identification of the avian homologues of mammalian CD4 and CD8 antigens. J Immunol 140, 21332138.
Cirone, M., Campadelli-Fiume, G., Foa-Tomasi, L., Torrisi, M. R. & Faggioni, A. (1994). Human herpesvirus 6 envelope glycoproteins B and HL complex are undetectable on the plasma membrane of infected lymphocytes. AIDS Res Hum Retroviruses 10, 175179.[Medline]
Djeraba, A., Bernardet, N., Dambrine, G. & Quere, P. (2000). Nitric oxide inhibits Marek's disease virus replication but is not the single decisive factor in interferon--mediated viral inhibition. Virology 277, 5865.[CrossRef][Medline]
Epstein, M. A. (2001). Historical background. Philos Trans R Soc Lond B Biol Sci 356, 413420.[Medline]
Gandon, S., Mackinnon, M. J., Nee, S. & Read, A. F. (2001). Imperfect vaccines and the evolution of pathogen virulence. Nature 414, 751756.[CrossRef][Medline]
Gupta, M. K., Chauhan, H. V., Jha, G. J. & Singh, K. K. (1989). The role of the reticuloendothelial system in the immunopathology of Marek's disease. Vet Microbiol 20, 223234.[CrossRef][Medline]
Haffer, K. & Sevoian, M. (1979). In vitro studies on the role of the macrophages of resistant and susceptible chickens with Marek's disease. Poult Sci 58, 295297.[Medline]
Haffer, K., Sevoian, M. & Wilder, M. (1979). The role of the macrophage in Marek's disease: in vitro and in vivo studies. Int J Cancer 23, 648656.[Medline]
Knipe, D. M., Senechek, D., Rice, S. A. & Smith, J. L. (1987). Stages in the nuclear association of the herpes simplex virus transcriptional activator protein ICP4. J Virol 61, 276284.[Medline]
Lee, L. F. (1979). Macrophage restriction of Marek's disease virus replication and lymphoma cell proliferation. J Immunol 123, 10881091.[Abstract]
Lee, L. F., Sharma, J. M., Nazerian, K. & Witter, R. L. (1978a). Suppression of mitogen-induced proliferation of normal spleen cells by macrophages from chickens inoculated with Marek's disease virus. J Immunol 120, 15541559.[Abstract]
Lee, L. F., Sharma, J. M., Nazerian, K. & Witter, R. L. (1978b). Suppression and enhancement of mitogen response in chickens infected with Marek's disease virus and the herpesvirus of turkeys. Infect Immun 21, 474479.[Medline]
Li, D., Green, P. F., Skinner, M. A., Jiang, C. & Ross, N. (1994). Use of recombinant pp38 antigen of Marek's disease virus to identify serotype 1-specific antibodies in chicken sera by Western blotting. J Virol Methods 50, 185195.[CrossRef][Medline]
Mast, J. & Goddeeris, B. M. (1999). Development of immunocompetence of broiler chickens. Vet Immunol Immunopathol 70, 245256.[CrossRef][Medline]
Mast, J., Goddeeris, B. M., Peeters, K., Vandesande, F. & Berghman, L. R. (1998). Characterisation of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KULO1. Vet Immunol Immunopathol 61, 343357.[CrossRef][Medline]
Morahan, P. S., Mama, S., Anarki, F. & Leary, K. (1989). Molecular localization of abortive infection of resident peritoneal macrophages by herpes simplex virus type 1. J Virol 63, 23002307.[Medline]
Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F. & Riccardi, C. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139, 271279.[CrossRef][Medline]
Payne, L. N. (editor) (1985). Pathology. In Marek's Disease, pp. 4376. Boston: Martinus Nijhoff.
Powell, P. C., Hartley, K. J., Mustill, B. M. & Rennie, M. (1983). Studies on the role of macrophages in Marek's disease of the chicken. J Reticuloendothel Soc 34, 289297.[Medline]
Reddy, S. M., Lupiani, B., Gimeno, I. M., Silva, R. F., Lee, L. F. & Witter, R. L. (2002). Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: use of a pp38 mutant to validate the technology for the study of gene function. Proc Natl Acad Sci U S A 99, 70547059.
Roizman, B. (1996). Herpesviridae. In Fields Virology, 3rd edn, pp. 22212230. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
Ross, L. J., Sanderson, M., Scott, S. D., Binns, M. M., Doel, T. & Milne, B. (1989). Nucleotide sequence and characterization of the Marek's disease virus homologue of glycoprotein B of herpes simplex virus. J Gen Virol 70, 17891804.[Abstract]
Ross, N., O'Sullivan, G., Rothwell, C., Smith, G., Burgess, S. C., Rennie, M., Lee, L. F. & Davison, T. F. (1997). Marek's disease virus EcoRI-Q gene (meq) and a small RNA antisense to ICP4 are abundantly expressed in CD4+ cells and cells carrying a novel lymphoid marker, AV37, in Marek's disease lymphomas. J Gen Virol 78, 21912198.[Abstract]
Rothwell, C. J., Vervelde, L. & Davison, T. F. (1996). Identification of chicken Bu-1 alloantigens using the monoclonal antibody AV20. Vet Immunol Immunopathol 55, 225234.[CrossRef][Medline]
Schat, K. A. & Xing, Z. (2000). Specific and nonspecific immune responses to Marek's disease virus. Dev Comp Immunol 24, 201221.[CrossRef][Medline]
Schat, K. A., Chen, C. L., Calnek, B. W. & Char, D. (1991). Transformation of T-lymphocyte subsets by Marek's disease herpesvirus. J Virol 65, 14081413.[Medline]
Sowder, J. T., Chen, C. L., Ager, L. L., Chan, M. M. & Cooper, M. D. (1988). A large subpopulation of avian T cells express a homologue of the mammalian T /
receptor. J Exp Med 167, 315322.[Abstract]
Taylor, G., Stott, E. J., Bew, M., Fernie, B. F., Cote, P. J., Collins, A. P., Hughes, M. & Jebbett, J. (1984). Monoclonal antibodies protect against respiratory syncytial virus infection in mice. Immunology 52, 137142.[Medline]
Tenney, D. J. & Morahan, P. S. (1991). Differentiation of the U937 macrophage cell line removes an early block of HSV-1 infection. Viral Immunol 4, 91102.[Medline]
Torrisi, M. R., Gentile, M., Cardinali, G., Cirone, M., Zompetta, C., Lotti, L. V., Frati, L. & Faggioni, A. (1999). Intracellular transport and maturation of human herpesvirus 6. Virology 257, 460471.[CrossRef][Medline]
Van Immerseel, F., De Buck, J., De Smet, I., Mast, J., Haesebrouck, F. & Ducatelle, R. (2002a). The effect of vaccination with a Salmonella enteritidis aroA mutant on early cellular responses in caecal lamina propria of newly-hatched chickens. Vaccine 20, 30343041.[CrossRef][Medline]
Van Immerseel, F., De Buck, J., De Smet, I., Mast, J., Haesebrouck, F. & Ducatelle, R. (2002b). Dynamics of immune cell infiltration in the caecal lamina propria of chickens after neonatal infection with a Salmonella enteritidis strain. Dev Comp Immunol 26, 355364.[CrossRef][Medline]
van Regenmortel, M. H. V., Fauqet, C. M., Bishop, D. H. L., Carstens, E., Estes, M. K., Lemon, S., Maniloff, J., Mayo, M. A., McGeoch, D., Pringle, C. R. & Wickner, R. B. (editors) (1999). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Volpini, L. M., Calnek, B. W., Sekellick, M. J. & Marcus, P. I. (1995). Stages of Marek's disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression. Vet Microbiol 47, 99109.[CrossRef][Medline]
Volpini, L. M., Calnek, B. W., Sneath, B., Sekellick, M. J. & Marcus, P. I. (1996). Interferon modulation of Marek's disease virus genome expression in chicken cell lines. Avian Dis 40, 7887.[Medline]
von Bülow, V. & Klasen, A. (1983). Effects of avian viruses on cultured chicken bone-marrow-derived macrophages. Avian Path 12, 179198.
Witter, R. L. (2001). Protective efficacy of Marek's disease vaccines. Curr Top Microbiol Immunol 255, 5790.[Medline]
Wu, L., Morahan, P. S. & Leary, K. (1993). Regulation of herpes simplex virus type 1 gene expression in nonpermissive murine resident peritoneal macrophages. J Leukoc Biol 53, 6165.[Abstract]
Xing, Z. & Schat, K. A. (2000a). Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures. Immunology 100, 7076.[CrossRef][Medline]
Xing, Z. & Schat, K. A. (2000b). Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus. J Virol 74, 36053612.
Xing, Z., Xie, Q., Morgan, R. W. & Schat, K. A. (1999). A monoclonal antibody to ICP4 of MDV recognizing ICP4 of serotype 1 and 3 MDV strains. Acta Virol 43, 113120.[Medline]
Zhu, Z. & Schaffer, P. A. (1995). Intracellular localization of the herpes simplex virus type 1 major transcriptional regulatory protein, ICP4, is affected by ICP27. J Virol 69, 4959.[Abstract]
Received 6 March 2003;
accepted 2 July 2003.