Marek's Disease Virus Infection Research Articles

Antiviral activity of ovotransferrin discloses an evolutionary strategy for the defensive activities of lactoferrin.

Ovotransferrin (formerly conalbumin) is an iron-binding protein present in birds. It belongs to the transferrin family and shows about 50% sequence homology with mammalian serum transferrin and lactoferrin. This protein has been demonstrated to be capable of delivering iron to cells and of inhibiting bacterial multiplication. However, no antiviral activity has been reported for ovotransferrin, although the antiviral activity of human and bovine lactoferrins against several viruses, including human herpes simplex viruses, has been well established. In this report, the antiviral activity of ovotransferrin towards chicken embryo fibroblast infection by Marek's disease virus (MDV), an avian herpesvirus, was clearly demonstrated. Ovotransferrin was more effective than human and bovine lactoferrins in inhibiting MDV infection and no correlation between antiviral efficacy and iron saturation was found. The observations reported here are of interest from an evolutionary point of view since it is likely that the defensive properties of transferrins appeared early in evolution. In birds, the defensive properties of ovotransferrin remained joined to iron transport functions; in mammals, iron transport functions became peculiar to serum transferrin, and the defensive properties towards infections were optimised in lactoferrin.

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression.

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.

Differential attenuation of the induction by Marek's disease virus of transient paralysis and persistent neurological disease: A model for pathogenesis studies

Since different biological characteristics of Marek's disease virus (MDV) are attenuated at different passage levels in cell culture, an analysis of attenuation times provides, in theory, a model for establishing the presence or absence of relationships between characteristics, thus providing a basis to link them to genetic changes in the causative virus. We have used this model to better understand the pathogenesis of the central nervous system infection as well as to evaluate the relationship of clinical neurological disease to various other parameters of MDV infection. Inoculation of 15 2 7 crossbred chickens with strain 648A of very virulent plus MDV at different passage levels (between 10 and 100) showed that two neurological syndromes (transient paralysis (TP) and persistent neurological disease), were attenuated at different passage levels. While strain 648A lost the ability to induce TP between 30 and 40 passages in chicken embryo fibroblast cultures, an event closely related with all parameters of MDV infection involving viral replication (early cytolytic infection in lymphoid organs and viral replication in the feather follicle epithelium), the ability to induce persistent neurological disease was lost between 80 and 90 passages in chicken embryo fibroblasts, coincident with the loss of neoplastic lesions in peripheral nerves and other visceral organs. These data strongly suggest that transient paralysis and persistent neurological disease are unrelated and differently regulated. Moreover, comparison of brain changes induced by strain 648A at passage level 30 (TP) and at passage level 40 (no TP) also contributed to a better understanding of which brain alterations are associated with the onset of TP. The use of viruses at different passage levels with varying degrees of attenuation is presented as a useful tool for studying pathogenesis of MDV infection.

Resistance to Marek's disease herpesvirus-induced lymphoma is multiphasic and dependent on host genotype.

Genotype-dependent differences in Marek's disease (MD) susceptibility were identified using 14-day-old line N and 6(1) (resistant) and 151 and 7(2) (susceptible) inbred chickens infected with HPRS-16 MD virus (MDV). All line 72 chickens developed progressive MD. Line 15I had fluctuating MD-specific clinical signs and individuals recovered. A novel histologic scoring system enabled indices to be calculated for lymphocyte infiltration into nonlymphoid organs. All genotypes had increased mean lesion scores (MLSs) and mean total lesion scores after MDV infection. These differed quantitatively and qualitatively between the genotypes. Lines 6(1) and 7(2) had a similar MLS distribution in the cytolytic phase, although scores were greater in line 7(2). At the time lymphomas were visible in line 7(2), histologic lesions in line 6(1) were regressing. AV37+ cells were present in similar numbers in all genotypes in the cytolytic phase, suggesting that neoplastically transformed cells were present in all genotypes regardless of MD susceptibility. After the cytolytic phase, AV37+ cell numbers increased in lines 7(2) and 15I but decreased in lines 6(1) and N. In the cytolytic and latent phases, in all genotypes, most infiltrating cells were CD4+. After this time, line 7(2) and 15I lesions increased in size and most cells were CD4+; line 6(1) and N lesions decreased in size and most cells were CD8+. In all genotypes, AV37 immunostaining was weak in lesions with many CD8+ cells, suggesting that AV37 antigen expression or AV37+ cells were controlled by CD8+ cells. The rank order, determined by clinical signs and pathology, for MD susceptibility (highest to lowest) was 7(2) > 15I > 6(1) > N.

Immune responses to Marek's disease virus infection.

Marek’s disease (MD), a herpesvirus-induced lymphomatous disease in chickens, has attracted the interest of immunologists since MD virus (MDV) was isolated in 1968 and vaccines became available shortly afterwards (Witter 1985,Witter 2000). The pathogenesis of MD has been reviewed extensively (Calnek 1986,Calnek 1998,Calnek 2000; Schat 1987b). Infection of chickens with MDV is characterized by several distinct phases in which innate and acquired immune responses play important roles. The first phase is characterized by the replication of MDV in lymphoid cells, which are mostly B lymphocytes. The consequence of the lytic infection is that T lymphocytes become activated. Only activated, but not resting, T cells can be infected with MDV. Schat and Xing (2000) hypothesized recently that a viral homologue of interleukin (IL)-8 (vIL-8) (LIu et al. 1999) may be involved in attracting the activated T cells to the infected B cells (see Sects. 2.1, 6.2) to facilitate the transfer of virus. Intimate contact between B and T cells is important for the transfer of infectious virus from cell to cell (Kaleta 1977), because MDV is highly cell-associated (Schat 1984). Temporal immunosuppression of the humoral immune responses is often one of the consequences of the lytic infection in B lymphocytes. During the second phase, a latent infection is normally established in these activated T cells, although some activated CD4+ and CD8+ T cells may undergo a lytic infection (Baigent et al. 1998). The actual process of establishment and maintenance of latency has not been elucidated, but it is likely that a complex set of interactions, including immune responses and specific cellular and viral genes, is responsible.

In vivo Events of Retroviral Long Terminal Repeat Integration into Marek's Disease Virus in Commercial Poultry: Detection of Chimeric Molecules as a Marker

The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV. A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HS-cPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.

Coinfection of Specific-Pathogen-Free Chickens with Marek's Disease Virus (MDV) and Chicken Infectious Anemia Virus: Effect of MDV Pathotype

Both Marek's disease virus (MDV) and chicken infectious anemia virus (CIAV) infections are prevalent in chickens throughout the world. In the past decade, MDV strains with increased virulence (very virulent plus MDV pathotype [vv+MDV]) have been isolated. The purpose of this experiment was to determine the effects of coinfection of chickens with CIAV and a vv+MDV isolate. Specific-pathogen-free chickens were inoculated at 1 day posthatch with RB1B (very virulent MDV pathotype [vvMDV]) only, 584A (vv+MDV) only, CIAV only, RB1B + CIAV, 584A + CIAV, or nothing. Samples of spleen, thymus, and bursa of Fabricius were collected at 4, 7, 10, and 13 days postinoculation (DPI). Thymic and bursal atrophy at 13 DPI and final mortality at 30 DPI were significantly greater in chickens inoculated with 584A with or without added CIAV, or with RB1B plus CIAV, compared with birds inoculated with RB1B alone. Both amounts of virus reisolated and levels of virus detected by quantitative-competitive polymerase chain reaction were greater at 4 DPI in 584A inoculates compared with RB1B inoculates. To monitor the early cytolytic infection, northern analysis was done with a probe for the MDV immediate early gene ICP4 (infected cell protein 4). In the absence of CIAV, ICP4 expression was more apparent in chickens inoculated with 584A than in those inoculated with RB1B. CIAV coinfection increased ICP4 expression in the spleens of chickens infected with RB1B. These results indicated that inoculation of chickens with the 584A isolate caused a more robust early cytolytic infection compared with inoculation with RB1B alone and support the classification of 584A as a vv+MDV strain. Coinfection with CIAV exacerbated vvMDV strain RB1B infection. The extent of this exacerbation was less evident when birds were coinfected with 584A and CIAV.

Anti-viral and anti-tumor effects induced by an attenuated Marek's disease virus in CD4- or CD8-deficient chickens.

By vaccination with an attenuated Marek's disease virus (MDV), strain CVI988, chickens are protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8(+) T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4(+) or CD8(+) T cell-depleted chickens. These results suggest that specific CD8(+) T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV.

Lack of evidence for Marek's disease virus genomic sequences in leukocyte DNA from multiple sclerosis patients in Germany

Herpes viruses have repeatedly been associated with the etiology of multiple sclerosis (MS). In the course of investigations on a cluster of MS patients in Key West, Florida, it was suggested that Marek's disease virus (MDV), a cell-associated avian herpes virus, might be involved in the pathogenesis of the disease. The aim of our study was to investigate 107 well-defined MS patients with regard to latent MDV infection. Using a highly-sensitive polymerase chain reaction technique, we did not find MDV-related sequences in leukocyte DNA of any of the patients. The results of our study do not suggest an implication of MDV in the pathogenesis of MS.

Atherosclerosis in Marek's disease virus infected hypercholesterolemic roosters is reduced by HMGCoA reductase and ACE inhibitor therapy

Accelerated atherosclerosis is associated with herpesviral infection both in transplant patients and after balloon angioplasty. Marek's disease virus (MDV) is a herpesvirus that induces accelerated atherosclerosis associated with the development of an invasive lymphoma in hyperlipemic roosters. We have examined the effects of pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitor and quinapril, an angiotensin converting enzyme (ACE) inhibitor, on atherosclerosis development in MDV infected, cholesterol fed rooster chicks. The effects of these drugs on plaque growth after MDV infection were examined in two studies. In Study 1, MDV infected White Leghorn rooster chicks were divided into 4 groups assigned to normal or high cholesterol diet, and treated at three months of age with either pravastatin or saline. In Study 2, cholesterol fed rooster chicks infected with MDV were divided into 3 groups for treatment with either pravastatin, quinapril, or saline control. A significant decrease in plaque area was detected after 60 days of treatment with both pravastatin and quinapril in cholesterol fed chicks (P < 0.001). Lymphocyte infiltration into the arterial wall or target organs was not inhibited by treatment with either drug. (1) HMGCoA reductase inhibitor and ACE inhibitor therapy reduce atherosclerosis induced by virus infection and cholesterol diet, but this decrease in plaque growth is not due to a reduction in lymphocyte invasion. (2) MDV infection in cholesterol fed roosters provides a model for virus-induced arterial injury in atherogenesis.

Transformation of Primary Chick Embryo Fibroblasts by Marek's Disease Virus

Marek's disease virus (MDV) is an alphaherpesvirus, which can mediate the malignant transformation of lymphocytes to form lymphomas in chickens. In this study, we demonstrate that MDV can transform primary chick embryo fibroblasts (CEF). The cell line derived from primary CEF infected with the GA strain of MDV was called CEM(MDV). The fibroblast nature of CEM(MDV)was verified by absence of cytokeratin type II. The CEM(MDV)phenotype differed from either primary CEF or MDV-infected CEF. CEM(MDV)were extensively vacuolated, with unusual multilamellar structures in the cytoplasm. The nuclei were considerably larger than those in primary CEF and were uniformly positive for proliferating cell nuclear antigen. The cell line was subcultured for more than 10 generations; however, CEM(MDV)did not support a fully productive MDV infection, because complete nucleocapsids were not detected and infectivity assays showed that the cell line produced no infectious virus. PCR analyses demonstrated that this cell line carried both polypeptide 38 (pp38) and Meq DNA, MDV-specific genes associated with transformation. In addition, examination by laser scanning confocal microscopy revealed that CEM(MDV)constitutively produced MDV MEQ protein in nuclei and pp38 as well as glycoprotein B in the cytoplasm and on the plasma membrane. Growth in soft agar assay demonstrated that CEM(MDV)formed colonies, similar to HeLa and human melanoma cells. Retroviral insertion was not detected in DNA from the CEM(MDV)line.

Evidence That Marek's Disease Virus Exists in a Latent State in a Sustainable Fibroblast Cell Line

Previously, we reported the development of two fibroblastic cell lines (MDV OU2.1 and OU2.2) infected with Marek's disease virus (MDV). The two cell lines, in nonconfluent continuous cultures, displayed characteristics consistent with MDV existing in a latent state. However, presence of distinct plaques in confluent cell monolayers and the ability to transfer cytolytic infection to susceptible birds and primary chick embryo fibroblasts, suggest that, if latent, the virus is easily reactivated from MDV OU2 cell lines. In this report, we present evidence which supports the hypothesis that MDV genomes in MDV OU2 cells are latent. PCR analyses andin vivoexperiments demonstrate that CHCC-OU2 cells stabilize MDV so that serialin vitropassage does not attenuate the virus. Following two years of active culture, MDV genomes in MDV OU2 cells are still oncogenic, similar to that seen in MDV-lymphoblastoid cell lines. Expansion of the 132-bp repeat within MDVBamHI fragments H and D, typical of highly passaged serotype 1 MDV, has not been observed beyond two copies in MDV OU2 cells. Indirect immunofluorescence assays clearly demonstrate that MDV OU2 cells do not express glycoproteins B and I when subconfluent. However, upon reaching confluence these proteins are expressed in readily detectable amounts. Using RT-PCR we demonstrate that glycoproteins E and D are highly expressed in confluent MDV OU2 cells but absent from subconfluent cells, and MDV latency-associated transcripts (LATs), which are antisense to ICP4 transcripts and have been associated with latent MDV infection, are expressed in subconfluent MDV OU2 cells. Coincident with an increase in ICP4 expression, MDV LAT expression is down regulated when MDV OU2 cells become confluent.

Syngeneic Marek's Disease Virus (MDV)-Specific Cell-Mediated Immune Responses against Immediate Early, Late, and Unique MDV Proteins

Marek's disease (MD) infection has been controlled effectively by vaccination using nononcogenic and/or attenuated oncogenic Marek's disease virus (MDV) vaccines. Thus far, there is little knowledge on the role of cell-mediated immune (CMI) responses during MDV infection or vaccination. To elucidate the importance of MDV proteins in CMI responses, the pp38, Meq, ICP4, or ICP22 genes of an oncogenic strain, GA and the gB, ORF A, A41, or L1 genes of a highly oncogenic strain, RB1B were stably transfected into reticuloendotheliosis virus (REV)-transformed lymphoblastoid cells, CU-91 (MHC:B19B19) and CU-205 (MHC:B21B21). Cell lines positive for MDV gene transcription and/or protein expression were used in a standard 4-hr chromium release assay. Effector cells for this assay were obtained from splenocytes of chickens infected with the oncogenic strain, JM-16/13 or the nononcogenic vaccine strain, SB-1/12. Cell lines expressing MDV pp38, Meq, or gB were lysed by syngeneic but not allogeneic MDV-sensitized splenocytes obtained from chickens ofB19B19andB21B21haplotypes. However, syngeneic CMI responses against ICP4 were detected only inB21B21chickens. CMI responses were not detected againstB19B19andB21B21cell lines expressing A41, L1, ORF A, or ICP22. This report suggests that syngeneic CMI responses against pp38, Meq, ICP4, and gB of GA and RB1B strains, respectively, can be induced in chickens inoculated with JM16/13 or SB-1/12. The difference in CMI response to ICP4 in genetically susceptible (B19B19) and genetically resistant (B21B21) chickens may be an important factor in genetic resistance.

Influence of Marek's Disease Virus Strain AC-1 on Cellular Immunity in Birds Carrying Endogenous Viral Genes

The effects of Marek's disease virus (MDV) strain AC-1 on humoral and cellular immune responses were investigated in two lines of chickens segregating for the endogenous viral (ev) genes ev-6 and ev-12. All birds were vaccinated at 14 days of age against Newcastle disease virus (NDV). At 3 wk of age, 48 birds per line received an intraperitoneal injection of MDV (AC-1 isolate), and 24 were injected with saline. Birds of each group were killed on days 5, 7, 12, and 14 postinfection. Data were first analyzed for each day of testing. Results showed that, for variable measured, treatment effects were similar on days 5 and 7, and on days 12 and 14. Therefore, day 5 and day 7 data, and day 12 and day 14 data were pooled and analyzed. In MDV-infected chickens, proliferative lymphocyte responses to mitogens were suppressed (P < 0.001) after the first and second week of infection, whereas responses to NDV antigen were enhanced (P < 0.001) after the first week and then reduced (P < 0.01) by the end of the second week when compared to uninfected birds. The percentage of CD4+ T cells was higher (P < 0.01), and the percentage of cells expressing major histocompatibility complex (MHC) class II antigens was lower (P < 0.001), in MDV-infected chickens than in uninfected birds. The cytotoxic activity of natural killer (NK) cells was also enhanced (P < 0.001) in MDV-infected birds when compared to uninfected birds. Antibody responses to NDV were not different among groups, and the presence of ev-6 or ev-12 genes did not influence the immune response parameters measured in both infected and uninfected chickens. In conclusion, a marked increase in the CD4+ T lymphocyte population occurred in the early stage of MDV infection in all chickens regardless of the presence of ev genes, whereas the number of cells expressing MHC class II antigen was severely reduced.

Genetic Susceptibility to Marek's Disease Virus of Local Chickens in Taiwan

Marek's disease is a contagious disease in chickens caused by Marek's disease virus (MDV) infection. Invasion of very virulent MDV (vvMDV) was considered to be a major cause of vaccine break, resulting in a large economic loss in the poultry industry. Two strains of the vvMDV (strains LTB-1 and LTS-1) have been reported in Taiwan, causing early mortality and formation of lymphoid tumors in broilers and layers. In this study, we report the susceptibility of local chickens in Taiwan to inoculation with strains LTB-1 and LTS-1 at 1 day of age. Five lines of Taiwanese local chickens (lines B, D, L2, KM, and BG) were used to compare the susceptibility to vvMDV. The chicks were inoculated via intraperitoneal route at 1 day of age. All MDV-inoculated chickens showed atrophy of various lymphoid organs. Protection tests were also conducted in local chickens, using vaccine of herpesvirus of turkey (HVT) at 1 day of age followed by vvMDV challenge at 10 days of age. Among the vaccinated chickens, 0-20.0% showed early mortality, and tumor occurrences in visceral organs and in peripheral nerves were 66.7%-100% and 0-27.2%, respectively. From these results, it could be concluded that local chickens in Taiwan have different susceptibilities to vvMDVs. Furthermore, vaccination with HVT showed no protective effect against Taiwanese vvMDV isolate challenge in this experiment.

Association between the Rfp-Y haplotype and the incidence of Marek's disease in chickens.

Certain haplotypes at the major histocompatibility (B) complex (Mhc) of the chicken provide an easily demonstrated influence on tumor formation following infections with Marek's disease virus (MDV). Recognition that there is a second histocompatibility complex of genes in the chicken, Rfp-Y, comprised of Mhc class I and class II genes, some of which are at least transcribed, evokes the question of whether this gene complex might also influence the outcome of MDV infections. To test this hypothesis, pedigree-hatched chicks in families from the original Rfp-Y-defining stock in which three Rfp-Y and two B system haplotypes are segregating were challenged with the RB1B strain of MDV. Birds with the Y3/Y3 genotype were found to have 2.3 times the risk of developing a tumor compared with birds with other Rfp-Y genotypes combined (P <0.02). Additionally, birds carrying the BR9/B11 genotype had 2.3 times the risk of tumor formation, relative to birds with the B11/B11 genotype (P <0.02). We found no evidence for an interaction between genotypes within the B and Rfp-Y systems. These data provide evidence that Rfp-Y haplotypes, as well as B haplotypes, can significantly influence the outcome of infection with MDV.

Development of a Sustainable Chick Cell Line Infected with Marek's Disease Virus

Marek's disease virus (MDV) is a highly infectious and cell-associated avian herpesvirus. Fully productive infections with MDV are restricted to feather follicle epithelium of afflicted birds. In culture, MDV infection of primary chick (CEF) and duck embryo fibroblast cells is semiproductive. Passage of MDV and production of MDV vaccines is limited to these primary cell-associated systems. The finite life span of primary avian cell cultures has hampered efforts to use positive selection in generation of recombinant MDV and complicates studies of temporal gene regulation. In this report, we describe continuous chick fibroblast cell lines (MDV OU2.2 and MDV OU2.1) which support MDV replication. Southern blot and PCR analyses demonstrate that these cell lines harbor MDV DNA. Western blot analyses indicate that MDV OU2.2 cells express at least a limited set of viral proteins, pp38 and pp14, similar to that seen in MDV-lymphoblastoid cells. Presence of distinct plaques in confluent MDV OU2.2 cell monolayers is consistent with cytolytic semiproductive infection, similar to that observed in primary CEF. MDV OU2.2 cells are capable of transferring MDV infection to primary CEF cultures and inducing clinical signs of Marek's disease in susceptible birds. MDV OU2.2 cells have maintained a MDV-positive phenotype for over 16 months of active culture. Southern blot hybridization of MDV OU2.2 cell DNA reveals a distinct expansion of the MDVBamHI H fragment in a subset of viral genomes following long-term cultivation.

Immunomodulation of peripheral T cells in chickens infected with Marek's disease virus: involvement in immunosuppression.

Marek's disease virus (MDV) causes T cell immunosuppression in chickens during latent infection. Morphological changes specific to apoptosis were demonstrated in peripheral blood mononuclear cells (PBMC) of MDV-infected chickens at 2-3 weeks post-inoculation (p.i.). Analysis of DNA fragmentation in T cell subsets in the peripheral blood revealed that CD4+ T cells but not CD8+ T cells underwent apoptosis after MDV infection. The proportion of CD4+ T cells, but not that of CD8+ T cells, in the peripheral blood expanded transiently at 16 days p.i., and rapidly decreased 1 week later. The decrease in CD4+ T cells might be mediated by apoptosis, because a rapid reduction in CD4+ T cells was observed when these cells underwent apoptosis. Analysis of the T cell-receptor (TCR) repertoire of the peripheral blood showed that V beta 1 but not V beta 2-alpha beta TCR-bearing cells expanded at 16 days p.i., when the transient expansion of the CD4+ T cell population was observed in these chickens. Flow cytometric profiles also showed that the expression of CD8 was down-regulated after infection with MDV, but there was no difference in the expression level of CD4 molecules between normal and infected chickens. Northern blot analysis indicated that the down-regulation of CD8 occurred at the transcriptional level. These results suggest that both apoptosis of CD4+ T cells and down-regulation of CD8 molecules could contribute to the immunosuppression caused by MDV.

Endothelial MHC class II antigen expression and endarteritis associated with Marek's disease virus infection in chickens.

Experimental Marek's disease virus (MDV) infection in chickens was used to study the early pathogenesis of virus-induced atherosclerosis. Previous investigations using this model have reported the occurrence of atherosclerotic lesions after approximately 7 months postinfection. In this study, a total of 75 susceptible Cornell P-line chickens were inoculated intraperitoneally with the CU-2 strain of MDV at 3 days of age and subsequently perfused for histologic examination. At 2, 4, 8, 13, and 20 weeks postinoculation, the ascending aorta and the brachiocephalic and coronary arteries were evaluated for early changes. Expression of class II major histocompatibility complex (Ia) antigen by the vascular endothelium was demonstrated by indirect immunodetection as early as 2 weeks after virus inoculation. This change was followed by significant thickening of the intimal layer associated with mononuclear cell infiltration. All the arteries examined from the MDV-infected chickens were affected. Preliminary immunohistochemical staining showed the presence of CD3+ CD4+, and CD8+ cells among the infiltrating cells. The results suggest that an immunopathologic mechanism may be involved in the early pathogenesis of MDV-induced atherosclerosis in chickens.

Kinetic analysis of T cells and antibody production in chickens infected with Marek's disease virus.

In chickens inoculated with a Marek's disease (MD) vaccine and subsequently with virulent MD virus (MDV), CD4+ T cell population was drastically decreased following a transient increase at 21 days after hatching (16 days after MDV infection). To elucidate the immune response after the decrease of CD4+ T cell population, the antibody production against sheep red blood cells (SRBC) was examined in these chickens. Chickens challenged with a virulent MDV after MD vaccination produced lower titers, of anti-SRBC antibody than untreated control chickens. Antibody production against SRBC was also lowered in vaccinated chickens or chickens challenged with a virulent MDV.