Marek's Disease Virus-infected Chickens Research Articles

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-βeta (IFN-β), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

Immune Responses in Cecal Tonsils of Marek's Disease Virus-Infected Chickens.

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens that is caused by a highly cell-associated oncogenic α-herpesvirus, Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. Clinical signs of MD include depression, crippling, weight loss, bursal/thymic atrophy, neurologic disorders, and rapid onset of T cell lymphomas that infiltrate lymphoid tissues, visceral organs, and peripheral nerves. The cecal tonsils (CTs) are considered the largest lymphoid aggregates of avian gut-associated lymphoid tissue. Along with Peyer's patches, CTs elicit protective immune responses against bacterial and viral pathogens in the intestinal tract of avian species. In this study, we investigated the effect of MDV infection on toll-like receptor (TLR) gene expression in CTs of MD-susceptible (72) and resistant (63) chicken lines. Real-time PCR gene expression profiling revealed that of the 10 TLRs tested, TLR2A, TLR3, TLR5, and TLR15 displayed significant differential expression patterns at different time points postinoculation. The expression levels of the remaining six genes were minimally affected by MDV infection in either line. Immunohistochemical analysis showed a severe depletion of B cells and CD4+ T cells in the CTs of susceptible line at 5 days postinfection (dpi), which recovered by 21 dpi. The destruction of B and T cells in the CTs of the resistant line was minimal at 5 dpi, which also recovered by 21 dpi. A significant infiltration of macrophages was observed after the depletion of B and T cells in the infected birds of both lines that could account for the differential TLR gene expression in the infected birds. The data presented provide further insight into the mechanism of MDV pathogenesis and tissue-specific immunologic responses to viral infection.

Systems analysis of immune responses in Marek's disease virus-infected chickens identifies a gene involved in susceptibility and highlights a possible novel pathogenicity mechanism.

Marek's disease virus (MDV) is a highly contagious oncogenic alphaherpesvirus that causes disease that is both a cancer model and a continuing threat to the world's poultry industry. This comprehensive gene expression study analyzes the host response to infection in both resistant and susceptible lines of chickens and inherent expression differences between the two lines following the infection of the host. A novel pathogenicity mechanism, involving the downregulation of genes containing HIC1 transcription factor binding sites as early as 4 days postinfection, was suggested from this analysis. HIC1 drives antitumor mechanisms, suggesting that MDV infection switches off genes involved in antitumor regulation several days before the expression of the MDV oncogene meq. The comparison of the gene expression data to previous QTL data identified several genes as candidates for involvement in resistance to MD. One of these genes, IRG1, was confirmed by single nucleotide polymorphism analysis to be involved in susceptibility. Its precise mechanism remains to be elucidated, although the analysis of gene expression data suggests it has a role in apoptosis. Understanding which genes are involved in susceptibility/resistance to MD and defining the pathological mechanisms of the disease gives us a much greater ability to try to reduce the incidence of this virus, which is costly to the poultry industry in terms of both animal welfare and economics.

Expression profiling of genes associated with regulatory functions of T-cell subsets in Marek's disease virus-infected chickens

The environment of tumours caused by Marek's disease virus (MDV) in chickens has been shown to have an immunoregulatory phenotype. The objective of the present study was to examine the expression of key T-regulatory markers during various stages of MDV pathogenesis. Specific-pathogen free (SPF) as well as major histocompatibility complex-defined chickens were infected with the RB1B and JM-16 strains of MDV, respectively. CD4+ and CD8+ T cells from the spleens of infected as well as age-matched controls were sorted by flow cytometry at 4, 10, and 21 days post infection (d.p.i.). The expression of molecules such as CTLA-4, IL-2aR (CD25), PD-1 and PDL-1 was quantified by real-time, quantitative, reverse-transcription polymerase chain reaction. There was an up-regulation of CTLA-4 in CD4+ T cells at 4 d.p.i. The expression of PD-1 was also up-regulated in the CD4+ T-cell subset of SPF birds at 21 d.p.i. Furthermore, the expression of PD-1 was enhanced in CD4+ and CD8+ T cells of genetically susceptible chickens, linking this molecule to susceptibility to disease. The expression of CD25 was down-regulated in both SPF and genetically defined birds after infection. This may be a mechanism through which the virus exerts its immunosuppressive effects. In conclusion, the results of the present study provide more insight into immunomodulatory processes that occur in the lymphoid tissues of MDV-infected chickens.

Expression of Cytotoxicity-Associated Genes in Marek's Disease Virus—Infected Chickens

Cytotoxic host responses to Marek's disease virus (MDV) have been attributed to both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). However, the mechanisms of cell lysis initiated by these cytotoxic responses during MDV infection are not well defined. Therefore, the current study was aimed at elucidating the molecular mechanisms of host cytotoxic responses to MDV infection by investigating the expression of genes in the cell lysis pathway involving granzyme A. Genes encoding cytolytic proteins, NK lysin, and granzyme A were upregulated during early stages of infection, whereas the genes encoding major histocompatibility complex (MHC) class I and the DNA repair and apoptosis protein, poly(ADP-ribose) polymerase (PARP), were downregulated. These findings shed more light on the mechanisms of host response to MDV infection in chickens.

Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαβ1+ CD8+ and CD4+ lymphocytes, bordered by a band of TCRγδ+ lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαβ1+ CD4+ and CD8+ lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.

Development of PCR method specific for Marek's Disease Virus

A rapid polymerase chain reaction (PCR) assay specific for Marek's Disease Virus (MDV) was developed. This assay was able to detect MDV in inoculated chick kidney cells at dilutions of 10 -5. Negative PCR results were obtained using uninoculated chick cells, Marek's Disease Vaccine (SB), Herpesvirus of Turkeys (HVT) and Fowl Laryngotracheitis Vaccine (LT). Bursae, feathers and kidneys from MDV infected chickens were positive in the PCR assay. The same tissues from normal chickens were negative. This method required only 0·5 h for sample preparatory, 3 h for PCR application and 1 h for electrophoresis. Internal probe hybridization confirmed that the PCR products are from MDV, but this hybridization will not be necessary for future MDV detection.

Antibody directed against Marek's disease-associated tumor surface antigen can be eluted from Marek's disease tumor cells

Antibody directed against Marek's disease-associated tumor surface antigen (MATSA) was eluted from tumor cells of lymphomas and peripheral blood lymphocytes that were isolated from Marek's disease virus-infected chickens. Feather follicular Marek's disease virus (MDV) antigen could not be demonstrated with this antibody by indirect immunofluorescent (IF) staining. Monoclonal antibody directed against MATSA could completely block the activity of eluted antibody and vice versa. By indirect IF staining using eluted antibody and fluorescein isothiocyanate (FITC) labelled antichicken globulin conjugate, MATSA-bearing cells were detected in MDV infected and herpes virus of turkey (HVT) vaccinated birds. Blocking of immunoglobulin molecules present on B-cells by anti-chicken globulin is critical in this test.

Growth inhibition of marek's disease T‐lymphoblastoid cell lines by chicken bone‐marrow‐derived macrophages activated in vitro

Studies have been performed on the induction of cytostatic activity of cultured chicken bone-marrow-derived macrophages. Cultured macrophages were exposed to supernatants of ConA-stimulated spleen cell cultures (lymphokines), lipopolysaccharide (LPS), ConA or infectious bursal disease virus (IBDV). Cytostatic activity of macrophages was examined by testing their growth-inhibiting effect on T-lymphoblastoid Marek's disease cell lines RP-1, HP-2 and MSB-1 as target cells. Lymphokines, LPS and IBDV caused considerable enlargement of adherent macrophages. Results of cytostasis assays suggested that morphological signs of macrophage activation were not correlated with the cytostatic potency of activated macrophages. Lymphokine-activated macrophages caused at least an 80% growth inhibition of target cells, whereas LPS, ConA or IBDV-activated macrophages exhibited only a marginal cytostatic effect in the range of 20%. Attempts to detect soluble cytostatic factors in supernatants of activated macrophage cultures failed or had equivocal results. Untreated macrophages were rarely cytostatic at an effector:target cell ratio of I:1 to 4:1, and they stimulated RP-1 cell growth if these cells were seeded at suboptimal concentrations. The results suggest that the suppressor activity of macrophages from Marek's disease virus-infected chickens, as demonstrated by other authors in vitro, is probably a result of nonspecific immunomodulation in vivo where lymphokines may also play a major role.

Preservation of a T Cell-Mediated Immune Response in Marek's Disease Virus-Infected Chickens by Vaccination with a Related Virus

Abstract The oncogenic effect of Merek's disease virus (MDV)3 is manifested by a lymphoproliferative neoplastic condition primarily affecting nerves and visceral organs (For reviews see References 1, 2). Necrosis and atrophy of both the thymus and bursa of Fabricius are frequent findings in MDV-infected birds and degenerative changes are found in feather follicle epithelium where complete enveloped virions are formed. The necrobiosis in the thymus and bursa is associated with the presence of viral antigens but enveloped virions are very rare in these organs. There is evidence that the lymphoid tumors are composed of thymus-derived cells (3, 4) which possess tumor-associated antigens (5). Suppression of both cellular (6) and humoral (7) immune responses in MDV-infected chickens has been reported. In this regard, we have shown that spleen cells from birds infected with MDV are markedly depressed in their responsiveness to the T cell mitogen phytohemagglutinin (PHA) (8).