Marek's Disease Virus Latency Research Articles

Latency and Tumorigenesis in Marek's Disease

Despite the remarkable progress in our understanding of Marek's disease (MD) and the causative Marek's disease virus (MDV) biology, a number of major features of this complex viral disease remain unknown. Significant information on critical aspects of virus latency in lymphoid cells, and the virus-host interaction in MDV-induced lymphoma, remains to be identified. Moreover, the nature of the unique milieu of the feather follicle epithelial cell that allows cytolytic infection to continue, despite maintaining the latent infection in the lymphoid cells, is not fully understood. Although there has been significant progress in our understanding of the functions of a number of viral genes in the pathogenesis of the disease, the characteristics of the latent infection, how it differs from tumor phase, and whether latency is a prerequisite for the tumor phase are all important questions still to be answered. Reticuloendotheliosis virus-transformed cell lines have been shown to support MDV latency in a manner almost identical to that seen in MDV-transformed cell lines. There are increasing data on the role of epigenetic regulation, including DNA methylation and histone modifications, in maintaining viral latency. Onset of MD tumor is relatively rapid, and recent studies based on chromosomal integration and T-cell repertoire analysis demonstrated the clonal nature of MD lymphomas. Among the viral determinants of oncogenicity, the basic leucine zipper protein Meq is considered to be the most important and the most extensively studied. Deleting the Meq proteins or abolishing some of the important interactions does affect the oncogenicity of the virus. In addition, the noncoding sequences in the viral genome, such as the viral telomerase RNA and the virus-encoded microRNAs, also have significant influence on MDV-encoded oncogenesis.

Latency of Marek's Disease Virus (MDV) in a Reticuloendotheliosis Virus–Transformed T-Cell Line. II: Expression of the Latent MDV Genome

Marek's disease virus (MDV) is an alphaherpesvirus of chickens that causes the paralysis and rapid lymphoma formation known as Marek's disease. MDV establishes latent infection in activated CD4+ T-cells, and these cells are also the target for transformation. MDV latency has been studied using MDV lymphoma-derived cell lines and T-cells isolated from infected chickens. Each of these models has limitations because MDV-transformed cell lines require the use of oncogenic viruses; conversely, pools of latently infected cells are in relatively low abundance and invariably contain cells undergoing reactivation to lytic infection. In this study we have examined the spontaneous and induced expression of the MDV genome, the effect of genome uptake on cellular proliferation and apoptosis resistance, and differences in cellular surface antigen expression associated with MDV genome uptake in a reticuloendotheliosis virus (REV)-transformed T-cell model. We report that the MDV genome is highly transcribed during this latent infection, and that the expression of Marek's EcoRI-Q-encoded protein (Meq) transcripts is similar to that of MDV-transformed cells, but is somewhat lower than MDV-transformed cells at the protein level. Uptake of the MDV genome was associated with an increased growth rate and resistance to serum starvation-induced apoptosis. Treatment of cells with bromodeoxyuridine induced the expression of MDV lyric antigens in a manner similar to MDV-transformed cells. Uptake of the MDV genome, however, was not consistently associated with alteration ofT-cell surface antigen expression. Overall, our data show that the REV-transformed cell line model for MDV latency mimics many important aspects of latency also observed in MDV-transformed cells and provides an additional tool for examining MDV latent infection.

Latency of Marek's Disease Virus (MDV) in a Reticuloendotheliosis Virus–Transformed T-Cell Line. I: Uptake and Structure of the Latent MDV Genome

Marek's disease virus (MDV) is an acute transforming alphaherpesvirus of chickens that causes Marek's disease. During the infection of chickens, MDV establishes latency in CD4+ (T-helper) cells, which are also the target of transformation. The study of MDV latency has been limited to the use of MDV tumor-derived cell lines or blood cells isolated from chickens during presumed periods of latent infection. In 1992 Pratt et al. described the uptake of the MDV genome by a reticuloendotheliosis-transformed T-cell line (RECC-CU91). They reported that MDV established latency in CU91 cells, but that MDV genome expression was very limited. In this report we have examined the uptake of oncogenic, recombinant, and vaccine strain MDVs. We report that the entire MDV genome is taken up by CU91 cells, is hypomethylated, and readily reactivates from this latent state in a manner similar to MDV-transformed cell lines. Notably, virus could not be recovered from cell lines harboring vaccine virus CVI988 or the JM102 strain of MDV. Overall these cell lines present a useful model for the further study of MDV latency, particularly for those viruses having mutations that may affect replication or fitness of the virus in vivo. In addition, these cell lines offer an attractive means to study the latency of vaccine viruses, which establish relatively low levels of latent infection in vivo.

Marek's disease virus reactivation from latency: changes in gene expression at the origin of replication

Marek's disease is a contagious lymphoma of chickens caused by Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes latency within and transforms chicken CD4+ T-cells. Transformed T-cells are seen as skin leukosis or as lymphomas in visceral organs. A major focus of our laboratory is the functional study of genes flanking the origin of replication. This origin (OriLyt) is contained within the repeats flanking the unique long (UL) region of the genome (IRL and TRL). To the left of this Ori are genes associated with MDV latent/transforming infection [1.8-kb RNA family, pp14, Meq), and to the right (UL) are genes associated with early stages of MDV lytic infection [BamHI-H-encoded protein (Hep), pp38/pp24, Mys]. During latency, MDV suppresses lytic gene expression and has evolved mechanisms for blocking the apoptosis of latently-infected CD4+ T-cells. Of the genes expressed during MDV latency and in the transformed cell, the Meq (Marek's EcoRI-Q-encoded protein) has been shown to block apoptosis and transactivate gene expression. Upon reactivation to lytic infection, we have found that splice variants of Meq predominate and that these forms lack several of the domains important to Meq trans-activation and trans-repression. We have found that rightward from the origin of replication, a family genes, including phosphoprotein 38 (pp38) are expressed during early stages of reactivation. Three separate open reading frames (Hep, Mys, and pp38) are encoded by distinct transcripts from this region. We are now determining the kinetics of expression of these transcripts and their relative abundance during reactivation.

Differential cytokine responses following Marek's disease virus infection of chickens differing in resistance to Marek's disease.

The production of cytokine mRNAs, in addition to viral DNA, was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) (cytokines) or PCR (virus) in splenocytes during the course of Marek's disease virus (MDV) infection in four inbred chicken lines: two resistant (lines 6(1) and N) and two susceptible (lines 7(2) and P). Virus loads were only different after 10 days postinfection (dpi), increasing in susceptible lines and decreasing in resistant lines. Gamma interferon (IFN-gamma) mRNA was expressed by splenocytes from all infected birds between 3 and 10 dpi, associated with increasing MDV loads. For other cytokines, differences between lines were only seen for interleukin-6 (IL-6) and IL-18, with splenocytes from susceptible birds expressing high levels of both transcripts during the cytolytic phase of infection, whereas splenocytes from resistant birds expressed neither transcript. These results indicate that these two cytokines could play a crucial role in driving immune responses, which in resistant lines maintain MDV latency but in susceptible lines result in lymphomas.

Stages of Marek's disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression

Cytokines in conditioned medium can suppress expression of viral internal antigens (VIA) in lymphocytes latently infected with Marek's disease virus. In the present study, conditioned media produced by spleen cells stimulated with concanavalin A or by mixed-lymphocyte reaction had significantly greater ( P < 0.05) VIA-suppressive activity with lymphocytes harvested from birds at 14 days post infection than with those collected at 7 days. This finding defines two stages during the latent period in which sensitivity of lymphocytes to cytokine modulation of viral expression differs. Suppression involved proteins representing immediate-early, early and late viral antigens. Physico-chemical characterization of the suppressive factor in conditioned medium was consistent with that expected of interferon. Indeed, natural interferon prepared from avian reovirus-exposed chicken embryo cells, and recombinant chicken interferon, both mimicked the activity of conditioned medium and were more suppressive with lymphocytes from the later stage of latency.

Latency and reactivation of Marek's disease virus in B lymphocytes transformed by avian leukosis virus.

The physical and biological state of the Marek's disease virus (MDV) genome in avian leukosis virus (ALV)-transformed cells is characterized using cell lines established from ALV tumours co-infected with the SB-1 strain of MDV. The MDV genome within the ALV-transformed cells was found to be methylated at 5' CpG 3' dinucleotides. Less than 2% of the tumour cells expressed MDV antigen and only one virus plaque that was characteristic of an MDV infection was noted when tumour cells were cocultured with fibroblasts permissive for a productive MDV infection. However, when methylation of the MDV genome was prevented by culturing the tumour cell lines in the presence of 5-azacytidine, both MDV antigen expression and viral replication increased. Based on these results, it appears that MDV resides within the ALV-transformed cells in a latent state and that MDV latency might be influenced, to some extent, by methylation of the MDV genome.

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.

Effect of reticuloendotheliosis virus and infectious bursal disease virus on MAREK'S disease herpesvirus latency

Birds infected with reticuloendotheliosis virus (REV) were exposed to Marek's disease virus (MDV) to determine if the establishment of MDV latency was affected by REV-induced immunosuppression, while other chickens, already latently infected with MDV, were challenged with REV or infectious bursal disease virus (IBDV) to determine if the consequent immunosuppression caused a return to cytolytic infection. Immunosuppression was assessed by in vitro mitogen stimulation assays with spleen cells. Latently MDV-infected cells were free of viral internal antigen(s) (VIA) but could be identified by their ability to produce VIA after in vitro cultivation. The results were unexpected: chickens infected with either of these viruses had very low, and often undetectable, levels of MDV infection when compared with appropriate controls. REV infection interfered with early cytolytic MDV infection, and IBDV and REV both failed to activate latent MDV infection in the face of inferred (IBDV) or demonstrated (REV) immunosuppression by these viruses. Apparently, both viruses reduced the number of MDV infected cells since neither cytolytic nor latent infection could be demonstrated. This was based on an absence of cells with VIA either before or after cultivation and, in the case of REV infection, on failure to detect MDV-DNA using a dot-blot hybridisation technique.