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.

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.

A deletion within glycoprotein L of Marek's disease virus (MDV) field isolates correlates with a decrease in bivalent MDV vaccine efficacy in contact-exposed chickens.

We examined the functional role of a naturally occurring deletion within the glycoprotein L (gL) gene of Marek's disease virus (MDV) field isolates. We previously showed that this mutation incrementally increased the virulence of an MDV in contact-exposed SPF leghorn chickens, when chickens shedding this virus were co-infected with herpesvirus of turkeys (HVT). In our present study, we examined this mutation using two stocks of the very virulent plus (vv+)MDV strain TK, one of which harbored this deletion (TK1a) while the other did not (TK2a). We report that TK1a replicating in vaccinated chickens overcame bivalent (HVT/SB1) vaccine protection in contact-exposed chickens. Treatment groups exposed to vaccinated chickens inoculated with a 1:1 mix of TK1a and TK2a showed decreased bivalent vaccine efficacy, and this decrease correlated with the prevalence of the gL deletion indicative of TK1a. These results were also found using quadruplicate treatment groups and bivalently vaccinated chickens obtained from a commercial hatchery. As this deletion was found in 25 out of 25 recent field isolates from Delaware, Maryland, North Carolina, Pennsylvania, and Virginia, we concluded that there is a strong selection for this mutation, which appears to have evolved in HVT or bivalently vaccinated chickens. This is the first report of a mutation in a vv+MDV field strain for which a putative biological phenotype has been discerned. Moreover, this mutation in gL has apparently been selected in MDV field isolates through Marek's disease vaccination.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis.

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.