Robert L. Dienglewicz

Marek's Disease Virus (MDV) Encodes an Interleukin-8 Homolog (vIL-8): Characterization of the vIL-8 Protein and a vIL-8 Deletion Mutant MDV

ABSTRACT Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Mareks disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to theBamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (γ2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8ΔsmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8ΔsmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8ΔsmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.

Understanding mechanisms of vitiligo development in Smyth line of chickens by transcriptomic microarray analysis of evolving autoimmune lesions

BackgroundThe Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather) of SL chickens at different vitiligo (SLV) states.ResultsCompared to the reference sample, which was from Brown line chickens (the parental control), 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE) (P ≤ 0.05) in feather samples collected from SL chickens that never developed SLV (NV), from SLV chickens prior to SLV onset (EV), during active loss of pigmentation (AV), and after complete loss of melanocytes (CV). Comparisons of gene expression levels within SL samples (NV, EV, AV and CV) revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death.ConclusionsThe microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune response plays a predominant role in melanocyte loss.

Examination of the Effect of a Naturally Occurring Mutation in Glycoprotein L on Marek's Disease Virus Pathogenesis

Abstract We recently reported a comparison of glycoprotein-encoding genes of different Mareks disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four–amino acid) deletion in the glycoprotein L (gL)–encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK/HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test groups, these data suggest that replication of the TK strain and HVT, when coadministered, might incrementally affect the virulence of MDV-1 strains. The strict correlation of this enhancement of virulence with the mutation in gL, however, requires additional experiments with genetically identical MDV background strains.

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

Abstract Mareks disease virus (MDV) is an alphaherpesvirus of chickens that causes the paralysis and rapid lymphoma formation known as Mareks 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 Mareks 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 lytic antigens in a manner similar to MDV-transformed cells. Uptake of the MDV genome, however, was not consistently associated with alteration of T-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.

Construction and Characterization of Marek's Disease Viruses Having Green Fluorescent Protein Expression Tied Directly or Indirectly to Phosphoprotein 38 Expression

Abstract Mareks disease (MD) is caused by Mareks disease virus (MDV), a highly cell-associated alphaherpesvirus. MD is primarily characterized by lymphocyte infiltration of the nerves and the development of lymphomas in visceral organs, muscle, and skin. MDV encodes two phosphoproteins, pp24 and pp38, that are highly expressed during lytic infection. These proteins were initially identified in MDV-induced tumors but are now known to be linked primarily to MDV lytic infection. Despite the recent characterization of a pp38 deletion mutant MDV, the functions of these phosphoproteins remain unknown. The goal of this work was to construct recombinant MDVs having direct fusions of a marker gene, the green fluorescent protein (GFP), to pp38 in order to study the expression patterns and localization of this protein during stages of MDV infection. We report the construction of two recombinant viruses, one having the enhanced green fluorescent protein (eGFP) fused in-frame to the pp38 open reading frame (ORF) (RB1Bpp38/eGFP) and the other having soluble-modified GFP (smGFP) downstream but out-of-frame with pp38 (RB1Bpp38/smGFP). During construction of RB1Bpp38/eGFP, an ORF located downstream of pp38 (LORF12) was partially deleted. In RB1Bpp38/smGFP, however, LORF12 and its immediate 5′ upstream sequence was left intact. This report describes the construction, cell culture, and in vivo characterization of RB1Bpp38/eGFP and RB1Bpp38/smGFP. Structural analysis showed that the virus stocks of RB1Bpp38/eGFP and RB1Bpp38/smGFP had incorporated the GFP cassette and were free of contaminating parent virus (RB1B). Moreover, RB1Bpp38/eGFP and RB1Bpp38/smGFP contained two and three and four and five copies of the 132-bp repeats, respectively. Expression analysis showed that the transcription of genes in RB1Bpp38/eGFP-and RB1Bpp38/smGFP-infected chicken embryo fibroblasts (CEFs) were similar to RB1B-infected CEFs, with the notable exception of deletion of a LORF12-specific transcript in RB1Bpp38/eGFP-infected cells. In CEFs, RB1Bpp38/eGFP and RB1Bpp38/smGFP showed comparable one-step growth kinetics to parental virus (RB1B). RB1Bpp38/eGFP and RB1Bpp38/smGFP, however, showed quite distinct growth characteristics in vivo. Two independent clones of RB1Bpp38/eGFP were highly attenuated, whereas RB1Bpp38/smGFP exhibited pathogenesis similar to parent virus and retained oncogenicity. Our results suggest that the RB1Bpp38/eGFP phenotype could be due to an interference with an in vivo-specific pp38 function via GFP direct fusion, to the deletion of LORF12, or to a targeting of the immune response to eGFP. Because deletion of pp38 was recently found not to fully attenuate very virulent MDV strain MD-5, it is possible that deletion of LORF12 may be at least partially responsible for the attenuation of RB1Bpp38/eGFP. The construction of these viruses and the establishment of cell lines from RB1Bpp38/smGFP provide useful tools for the study of MDV lytic infection in cell culture and in vivo, in studies of the reactivation of MDV from latency, and in the functional analysis of LORF12.

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

Abstract Mareks disease virus (MDV) is an acute transforming alphaherpesvirus of chickens that causes Mareks 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.