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

BACKGROUND: The 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. RESULTS: Compared 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. CONCLUSIONS: The 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.

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.

Examination of the effect of a naturally occurring mutation in glycoprotein L on Marek's disease virus pathogenesis.

We recently reported a comparison of glycoprotein-encoding genes of different Marek's 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 TK1HVT-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.

Construction and characterization of Marek's disease viruses having green fluorescent protein expression tied directly or indirectly to phosphoprotein 38 expression.

Marek's disease (MD) is caused by Marek's 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 lyric infection in cell culture and in vivo, in studies of the reactivation of MDV from latency, and in the functional analysis of LORF12.

Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence.

Marek's disease (MD) is a highly contagious lymphoproliferative and demyelinating disorder of chickens. MD is caused by Marek's disease virus (MDV), a cell-associated, acute-transforming alphaherpesvirus. For three decades, losses to the poultry industry due to MD have been greatly limited through the use of live vaccines. MDV vaccine strains are comprised of antigenically related, apathogenic MDVs originally isolated from chickens (MDV-2), turkeys (herpesvirus of turkeys, HVT) or attenuated-oncogenic strains of MDV-1 (CVI-988). Since the inception of high-density poultry production and MD vaccination, there have been two discernible increases in the virulence of MDV field strains. Our objectives were to determine if common mutations in the major glycoprotein genes, a major lytic antigen phosphoprotein 38 (pp38) or a major latency/transformation antigen Meq (Marek's EcoRI-Q-encoded protein) were associated with enhanced MDV virulence. To address this, we cloned and sequenced the major surface glycoprotein genes (gB, gC, gD, gE, gH, gI, and gL) of five MDV strains that were representative of the virulent (v), very virulent (vv) and very virulent plus (vv+) pathotypes of MDV. We found no consistent mutations in these genes that correlated strictly with virulence level. The glycoprotein genes most similar among MDV-1, MDV-2 and HVT (gB and gC, approximately 81 and 75%, respectively) were among the most conserved across pathotype. We found mutations mapping to the putative signal cleavage site in the gL genes in four out of eleven vv+MDVs, but this mutation was also identified in one vvMDV (643P) indicating that it did not correlate with enhanced virulence. In further analysis of an additional 12 MDV strains, we found no gross polymorphism in any of the glycoprotein genes. Likewise, by PCR and RFLP analysis, we found no polymorphism at the locus encoding the pp38 gene, an early lytic-phase gene associated with MDV replication. In contrast, we found distinct mutations in the latency and transformation-associated Marek's EcoRI-Q-encoded protein, Meq. In examination of the DNA and deduced amino acid sequence of meq genes from 26 MDV strains (9 m/vMDV, 5 vvMDV and 12 vv+MDVs), we found distinct polymorphism and point mutations that appeared to correlate with virulence. Although a complex trait like MDV virulence is likely to be multigenic, these data describe the first sets of mutations that appear to correlate with MDV virulence. Our conclusion is that since Meq is expressed primarily in the latent/transforming phase of MDV infection, and is not encoded by MDV-2 or HVT vaccine viruses, the evolution of MDV virulence may be due to selection on MDV-host cell interactions during latency and may not be mediated by the immune selection against virus lytic antigens such as the surface glycoproteins.