Identification of Marek''s disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.     

Histone Methylation Analysis and Pathway Predictions in Chickens after MDV Infection

Marek's disease (MD) is a lymphoproliferative disease in chicken induced by Marek's disease virus (MDV). Although studies have focused on the genetic differences between the resistant and susceptible chicken, less is known about the role of epigenetic factors in MD. In this study, genome-wide histone modifications in the non-MHC-associated resistant and susceptible chicken lines were examined. We found that tri-methylation at histone H3 Lys4 (H3K4me3) enrichment is positively correlated with the expression of protein coding genes as well as microRNA (miRNA) genes, whereas tri-methylation at histone H3 Lys27 (H3K27me3) exhibits a negative correlation. By identifying line-specific histone modifications in MDV infection, we found unique H3K4me3 islands in the resistant chicken activated genes, which are related to immune response and cell adhesion. Interestingly, we also found some miRNAs from unique H3K27me3 patterns in the susceptible chickens that targeted genes involved in 5-hydroxytryptamine (5-HT)-receptor and adrenergic receptor pathways. In conclusion, dynamic line-specific histone modifications in response to MDV infection suggested that intrinsic epigenetic mechanisms may play a role in MD-resistance and -susceptibility.     

The safety of the preparation viroden for vertebrate animals]

Toxicity-pathogenicity test of viroden, a new preparation, and its acting agent--a mosquito densonucleosis virus (MDV) has been carried out on warm-blooded animals. It is shown that the preparation is not toxic for laboratory animals (white common mice, rats, guinea pigs, rabbits), chicken embryos and cell cultures of warm-blooded animals. The MDV is not adapted to a warm-blooded organism with different ways of introduction and in passages. Using electron and luminescent microscopy, serological reactions, specific test systems and a biological test for sensitive insects no explicit or latent infection was found in animals, chicken embryos and cell cultures of vertebrates with primary infection and in passages. Sensibilized animals shown an immunological rearrangement of the organism proceeding by the retarded hypersensitivity type.     

Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells.

Marek's disease virus (MDV), a lymphotropic herpesvirus, induces T-cell lymphomas in chicken, its natural host. The lymphoma cells are latently infected with MDV but the viral contribution to the transformed phenotype is not understood. To investigate the virus-cell interaction, we focused on the status of MDV in the transformed cells. By the use of highly sensitive fluorescent in situ hybridization with metaphase chromosomes, we found (i) MDV DNA to be randomly integrated at multiple sites in the chromosomes of primary lymphoma cells from chicken tissues; (ii) extrachromosomal, circular MDV genomes were absent and linear virion DNA was usually not detectable in the latently infected lymphoma cells; (iii) the pattern of integration sites revealed the clonal origin of the tumour cells; which (iv) was retained in in vitro established cell lines derived from primary lymphomas; (v) activation of the lytic phase of MDV's life cycle occurred in vitro suggesting that MDV can escape from its integrated status by an unknown mechanism.     

A Systematic Analysis of miRNA Transcriptome in Marek’s Disease Virus-Induced Lymphoma Reveals Novel and Differentially Expressed miRNAs

Marek’s disease is a lymphoproliferative neoplastic disease of the chicken, which poses a serious threat to poultry health. Marek’s disease virus (MDV)-induced T-cell lymphoma is also an excellent biomedical model for neoplasia research. Recently, miRNAs have been demonstrated to play crucial roles in mediating neoplastic transformation. To investigate host miRNA expression profiles in the tumor transformation phase of MDV infection, we performed deep sequencing in two MDV-infected samples (tumorous spleen and MD lymphoma from liver), and two non-infected controls (non-infected spleen and lymphocytes). In total, 187 and 16 known miRNAs were identified in chicken and MDV, respectively, and 17 novel chicken miRNAs were further confirmed by qPCR. We identified 28 down-regulated miRNAs and 11 up-regulated miRNAs in MDV-infected samples by bioinformatic analysis. Of nine further tested by qPCR, seven were verified. The gga-miR-181a, gga-miR-26a, gga-miR-221, gga-miR-222, gga-miR-199*, and gga-miR-140* were down-regulated, and gga-miR-146c was up-regulated in MDV-infected tumorous spleens and MD lymphomas. In addition, 189 putative target genes for seven differentially expressed miRNAs were predicted. The luciferase reporter gene assay showed interactions of gga-miR-181a with MYBL1, gga-miR-181a with IGF2BP3, and gga-miR-26a with EIF3A. Differential expression of miRNAs and the predicted targets strongly suggest that they contribute to MDV-induced lymphomagenesis.     

The RNA subunit of telomerase is encoded by Marek's disease virus

Marek's disease virus (MDV) is a herpesvirus of chickens that induces T lymphomas and tumors within 4 to 5 weeks of infection. Although the ability of MDV to induce tumors was demonstrated many years ago and although a number of viral oncogenic proteins have been identified, the mechanism by which the MDV is implicated in tumorigenesis is still unknown. We report the identification of a virus-encoded RNA telomerase subunit (vTR) within the genome of MDV. This gene is found in the genomic DNA of the oncogenic MDV strains, whereas it is not carried by the nononcogenic MDV strains. The vTR sequence exhibits 88% sequence identity with the chicken gene (cTR). Our functional analysis suggests that this telomerase RNA can reconstitute telomerase activity in a heterologous system (the knockout murine TR(-/-) cell line) by interacting with the telomerase protein component encoded by the host cell. We have also demonstrated that the vTR promoter region is efficient whatever the species of cell line considered and that vTR is expressed in vivo in peripheral blood leukocytes from chickens infected with the oncogenic MDV-RB1B and the vaccine MDV-Rispens strains. The functionality of the vTR gene and the potential implication of vTR in the oncogenesis induced by MDV is discussed.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

Microsatellite Instability in Marek's Disease Virus Infected Primary Chicken Embryo Fibroblasts

ABSTRACT: BACKGROUND: Marek's disease virus (MDV), an oncogenic alpha-herpesvirus, causes a devastating disease in chickens characterized by development of lymphoblastoid tumors in multiple organs. Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, is a form of genomic instability frequently detected in many types of tumors. However, the involvement of MSI in MDV-infected cells has not been investigated. In this study, we determined the presence and frequency of MSI in primary chicken embryo fibroblasts infected with or without virulent RB-1B strain of MDV in vitro. RESULTS: 118 distinct microsatellite markers were analyzed by polymerase chain reaction (PCR) in 21 samples. MSI was found in 91 of 118 markers, and 12 out of 118 demonstrated frequency of MSI at [greater than or equal to] 40%. 27 of 118 microsatellite loci did not show microsatellite instability. CONCLUSIONS: These findings showed that MSI was a real event occurring in primary chicken embryo fibroblasts infected with MDV in vitro as evidenced by the high frequency of MSI, and may be specifically associated with genome alteration of host cells during MDV infected.     

Cloning, sequencing, and functional analysis of a Marek''s disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.     

Modeling the proteome of a Marek's disease transformed cell line: a natural animal model for CD30 overexpressing lymphomas

Marek's disease (MD) in the chicken, caused by the highly infectious MD alpha-herpesvirus (MDV), is both commercially important and a unique, naturally occurring model for human T-cell lymphomas overexpressing the Hodgkin's disease antigen, CD30. Here, we used proteomics as a basis for modeling the molecular functions and biological processes involved in MDV-induced lymphomagenesis. Proteins were extracted from an MDV-transformed cell line and were then identified using 2-D LC-ESI-MS/MS. From the resulting 3870 cellular and 21 MDV proteins we confirm the existence of 3150 "predicted" and 12 "hypothetical" chicken proteins. The UA-01 proteome is proliferative, differentiated, angiogenic, pro-metastatic and pro-immune-escape but anti-programmed cell death, -anergy, -quiescence and -senescence and is consistent with a cancer phenotype. In particular, the pro-metastatic integrin signaling pathway and the ERK/MAPK signaling pathways were the two predominant signaling pathways represented. The cytokines, cytokine receptors, and their related proteins suggest that UA-01 has a regulatory T-cell phenotype.     

Rho-ROCK and Rac-PAK Signaling Pathways Have Opposing Effects on the Cell-to-Cell Spread of Marek's Disease Virus

Marek's Disease Virus (MDV) is an avian alpha-herpesvirus that only spreads from cell-to-cell in cell culture. While its cell-to-cell spread has been shown to be dependent on actin filament dynamics, the mechanisms regulating this spread remain largely unknown. Using a recombinant BAC20 virus expressing an EGFPVP22 tegument protein, we found that the actin cytoskeleton arrangements and cell-cell contacts differ in the center and periphery of MDV infection plaques, with cells in the latter areas showing stress fibers and rare cellular projections. Using specific inhibitors and activators, we determined that Rho-ROCK pathway, known to regulate stress fiber formation, and Rac-PAK, known to promote lamellipodia formation and destabilize stress fibers, had strong contrasting effects on MDV cell-to-cell spread in primary chicken embryo skin cells (CESCs). Inhibition of Rho and its ROCKs effectors led to reduced plaque sizes whereas inhibition of Rac or its group I-PAKs effectors had the adverse effect. Importantly, we observed that the shape of MDV plaques is related to the semi-ordered arrangement of the elongated cells, at the monolayer level in the vicinity of the plaques. Inhibition of Rho-ROCK signaling also resulted in a perturbation of the cell arrangement and a rounding of plaques. These opposing effects of Rho and Rac pathways in MDV cell-to-cell spread were validated for two parental MDV recombinant viruses with different ex vivo spread efficiencies. Finally, we demonstrated that Rho/Rac pathways have opposing effects on the accumulation of N-cadherin at cell-cell contact regions between CESCs, and defined these contacts as adherens junctions. Considering the importance of adherens junctions in HSV-1 cell-to-cell spread in some cell types, this result makes of adherens junctions maintenance one potential and attractive hypothesis to explain the Rho/Rac effects on MDV cell-to-cell spread. Our study provides the first evidence that MDV cell-to-cell spread is regulated by Rho/Rac signaling.     

Identification of chicken lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek's disease resistance gene via a virus-host protein interaction screen

Marek's disease virus (MDV) is a naturally occurring oncogenic avian herpesvirus that causes neurological disorders and T cell lymphoma disease in domestic chickens. Identification and functional characterization of the individual factors involved in Marek's disease (MD) resistance or pathogenesis will enhance our understanding of MDV pathogenesis and further genetic improvement of chickens. To study the genetic basis for resistance to MD, a strategy that combined protein-protein interaction screens followed by linkage analysis was performed. The MDV protein US10 was used as the bait in an E. COLI two-hybrid screening of a cDNA library derived from activated splenic T cells. The chicken LY6E, also known as SCA2 and TSA1, was found to specifically interact with US10. This interaction was confirmed by an in vitro protein-binding assay. Furthermore, LY6E was found to be significantly associated with MD traits in an MD resource population comprised of commercial chickens. Previously, LY6E was implicated in two independent DNA microarray experiments evaluating differential gene expression following MDV infection. Given that LY6E is involved in T cell differentiation and activation, we suggest that LY6E is a candidate gene for MD resistance and deserves further investigation on its role in MDV pathogenesis, especially with respect to the binding of US10.     

利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

Changes in the Gallus gallus proteome induced by Marek's disease virus

Marek's disease virus (MDV) is a highly oncogenic avian herpesvirus. We have used a modified MudPIT analysis to examine the effect of MDV infection on the chicken proteome. We identified 3561 unique nonphosphorylated peptides, representing 1460 chicken proteins, in a mock-infected sample versus 4240 unique nonphosphorylated peptides, representing 1676 proteins, in an MDV-infected sample. Of these unique peptides, 59.1% from the mock- and 49.6% from the MDV-infected samples were detected in both samples, and for the represented proteins, 69.1% from the mock- and 60.2% from the MDV-infected samples were common to both samples. In terms of phosphorylation, 357 and 506 phosphopeptides, representing 342 and 483 proteins, were detected in the mock- and MDV-infected samples, respectively. At the phosphopeptide level, 10.1% from the mock- and 7.1% from the MDV-infected samples overlapped, and for the represented phosphoproteins, 12.0% from the mock- and 8.5% from the MDV-infected samples were common to both samples. There were no significant differences in the hydropathicity values and number of transmembrane domains of the identified protein sets. Subtle differences were observed for subcellular localizations of the identified proteins. These results suggest that MDV infection may alter host cell biochemistry by perturbing the host's proteomic composition.     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.