Publications (16) View all
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Article: Differential expression of microRNAs in Marek's disease virus-transformed T-lymphoma cell lines.
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ABSTRACT: MicroRNAs (miRNAs) are increasingly recognized to play crucial roles in regulation of gene expression in different biological events, including many sporadic forms of cancer. However, despite the involvement of several viruses in inducing cancer, only a limited number of studies have been carried out to examine the miRNA expression signatures in virus-induced neoplasia, particularly in herpesvirus-induced tumours where virus-encoded miRNAs also contribute significantly to the miRNome of the tumour cell. Marek's disease (MD) is a naturally occurring, rapid-onset CD4+ T-cell lymphoma of poultry, induced by the highly contagious Marek's disease virus (MDV). High levels of expression of virus-encoded miRNAs and altered expression of several host-encoded miRNAs were demonstrated in the MDV-transformed lymphoblastoid cell line MSB-1. In order to identify the miRNA expression signature specific to MDV-transformed cells, we examined the global miRNA expression profiles in seven distinct MDV-transformed cell lines by microarray analysis. This study revealed that, in addition to the high levels of MDV-encoded miRNAs, these MD tumour-derived lymphoblastoid cell lines showed altered expression of several host-encoded miRNAs. Comparison of the miRNA expression profiles of these cell lines with the MDV-negative, retrovirus-transformed AVOL-1 cell line showed that miR-150 and miR-223 are downregulated irrespective of the viral aetiology, whereas downregulation of miR-155 was specific for MDV-transformed tumour cells. Thus, increased expression of MDV-encoded miRNAs with specific downregulation of miR-155 can be considered as unique expression signatures for MD tumour cells. Analysis of the functional targets of these miRNAs would contribute to the understanding of the molecular pathways of MD oncogenicity.Journal of General Virology 04/2009; 90(Pt 7):1551-9. · 3.36 Impact Factor -
Article: MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1.
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ABSTRACT: MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27(Kip1). Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27(Kip1) 3'-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27(Kip1) in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27(Kip1) levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27(Kip1) in the induction and progression of T-cell lymphomas.Journal of General Virology 04/2009; 90(Pt 5):1164-71. · 3.36 Impact Factor -
Article: Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.
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ABSTRACT: We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Marek's disease virus in feather tips of chickens, and this has been used clinically to monitor a flock's response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.Avian Pathology 01/2008; 36(6):467-74. · 1.71 Impact Factor -
Article: Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC.
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ABSTRACT: Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.Journal of Virology 11/2007; 81(19):10575-87. · 5.40 Impact Factor -
Article: Vaccinal control of Marek's disease: current challenges, and future strategies to maximize protection.
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ABSTRACT: Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research.Veterinary Immunology and Immunopathology 08/2006; 112(1-2):78-86. · 2.08 Impact Factor
