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MSphere Jan 2019The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected...
The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors. Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.
Topics: Animals; Biomarkers, Tumor; Chickens; Image Processing, Computer-Assisted; Isotope Labeling; Laser Capture Microdissection; Lymphoma; Marek Disease; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 30651403
DOI: 10.1128/mSphere.00569-18 -
Journal of Virological Methods Dec 2019The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing... (Review)
Review
The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing highly sensitive and fast methods of diagnosis. The present review will discuss the biotic factors and the complexities that became evident with the evolution of the novel molecular diagnostic assays with emphasis on 4 avian viruses, chicken anemia, infectious laryngotracheitis, turkey meningoencephalitis, but mainly on Marek's disease virus. To create a biologically meaningful diagnosis, attention should be dedicated to various biotic factors and not only of the diagnostic assay. Included among the important factors are, (a) the sample examined and the sampling strategy, (b) the outcomes of the pathogen amplification ex vivo, (c) the sampling time and its reflection on the disease diagnosis, (d) the impact of simultaneous multiple virus-infections regarding the ability to demonstrate all pathogens and inter- and intra-interactions between the pathogens. A concerted consideration of the relevant factors and the use of advanced molecular diagnostic assay would yield biologically significant diagnosis in real-time that would beneficiate the poultry industry.
Topics: Animals; Chickens; Circoviridae Infections; Encephalitis, Viral; Herpesviridae Infections; Marek Disease; Molecular Diagnostic Techniques; Poultry Diseases; Specimen Handling; Turkey; Veterinary Medicine
PubMed: 31351169
DOI: 10.1016/j.jviromet.2019.113708 -
Frontiers in Veterinary Science 2023Marek's disease virus (MDV) establishes latency in chicken T lymphocytes that can lead to T cell transformation and cancer. Transformed Marek's disease chicken cell...
Marek's disease virus (MDV) establishes latency in chicken T lymphocytes that can lead to T cell transformation and cancer. Transformed Marek's disease chicken cell lines (MDCCs) can be expanded and provide a valuable model to study latency, transformation, and reactivation. Here, we developed MDCCs from chickens infected with MDV that fluoresce during lytic replication and reactivation. Sodium butyrate treatment increased fluorescent protein expression as evidenced by fluorescent microscopy, flow cytometry, and western blotting; however, it caused significant apoptosis and necrosis. Treatment of MDCCs by decreasing the temperature resulted in robust MDV reactivation without significant induction of apoptosis and necrosis. Furthermore, MDV reactivation was significantly affected by the time in culture that can affect downstream reactivation analyses. In all, our data show that fluorescent protein expression during reactivation is a robust tool to examine viral replication in live cells , and temperature treatment is an efficient technique to induce reactivation without punitive effects on cell viability seen with chemical treatment.
PubMed: 36968465
DOI: 10.3389/fvets.2023.1145757 -
Avian Pathology : Journal of the W.V.P.A Dec 2023Marek's disease (MD) is caused by oncogenic MD virus serotype 1 (MDV1) and is characterized by lymphoproliferative lesions resulting in high morbidity and mortality in...
Marek's disease (MD) is caused by oncogenic MD virus serotype 1 (MDV1) and is characterized by lymphoproliferative lesions resulting in high morbidity and mortality in chickens. Despite being ubiquitous on poultry farms, there is a dearth of information on its molecular characteristics in Nigeria. This study aimed at characterizing three virulence genes ( and ) of MDV1 from chickens in Ogun state, Nigeria. Blood, feather quill, and tumour samples of chickens from different commercial poultry farms in Ogun State were pooled, spotted on 107 FTA cards, and screened for MDV1 by polymerase chain reaction (PCR). Phylogenetic analysis was carried out to compare Nigerian MDV1 , and genes sequences with the published references. Thirteen samples were MDV1-positive and the , as well as and genes from the different samples were 100% identical. The genes contained 339 amino acids (aa) with three PPPP motifs in the transactivation domain and two interruptions of the PPPP motifs due to proline-to-arginine substitutions at positions 176 and 217 resulting in a 20.88% proline composition. Phylogenetic analysis revealed that the gene clustered with strains from Egypt and very virulent ATE2539 strain from Hungary. Mutations were observed in the pp38 protein (at positions 107 and 109) and protein (at positions 4 and 31). Based on the molecular analysis of the three genes, the results indicate the presence of MDV1 with virulence signatures; therefore, further studies on pathotyping of Nigerian MDV1 from all states should be performed. genes were 100% identical between Nigerian MDV strains.Proline content in Nigerian gene was 20.88% with two PPPP motifs interruptions. genes of Nigerian MDV were similar to Egyptian and Indian strains.
Topics: Animals; Chickens; Phylogeny; Oncogene Proteins, Viral; Marek Disease; Nigeria; Herpesvirus 2, Gallid; Poultry; Proline; Poultry Diseases
PubMed: 37605844
DOI: 10.1080/03079457.2023.2243838 -
Cellular and Molecular Life Sciences :... Aug 2020Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens.... (Review)
Review
Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. Although Marek's disease (MD) is well controlled by current vaccines, the evolution of MDV field viruses towards increasing virulence is concerning as a better vaccine to combat very virulent plus MDV is still lacking. Our understanding of molecular and cellular immunity to MDV and its immunopathogenesis has significantly improved, but those findings about cellular immunity to MDV are largely out-of-date, hampering the development of more effective vaccines against MD. T-cell-mediated cellular immunity was thought to be of paramount importance against MDV. However, MDV also infects macrophages, B cells and T cells, leading to immunosuppression and T-cell lymphoma. Additionally, there is limited information about how uninfected immune cells respond to MDV infection or vaccination, specifically, the mechanisms by which T cells are activated and recognize MDV antigens and how the function and properties of activated T cells correlate with immune protection against MDV or MD tumor. The current review revisits the roles of each immune cell subset and its effector mechanisms in the host immune response to MDV infection or vaccination from the point of view of comparative immunology. We particularly emphasize areas of research requiring further investigation and provide useful information for rational design and development of novel MDV vaccines.
Topics: Animals; Chickens; Herpesvirus 2, Gallid; Humans; Immunity, Cellular; Marek Disease; Oncogenic Viruses; T-Lymphocytes; Virulence
PubMed: 32080753
DOI: 10.1007/s00018-020-03477-z -
Journal of Virology Nov 2020Marek's disease virus (MDV) transforms CD4 T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in...
Marek's disease virus (MDV) transforms CD4 T cells and causes a deadly neoplastic disease that is associated with metabolic dysregulation leading to atherosclerosis in chickens. While MDV-infected chickens have normal serum concentrations of cholesterol, their aortic tissues were found to have elevated concentrations of free and esterified cholesterol. Here, we demonstrate that infection of chicken embryonated fibroblasts (CEFs) with highly pathogenic MDV-RB1B increases the cellular cholesterol content and upregulates the genes involved in cholesterol synthesis and cellular cholesterol homeostasis using comprehensive two-dimensional gas chromatography-mass spectrometry and real-time PCR (RT-PCR), respectively. Using small pharmacological inhibitors and gene silencing, we established an association between MDV-RB1B replication and mevalonic acid, sterol, and cholesterol biosynthesis and trafficking/redistribution. We propose that MDV trafficking is mediated by lysosome-associated membrane protein 1 (LAMP-1)-positive vesicles based on short hairpin RNA (shRNA) gene silencing and the colocalization of LAMP-1, glycoprotein B (gB) of MDV, and cholesterol (filipin III) fluorescence signal intensity peaks. In conclusion, our results demonstrate that MDV hijacks cellular cholesterol biosynthesis and cholesterol trafficking to facilitate cell-to-cell spread in a LAMP-1-dependent mechanism. MDV disrupts lipid metabolism and causes atherosclerosis in MDV-infected chickens; however, the role of cholesterol metabolism in the replication and spread of MDV is unknown. MDV-infected cells do not produce infectious cell-free virus , raising the question about the mechanism involved in the cell-to-cell spread of MDV. In this report, we provide evidence that MDV replication depends on cholesterol biosynthesis and uptake. Interruption of cholesterol trafficking within multivesicular bodies (MVBs) by chemical inhibitors or gene silencing reduced MDV titers and cell-to-cell spread. Finally, we demonstrated that MDV gB colocalizes with cholesterol and LAMP-1, suggesting that viral protein trafficking is mediated by LAMP-1-positive vesicles in association with cholesterol. These results provide new insights into the cholesterol dependence of MDV replication.
Topics: Animals; Antigens, Viral; Chickens; Cholesterol; Herpesvirus 2, Gallid; Homeostasis; Lanosterol; Lipid Metabolism; Lipogenesis; Lysosomal Membrane Proteins; Marek Disease; Mevalonic Acid; Protein Transport; Transcription Factors; Viral Envelope Proteins; Viral Proteins; Virus Replication
PubMed: 32999035
DOI: 10.1128/JVI.01001-20 -
Scientific Reports Apr 2023Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However,...
Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek's disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.
Topics: Animals; Chickens; Marek Disease; Herpesvirus 2, Gallid; Disease Susceptibility; Spleen
PubMed: 37005445
DOI: 10.1038/s41598-023-32308-x -
International Journal of Molecular... Nov 2019MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of various biological processes including cell development and... (Review)
Review
MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of various biological processes including cell development and differentiation, apoptosis, tumorigenesis, immunoregulation and viral infections. Avian immunosuppressive diseases refer to those avian diseases caused by pathogens that target and damage the immune organs or cells of the host, increasing susceptibility to other microbial infections and the risk of failure in subsequent vaccination against other diseases. As such, once a disease with an immunosuppressive feature occurs in flocks, it would be difficult for the stakeholders to have an optimal economic income. Infectious bursal disease (IBD), avian leukemia (AL), Marek's disease (MD), chicken infectious anemia (CIA), reticuloendotheliosis (RE) and avian reovirus infection are on the top list of commonly-seen avian diseases with a feature of immunosuppression, posing an unmeasurable threat to the poultry industry across the globe. Understanding the pathogenesis of avian immunosuppressive disease is the basis for disease prevention and control. miRNAs have been shown to be involved in host response to pathogenic infections in chickens, including regulation of immunity, tumorigenesis, cell proliferation and viral replication. Here we summarize current knowledge on the roles of miRNAs in avian response to viral infection and pathogenesis of avian immunosuppressive diseases, in particular, MD, AL, IBD and RE.
Topics: Animals; Bird Diseases; Chickens; Immune Tolerance; Immunity; Infectious bursal disease virus; Marek Disease; MicroRNAs; Virus Diseases
PubMed: 31683847
DOI: 10.3390/ijms20215454 -
Journal of Veterinary Research Dec 2020Marek's disease (MD) is a tumourous disease caused by Marek's disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the...
INTRODUCTION
Marek's disease (MD) is a tumourous disease caused by Marek's disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the occurrence of Marek's disease virus infections in Poland and analyse clinical cases in the years 2015-2018.
MATERIAL AND METHODS
The birds for diagnostic examination originated from 71 poultry flocks of various types of production. Birds were subjected to anatomopathological examination post mortem, during which liver and spleen sections and other pathologically changed internal organs were taken. These sections were homogenised with generally accepted methods, then total DNA was isolated and amplified with a real-time PCR. A pair of primers complementary to the MDV genome region encoding the gene were used.
RESULTS
MDV infection was found predominantly in broiler chicken flocks (69.01%), and also in layer breeder (9.85%) and commercial layer flocks (7.04% each).
CONCLUSION
The results of research conducted in the years 2015-2018 clearly indicate that the problem of MDV infections is still current.
PubMed: 33367138
DOI: 10.2478/jvetres-2020-0079 -
Genes Feb 2019Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell...
Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IR/TR) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.
Topics: Animals; Chickens; Exosomes; Female; Lymphoma; Male; Marek Disease; Poultry Diseases; Proteome; Transcriptome; Vaccination
PubMed: 30764491
DOI: 10.3390/genes10020116