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Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

ABSTRACT: Marek's disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been successful. The aim of this study was to develop a model for infecting phagocytes [macrophages and dendritic cells (DCs)] with MDV in vitro and to characterize the infected cells. Chicken bone marrow cells were cultured with chicken CSF-1 or chicken IL-4 and chicken CSF-2 for 4 days to produce macrophages and DCs, respectively, and then co-cultured with FACS-sorted chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing EGFP. Infected phagocytes were identified and sorted by FACS using EGFP expression and phagocyte-specific mAbs. Detection of MDV-specific transcripts of ICP4 (immediate early), pp38 (early), gB (late) and Meq by RT-PCR provided evidence for MDV replication in the infected phagocytes. Time-lapse confocal microscopy was also used to demonstrate MDV spread in these cells. Subsequent co-culture of infected macrophages with CEFs suggests that productive virus infection may occur in these cell types. This is the first report of in vitro infection of phagocytic cells by MDV.

SUBMITTER: Chakraborty P

PROVIDER: S-EPMC5656796 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

Chakraborty Pankaj P Vervelde Lonneke L Dalziel Robert G RG Wasson Peter S PS Nair Venugopal V Dutia Bernadette M BM Kaiser Pete P

The Journal of general virology 20170526 5

Marek's disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been succ ...[more]

PMID: 28548038

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Project description:Marek's disease virus (MDV) causes deadly lymphoma and induces an imbalance of the lipid metabolism in infected chickens. Here, we discovered that MDV activates the fatty acid synthesis (FAS) pathway in primary chicken embryo fibroblasts (CEFs). In addition, MDV-infected cells contained high levels of fatty acids and showed increased numbers of lipid droplets (LDs). Chemical inhibitors of the FAS pathway (TOFA and C75) reduced MDV titers by approximately 30-fold. Addition of the downstream metabolites, including malonyl-coenzyme A and palmitic acid, completely restored the inhibitory effects of the FAS inhibitors. Furthermore, we could demonstrate that MDV infection activates the COX-2/prostaglandin E2 (PGE2) pathway, as evident by increased levels of arachidonic acid, COX-2 expression, and PGE2 synthesis. Inhibition of the COX-2/PGE2 pathway by chemical inhibitors or knockdown of COX2 using short hairpin RNA reduced MDV titers, suggesting that COX-2 promotes virus replication. Exogenous PGE2 completely restored the inhibition of the COX-2/PGE2 pathway in MDV replication. Unexpectedly, exogenous PGE2 also partially rescued the inhibitory effects of FAS inhibitors on MDV replication, suggesting that there is a link between these two pathways in MDV infection. Taken together, our data demonstrate that the FAS and COX-2/PGE2 pathways play an important role in the replication of this deadly pathogen.IMPORTANCE Disturbances of the lipid metabolism in chickens infected with MDV contribute to the pathogenesis of disease. However, the role of lipid metabolism in MDV replication remained unknown. Here, we demonstrate that MDV infection activates FAS and induces LD formation. Moreover, our results demonstrate that MDV replication is highly dependent on the FAS pathway and the downstream metabolites. Finally, our results reveal that MDV also activates the COX-2/PGE2 pathway, which supports MDV replication by activating PGE2/EP2 and PGE2/EP4 signaling pathways.

Project description: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.IMPORTANCE 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 in vitro, 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 de novo 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.

Project description:Marek's disease virus (MDV) strain GX0101 was the first reported field strain of recombinant gallid herpesvirus type 2 (GaHV-2). However, the splenic proteome of MDV-infected chickens remains unclear. In this study, a total of 28 1-day-old SPF chickens were intraperitoneally injected with chicken embryo fibroblast (CEF) containing 2000 PFU GX0101. Additionally, a control group, consisting of four one-day-old SPF chickens, received intraperitoneal equal doses of CEF. Blood and various tissue samples were collected at different intervals (7, 14, 21, 30, 45, 60, and 90 days post-infection; dpi) for histopathological, real-time PCR, and label-free quantitative analyses. The results showed that the serum expressions of MDV-related genes, meq and gB, peaked at 45 dpi. The heart, liver, and spleen were dissected at 30 and 45 dpi, and their hematoxylin-eosin staining indicated that virus infection compromised the normal organizational structure at 45 dpi. Particularly, the spleen structure was severely damaged, and the lymphocytes in the white medulla were significantly reduced. Furthermore, liquid chromatography-mass spectrometry (LC-MS) and label-free techniques were used to analyze the difference in splenic proteome profiles of the experimental and control groups at 30 and 45 dpi. Proteomic analysis identified 1660 and 1244 differentially expressed proteins (DEPs) at 30 and 40 dpi, respectively, compared with the uninfected spleen tissues. According to GO analysis, these DEPs were involved in processes such as organelle organization, cellular component biogenesis, cellular component assembly, anion binding, small molecule binding, metal ion binding, cation binding, cytosol, nuclear part, etc. Additionally, KEGG analysis indicated that the following pathways were linked to MDV-induced inflammation, apoptosis, and tumor: Wnt, Hippo, AMPK, cAMP, Notch, TGF-β, PI3K-Akt, Rap1, Ras, Calcium, NF-κB, PPAR, cGMP-PKG, Apoptosis, VEGF, mTOR, FoxO, TNF, JAK-STAT, MAPK, Prion disease, T cell receptor, and B cell receptor. We finally screened 674 DEPs that were linked to MDV infection in spleen tissue. This study improves our understanding of the MDV response mechanism in the spleen.

Project description:Marek's disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek's disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious bacterial artificial chromosome (BAC) clone. All replicates became completely or highly attenuated, indicating that de novo mutation, and not selection among quasispecies existing in a strain, is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41 to 95 single-nucleotide variants (SNVs) at 2% or higher frequency in each population and several candidate genes containing high-frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0 to 11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines.Marek's disease virus (MDV) is a very important pathogen in chickens that costs the worldwide poultry industry $1 billion to $2 billion annually. Marek's disease (MD) vaccines, the primary control method, are often produced by passing virulent strains in cell culture until attenuated. To understand this process, we identified all the changes in the viral genome that occurred during repeated cell passage. We find that a single mutation in the UL5 gene, which encodes a viral protein necessary for DNA replication, reduces disease incidence by 90% or more. In addition, other candidate genes were identified. This information should lead to the development of more effective and rationally designed MD vaccines leading to improved animal health and welfare and lower costs to consumers.

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Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

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Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

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