Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus

Denesvre, Caroline; Rémy, Sylvie; Trapp-Fragnet, Laëtitia; Smith, Lorraine P.; Georgeault, Sonia; Vautherot, Jean-François; Nair, Venugopal

doi:10.1099/jgv.0.000354

Cited by 14 publications

(12 citation statements)

References 30 publications

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“…However, we found that a small proportion of cells spontaneously expressed EGFP at levels similar to those of MDV lytic genes such as pp38 seen in cells with reactivating virus [ 22, 29 ]. We have also recently shown that these cells produce virus particles [ 28 ]. The ability to isolate these spontaneously reactivating fluorescent cells presented an ideal resource to analyse, with minimal manipulation, cell populations showing viral reactivation in relation to the majority latent cell population.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, we established two LCLs from tumours induced by pRB1B-UL47eGFP virus [ 27 ] as a tool to study spontaneous lytic switch of MDV. We have recently used transmission electron microscopy to demonstrate morphogenesis of herpesvirus particles in the enhanced green fluorescent protein (EGFP)-expressing cells of one of these cell lines [ 28 ]. Global gene expression profiling using RNA-seq was used to examine the transcriptome changes associated with lytic switch.…”

Section: Introductionmentioning

confidence: 99%

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Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling

Mwangi

Vasoya

Kgosana

et al. 2017

Journal of General Virology

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Marek’s disease virus (MDV), an alphaherpesvirus of poultry, causes Marek’s disease and is characterized by visceral CD4+TCRαβ+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus–host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling

Mwangi

Vasoya

Kgosana

et al. 2017

Journal of General Virology

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“…Primary chicken embryonic skin cells (CESCs) were prepared from 12-day-old specific-pathogen-free (SPF) White Leghorn (LD1) chicken embryos and maintained in culture as previously described (48). The MDCC-3867K cell line was derived from a renal lymphoma induced upon infection of a chicken with the highly pathogenic recombinant vRB-1B 47EGFP virus encoding the UL47 gene fused to the enhanced green fluorescent protein (EGFP) (47). 3867K cells were cultured in RPMI 1640 supplemented with 2 mM glutamine, 1% pyruvate, 1% nonessential amino acids, 1% glucose, 10% tryptose phosphate broth, and 10% fetal bovine serum (FBS) and maintained at 41°C in a 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

“…Next, we set out to determine if DNA damage is induced upon MDV reactivation. We used a lymphoblastoid cell line that expresses GFP fused to the tegument protein UL47 upon reactivation (3867K cells), as described previously (47). We sorted EGFP-positive and -negative cells and assessed DNA damage by comet assays.…”

Section: Fig 2 Dna Damage Induction Enhances MDV Replication (A To D)mentioning

confidence: 99%

Induction of DNA Damages upon Marek's Disease Virus Infection: Implication in Viral Replication and Pathogenesis

Bencherit

Rémy

Vern

et al. 2017

J Virol

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Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage and that this likely contributes to viral oncogenesis. Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication and DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

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“…Spleen samples were collected from all infected groups at 20 th , 25 th , 30 th , 35 th , and 40 th weeks post-infection from different groups and tested by PCR assay for the detection of MDV [ 18 ]. Furthermore, at 40 th week post-infection, tumorized spleen sample of Bovans breed was collected and prepared for examination by transmission electron microscope (TEM) to confirm the presence of MDV [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

Genetic resistance of eight native Egyptian chicken breeds having chicken B-cell marker 6 gene post-challenge with field strain of Marek's disease-induced tumor virus

et al. 2018

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Aim:The aim of this work was to detect chicken B-cell marker 6 (ChB6) gene in some native breeds in Egypt and find the relationship between founded genes in these different breeds to determine the resistance of native Egyptian breeds of chicken to Marek’s disease (MD).Materials and Methods:A total of 14 different chicken breeds (30 each) including ten native breeds in addition to SPF Lohmann, High Line, Bovans, and Roodiland were used. Blood samples were collected for the detection of (ChB6) by polymerase chain reaction (PCR) assay and sequenced to determine the presence or absence of ChB6 gene. Experimental infection was done using local field isolated MD virus (MDV) of 11 (1 day old) unvaccinated chick breeds having no maternal antibodies against MDV. Ten breeds of them carry ChB6 gene, eight breeds were native, and the rest two breeds were SPF Lohmann and High Line in addition to a group of ChB6 gene-lacking breed (Bovans) were infected. Spleen samples were collected from all infected breeds at 20th, 25th, 30th, 35th, and 40th weeks post-infection and tested by PCR assay for the detection of MDV. Furthermore, at 40th week post-infection, tumorized spleen sample of Bovans breed was collected and prepared for examination by transmission electron microscope (TEM) to confirm the presence of MDV.Results:Our results revealed the positivity of 10 out of 14 breeds (Gimmizah, Sinai, Dandarawi, Fayoumi, Golden Montazah, Matrouh, Beheri, Dokki, SPF Lohmann, and High Line) to the presence of ChB6 gene and resistance to MDV infection, while the Bovans, Mandarah, Inshas and Roodiland breeds lack the ChB6 gene and are susceptible to MDV infection. The collected spleen samples revealed negative for the presence of challenged MDV by PCR in 10 breeds (Gimmizah, Sinai, Dandarawi, Fayoumi, Golden Montazah, Matrouh, Beheri, Dokki, SPF Lohmann, and High Line) and positive for Bovans breed. TEM is used to confirm MDV infection in Bovans group which demonstrated tumors.Conclusion:The study confirms the relationship between the presence of ChB6 gene in our native breeds and the absence of tumors.

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Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus

Cited by 14 publications

References 30 publications

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling

Induction of DNA Damages upon Marek's Disease Virus Infection: Implication in Viral Replication and Pathogenesis

Genetic resistance of eight native Egyptian chicken breeds having chicken B-cell marker 6 gene post-challenge with field strain of Marek's disease-induced tumor virus

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