scholarly journals Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines

2003 ◽  
Vol 77 (16) ◽  
pp. 8712-8718 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Ken Howes ◽  
Susan J. Baigent ◽  
Melanie A. Sacco ◽  
Simon Evans ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Marek's Disease ◽  
Published Data ◽  
Marek’S Disease ◽  
Bacterial Artificial Chromosomes ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the meq gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-meq of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.

scholarly journals Oncogenicity of Virulent Marek's Disease Virus Cloned as Bacterial Artificial Chromosomes

2004 ◽  
Vol 78 (23) ◽  
pp. 13376-13380 ◽  
Author(s):  
Lawrence Petherbridge ◽  
Andrew C. Brown ◽  
Susan J. Baigent ◽  
Ken Howes ◽  
Melanie A. Sacco ◽  
...  
Keyword(s):  
Disease Virus ◽  
Artificial Chromosome ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Wild Type Virus ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Bac Clone ◽  
Artificial Chromosomes ◽  
Bac Clones

ABSTRACT Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the US2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.

scholarly journals Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 466 ◽  
Author(s):  
Jun Luo ◽  
Man Teng ◽  
Xusheng Zai ◽  
Na Tang ◽  
Yaoyao Zhang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Gene Editing ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Bac Clone ◽  
Virus Growth ◽  
Virus Serotype

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek’s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

scholarly journals Macrophages from Susceptible and Resistant Chicken Lines have Different Transcriptomes following Marek’s Disease Virus Infection

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 74 ◽  
Author(s):  
Pankaj Chakraborty ◽  
Richard Kuo ◽  
Lonneke Vervelde ◽  
Bernadette M. Dutia ◽  
Pete Kaiser ◽  
...  
Keyword(s):  
Disease Virus ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Activated T Cells ◽  
Defense Strategies ◽  
Infection Resistance

Despite successful control by vaccination, Marek’s disease (MD) has continued evolving to greater virulence over recent years. To control MD, selection and breeding of MD-resistant chickens might be a suitable option. MHC-congenic inbred chicken lines, 61 and 72, are highly resistant and susceptible to MD, respectively, but the cellular and genetic basis for these phenotypes is unknown. Marek’s disease virus (MDV) infects macrophages, B-cells, and activated T-cells in vivo. This study investigates the cellular basis of resistance to MD in vitro with the hypothesis that resistance is determined by cells active during the innate immune response. Chicken bone marrow-derived macrophages from lines 61 and 72 were infected with MDV in vitro. Flow cytometry showed that a higher percentage of macrophages were infected in line 72 than in line 61. A transcriptomic study followed by in silico functional analysis of differentially expressed genes was then carried out between the two lines pre- and post-infection. Analysis supports the hypothesis that macrophages from susceptible and resistant chicken lines display a marked difference in their transcriptome following MDV infection. Resistance to infection, differential activation of biological pathways, and suppression of oncogenic potential are among host defense strategies identified in macrophages from resistant chickens.

scholarly journals IFNα and IFNγ Impede Marek’s Disease Progression

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1103 ◽  
Author(s):  
Luca D. Bertzbach ◽  
Olof Harlin ◽  
Sonja Härtle ◽  
Frank Fehler ◽  
Tereza Vychodil ◽  
...  
Keyword(s):  
Immune Responses ◽  
Disease Onset ◽  
Antiviral Effect ◽  
Lymphoproliferative Disease ◽  
Innate Immune ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Innate Immune Responses

Marek’s disease virus (MDV) is an alphaherpesvirus that causes Marek’s disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek’s disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus.

scholarly journals Horizontal Transmission of Marek's Disease Virus Requires US2, the UL13 Protein Kinase, and gC

2007 ◽  
Vol 81 (19) ◽  
pp. 10575-10587 ◽  
Author(s):  
Keith W. Jarosinski ◽  
Neil G. Margulis ◽  
Jeremy P. Kamil ◽  
Stephen J. Spatz ◽  
Venugopal K. Nair ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Disease Virus ◽  
Horizontal Transmission ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Artificial Chromosomes ◽  
Multiple Organs

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.

scholarly journals A DNA vaccine containing an infectious Marek’s disease virus genome can confer protection against tumorigenic Marek’s disease in chickens

2002 ◽  
Vol 83 (10) ◽  
pp. 2367-2376 ◽  
Author(s):  
B. Karsten Tischer ◽  
Daniel Schumacher ◽  
Martin Beer ◽  
Jörg Beyer ◽  
Jens Peter Teifke ◽  
...  
Keyword(s):  
Disease Virus ◽  
Dna Vaccine ◽  
Chitosan Nanoparticles ◽  
Vaccine Virus ◽  
Marek's Disease Virus ◽  
Challenge Infection ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek’s disease virus (MDV) was tested for its potential to protect against Marek’s disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.

scholarly journals Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens

2008 ◽  
Vol 83 (2) ◽  
pp. 859-869 ◽  
Author(s):  
Paulette F. Suchodolski ◽  
Yoshihiro Izumiya ◽  
Blanca Lupiani ◽  
Dharani K. Ajithdoss ◽  
Oren Gilad ◽  
...  
Keyword(s):  
Disease Virus ◽  
Leucine Zipper ◽  
Rapid Onset ◽  
Etiologic Agent ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease

ABSTRACT Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.

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