Virulent Marek's disease virus generated from infectious bacterial artificial chromosome clones with complete DNA sequence and the implication of viral genetic homogeneity in pathogenesis

Niikura, Masahiro; Kim, Taejoong; Silva, Robert F.; Dodgson, Jerry B.; Cheng, Hans H.

doi:10.1099/vir.0.026864-0

Cited by 47 publications

(26 citation statements)

References 35 publications

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“…Targeted resequencing of candidate single-nucleotide polymorphisms allowed for the frequency of each mutation to be determined from the earliest passage in which the mutation occurred until attenuation. of genetically diverse genotypes existing within a strain population (8,11). Thus, while the viral strain may be described as a virulent, there can be unique subpopulations that do not share the same degree of virulence compared to the whole population.…”

Section: Figmentioning

confidence: 99%

Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

Hildebrandt

Dunn²,

Perumbakkam

et al. 2014

J Virol

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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. IMPORTANCEMarek'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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Section: Figmentioning

confidence: 99%

Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

Hildebrandt

Dunn²,

Perumbakkam

et al. 2014

J Virol

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“…Similarly, a continuous Vero cell line was reported to produce low titres of MDV virus after long adaptation (Jaikumar et al, 2001). A spontaneously immortalized CEF cell line, DF-1 (Himly et al, 1998;Schaefer-Klein et al, 1998), was shown to support growth of virulent MDV (Niikura et al, 2011).…”

Section: Introductionmentioning

confidence: 98%

Efficient Marek's disease virus (MDV) and herpesvirus of turkey infection of the QM7 cell line that does not contain latent MDV genome

2014

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Marek's disease virus (MDV; also known as Gallid herpesvirus 2, MDV-1) causes oncogenic disease in chickens producing clinical signs that include lymphomas, visceral tumours, nerve lesions, and immunosuppression. MDV vaccines are widely used and mostly produced using primary cells: chicken embryo fibroblast cells, duck embryo fibroblast cells, chicken embryo kidney cells or chicken kidney cells. An immortalized cell line that can be used to manufacture the virus has long been desired. In this report, we demonstrate that QM7 cells were susceptible to infection with either MDV or herpesvirus of turkey (HVT; also known as Meleagrid herpesvirus 1, MDV-3). Polymerase chain reaction analysis with primers amplifying selected MDV genes revealed that QM7 cells did not contain these sequences. However, MDV genes were detected in QT35 cells, which have been reported to harbour latent MDV virus. Transfection of naked MDV DNA initiated efficient infection of QM7 cells. In addition, QM7 cell lysate, clarified supernatant, and QM7 cell pellet infected with MDV were negative for reverse transcriptase activity, indicating absence of endogenous retrovirus. QM7 cells were also found to be free of other avian pathogens in a chick embryo inoculation test. In vivo studies of MDV growing in QM7 cells showed the virus retained its pathogenicity and virulence. In ovo experiments demonstrated that both HVT and MDV propagated in QM7 cells did not interfere with hatchability of injected eggs, and viruses could be re-isolated from hatched chicks. The results suggest that QM7 could be a good host cell line for growing both MDV and HVT.

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“…The insertion of the REV LTR sequences into the rMd5 BAC clone was previously published (Kim et al, 2010). Briefly, REV LTR sequences from the IR S region in RM1 (Jones et al, 1996) were inserted using Red-mediated recombination techniques (Tischer et al, 2006) into the IR S region of the rMd5 BAC clone (Niikura et al, 2011) at the same location as the LTR in RM1 (Jones et al, 1996).…”

Section: Methodsmentioning

confidence: 99%

“…We recently cloned the LTR from the IR S of RM1 and inserted the LTR into the IR S of a BAC clone of a very virulent strain of MDV, Md5 (Niikura et al, 2011), generating rMd5-RM1-LTR . The LTR insertion site of the rMd5-RM1-LTR BAC is nearly identical to the insertion site of the LTR in the RM1 virus.…”

Section: Introductionmentioning

confidence: 99%

“…Since the original construction of the bacterial artificial chromosome (BAC) vector (Shizuya et al, 1992), the BAC technology has been used to clone and characterize several herpesviruses (Messerle et al, 1997;Chaundhuri et al, 2008) including MDV (Schumacher et al, 2000;Petherbridge et al, 2003Petherbridge et al, , 2004Baigent et al, 2006;Silva et al, 2010;Singh et al, 2010;Niikura et al, 2011;Smith et al, 2011) and naturally occurring field strains of MDV containing a REV LTR insertion (Sun et al, 2009). We recently cloned the LTR from the IR S of RM1 and inserted the LTR into the IR S of a BAC clone of a very virulent strain of MDV, Md5 (Niikura et al, 2011), generating rMd5-RM1-LTR .…”

Section: Introductionmentioning

confidence: 99%

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Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity

et al. 2012

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Virulent Marek's disease virus generated from infectious bacterial artificial chromosome clones with complete DNA sequence and the implication of viral genetic homogeneity in pathogenesis

Cited by 47 publications

References 35 publications

Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

Efficient Marek's disease virus (MDV) and herpesvirus of turkey infection of the QM7 cell line that does not contain latent MDV genome

Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity

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