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.

UNLABELLED: 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. IMPORTANCE: 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.

Addition of a UL5 helicase-primase subunit point mutation eliminates bursal-thymic atrophy of Marek's disease virus ∆Meq recombinant virus but reduces vaccinal protection.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus and the causative agent of Marek's disease (MD), characterized by immunosuppression, paralysis, nerve enlargement and induction of T-cell lymphomas in chickens. Despite widespread usage of vaccines since the 1970s to control MD, more virulent field strains of MDV have emerged that overcome vaccinal protection, necessitating the development of new and more protective MD vaccines. The ∆Meq virus, a recombinant Md5 strain MDV lacking the viral oncogene Meq, is one candidate MD vaccine with great potential but unfortunately it also causes bursal-thymic atrophy (BTA) in maternal antibody negative chickens, raising concerns that impede commercial use as a vaccine. Previously, we identified a point mutation within UL5 that reduced in vivo replication in attenuated viruses. We proposed that introduction of the UL5 point mutation into the ∆Meq virus would reduce in vivo replication and eliminate BTA yet potentially retain high protective abilities. In birds, the ∆Meq+UL5 recombinant MDV had reduced replication compared to the original ∆Meq virus, while weights of lymphoid organs indicated that ∆Meq+UL5 did not induce BTA, supporting the hypothesis that reduction of in vivo replication would also abolish BTA. Vaccine trials of the ∆Meq+UL5 virus compared to other ∆Meq-based viruses and commercial vaccines show that, while the ∆Meq+UL5 does provide vaccinal protection, this protection was also reduced compared to the original ∆Meq virus. Therefore, it appears that a very delicate balance is required between levels of replication able to induce high vaccinal protection, yet not so high as to induce BTA.

The Mut UL5-I682R Marek's Disease Virus with a Single Nucleotide Mutation Within the Helicase-Primase Subunit Gene not only Reduces Virulence but also Provides Partial Vaccinal Protection Against Marek's Disease.

Marek's disease virus (MDV) is an oncogenic herpesvirus that afflicts chickens with the disease known as Marek's disease (MD). This virus induces tumors, nerve lesions, immunosuppression, and death of affected birds. Vaccines are the primary control method for MD but, due to the periodic evolution of field strains, it is necessary to explore the development of new MD vaccines. MD vaccines are often attenuated MDV strains generated through serial passage in vitro. We previously used experimental evolution of MDV to provide a better understanding of the genetic basis of attenuation. During complete genome sequencing of evolved MDV populations, we identified a point mutation within the UL5 helicase-primase gene and created a UL5 recombinant virus that significantly reduced disease incidence by 89%-100%. To determine if experimental evolution also identifies mutations that provide protective qualities as potential vaccine candidates, we tested the UL5 recombinant virus as a vaccine and compared its protection to commercial herpesvirus of turkey (HVT) and bivalent (HVT + SB-1) vaccines. Both commercial vaccines resulted in higher protection against MD than did the UL5 recombinant virus, although the UL5 virus did provide protection against developing MD in 46%-70% of birds challenged. This indicates that a mutation within the UL5 helicase-primase gene not only reduces virulence but also confers protection against challenge with virulent MDV, providing support that not only can experimental evolution identify candidate mutations involved in attenuation but can also identify potential candidates for use in vaccine development.

Identification and Validation of Ikaros (IKZF1) as a Cancer Driver Gene for Marek's Disease Virus-Induced Lymphomas.

Marek's disease virus (MDV) is the causative agent for Marek's disease (MD), which is characterized by T-cell lymphomas in chickens. While the viral Meq oncogene is necessary for transformation, it is insufficient, as not every bird infected with virulent MDV goes on to develop a gross tumor. Thus, we postulated that the chicken genome contains cancer driver genes; i.e., ones with somatic mutations that promote tumors, as is the case for most human cancers. To test this hypothesis, MD tumors and matching control tissues were sequenced. Using a custom bioinformatics pipeline, 9 of the 22 tumors analyzed contained one or more somatic mutation in Ikaros (IKFZ1), a transcription factor that acts as the master regulator of lymphocyte development. The mutations found were in key Zn-finger DNA-binding domains that also commonly occur in human cancers such as B-cell acute lymphoblastic leukemia (B-ALL). To validate that IKFZ1 was a cancer driver gene, recombinant MDVs that expressed either wild-type or a mutated Ikaros allele were used to infect chickens. As predicted, birds infected with MDV expressing the mutant Ikaros allele had high tumor incidences (~90%), while there were only a few minute tumors (~12%) produced in birds infected with the virus expressing wild-type Ikaros. Thus, in addition to Meq, key somatic mutations in Ikaros or other potential cancer driver genes in the chicken genome are necessary for MDV to induce lymphomas.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Evolution of dependoparvoviruses across geological timescales-implications for design of AAV-based gene therapy vectors.

Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyse the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha (whales and hippos), Vespertilionidae (smooth-nosed bats), and Lagomorpha (rabbits, hares, and pikas), indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77 to 23 million years ago. These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modelling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions, especially in those related to the three- and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus 'fossils' relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based on paleovirology data.

Characterizing in vivo stability and potential interactions of a UL5 helicase-primase mutation previously shown to reduce virulence and in vivo replication of Marek's disease virus.

The unpredictable yet recurrent emergence of more virulent field strains of Marek's disease virus (MDV) in Marek's disease (MD) vaccinated flocks of chickens has prompted concerns regarding the sustainability of MD vaccines. A single non-synonymous point mutation (I682R) within the UL5 helicase-primase unit was shown to reduce virulence by over 90%. Considering in vitro attenuation is commonly used to generate MD vaccines, this result prompted further characterization of this mutation, particularly to better understand the potential of point mutations for use in vaccine development. Incorporation of a second non-synonymous point mutation (UL46-Q117R; tegument) found at high frequencies in the same attenuated MDV as the UL5 mutation did not further reduce virulence compared to the single UL5 mutation alone. Furthermore, when the UL5-containing MDV was serially passed three times in vivo, the resulting viruses did not show increases in replication or virulence, and no revertant viruses could be detected. This suggests that point mutations that reduce fitness and in vivo replication may be more stable than initially anticipated, which may alleviate some concerns regarding rationally designed MD vaccines based upon point mutations.