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

Abstract

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.

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

“…Md5B40ΔMeq BAC generated and characterized by Silva et al (2010) was used for incorporation of the UL5 point mutation using Red-mediated recombineering protocols and mutational primers as previously described (Hildebrandt et al, 2014). The resulting Md5B40BACΔMeq+UL5 (ΔMeq+UL5) recombinant mutant contained deletion of both copies of Meq and the nonsynonymous point mutation of amino acid I682R within UL5.…”

“…The two MD vaccines compared were (1) bivalent vaccine consisting of HVT (p10) and SB-1 (p14) strains and (2) Rispens (p43); viruses originated from ADOL stocks. Cells and viruses were cultured in a 1:1 mixture of Leibovitz's L-15 and 100 McCoy's 5A (LM) media containing 1-4% of foetal bovine serum with antibiotics as previously described (Hildebrandt et al, 2014).…”

“…DNA extracted from PBLs was used for qPCR analysis to compare in vivo replication among vaccines. The Taqman Fast Universal PCR kit (Applied Biosciences, Foster City, CA, USA) and primers for amplification of chicken GAPDH and MDV gB were used to calculate the relative ratio of the number of MDV genomes versus chicken genome numbers as previously described Hildebrandt et al, 2014).…”

“…During previous efforts to identify the driving factors for MDV attenuation at a genetic level, we identified a point mutation within UL5, the helicase primase subunit, that resulted in significant reduction of in vivo replication of the virus, while significantly reducing MD incidence to 0-11% as well as providing protection against challenge with virulent MDV (Hildebrandt et al, 2014). Considering the hypothesis that loss of BTA seen in the high-passage rMd5ΔMeq resulted from considerable reduction in replication after passage, we predicted that addition of the UL5 point would reduce replication of ΔMeq to low levels seen in the UL5 point mutant, while simultaneously eliminating BTA characteristic of low passage ΔMeq viruses, but still confer vaccinal protection.…”

“…Therefore, use of the BAC-cloned MDV allowed us to use Red-mediated recombineering to create a double mutant of Md5B40BACΔMeq+UL5 (Hildebrandt et al, 2014). We compared in vivo replication and ratios of lymphoid organ weights for low passage ΔMeq, high passage ΔMeq (p41), and the double mutant, Md5B40BACΔMeq+UL5, against uninfected birds.…”