Effects of Meq mutations on Marek's disease virus (MDV): pathogenecity and oncogenicity
Date
2017
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Marek’s disease (MD) is a highly transmissible lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek’s disease virus-1 (MDV-1). MD remains a constant problem to poultry production worldwide, due to the cost of vaccination and the continuous evolution of more virulent field strains. Upon analyzing MDV-1 strains of distinct pathotypes, we, and others, have found that the major oncoprotein (Meq) of MDV-1 strains has specific coding mutations that correlate with virulence level. We, and others, have previously reported differences in the transcriptional activation potential and the cellular binding profiles of the different Meq isoforms. We therefore hypothesized that these mutations could directly affect the virulence of MDV-1 strains. Using RB-1B as a well characterized genetic backbone, we constructed recombinant MDVs that harbor the meq genes of the vaccine strain CVI988 (399 aa form), and the very virulent plus N strain (339 aa, vv+ form). Contrary to our hypothesis, pathogenicity studies in SPF chickens revealed that there was no effect of the replacement of the meq genes on MD, mortality or tumor incidence. These data therefore suggest that in unvaccinated, maternal antibody-free chickens, changes in the Meq coding sequence conferred no inherent pathogenicity increase (or decrease) for the mutations examined. Experimental validation via Southern blotting showed that both copies of meq were restored in all the recombinant viruses upon replication in CEF in vitro, SPC in vivo or in established cell lines. Similarly, PCR and sequencing of the meq loci and sequencing of meq loci ensured that viruses had not undergone significant mutations (duplications, deletions, etc.) during propagation in cell culture or in vivo, or had not been mixed during the study. ☐ A follow up hypothesis we formulated based on the aforementioned observations was that the mutations in Meq have occurred upon selection of viruses that have the ability to overcome the innate immune responses and signaling elicited from vaccination. Vaccines are presumed to be the main drivers of MDV evolution towards higher virulence. Since, the vaccines do not elicit sterilizing immunity, but only prevent MDV-induced lymphoma formation, this provides an opportunity to the virus to continually evolve in the presence of selective pressure exerted by vaccinal immunity on the full length Meq form. To address this hypothesis, we simulated innate immune activation analogous to early innate immune responses post-vaccination, using various innate immune agonists (LPS, Poly I:C, cGAMP). Replication of the recombinant viruses in CEF showed that cGAMP treatment (inducer of STING and type I interferon expression) caused a decrease in plaque number and plaque area size for RB-1B CVI Meq, but did not affect the RB-1B parent or RB-1B N Meq viruses, suggesting that Meq from mildly virulent MDV CVI988 lacks the ability to overcome cGAS-STING anti-viral signaling. On the other hand, vv and vv+ Meq forms displayed an ability to overcome cGAS-STING anti-viral signaling. Plaque number and plaque area analysis for the RB-1B N meq recombinant showed that its replication was least affected by any of the agonist treatments in both CEF and spleen cells (SPC). Thus, the N strain-meq, seems to have overcome innate signaling triggered via either of TLR3, TLR4 or cGAS-STING pathways. Agonist-treated, virus-infected SPC co-cultured with CEF ex vivo, showed a profound reduction in plaque number in the order: RB-1B CVI meq virus (~30% for each treatment) > RB-1B parent virus (~10% for each treatment) > RB-1B N meq virus (~4% for each treatment). ☐ Our work therefore suggests that mutations in the Meq oncoprotein are not directly responsible for the observed differences in MDV virulence levels, but that these Meq mutations have likely been selected indirectly based upon the pressure exerted by the innate immune responses to vaccines and it is through this evasion that they have a role in the increased MDV virulence.
Description
Keywords
Biological sciences, Marek, MDV, Meq, Mutations, Oncogenicity, Pathogenecity, Virus