The role of a deletion in the glycoprotein l (gL) gene of Marek’s disease virus (MDV) on MDV virulence
Date
2011
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Publisher
University of Delaware
Abstract
Marek’s Disease is a lymphoproliferative, immunosuppressive, and demyelinating disorder of chickens caused by a cell-associated alphaherpesvirus, Marek’s Disease Virus (MDV). MD is considered to be the most expensive disease to control in poultry production, due to the cost of vaccination and the immunosuppressive nature of the causative agent. MD is mainly controlled through the use of avirulent vaccines and management practi. Vaccines can be comprised of three viruses, and are used in various combinations: attenuated MDV-1 strains (CV1988, Rispens), non-oncogeneic viruses MDV-3 and herpesvirus of turkeys (HVT). A bivalent combination of HVT and MDV-2 (strain SB-1) is the most commonly used vaccine for broiler chickens in the US. Due to the evolutionary pressures generated by extensive vaccine use and the selection of rapidly-growing chickens, field strains of MDV have continued to evolve in virulence. There have been no genetic mutations directly associated with the increased virulence of MDV strains that can explain the ability of virus to overcome vaccine protection. We have previously identified a novel mutation in the glycoprotein L (gL) genes of highly virulent field isolates of MDV. This mutation is 12 nucleotide deletion within the coding sequence of gL and is found to be common to all the field isolates we have obtained from DE, MD, VA, PA and NC since 2005. In previous work, we proposed that the gL mutation directly affects bivalent vaccine efficacy in naturally-
challenged chickens, when these chickens were exposed to vaccinated chickens infected with a gL mutation-containing MDV. Although these data suggested that this mutation was responsible for this observation, it is unclear as to how this occurs, mechanistically. To confirm the observation using a genetically-identical background strain, and to test the mechanism of the observed effect, we have introduced this deletion in the gL gene of pRB1B BAC, an infectious clone of the RB1B strain of MDV. We introduced this mutation using a two-step recombination method resulting in markerless mutants. To confirm our analysis of this recombinant, we planned to generate a revertant virus using the same approach, but were unsuccessful. The parental (pRB1B) and mutant (pRB1BgL?) were compared with respect to: (1) their ability to replicate in cell culture, (2) differences in expression of gL, gH and the gH/gL complex on the surface of infected cells, (3) selection for the mutant at the level of cellular replication, (4) their ability to replicate, transmit, and cause disease in specific pathogen free (SPF) chickens, (5) their ability to overcome vaccine protection in contact-exposed chickens, and (6) their differences in their abilities to overcome antibody neutralization. We found that the gL mutation in the context of pRB1B did not confer increased replication or competitive advantage at the level of replication in cell culture, although the mutant did show some increase in the plaque size in chicken embryo fibroblasts. In chickens, both parental and mutant viruses replicated to high titers and caused some mortality and tumor incidence, however, this was notably less than the level of disease caused by non-BAC-based RB1B. Due to the decrease in pathogenicity of the pRB1B-based viruses, we found no loss of vaccine protection in contact-exposed chickens, nor-did the gL mutation confer increased resistance to neutralization by maternal antibodies. Our data suggest that the gL mutation may have arisen in the field after mutations arose that have more directly affected increased virulence, and that this mutation may confer advantage only in this context.