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Title: Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL 41 gene via an in vitro competition assay

item Silva, Robert
item Cheng, Hans

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2007
Publication Date: 1/1/2008
Citation: Mao, W., Niikura, M., Silva, R.F., Cheng, H.H. 2008. Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay. Journal of Virological Methods. 148:125-131.

Interpretive Summary: Marek’s disease (MD) is an economically-important disease of chickens caused by a pathogenic virus known as MD virus (MDV). Vaccines have controlled the problem but new emerging viral strains that result in disease outbreaks are being encountered more frequently. Thus, understanding how mutations in the MDV genome influence virulence is important for long-term control. Currently, it is possible to make recombinant MDVs that help scientists understand how genetic changes observed in new field strains might influence virus traits. However, there is no quick and sensitive method to evaluate many viruses, which is compounded by the need for animal experimentation. In this paper, we describe a head-to-head competition assay, which can detect subtle changes in the ability of MDV to replicate in cell culture. This method and the resulting information enhance our understanding of MDV genes and biology, and suggests possibilities for improved disease control.

Technical Abstract: Marek’s disease (MD), a T cell lymphoma, is an economically important disease of poultry caused by the Marek’s disease virus (MDV), a highly cell-associated alpha-herpesvirus. A greater understanding of viral gene function and the contribution of sequence variation to virulence should facilitate efforts to control MD in chickens. To characterize a naturally-occurring single nucleotide polymorphism (SNP) in the MDV UL41 gene that resulted in a missense mutation (Arg to Cys), BAC-derived MDVs that differed only in the UL41 SNP were evaluated using a head-to-head competition assay in vitro. Monitoring the frequency of each SNP by pyrosequencing during virus passage determined the ratio of each viral genome in a single monolayer, which is a very sensitive method to monitor viral fitness. MDV with the UL41*Cys allele showed enhanced fitness in vitro. To evaluate the mechanism of altered viral fitness caused by this SNP, the virion-associated host shutoff (vhs) activity of both UL41 alleles was determined. The UL41*Cys allele had no vhs activity as predicted, which suggests that enhanced fitness in vitro for MDV with inactive vhs was due to reduced degradation of viral transcripts. The in vitro competition assay should be applicable to other MDV genes and mutations.