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Title: POLYMORPHISMS IN THE LONG REPEAT REGIONS OF ONCOGENIC AND ATTENUATED PATHOTYPES OF MAREK'S DISEASE VIRUS 1

Author
item Spatz, Stephen
item Silva, Robert

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2006
Publication Date: 8/7/2007
Citation: Spatz, S.J., Silva, R.F. 2007. Polymorphisms in the long repeat regions of oncogenic and attenuated pathotypes of Marek's disease virus 1. Virus Genes. 35(1):41-53.

Interpretive Summary: Marek’s disease is a highly contagious disease of chicken caused by a herpesvirus known as Marek’s disease virus or MDV. Birds infected with this virus develop leukemia of T-lymphocytes, tumor of lymphoid tissues, paralysis and die shortly after infection. Since the late 1960s this disease is prevented through mass vaccination of poultry and represents the first instance in which a vaccine can prevent cancer. Because of this rigorous vaccine program and high capacity chicken rearing, the MD virus found in the field has been evolving and every decade or so newer vaccines have to be introduced to keep the disease in check. Three difference vaccines have been introduced over the last 35 years and most recently the field virus as evolved to greater disease capability or virulence. It is feared that current vaccine programs will fail to protect poultry against these newer “superbugs.” To understand the reason why the current vaccines are becoming less efficacious we began a research project to determine the genetic differences between the vaccine (non disease producing) and disease-causing or virulent field strains. To this end, 12 strains of varying disease capability or virulence were characterized in a specific bred of chickens. The genetic material (DNA) was isolated from these 12 strains and a region termed long repeat (LR), highly suspected in encoding determinants important to virulence was prepared in order to determine the DNA sequence. A detailed analysis of the LR DNA sequence from these 12 strains indicated genetic changes that differed between vaccine strains and strains that produce clinical signs and death in infected chickens. This analysis has indicated three determinants or genes are important in the disease process, two of which have been implement in causing tumors in infected birds. This information will be instrumental in genetic engineering new vaccines that will protect chickens against these ever evolving virulent field viruses.

Technical Abstract: The nucleotide sequence of the long repeat (LR) regions in the genomes of 12 strains of Marek’s disease virus type 1 (MDV-1) was determined and represent the largest collection of sequencing data from a contiguous region in the serotype 1 genomes. The collection of strains used in this study has been well characterized with respect to their virulence and contains members of each pathotype (4 attenuated, 1 mildly virulent, 2 virulent, 3 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the A level.LR region are likely to encode virulence factors based on comparative genomic studies involving vaccine and virulent strains. Additional studies using knockout mutants have provided stronger evidence that indeed RLORF4 and meq or the overlapping genes 23 kD and RLORF6 are involved in virulence. In this report we provide evidence that additional open reading frames (ORFs) in the LR region differ significantly between the extremes of the pathotypes (attenuated vs nonattenuated). A deletion of 39 base pairs has been identified in RLORF12 from three attenuated strains: CVI988 (Intervet), p27; CVI988 BP-5, p48 and RM-1, p40. A deletion of 40 bps was also identified in RLORF4 of the vaccine strain R2/23, passage 107. Insertion-deletion polymorphisms in the meq loci have also been identified in all of the attenuated pathotypes examined. The locus encoding meq, 23kD and RLORF6 from strains CVI988 (Intervet), CVI988-BP5, CU-2 and RM-1 all contain a 177 bp insertion. Surprisingly attenuated strain R2/23 did not contain this insertion in the meq locus but instead truncated proteins are predicted for the three overlapping proteins due to a frame shift mutation. Single nucleotide polymorphisms that generally partition between attenuated and nonattenuated pathotypes have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG .The use of these single nucleotide, insertion and deletion polymorphisms may enable researchers the ability to predict virulence shifts at the DNA level.