Characterization of the a-like sequences of attenuated and nonattenuated strains of Marek's disease virus

Authors: Spatz, Stephen; Rue, Cary

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 11/15/2007
Publication Date: 12/2/2007
Citation: Spatz, S.J., Rue, C.A. 2007. Characterization of the a-like sequences of attenuated and nonattenuated strains of Marek's disease virus. Research Workers in Animal Diseases Conference Proceedings. p. 115.

Interpretive Summary:

Technical Abstract: Analysis of the genomic sequences of four virulent strains (GA, Md5, RB-1B and Md11) has identified sequence differences within the internal repeat long/internal repeat short junction, specifically the a-like sequence. We have determined the sequence of this region from 13 strains representing the 5 pathotypes of MDV-1 in hope of identifying cis-acting sites or genes involved in virulence. The junction regions were PCR-amplified from DNA preps of low-passaged strains, cloned and sequenced. The sizes of the PCR products varied extensively from 3453 to 5761 bp. Based on multiple alignments, we identified sequences similar to the pac1 and pac2 sites of HSV-1 which are bracketed by DR-1 sites (GGCCGCGAGAGC). Internal to these sites are telomeric repeat sequences (TRS) containing (GGGTTA) motifs inundated with islands of 13 bp repeats (GGGTTCAGGCCTA). Downstream of this region is a hypervariable region containing 60 bp repeats. In addition to the variations in the telomeric and 60 bp repeats, some attenuated and mildly virulent pathotypes contain deletions in a region immediately downstream of their 60 bp repeats. These deletions are in the promoter region of the large LAT and spliced LAT and in some strains encompass the first exon of the spliced LAT and recently identified LAT microRNA miR8. The presence of cis-acting sites (e.g. TRS and pac sites) in other lymphotropic herpesvirus (HHV-6, HHV-7 and EHV-2) suggests these sequences are functionally important. What has not been reported is whether these sequences are present at the genomic termini of MDV-1. To investigate this, 5’ and 3’ adaptor mediated- PCR was used to determine the terminal sequences of the mildly virulent strain CU-2. The results indicate that the right terminus ends in varying numbers of TRS downstream from a pac2 site. However these TRS are not present at the left terminus. Instead this terminus ends predominantly with sequences adjacent to the pac1 site. Our research has identified deletions downstream of the a-like sequence (IRL/IRS junction) in attenuated strains of MDV-1 which may play a role in virulence. Furthermore we have determined that only the right terminus of MDV-1 ends with telomeric repeats.