Polymorphisms in the repeat long regions of oncogenic and attenuated pathotypes of Marek’s disease virus 1

Abstract

The nucleotide sequences of the terminal repeat long (TR(L)) and internal repeat long regions (IR(L)) in the genomes of 13 strains of Marek's disease virus type 1 (MDV-1) were determined and represent the largest collection of sequencing data from a contiguous region (12.8 kb) 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, 3 virulent, 2 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the RL regions 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 RL regions differ significantly between the extremes of the pathotypes (attenuated vs. nonattenuated). A deletion of 10 base pairs has been identified in RLORF12 from two attenuated strains CVI988 BP-5, p48 and RM-1, p40; and the lower virulence strain JM/102W. A deletion of 40 bp was also identified in RLORF4 of the attenuated strain R2/23, passage 106. A 177 bp insertion within the meq loci has been identified in most of the attenuated strains examined. Interestingly, R2/23 did not contain this insertion but instead truncated proteins are predicted for the three overlapping ORFs (meq, 23 kD and RLORF6) due to a frameshift mutation. Single nucleotide polymorphisms (SNPs), which loosely partition between attenuated and nonattenuated strains, have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23 kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG. Although no single nucleotide polymorphism in the RL regions could predict virulence, their overall contribution to virulence can now be examined in defined mutants containing additional insertions or deletions in ORFs, suspected of encoding virulence factors, identified by this research.

A p53-dependent promoter associated with polymorphic tandem repeats controls the expression of a viral transcript encoding clustered microRNAs

“…This region of tandem repeats in GaHV-2 isolates is hypervariable (Spatz and Silva 2007a): there are between 0 and 13 copies of the 60-bp direct repeat, depending on the viral strain. The P1D39 construct obtained from RB-1B Bacmide DNA contains three copies, with 63 nt of upstream and 10 nt of downstream sequences.…”

“…Several strains of GaHV-2 with different oncogenic capacities, correlated with their level of pathogenicity, have been isolated and partially sequenced. Recent published alignments of the sequences from a panel of 14 strains, including the promoter and part of the LAT gene, have shown that all the virulent, very virulent, and hypervirulent oncogenic GaHV-2 strains harbor two or more 60-bp tandem repeats (Spatz and Silva 2007a). By contrast, avirulent, mildly virulent, and vaccine strains have no copies of the tandem repeat.…”

Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity withmeqgene polymorphism

“…The 177 bp insertion in the meq gene has been associated with isolates of lower virulence (Shamblin et al, 2004;Spatz & Silva, 2007). However, all five Australian isolates showed an insertion of 177 bp but, based on the results of the pathotyping experiment, were classified as virulent or very virulent according to the classification system of Witter (1997), inducing MD tumours in 53 to 94% of challenged chickens.…”

“…In addition, sequences were compared with the meq gene of cloned isolate of the vaccinal MDV1 isolate Rispens/CVI988 vaccine strain (Spatz & Silva, 2007; GenBank accession number DQ534538). This isolate had been passaged 27 times in chicken embryo fibroblast cell culture and was obtained from Intervet (Boxmeer, the Netherlands) (Spatz & Silva, 2007).…”

“…Differences in the meq gene between oncogenic and attenuated isolates of MDV1 have been reported with a 177 to 180 bp insertion in the meq gene of the attenuated vaccinal isolate CVI988 being possibly responsible for its reported non-oncogenicity (Lee et al, 2000;Chang et al, 2002b;Spatz & Silva, 2007). There are meq sequences in many isolates of MDV1 available from GenBank.…”