Marek’s Disease virus (MDV) is a highly contagious, widespread and persistent neoplastic
α-herpesvirus causing extensive lymphoblastic tumours in chickens. The virus is shed in feather
dust and spread through inhalation. Vaccines are available to protect against the effects of MDV
but not replication of the virus and subsequent contamination of the environment leading to flock
exposure. Increased virulence in strains of MDV has been identified and currently available
vaccines may not offer protection from the disease. Disease outbreaks result in economic
losses as well as welfare issues. To break this cycle better methods of controlling MDV
preventing both tumourogenesis and shedding of infectious virus must be developed. Targeting
specific MDV genes key to maintaining latency and viral replication using siRNA could potentially
be used as a control strategy. At the present time, many of the unique genes in MDV are largely
uncharacterised.
15 uncharacterised open reading frames (ORFs) were screened for expression in a MDV latent
infection model in a non-producer MDV transformed chicken lymphoblast cell line, RPL-1. Of
these uncharacterised ORFs LORF1, LORF3, LORF11, LORF12, ANTISENSE, US2, MLTI,
RLORF11, RLORF12, 23kDa and RLORF6 were expressed during latency. To investigate the
effect of post-transcriptional knockdown of these ORF products two 25-mer siRNA
oligonucleotides were designed for each gene, transfected into RPL-1 cells and analyzed using
growth rate as an indicator of changed phenotype over a period of 120 hours post-transfection.
RPL-1 cells transfected with a nonsense siRNA oligonucleotide were used as the control group.
No significant changes in transfected cell growth over the controls were identified in LORF3,
ANTISENSE, RLORF12, LORF1, LORF11 or MLTI. Increased RPL-1 cell growth was observed
(adjusted p-value of 0.0094) in one of the two siRNA oligonucleotides specific for RLORF6 at 72
hours post-transfection. RLORF6 was further characterised using confocal microscopy
techniques and was found to be localized in the nucleus but not the nucleolus of chicken embryo
fibroblasts and chicken lymphoblastic cells
