Woźniakowski et al. Acta Veterinaria Scandinavica 2011, 53:10 http://www.actavetscand.com/content/53/1/10 Page 5 of 7 Figure 3 PCR for the detection of reticuloendotheliosis virus (REV) long terminal repeats. M-Mass Ruller 100 kb Plus (80 base pairs - 10,000 base pairs), Fermentas, Neg- negative DNA control extracted from non-infected chicken embryo kidney cells, Pos - replicates of DNA control extracted from the infected chicken embryo kidney cell cultures with REV-T strain 5 days post inoculation. All three positives are derived from the same original strain. 1 - 23_07_PL, 2 - 25_07_PL, 3 - 29_07_PL, 4 - 30_07_PL, 5 - 31_07_PL, 6 - 32_07_PL, 7 - 2_08_PL, 8 - 3_08_PL, 9 - 7_08_PL, 10 - 8_08_PL, 11 - 45_08_PL, 12 - 48_08_PL, 13 - 56_08_PL, 14 - 73_08_PL, 15 - 8_09_PL, 16 - 23_09_PL, 17 - 26_09_PL, 18 - 40_09_PL, 19 - 42_09_PL, 20 - 45_09_PL, 21 - 51_09_PL, 22 - 81_09_PL, 23 - 82_09_PL, 24 - 86_09_PL, 25 - 8_10_PL, 26 - 10_10_PL, 27 - 11_10_PL, 28 - 30_10_PL, 29 - 61_10_PL, 30 - BAC_31_07Δmeq, 31 - BAC_7_08, 32 - HPRS16, 33 - Rispens, random insertions into Marek’s disease virus genomes are marked with white boxes. between MDV and REV is possible as previously dis- cussed by Jones et al. [4]. In vivo studies on the molecular and biological features of MDV-REV BAC clone GX0101 and its virulence for chickens were studied by Sun et al. [13,14]. However the consequences of a LTR insertion into the IRs and Us junction of MDV still remains unknown. The presented study of Polish MDV strains was done using heterogeneous isolates. The obtained two BACs constructed from these strains will be a good model for further study of the effects of these insertions. In spite of detection of parallel changes in meq and LTR insertions into the MDV genome, their role remains Figure 4 Nucleotide alignment of long terminal repeat inserts from 10 Polish Marek’s disease virus field strains (Geneious ver 5.1). unexplained. However, LTR insertions may affect genes of importance for MDV infection mechanisms. We will investigate this in future studies using BACs. The inser- tions present in 12 of the 24 Polish field strains suggest that this is a common event in the high density poultry industry. In this study real-time PCR for the detection of the MDV specific amplicons like ICP4, pp38 and a part of meq revealed no influence of LTR insertion on the molecular features of these genes. However LTR inser- tions were also present 100-200 bp downstream from the ICP4 gene [15]. Therefore a LTR insertion may alter also basis transactivation function by ICP4. Moreover, the full sequencing of meq oncogene conducted in our study and the analysis of the assembled sequences showed a 68 bp mutation within this gene. The insertion was located at the nucleotide 565 of meq. Similarly, in the same position a longer 180 bp insertion was observed in attenuated strains (CU-2, JM/102W and CVI988 Rispens). Interest- ingly, MDV strains with the 68 bp insertion were isolated in the south-western provinces of Poland where retroviral infections in poultry have been recognized previously. It is however possible that the phenomena occurred inde- pendently. But it might also suggest an association of the insertion with changes in virulence. The virulence of the