Woźniakowski et al. Acta Veterinaria Scandinavica 2011, 53:10 http://www.actavetscand.com/content/53/1/10 Table 2 Sequences of meq oncogene and long terminal repeats sequence fragment of reticuloendotheliosis virus genome submitted to NCBI Genebank database Strain 25_07_PL 8_10_PL 10_10_PL 11_10_PL 30_10_PL 8_09_PL 23_09_PL 40_09_PL 42_09_PL 45_09_PL 51_09_PL 81_09_PL 82_09_PL 29_07_PL 31_07_PL 32_07_PL 2_08_PL Accession number Genebank:HQ204792 Genebank:HQ204793 Genebank:HQ204794 Genebank:HQ204795 Genebank:HQ204796 Genebank:HQ204797 Genebank:HQ204798 Genebank:HQ204799 Genebank:HQ204800 Genebank:HQ204801 Genebank:HQ204802 Genebank:HQ204803 Genebank:HQ204804 Genebank:HQ204805 Genebank:HQ204806 Genebank:HQ204807 Genebank:HQ204808 Strain 3_08_PL 7_08_PL 8_08_PL 12_08_PL 45_08_PL 56_08_PL 73_08_PL LTR 7_08 LTR 11_10 LTR 29_07 LTR 31_07 LTR 30_10 LTR 42_09 LTR 45_08 LTR 45_09 LTR 51_09 LTR 82_09 Accession number Genebank:HQ204809 Genebank:HQ204810 Genebank:HQ204811 Genebank:HQ204812 Genebank:HQ204813 Genebank:HQ204814 Genebank:HQ204815 Genebank:HQ204816 Genebank:HQ204817 Genebank:HQ204818 Genebank:HQ204819 Genebank:HQ204820 Genebank:HQ204821 Genebank:HQ204822 Genebank:HQ204823 Genebank:HQ204824 Genebank:HQ204825 substitutions were also detected in 12 strains without influencing the amino-acid composition of MEQ. Nucleotide alignment with meq sequences accessible in Genebank was performed. DNA sequences of meq shared 78.9% homolology of gene sites. Figure 2 Nucleotide alignment for meq oncogene of Polish Marek’s disease virus (MDV) field strains and reference strains. The difference between strains isolated between 2007 and 2009 and 12 of 24 MDV strains (isolated 2009-2010) are marked with a red box (Geneious ver 5.1). Page 4 of 7 LTR analysis PCR for LTR of REV was specific for DNA extracted from CEKs infected with the REV-T strain and showed the presence of an about 0.8 kb insertion in 28 MDV strains (Figure 3). The fragment was not observed in two strains (Figure 3, lanes 14 and 30). An additional insertion of LTR-REV about 0.78 kb into the MDV gen- ome was detected in two strains (Figure 3, lanes 4 and 16). Two other strains showed the presence of an addi- tional 1.6 kb PCR product (Figure 3, lanes 11 and 12). Moreover in two strains (Figure 3, lanes 10 and 13) the LTR insertion was 20 bp longer than in other strains harboring the 0.8 bp fragment. Except for the 0.8 kb product in DNA of the 21 strains, a fine PCR ladder of PCR products ranging from about 0.8 kb to 2.0 kb was observed in the background. LTR fragments were sequenced and submitted to NCBI Genebank. The accession numbers are given in Table 2. The sequences were aligned with other REV sequences accessible in NCBI Genebank. Sequencing of 10 out of 28 MDV recombinant sam- ples confirmed the finding that MDV strains occurring in Poland have a LTR-REV insertion. These sequences shared 70.1% of LTR-REV sequence homology. An alignment of MDV-LTR fragments is shown on Figure 4. Inserts of two chosen strains were significantly longer than in case of the other 8 strains. Discussion Our previous study on the molecular characteristics of Polish MDV strains isolated during 2007-2009 demon- strated a close correlation between changes in nucleotide and amino-acid sequences and the strain virulence [10]. The present study expands the knowledge on the mole- cular characteristics of Polish MDV strains. The obtained results from sequencing of genes in 24 strains suggested a possible frame shift being detected in 12 of the recently isolated field strains. Interestingly, these strains were mainly found in chickens from the south-western parts of Poland, where cases of chicken lymphomas have been observed in the past [personal observation E Samorek- Salamonowicz]. Strains not having a frame shift in their genome originated from the western regions of Poland close to the German border. The incidence of REV infec- tions in Poland is unknown. However it is assumed, e.g. based on veterinary reports that the incidence is higher in southern Poland where poultry farming is most inten- sive. It is known that MDV may co-exist with other ret- roviruses, especially ALV and REV in chicken cells [1,5,6]. In vitro recombination of these viruses and MDV in chicken and duck embryo fibroblasts has been reported [4,8]. The 3 passages of the strains isolated from chickens with clinical MD revealed a persistent presence of LTR in the MDV genomes. A stable integration