Page 2 of 7 Table 1 Marek’s disease virus strains used in the study Number Name of strain Origin of strain Woźniakowski et al. Acta Veterinaria Scandinavica 2011, 53:10 http://www.actavetscand.com/content/53/1/10 are classified into Alpharetrovirus [6] whereas the REV group is in the separate Gammaretrovirus genus. How- ever the structure and organization of their genome are common. The genomes of retroviruses consist of single- stranded RNA with positive polarity and present in two copies. REV is an immunosuppressive virus affecting both humoral and cellular immunity [7]. An additional pro- blem with REV is their ability to integrate a part or entire genome into the DNA of MDV [3,8]. This phe- nomenon occurs under field conditions [9]. The influ- ence of a LTR-REV insertion into the MDV genome may change viral oncogenity or attenuation. The main objective of this study was to determine molecular characteristics of recently isolated Polish strains of MDV and to identify factors responsible for changes of MDV virulence; changes that are commonly associated with mutation of the nucleotide sequence of meq oncogene [10]. Methods Viruses A total of 29 MDV field strains isolated from spleens of spontaneously infected vaccinated chickens, the HPRS16 reference strain (Houghton Poultry Research Station, Huntingdon. UK), the CVI988 Rispens strain from a commercial vaccine (Mérial, Lyon, France) and two BAC clones of Polish strains were propagated in chicken embryo kidney cells (CEKs) prepared from SPF chicken embryos (LTZ, Cuxhaven, Germany). A REV-T strain (National Veterinary Research Institute, Pulawy, Poland) was included in the study. The growth medium MEM (Gibco, Paisley, Scotland) supplemented with 10% bovine foetal serum and 1% antibiotics (Antibiotic-anti- mycotic, Gibco) was used, while a maintenance medium consisting of MEM with 1% mixture of antibiotics as above was used. MDV strains were incubated at 37.8°C in 5% CO2 until a cytophatic effect (CPE) was observed. The REV-T strain was also cultivated in CEKs and har- vested 5 days post inoculation Third passage of each strain in CEKs was suspended in preservative medium containing 10% DMSO, gradually chilled and finally fro- zen in liquid nitrogen. The strains are listed in Table 1. The purity of the MDV strains from REV contamination was tested by filtering supernatants from the 3rd passage of each MDV strain through the 0.2 μM syringe filters (Sartorious, Goettingen, Germany) and inoculating monolayers of CEKs. These cell cultures were incubated for 14 days in conditions as described above. Addition- ally DNA extracted from supernatants was tested with real-time polymerase chain reaction (real-time PCR) for the gag gene of REV according to the procedure devel- oped by Hauck et al. [11] for excluding incidental con- tamination with REV. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 23_07_PL 25_07_PL 29_07_PL 30_07_PL 31_07_PL 32_07_PL 2_08_PL 3_08_PL 7_08_PL 8_08_PL 45_08_PL 48_08_PL 56_08_PL 73_08_PL 8_09_PL 23_09_PL 26_09_PL 40_09_PL 42_09_PL 45_09_PL 51_09_PL 81_09_PL 82_09_PL 86_09_PL 8_10_PL 10_10_PL 11_10_PL 30_10_PL 61_10_PL BAC_7_08 HPRS16 16-w layers, spleen 20-w breeders, spleen 16-w layers, spleen 17-w breeders, spleen 27-w layers, spleen 27-w layers, spleen 28-w layers, spleen 20-w layers, spleen 26-w layers, spleen 29-w layers, spleen 19-w breeders, spleen 15-w breeders, spleen 21-w layers, spleen 6-w broilers, spleen 5-w broilers, spleen 17-w layers, spleen 32-w layers, spleen 18-w breeders, spleen 7-w broilers, spleen 18-w layers, spleen 18-w layers, spleen 26-w layers, spleen 25-w layers, spleen 28-w layers, spleen 24-w layers, spleen 19-w layers, spleen 25-w layers, spleen 27-w layers, spleen 6-w broilers, spleen BAC_31_07Δmeq BAC-clone from Escherichia coli GST1763 cells BAC clone from Escherichia coli GST1763 cells Houghton Poultry Research Station, Huntingdon UK CVI/988 Rispens Commercial vaccine (Mérial) All strains were passed 3 times in chicken embryo kidney cells. W: week-old, CEK: chicken embryo kidney cells, BAC_31_07Δmeq: deletive strain of 31_07 strain in bacterial artificial chromosome. DNA extraction Total DNA was extracted from infected CEKs using QIAamp Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s procedure. Real-time PCR Primers complementary to ICP4, pp38 and meq oncogene were used [2]. For the normalisation of results as well as for calculation of viral copy number per 104 infected cells,