Woźniakowski et al. Acta Veterinaria Scandinavica 2011, 53:10 http://www.actavetscand.com/content/53/1/10 isolated MDV strains were not determined in this study but will be examined in the future. However a rather high level of virulence is expected as the strains were iso- lated from chickens with severe clinical symptoms and MD lesions. The 0.8 kb LTR insertion into the MDV genome was detected in 28 strains while 2 strains also had an inser- tion being visible as a 1.6 kb band. This insertion could be assigned as a most efficiently transcribed LTR region within SORF2 region of MDV [4]. SORF2 is a homolo- gue of US22 gene of human cytomegalovirus and human herpesvirus 6 EPLF3 [4,16]. This insertion may alter transactivation of MDV genes as suggested in pre- vious studies [8,9]. The multiple bands observed in a few Polish field strains suggest different sites of LTR integration in the US MDV region. Jones et al. [8] described smeared bands as randomly integrated REV proviruses. This may influence the sequence and struc- ture of different MDV genes including meq. However the smears in the background of the deletive BAC_31_07Δmeq suggest that the insertions are not necessarily associated only with meq (Figure 1). The sequenced LTR fragments of 10 strains were new in NCBI Genebank. The changes detected within meq are confirmed by the finding that LTR insertions are often located near to proto-oncogenes thus being able to acti- vate tumoral processes [3]. An other scenario is that IRS and Us insertions may alter the recombination of chi- meric viruses in vitro and in vivo since the LTR-REV regions are known as strong enhancers of promoters in chicken embryo fibroblasts [17]. However confirmation of these hypotheses needs further studies. Previous reports on the effects of a LTR-REV insertion into RM1 [1] or GX0101 [13,14] strains of MDV suggest attenua- tion and reduction of their oncogenicity with intact replication abilities. However insertions in Polish MDV strains seemed not to influence their oncogenicity since they caused outbreaks of MD in vaccinated chickens. The exact effects of these insertions will be studied experimentally after establishing BAC clones lacking the LTR fragment. The LTR insertions in MDV could make the epidemiology of Marek’s disease more complex. These aspects will be investigated in future studies. Conclusions Significant insertions within meq oncogene sequence were detected among recently isolated virulent MDV strains from Poland. The detected insertional mutation may lead to structural and functional changes of multi- ple proteins and virus features. List of abbreviations ALV: Avian leukosis virus; BAC: bacterial artificial chromosome including MDV genome; bp: base pair; CEK: chicken embryo kidney cells; DMSO: dimethyl sulfoxide; EPLF3: gene of human herpesvirus 6; IRL: internal repeated long Page 6 of 7 sequence of MDV; IRs: internal repeated short sequence of MDV; kbp: thousand of base pairs; LTR: long terminal repeats of REV genome; LTZ: Lohmann Tierzucht Company, Germany; MEM: minimal essential medium; MEQ: main oncoprotein of MDV crucial in tumorigenesis; MDV: Marek’s disease virus; REV: reticuloendotheliosis virus; SPF: specific pathogen free chickens. The following pathogens are certified to be absent: avian adenoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, avianleukosis, avian reticuloendotheliosis and Marek’s disease virus; SORF2: short opened reading frame 2 of MDV; TRs: terminal repeated short sequence of MDV; TRL: terminal repeated long sequence of MDV; Us: unique short region of MDV; UL: unique long region of MDV; US22: gene located within unique short region of MDV; ver. -version. Acknowledgements Dr Peter Biggs, Houghton Poultry Research Station, Huntingdon, UK is acknowledged for providing reference strain HPRS16. Authors’ contributions GW carried out isolation of the used strains, DNA extraction, PCRs, sequencing of meq and LTR genes and nucleotide alignments. ESS and WK participated in DNA extraction, real-time PCRs, PCRs and assesment of the final results. ESS provided personal observations used in the present study. WK provided detailed information regarding origin of used MDV field strains. ESS and WK participated in coordination of the study. The final manuscript was read and approved by all authors. Competing interests The authors declare that they have no competing interests. Received: 24 November 2010 Accepted: 14 February 2011 Published: 14 February 2011 References 1. Witter RL, Li D, Jones D, Lee LF, Kung HJ: Retroviral insertional mutagenesis of a herpesvirus: a Marek’s disease virus mutant attenuated for oncogenicity but not for immunosuppression or in vivo replication. Avian Dis 1997, 41:407-421. 2. Woźniakowski G, Samorek-Salamonowicz E, Kozdruń W: Occurence of main Marek’s disease genes during infection of chickens. Bull Vet Inst Pulawy 2010, 54:123-127. 3. Isfort R, Jones D, Kost R, Witter R, Kung HJ: Retrovirus insertion into herpesvirus in vitro and in vivo. Proc Natl Acad Sci USA 1992, 89:991-995. 4. 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