Molecular indications for in vivo integration of the avian leukosis virus, subgroup J-long terminal repeat into the Marek's disease virus in experimentally dually-infected chickens

Marek's disease virus, a herpesvirus, and avian leukosis virus, subgroup J, a retrovirus, are oncogenic viruses of poultry. Both viruses may infect the same flock, the same bird and the same cell. In a double-infected cell, the retroviral DNA can integrate into the cellular or the Marek's disease virus (MDV) genome. The retroviral long terminal repeat (LTR) integration into MDV was first described by Isfort et al., (Proc Natl Acad Sci 89, 991-995, 1992) [1] following tissue culture co-infection. The recombinant virus isolated, RM1, had altered biological properties compared to the parental MDV (Witter R.L., Li D., Jones D., and Kung H.-J., Avian Dis 41, 407-421, 1997) [2]. The issue of retroviral sequence integration into herpesviruses in vivo, in cases of double-virus infection is of wide significance in general virology and veterinary medicine; it also represents a special case of gene transposition. Using the avian system, we aimed to determine occurrence of such integrations in vivo. Chickens were experimentally co-infected with both avian leukosis virus (ALV) subgroup J and with MDV. To demonstrate the presence of the retroviral LTR in the MDV genome we applied the Hot Spot-combined PCR assay (Borenshtain R. and Davidson I., J Virol Meth 82, 119-127, 1999) [3] that consisted of two consecutive steps of amplification. By that HS-cPCR assay, certain MDV genomic sites, defined as HS for integration were specifically amplified, the HS step, and then subjected to screening in an attempt to detect LTR inserts. The screening was achieved by amplification using heterologous primer sets, one for the MDV hot spot and the other for the retroviral LTR, the cPCR step. The products were Southern blotted and hybridized with MDV and ALV-LTR probes. Chimeric molecules were detected and evidenced by an intense signal in 3/10 chickens and weakly in other 3/10 birds. Detection was by LTR amplification, sequencing and multiple alignment to the ALV-J-LTR sequence. The present study indicated that chimeric molecules were produced in vivo.