In vivo Events of Retroviral Long Terminal Repeat Integration into Marek's Disease Virus in Commercial Poultry: Detection of Chimeric Molecules as a Marker

Abstract

The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV. A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HS-cPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.

Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity

“…The resultant virus, RM1, was highly attenuated; however, it induced severe BTA and immunosuppression similar to very virulent MDVs . Natural recombination is highly prevalent in commercial chicken and turkey flocks in Israel (Davidson et al, 1995(Davidson et al, , 2002Davidson & Borenshtain, 2001) and in China , further providing evidence that recombination between DNA and RNA viruses occurs readily in vivo in avian species. Understanding the mechanism of recombination events in vivo is important.…”

“…The inability to detect RM1 LTR after 3 weeks of infection could be as a result of either a mixed population of rMd5-RM1-LTR viruses including rMd5-RM1-LTR viruses containing no RM1 LTR inserts along with viruses containing two copies of RM1 LTR or due to the loss of the RM1 LTR insert during replication in chickens. Only recombinant viruses with integration at MDV sites not vital for in vivo replication would be retained during replication in the bird (Davidson & Borenshtain, 2001). Previous studies demonstrated the difficulty in isolation of recombinant viruses in chickens.…”

“…Integration of a REV long terminal repeat (LTR) into fowlpoxvirus in avian species demonstrated that natural recombination occurs in nature and can lead to unexpected changes in the retroviral spread pattern and pathogenicity of the resultant recombinant virus (Hertig et al, 1997). Furthermore, beginning in 1993, an extensive survey of Israeli commercial flocks evaluated the incidence and the prevalence of flocks and birds with multiple viral infections (Davidson et al, 1995;Davidson & Borenshtain, 2001). A total of 2926 DNA samples were analysed by Hot-Spot combined polymerase chain reaction (PCR) analysis (Borenshtain & Davidson, 1999), including 2428 chicken and 498 turkey DNA samples (Davidson & Borenshtain, 2001).…”

“…Furthermore, beginning in 1993, an extensive survey of Israeli commercial flocks evaluated the incidence and the prevalence of flocks and birds with multiple viral infections (Davidson et al, 1995;Davidson & Borenshtain, 2001). A total of 2926 DNA samples were analysed by Hot-Spot combined polymerase chain reaction (PCR) analysis (Borenshtain & Davidson, 1999), including 2428 chicken and 498 turkey DNA samples (Davidson & Borenshtain, 2001). Of these samples, 991 DNAs were from flocks with multiple virus infections including 48% (27/56) containing chimeric MDV and retroviral LTR molecules.…”

“…Of these samples, 991 DNAs were from flocks with multiple virus infections including 48% (27/56) containing chimeric MDV and retroviral LTR molecules. The majority of the blood samples from co-infected chickens were positive for MDV plus REV (17%), with co-infection of MDV plus avian leucosis virus (A 'C) (13%) and MDV plus avian leucosis virus (J) (3%) (Davidson & Borenshtain, 2001). Several field strains of MDV containing an REV LTR insert have been isolated in China .…”