Microsatellite instability in Marek’s Disease Virus infected primary chicken embryo fibroblasts

Marek’s disease (MD), caused by the highly oncogenic lymphotropic Marek’s disease virus (MDV), is a highly contagious neoplastic disease of poultry. MD is characterized by lymphomas and lymphoproliferative infiltration of their visceral organs, peripheral nerves, muscles and skin[1]. Although many different kinds of control measures have been implemented against the disease since 1907, MD remains a severe threat to poultry industry in many countries because of its high occurrence. Economically losses from MD is estimated at least US $1 billion annually[2].

The pathogenesis of MD is complex and is not fully understood. Susceptible genotypes of birds are infected by the infectious virus which replicates in the host lymphoid organs. B-cells and macrophages undergo a lytic infection, resulting in the activation of T-cells. The latent T-cells become targets for neoplastic transformation leading to the development of lymphomatous tumors in various visceral organs[3, 4]. Some of the factors that lead to MD lymphomas have been investigated. A study demonstrated that MDV might induce host cell DNA strand breaks. Accumulation of genomic alteration over time can lead to gene modifications in cells that might turn to be mutagenic or carcinogenic[5].

Genetic mutations may occur throughout the genome, involving gross chromosomal aberrations or only variations in the length of genomic DNA fragments carrying microsatellite sequences. Microsatellites are repetitive DNA sequences comprised of short reiterative motifs, which locates within the heterochromatin near chromosomal centromeres and telomeres[6]. Microsatellite sequences are stably inherited with high accuracy making them excellent genetic markers. The length of microsatellite sequences is unique to each individual and distinct among individuals. In addition, individual has identical microsatellite in different kinds of cells. Microsatellite mutations or microsatellite instability (MSI) leads to DNA replication error (RER) phenotype. If uncorrected, these errors are fixed after a next round of replication as addition or deletion of one or more microsatellite sequences. This mutated phenotype manifests as MSI, and appears to play an important role in tumorigenesis and/or tumor progression[7‐9].

A total of 21 samples were determined for the presence of MIS. All samples were tested at 118 microsatellite loci. Alternations in the size of microsatellites in the MDV-infected samples were revealed as differences in the electrophoretic migration of MDV-infected DNA as compared with control DNA. The MSI frequencies of MDV-infected chicken embryo fibroblasts are summarized in Table 1 and representative samples are illustrated in Figure 1. Twelve markers showed high frequency of MSI (>40%). These markers were ABR0052, ABR0392, LEI0099, ABR0123, ADL0199, ABR0007, ABR204, ABR0086, ABR0059, ABR0634, ABR0133 and ABR0026. 27 microsatellite markers showed no change in length of fragments and 79 microsatellite markers had the lower occurrence of MSI (<40%). Over all, the incidence of MSI in CEF samples of 21 chicken embryos induced by the virus was 100%, at least one microsatellite marker was demonstrated MSI (data not showed).

Marek’s disease is a very common disease and provides excellent model for the study of herpes virus-induced tumors in both experimental and natural conditions. In this study, chicken embryo fibroblasts were used to evaluate the effects of MDV on host cells genome in vitro. It was reported that not only lymphocytes but also primary chicken embryo fibroblasts could be targets for neoplastic transformation by the MDV[10]. Microsatellite instability (MSI), reflecting a cellular deficiency in DNA mismatch repair (MMR), is now regarded as an important biomarker to predict the cellular genome mutation.

Microsatellites are located in both non-coding and coding portions of the genome, and are thought to play a functional role in gene regulation or indirectly as hot spots for recombination. MSI phenotype mainly caused by mutations in the DNA mismatch repair (MMR) genes. This defect may alter gene expression and contribute to cancer development and progression by accelerating the accumulation of mutations in tumor-related genes[11]. MSI in human tumors development and progression has been extensively investigated, which was regarded to have a closed relationship with malignancies, such as endometrial, renal, colorectal, and gastric cancer[12‐15]. The present study indicates for the first time an association between MSI and MDV infection. It was showed that the high frequency of MSI in MDV-infected CEF specimens could be regarded as one of the fundamental feature of CEF transformation. Regulation from oncogenic genes derived from MDV could be a major driving force of the transformation[10]. It is envisaged MDV might lead to defect of MMR system during replication in host cells, which may result in dysregulation of cell division, imbalance between cell growth and death, and act as a trigger for the initiation of cell transformation.