Novel genetic factors in Marek’s disease virus pathogenesis and establishment of a cell culture-based viral integration system

Marek’s disease virus (MDV) is a highly contagious alphaherpesvirus that causes neurological disorders, immunosuppression, and deadly lymphomas in chickens. The virus causes substantial economic losses in poultry production worldwide due to high mortality rates and the costs of the vaccination. Despite the widespread use of live attenuated vaccines that provide excellent protection against clinical disease, the virus still infects vaccinated animals and continuously evolved towards a higher virulence. MDV encodes many viral factors, from proteins to non-coding RNAs, which orchestrate the complex viral lifecycle and/or contribute to pathogenesis. Several MDV-specific genes have been shown to be involved in viral pathogenesis and tumorigenesis. Comprehensive MDV transcriptome analyses revealed that the coding capacity of the MDV genome is far greater than previously anticipated and identified some putative MDV-specific genes and splice isoforms. In-depth characterization of the genetic factors of MDV is an essential step toward understanding the complex viral lifecycle, from viral infection, lytic replication, latency, to tumor formation. Here, we set out to assess three MDV-specific hypothetical genes (MDV082, RLORF11, and SORF6) and a putative vIL-8 exon (vIL-8-E3′). Using FLAG-tagged recombinant viruses based on the very virulent MDV strain RB1B, we identified a novel protein encoded by MDV082 and a novel spliced vIL-8-E3′protein. We demonstrate that the novel pMDV082 and vIL-8 splice variant are not essential for virus replication, spread, and tumor formation, but pMDV082 contributes to the rapid onset of Marek’s disease. These studies shed light on the expression of MDV-specific genes and the splice forms of the CXC chemokine and unraveled the role of pMDV082 in MDV pathogenesis. In addition to viral virulence factors, the lymphomagenic properties of oncogenic MDV is also directly linked to viral integration, a process which occurs during the establishment of viral latency. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres, but the integration mechanism remains poorly understood. To shed further light on the MDV integration mechanism, we developed and validated a cell culture-based integration assay, providing an optimal basis for investigating the role of viral and cellular factors that could be involved in the integration of MDV.