RNA-seq analysis of viral gene expression in the skin of Marek’s disease virus infected chickens

Authors: SUNKARA, LAKSHMI - Orise Fellow; AHMAD, MUDASIR - University Of Kashmir; Heidari, Mohammad

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2019
Publication Date: 7/1/2019
Citation: Sunkara, L., Ahmad, M., Heidari, M. 2019. RNA-seq analysis of viral gene expression in the skin of Marek’s disease virus infected chickens. Veterinary Immunology and Immunopathology. 213:1-9. https://doi.org/10.1016/j.vetimm.2019.109882.
DOI: https://doi.org/10.1016/j.vetimm.2019.109882

Interpretive Summary: Marek’s disease virus (MDV) is an alpha-herpesvirus that causes T cell lymphomas in domestic chickens. The skin and feather follicles are the only anatomical sites where infectious enveloped cell-free virus particles are produced and disseminated into the environment. The mechanisms of replication and assembly of virus particles in the feather follicle epithelial cells are poorly understood. To identify viral genes that might play a role in the process, we conducted global gene expression profiling in the skin tissues of MDV infected chickens at three time points post infection. We identified 42 viral genes that were differentially expressed in the skin tissues of Marek’s disease (MD) susceptible birds. Majority of the identified genes are involved in DNA replication, capsid, tegument, and envelop formation. We also employed Real-Time PCR to compare the expression patterns of the identified genes between the skin and spleen tissues of infected MD resistant and susceptible lines. Data revealed that the expression levels of the MDV genes were much higher in the skin of the susceptible line that those of the resistant line. Additionally, the expression levels of all the tested genes were significantly higher in the skin of either line when compared with the expression levels of the genes in the spleen tissues where no cell-free enveloped virus particles are produced. Deletion of specific gene(s) from the cloned MDV genome is in progress to characterize and determine the specific roles of the viral genes in the replication and assembly of virions in the skin of infected birds. This study provides the groundwork for development of mutant recombinant viruses as vaccine against the evolving and highly pathogenic strains of MDV.

Technical Abstract: Marek’s disease virus (MDV), a highly cell-associated oncogenic avian herpesvirus, is the causative agent of malignant transformation of T cells in domestic chickens. The latently infected CD4+ CD8- T cells carry the virus through the blood stream and establish lymphomas in the skin, visceral organs and peripheral nerves. The feather follicle epithelium (FFE) is the only anatomical site where fully infectious enveloped virions are produced and eventually disseminated into the environment to infect contact birds. Therefore, skin and FFE cells play a critical role as being the common source of re-infection of birds sharing the same habitat. The molecular mechanism involved in the replication and assembly of MDV in the FFE leading to the production and release of cell-free infectious virus particles is unknown and to date no viral or host gene has been implicated in the process. To examine alterations in the expression pattern of viral genes, we performed a comprehensive RNA-seq on the skin samples of Marek’s disease virus-infected susceptible chickens at 10, 20 and 30 days post infection. For comparative analysis of the expression patterns of viral genes between the skin and spleen of the MD-susceptible and resistant lines, Real-Time RT-PCR was employed. In total, RNA-seq based analysis identified 42 viral genes that were differentially expressed in the skin of infected birds. A Majority of the identified genes are involved in DNA replication, capsid, tegument, and envelop formation. Comparative analysis between the skin and spleen of MD-susceptible and resistant chicken lines, revealed significantly higher expression of the genes in the skin of either lines than the spleen. Furthermore, much higher expression of the genes was observed in the skin of the susceptible line than the resistant line.