Marek’s disease (MD) is a highly infectious economically important oncogenic viral disease of chickens. It is found throughout the world and is caused by an alphaherpesvirus, Marek’s disease virus (MDV). Though this disease can currently be successfully controlled by vaccination, the virus has continuously evolved to greater virulence over the last several decades. Hence, there is a need for alternative approaches to control MD. Selection and breeding of MD-resistant chickens presents an attractive option for prevention of this disease. MHC-congenic chicken inbred lines, 61 and 72, which are highly resistant and susceptible to MD, respectively, have been identified, but the cellular and genetic basis for these phenotypes is unknown. The overall aim of this study was to investigate the cellular basis of resistance to MD using an in vitro MDV infection model with the hypothesis that resistance is exerted by the innate immune cells. MDV is a highly cell-associated virus which makes in vitro studies difficult. In vivo, MDV infects APCs (antigen-presenting cells: macrophages and/or dendritic cells [DCs]), B cells and activated T cells. Though both B and T cells can be infected in vitro, co-culture infection models have not been described for APCs. Thus, the primary goal was to develop a model for infecting these cells with MDV in vitro and to characterise infected and uninfected cells. Developmental studies used APCs derived from outbred chickens. Chicken bone marrow cells were cultured with chCSF-1 (for macrophages) or chIL-4 and chCSF-2 (for DCs) for 4 days and then infected by the addition of chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing GFP. CEF preparations naturally contain a mixture of CEFs (92-98%) and macrophages (2-8%) and both appear to be infectable with MDV. Infected CEFs were therefore separated from infected macrophages by FACS before adding to the bone marrow-derived APCs. Infected and uninfected APCs were sorted by FACS using GFP expression and APC-specific mAb staining (KUL01 and anti-CD45). Characteristic virus-infected and uninfected APCs were revealed via examination with live cell confocal microscopy. The presence of herpesvirus specific immediate early (ICP4), early (pp38), late (gB) transcripts and MDV specific transcript, L-Meq, in infected APCs was confirmed by RT-PCR providing evidence for MDV replication. Hence, a new in vitro MDV infection model of APCs has been established. Using the infected macrophages to infect CEFs showed that the infection was productive. This model was then extended to infect APCs of lines 61 and 72. Flow cytometric analysis revealed that a higher percentage of macrophages were infected in the susceptible line (72) than in the resistant line (61). To analyse this in detail, RNA-Seq was carried out to identify differentially expressed (DE) genes between the two lines pre- and post-MDV infection. From these DE genes, potential candidate genes involved in MD resistance and susceptibility were identified. Functional analysis of DE genes support the hypothesis that resistance to MD is determined at the macrophage level of the resistant line (61) and the JAK-STAT signalling pathway is at least one anti-viral mechanism by which this signature is expressed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:702242 |
| Date | January 2015 |
| Creators | Chakraborty, Pankaj |
| Contributors | Kaiser, Peter ; Dutia, Bernadette |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/19525 |
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