الفهرس 
INTRODUCTION
IntroductionOnly 14 pages are availabe for public view
 |  | from | 154 |  |  |
| | | from | 154 | | |
Abstract
In the present study we successfully generated the fluorescently tagged vRB 1B ΔIRL UL49 P2A mRFP, as an indicator virus for the lytic MDV infection phase. The mutant was replicated in an indistinguishable level comparable with the parental virus vRB 1B ΔIRL in vitro and in vivo. This was followed by generation of a triple marked lytic fluorescent virus with a novel tool for tagging important viral genes such as UL49 with mRFP at the 3_ end (N-terminus), UL42 with E2 Crimson at the 3_ end (N-terminus) and UL54 with GFP at the 5_ end (C-terminus) in order to distinguish between different stages of lytic infection.
Furthermore, we used P2A (ribosomal skipping site) as a linker between the fluorescent proteins and MDV genes. The virus replicated well in CEC and the fluorescent proteins were expressed brightly and shade lights in lytic infected cells which distinguished between IE, early and late lytic infected cells in vitro. Plaque size assay and growth kinetics of the triple lytic fluorescent tagged virus indicated non significant differences with the parental virus.
In order to generate a marker virus for lytic and latent infection with an oncogenic background to study pathogenesis and tumorigenesis, vUL49-P2A mRFP_Meq-P2A eGFP was constructed and characterized in vitro. As the virus replicated well in vitro, our data confirmed that it is a suitable candidate to distinguish between lytic and latent infected cells.
In vivo, we infected chickens by vUL49-P2A mRFP which replicated and induced lymphoma in different organs very efficiently without any attenuation. In addition, pUL49-P2A mRFP was expressed in vivo in different levels with the exception of feather follicle epithelium where pUL49-P2A mRFP was highly expressed. Our results showed that this fluorescently labeled virus allows selecting MD cells in lytic cycle, providing new opportunities to examine MDV morphogenesis in various cell contexts.
Histopathologically, MD lesions, in skin and muscles, as well as visceral tumors in liver, spleen, kidney, intestine and testes showed either focal and/or diffuse pleomorphic lymphoid infiltrates, mainly lymphoblasts, small and large lymphocytes, and plasma cells.