Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

Pankaj Chakraborty,Lonneke Vervelde,Venugopal Nair,Bernadette M Dutia,Peter S Wasson,Robert G Dalziel,Pete Kaiser

doi:10.1099/jgv.0.000763

Pankaj Chakraborty, Lonneke Vervelde + Show 5 more

Open Access

https://doi.org/10.1099/jgv.0.000763

Copy DOI

Abstract

Marek’s disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been successful. The aim of this study was to develop a model for infecting phagocytes [macrophages and dendritic cells (DCs)] with MDV in vitro and to characterize the infected cells. Chicken bone marrow cells were cultured with chicken CSF-1 or chicken IL-4 and chicken CSF-2 for 4 days to produce macrophages and DCs, respectively, and then co-cultured with FACS-sorted chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing EGFP. Infected phagocytes were identified and sorted by FACS using EGFP expression and phagocyte-specific mAbs. Detection of MDV-specific transcripts of ICP4 (immediate early), pp38 (early), gB (late) and Meq by RT-PCR provided evidence for MDV replication in the infected phagocytes. Time-lapse confocal microscopy was also used to demonstrate MDV spread in these cells. Subsequent co-culture of infected macrophages with CEFs suggests that productive virus infection may occur in these cell types. This is the first report of in vitro infection of phagocytic cells by MDV.

Highlights

Marek’s disease (MD) is a highly infectious and economically important oncogenic disease of chickens caused by the lymphotropic alphaherpesvirus Marek’s disease virus (MDV) (Gallid alphaherpesvirus 2)
In order to study the infection of macrophages and dendritic cells (DCs) with MDV, we used a recombinant MDV which expresses EGFP under control of the murine phosphoglycerol kinase promoter, independently of viral gene expression
Flow cytometric analysis revealed that MDV-infected chicken embryo fibroblasts (CEFs) contained a significant percentage (8 %) of KUL01+ cells and approximately 0.1 % of these cells were infected with MDV

Summary

Introduction

Marek’s disease (MD) is a highly infectious and economically important oncogenic disease of chickens caused by the lymphotropic alphaherpesvirus Marek’s disease virus (MDV) (Gallid alphaherpesvirus 2). The ‘Cornell model’ of MDV infection [1] proposes that, in vivo, infection takes place when airborne cell-free virus wrapped in dander enters the respiratory tract and is engulfed by phagocytic cells, which carry the virus to the spleen and other lymphoid tissues. Virus is thought to pass to lymphocytes where it causes lytic infection of B lymphocytes and lytic or latent infection in T cells. Infected T cells are thought to play a crucial role in the spread of virus to the various visceral organs and peripheral nerves, where proliferating T cells cause pathological lesions. The role of innate immune cells, in particular phagocytes, in MDV infection remains unclear

Objectives

Methods

Results

Conclusion