Visualization of Marek's disease virus in vitro using enhanced green fluorescent protein fused with US10

Marek's disease virus (MDV) is an highly cell-associated avian alphaherpesvirus. Although viral replication is supported in chicken embryo fibroblasts (CEF) or duck embryo fibroblasts, identification of MDV-infected cells is quite cumbersome especially during the early stages of virus replication when plaques can be difficult to recognize. To visualize MDV replication in infected cells and characterize MDV US10 in vitro, rMd5-US10-EGFP, a recombinant MDV, was generated that expresses enhanced green fluorescent protein (EGFP) as a tagged protein fused with US10 at the C-terminal end. The expression of US10-EGFP was detected in infected CEF using fluorescent microscopy and the expression intensity was quantified using flow cytometry analysis. In addition, confocal microscopic analysis provided information on subcellular localization of US10-EGFP in virus-infected cells. In conclusion, rMd5-US10-EGFP virus can be used to help monitor virus activity in vitro.