Fluorescent tagging of VP22 in N-terminus reveals that VP22 favors Marek’s disease virus (MDV) virulence in chickens and allows morphogenesis study in MD tumor cells

Marek’s disease virus (MDV) is an alpha-herpesvirus causing Marek’s disease in chickens, mostly associated with T-cell
lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV
spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to
the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In
culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was
impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood
and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but
had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for
tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors
showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and
analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and
all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells.
These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic
cells in culture, albeit poorly efficient, than in feather follicle epithelial cells