Horizontal Transmission of Marek's Disease Virus Requires U _S 2, the U _L 13 Protein Kinase, and gC

Abstract

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the U S 2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of U S 2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the U L 13, U L 44, and U S 6 genes encoding the U L 13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that U S 2, U L 13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.Marek's disease virus (MDV), also known as Gallid herpesvirus 2, causes Marek's disease (MD), a lymphoproliferative disease in chickens characterized by the development of tumors in the viscera and other organs. The pathogenesis of infection with MDV can be divided into four phases (4). In the first phase, between 3 and 6 days postinfection (p.i.), the primary target cells of infection are bursa-derived (B) lymphocytes. Infection of activated CD4 ϩ thymus-derived (T) lymphocytes follows in the second phase. During the second phase, viral replication typically decreases and a latent infection is established between 5 and 10 days p.i. in activated CD4 ϩ T lymphocytes. The third phase is characterized by reactivation of MDV replication between 14 and 21 days p.i. and infection of feather follicle epithelium (FFE) cells. After the third phase, virus is shed from the chicken in dried FFE cells, and clinical MD symptoms and lymphomas may develop depending on the genetic susceptibility of the chickens and the virulence of the virus strain.The MDV genome consists of the unique short (U S ) and long (U L ) regions, flanked by the inverted repeat long (IR L ) and short (IR S ) regions, and the terminal repeat long (TR L ) and short (TR S ) regions. Through manipulation of the MDV genome using multiple approaches, specific genes or re...

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“…In stark contrast, none of the chickens infected with either of the two vC98A USP mutants presented with clinical signs of Marek's disease, even at 13 weeks p.i. At the termination of the experiment at 13 weeks p.i., ample time for Marek's disease to develop even with low-virulence strains (21), only five (three in the C98A-1 group and two in the C98A-2 group) of 27 chickens infected with the mutant virus contained tumors. However, these tumors were very small and isolated, with no signs of metastasis.…”

“…We used recombination-based mutagenesis of a modified infectious bacterial artificial chromosome (BAC) clone of MDV (pRB-1B), derived from the highly oncogenic RB-1B strain (21), to obtain two independent isolates of the C98A mutant in the USP domain of MDV (vC98A-1 and -2). In addition, we prepared revertants of Author contributions: K.J.…”

“…To measure viral DNA copies in chicken blood by using qPCR assays, DNA was extracted from chicken blood as follows: 40 l of blood was obtained from the wing vein and mixed with 20 l of 0.1 M EDTA and then frozen at Ϫ80°C until all samples were collected. Ten microliters of blood/EDTA mix was used to collect DNA by using the DNeasy 96 Tissue kit from Qiagen following the manufacturer's instructions as described (21). Quantification of MDV genomic copies by using qPCR was performed as described (21,26).…”

“…Two-step red-mediated recombination was performed exactly as described (21,35). See supporting information (SI) Fig.…”

“…Lymphoblastoid T cell lines (LCL) transformed with either MDV (21) or REV (25) were grown in RPMI medium 1640 plus 10% FBS at 41°C in a humidified atmosphere of 5% CO 2 . The MSB-1 cell line was kindly provided by Mark S. Parcells (University of Delaware, Newark, DE) whereas the CU91, CU205, and CU210 cell lines were kindly provided by Karel A. Schat (Cornell University, Ithaca, NY).…”