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...

Herpesvirus telomeric repeats facilitate virus integration into host telomeres, a process which is required for the establishment of virus latency.

Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterizedby the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1B⌬RLORF5a (rRB-1B⌬RLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1B⌬RLORF4 (rRB-1B⌬RLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1B⌬RLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1B⌬RLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1B⌬RLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1B⌬RLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.Marek's disease (MD) is a lymphoproliferative disease in chickens caused by a member of the Alphaherpesvirinae subfamily, MD virus (MDV). The pathogenesis of infection with MDV can be divided into four phases (3). Bursa-derived (B) lymphocytes are infected between 3 and 6 days postinfection (p.i.) and are the primary target cells for the first phase of lytic infection, which is followed by infection of activated CD4 ϩ thymus-derived (T) lymphocytes 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 CD4ϩ T lymphocytes. The third phase is characterized by reactivation of MDV replication between 14 and 21 days p.i. After the third phase, lymphomas may develop depending on the genetic susceptibility of the chickens and the virulence of the virus strain.The MDV genome consists of two unique regions, the unique short region (U S ) and the unique long region (U L ), flanked by inverted repeats known as the inverted repeat long (IR L ) and short (IR S ), and the terminal repeat long (TR L ) and short (TR S ). The MDV Eco Q (Meq or RLORF7) and viral interleukin-8 (IL-8) homologue genes and the open reading frames ...