Overexpression of cellular telomerase RNA enhances virus-induced cancer formation.

The telomerase RNA subunit (TR) is overexpressed in many tumors; however, the contribution of TR in cancer formation remains elusive. The most frequent clinically diagnosed cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a TR (vTR) that plays an important role in virus-induced tumorigenesis and shares 88% sequence identity with its cellular homologue. To determine if the cellular TR possesses pro-oncogenic activity, we replaced vTR with the cellular homologue in the virus genome. Insertion of cellular TR resulted in a strong overexpression in virus infected cells, while virus replication was not affected. Strikingly, cellular TR promoted tumor formation as efficient as vTR, while tumorigenesis was severely impaired in the absence of vTR. Our data provide the first evidence that overexpression of cellular TR can contribute to tumor formation in vivo using this natural virus-host model for herpesvirus-induced oncogenesis.

Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek's disease.

The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek's disease (MD) in the chicken is yet to be elucidated. Chicken embryo fibroblast (CEF) cells from MD susceptible and resistant lines were infected either with Marek's disease virus (MDV) or treated with polyionosinic-polycytidylic acid, a synthetic analog of dsRNA, and the expression of TLR and pro-inflammatory cytokines was studied at 8 and 36 h posttreatment by quantitative reverse transcriptase PCR. Findings of the present study reveal that MDV infection and polyionosinic-polycytidylic acid treatment significantly elevated the mRNA expression of TLR3, IL6, and IL8 in both susceptible and resistant lines. Furthermore, basal expression levels in uninfected CEF for TLR3, TLR7, and IL8 genes were significantly higher in resistant chickens compared with those of susceptible chickens. Our results suggest that TLR3 together with pro-inflammatory cytokines may play a significant role in genetic resistance to MD.

Characterization of cytotoxicity-related gene expression in response to virulent Marek's disease virus infection in the bursa of Fabricius.

Cell-mediated cytotoxic responses are critical for control of Marek's disease virus (MDV) infection and tumour development. However, the mechanisms of virus clearance mediated by cytotoxic responses in the bursa of Fabricius of chickens during MDV infection are not fully understood. In this study, the host cytotoxic responses during MDV infection in the bursa were investigated by examining the expression of genes in the cell lysis pathways. Partial up-regulation existed in the expression of the important cytolytic molecule granzyme A (GzmA), Fas, NK lysin and DNA repair enzyme Ape1, whereas little or no expression appeared in other cytolytic molecules, including perforin (PFN) and Fas ligand (FasL), and molecules involved in DNA repair and apoptosis in the bursa during MDV infection. These results suggest that less sustained cytotoxic activities are generated in the bursa of MDV-infected chickens. The findings of this study provide a more detailed insight into the host cytotoxic responses to MDV infection.

Cloning of Gallid herpesvirus 3 (Marek's disease virus serotype-2) genome as infectious bacterial artificial chromosomes for analysis of viral gene functions.

Marek's disease virus serotype 2 (Gallid herpesvirus 3) is a non-pathogenic alphaherpesvirus belonging to the Mardivirus genus, used widely in live vaccines against Marek's disease. Although the complete genome sequence of the MDV-2 strain HPRS-24 has been published, very little is known about the gene functions. As a first step for carrying out functional genomic analysis of MDV-2, the full-length genome of the MDV-2 vaccine strain SB-1 was cloned as an infectious bacterial artificial chromosome (BAC) clone pSB-1. Virus reconstituted from the pSB-1 clone showed morphological and growth characteristics in cell culture very similar to the parent virus. Generation of SB-1 constructs deleted in glycoprotein E and viruses expressing Citrine-UL35 fusion protein by the application of different BAC mutagenesis techniques demonstrated the amenability of the pSB-1 clone for reverse genetics approaches to identify molecular determinants associated with different biological features of this virus. The generation of replication-competent infectious clones of SB-1, together with those of CVI988 and herpesvirus of turkey strains described previously, completes the portfolio of generating infectious BAC clones of the MD vaccine strains belonging to all the three serotypes, paving the way for the application of reverse genetics for functional analysis of immunogenic determinants of these vaccines as well as for developing novel recombinant vectors.