Keith W. Jarosinski

Horizontal Transmission of Marek's Disease Virus Requires US2, the UL13 Protein Kinase, and gC

ABSTRACT Mareks 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 US2 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 Mareks 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 US2 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 UL13, UL44, and US6 genes encoding the UL13 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 US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.

Attenuation of Marek's Disease Virus by Deletion of Open Reading Frame RLORF4 but Not RLORF5a

ABSTRACT Mareks disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by 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-1BΔRLORF5a (rRB-1BΔRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BΔRLORF4 (rRB-1BΔRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BΔRLORF5a 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-1BΔRLORF4 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-1BΔRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BΔRLORF4 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.

Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation

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

Influence of Genetic Resistance of the Chicken and Virulence of Marek's Disease Virus (MDV) on Nitric Oxide Responses After MDV Infection

SUMMARY. Nitric oxide (NO), a free radical produced by the enzyme NO synthase (NOS), is a potent antiviral agent in addition to having immune regulating functions. Recently, it was reported that chickens resistant (N2a, MHC: B21B21) to the development of Mareks disease (MD) had a greater potential to produce NO than MD-susceptible chickens (P2a, MHC: B19B19). This difference was shown by measuring NO levels in chick embryo fibroblast cultures obtained from these chickens after treatment with lipopolysaccharide and recombinant chicken interferon-gamma (IFN-γ). To extend these results, the levels of NO in blood plasma from N2a and P2a chickens inoculated with the nonattenuated JM-16 strain of MD virus (MDV) were examined. In four out of five experiments, N2a chickens had increased NO levels at 7 days postinoculation (DPI). In contrast, P2a chickens challenged with JM-16 had a significant increase in NO in only one of four experiments, and in that experiment the increase was delayed (10 DPI) compared with N2a chickens. Attenuation abrogated MDV-induced NO in chickens. Inoculation with MDV strains ranging from mild to very virulent plus showed that the more virulent strains induced the highest level of NO in blood plasma, suggesting a role of NO in the pathogenesis of MD with more virulent strains. On the basis of quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) assays for analysis of mRNA expression, IFN-γ does not appear to be the primary inducer of inducible (i)NOS gene expression during MDV infection. iNOS gene expression and NO production are mediated during the cytolytic phase of MDV infection on the basis of real-time RT-PCR assays with primers specific for glycoprotein B, a late gene expressed only during the cytolytic phase of MDV infection. These findings implicate NO as a factor potentially involved in increasing virulence of MDV, possibly through immune suppression.

A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation

The herpesvirus ubiquitin-specific protease (USP) family, whose founding member was discovered as a protease domain embedded in the large tegument protein of herpes simplex virus 1 (HSV-1), is conserved across all members of the Herpesviridae. Whether this conservation is indicative of an essential function of the enzyme in vivo has not yet been established. As reported here, USP activity is conserved in Mareks disease virus (MDV), a tumorigenic alphaherpesvirus. A single amino acid substitution that abolishes the USP activity of the MDV large tegument protein diminishes MDV replication in vivo, and severely limits the oncogenic potential of the virus. Expression of the USP transcripts in MDV-transformed cell lines further substantiates this hypothesis. The herpesvirus USP thus appears to be required not only to maintain a foothold in the immunocompetent host, but also to contribute to malignant outgrowths.

Marek's disease virus: lytic replication, oncogenesis and control.

Marek’s disease (MD) is caused by a ubiquitous, lymphotropic alphaherpesvirus, MD virus (MDV). MD has been a major concern in the poultry industry owing to the emergence of increasingly virulent strains over the last few decades that were isolated in the face of comprehensive vaccination. The disease is characterized by a variety of clinical signs; among them are neurological symptoms, chronic wasting and, most notably, the development of multiple lymphomas that manifest as solid tumors in the viscera and musculature. Much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among the many genes encoded by MDV, a number have been shown recently to affect the development of tumors in chickens, one protein directly causing transformation of cells (Meq) and another being involved in maintaining transformed cells (vTR). Other MDV gene products modulate and are involved in early lytic in vivo replication, thereby increasing the chance of transformation occurring. In this review, we will summarize specific genes encoded by MDV that are involved in the initiation and/or maintenance of transformation and will focus mostly on current vaccination and control strategies against MD, particularly how modern molecular biological methods may be used to improve strategies to combat the disease in the future.

Expression and function of the protein tyrosine phosphatase SHP-1 in oligodendrocytes

Previous studies in this laboratory have shown that the SH‐2 domain‐containing protein tyrosine phosphatase SHP‐1 is expressed in CNS glia and functions to modulate cytokine activities in these cells. The present study demonstrates that SHP‐1 is expressed within multiple regions of the CNS in vivo, especially in white matter. Interestingly, we show that mice genetically lacking in SHP‐1 (motheaten mice) in the CNS displayed dysmyelination. We therefore examined the expression of SHP‐1 in the myelin‐forming oligodendrocytes. Oligodendrocytes present in either mixed glial cultures or pure cultures expressed high levels of SHP‐1 in the cytoplasm of cell bodies and processes. Oligodendrocytes isolated from motheaten mice did not express SHP‐1. To test possible functions for SHP‐1 in oligodendrocytes in controlling cytokine signaling, we compared the responsiveness of oligodendrocytes isolated from either motheaten or normal littermate mice with IL‐6. IL‐6 induced higher levels of STAT3 phosphorylation and STAT3‐responsive c‐fos gene expression in pure oligodendrocyte cultures of motheaten compared with normal littermate mice. These studies demonstrate that oligodendrocytes express SHP‐1 and that SHP‐1 functions to control IL‐6 signaling. SHP‐1 may therefore be a critical regulator of oligodendrocyte differentiation in response to IL‐6 family cytokines. Further, these findings may relate to dysmyelination in mice lacking SHP‐1. GLIA 29:376–385, 2000.

Cellular responses in chickens treated with IFN-α orally or inoculated with recombinant Marek's disease virus expressing IFN-α

Mammalian type I interferons (IFN- a/b ) are potent mediators of innate antiviral immune responses, in particular through enhancement of natural killer (NK) cell cytotoxicity. Recently, chicken IFN- a (ChIFN-a) has been identified and shown to ameliorate Newcastle disease virus (NDV) infection when given to chickens at relatively high concentrations in the drinking water. In this report, the effect of recombinant ChIFN- a (rChIFN-a) on NK cell cytotoxicity was examined using 51 Cr-release assays. NK cell cytotoxic activity was also analyzed following inoculation with attenuated Marek’ s disease virus (MDV) serotype 1 strain R2/23 and a recombinant MDV (parent strain R2/23)-expressing ChIFN- a [rMDV(IFN-a)]. Treatment of chickens with high doses of rChIFN- a in the drinking water significantly decreased NK cell cytotoxicity compared with untreated chickens over a 7-day period. Inoculation of chickens with R2/23 significantly decreased NK cell cytotoxicity as well, whereas the rMDV(IFN- a) had no effect on NK cell cytotoxicity. Treatment of chicken embryo cell cultures with rChIFN- a inhibited replication of the very virulent MDV RB-1B strain in vitro, and oral treatment of chickens with rChIFN- a reduced MDV R2/23 replication in vivo.

Impact of deletions within the Bam HI-L fragment of attenuated Marek's disease virus on vIL-8 expression and the newly identified transcript of open reading frame LORF4

Mareks disease (MD) in chickens is caused by MD herpesvirus (MDV), which induces T cell lymphomas. The early pathogenesis of MDV infection is characterized by a primary infection in B lymphocytes followed by infection of activated T lymphocytes. It has been speculated that a MDV-encoded homologue of interleukin-8 (vIL-8) may be important to attract activated T lymphocytes to infected B lymphocytes. Recently, more virulent strains of MDV have emerged, named very virulent plus (vv+)MDV, that cause earlier and more prolonged cytolytic infections compared to less virulent strains. In this report, it was found that vIL-8 mRNA expression in vivo was increased in very virulent (vv) and vv+MDV strains compared to mild (m) and virulent (v) strains, and could not be detected in two attenuated MDV strains examined using very sensitive real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays. In order to identify potential mechanisms for the increased vIL-8 mRNA expression in more virulent strains, and lack thereof in attenuated strains, the vIL-8 gene and putative promoter sequences upstream of the vIL-8 gene were compared from 10 different MDV strains, including attenuated derivatives. Only the JM-16 strain (both non-attenuated and attenuated) and attenuated 584A (584Ap80C) encoded a predicted vIL-8 gene sequence different from all other strains examined. Within the putative vIL-8 gene promoter sequence, there was little difference among the non-attenuated strains; however significant deletions were identified in the attenuated JM-16/p71, Md11 (R2/23), and 584Ap80C strains. Additionally, these deletions were located within a previously hypothetical open reading frame (ORF) named LORF4. Rapid amplification of cDNA ends identified a full-length transcript of LORF4 in the MDV-transformed lymphoblastoid cell line MSB-1, and deletions within this ORF caused truncated predicted proteins in 4 out of 6 attenuated MDV strains examined.

Fluorescently Tagged pUL47 of Marek's Disease Virus Reveals Differential Tissue Expression of the Tegument Protein In Vivo

ABSTRACT Mareks disease virus (MDV), a lymphotropic alphaherpesvirus, causes Mareks disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells.