Marek's disease is a natural model for lymphomas overexpressing Hodgkin's disease antigen (CD30).

Animal models are essential for elucidating the molecular mechanisms of carcinogenesis. Hodgkin's and many diverse non-Hodgkin's lymphomas overexpress the Hodgkin's disease antigen CD30 (CD30(hi)), a tumor necrosis factor receptor II family member. Here we show that chicken Marek's disease (MD) lymphoma cells are also CD30(hi) and are a unique natural model for CD30(hi) lymphoma. Chicken CD30 resembles an ancestral form, and we identify a previously undescribed potential cytoplasmic signaling domain conserved in chicken, human, and mouse CD30. Our phylogeneic analysis defines a relationship between the structures of human and mouse CD30 and confirms that mouse CD30 represents the ancestral mammalian gene structure. CD30 expression by MD virus (MDV)-transformed lymphocytes correlates with expression of the MDV Meq putative oncogene (a c-Jun homologue) in vivo. The chicken CD30 promoter has 15 predicted high-stringency Meq-binding transcription factor recognition motifs, and Meq enhances transcription from the CD30 promoter in vitro. Plasma proteomics identified a soluble form of CD30. CD30 overexpression is evolutionarily conserved and defines one class of neoplastic transformation events, regardless of etiology. We propose that CD30 is a component of a critical intracellular signaling pathway perturbed in neoplastic transformation. Specific anti-CD30 Igs occurred after infection of genetically MD-resistant chickens with oncogenic MDV, suggesting immunity to CD30 could play a role in MD lymphoma regression.

Characterization of a spontaneously transformed chicken mononuclear cell line.

We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 or 41 degrees C in culture medium containing either 5% FBS or 2% chicken serum. The HTC cells are acid phosphatase positive, show expressions of both class I and class II major histocompatibility complex (MHC), CD44, K1, and K55 cell surface antigens, and engulf latex beads, produce nitrite and interleukin-6 on stimulation with bacterial lipopolysaccharide (LPS). Treatment with phorbol myristate acetate (PMA) induces respiratory burst in HTC cells and the secretion of matrix metalloproteinase (MMP) into culture medium. Using gene-specific primers and reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of mRNA trancripts for interferon-gamma (IFN-gamma), interleukin-1 (IL-1), interleukin-6 (IL-6), nitric oxide synthase (NOS), matrix metalloproteinase-2 (MMP-2), and transforming growth factor-beta (TGF-beta) were detected. Lipopolysaccharide (LPS) treatment of HTC cells modulated IL-1, IL-6, IFN-gamma, NOS mRNA levels as detected by RT-PCR analyses. Using different avian tumor virus gene-specific primers and PCR, the HTC cells were positive for the presence of avian leukosis virus (ALV) and Marek's disease virus (MDV) but negative for reticuloendothelial virus (REV), chicken infectious anemia virus (CIAV), and herpes virus of turkeys (HVT). The production of ALV antigens by HTC cells was further confirmed using p27 gag protein ELISA. Collectively, these results show that the HTC cells belong to myeloid/macrophage lineage and were likely transformed by ALV and MDV but retain many interesting and useful biological activities.

Marek's disease virus reactivation from latency: changes in gene expression at the origin of replication.

Marek's disease is a contagious lymphoma of chickens caused by Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes latency within and transforms chicken CD4+ T-cells. Transformed T-cells are seen as skin leukosis or as lymphomas in visceral organs. A major focus of our laboratory is the functional study of genes flanking the origin of replication. This origin (OriLyt) is contained within the repeats flanking the unique long (UL) region of the genome (IRL and TRL). To the left of this Ori are genes associated with MDV latent/transforming infection [1.8-kb RNA family, pp14, Meq), and to the right (UL) are genes associated with early stages of MDV lytic infection [BamHI-H-encoded protein (Hep), pp38/pp24, Mys]. During latency, MDV suppresses lytic gene expression and has evolved mechanisms for blocking the apoptosis of latently-infected CD4+ T-cells. Of the genes expressed during MDV latency and in the transformed cell, the Meq (Marek's EcoRI-Q-encoded protein) has been shown to block apoptosis and transactivate gene expression. Upon reactivation to lytic infection, we have found that splice variants of Meq predominate and that these forms lack several of the domains important to Meq trans-activation and trans-repression. We have found that rightward from the origin of replication, a family genes, including phosphoprotein 38 (pp38) are expressed during early stages of reactivation. Three separate open reading frames (Hep, Mys, and pp38) are encoded by distinct transcripts from this region. We are now determining the kinetics of expression of these transcripts and their relative abundance during reactivation.

Marek's disease virus (MDV) encodes an interleukin-8 homolog (vIL-8): characterization of the vIL-8 protein and a vIL-8 deletion mutant MDV.

Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Marek's disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to the BamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (gamma2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8DeltasmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8DeltasmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8DeltasmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.

Marek's disease virus down-regulates surface expression of MHC (B Complex) Class I (BF) glycoproteins during active but not latent infection of chicken cells.

Infection of chicken cells with three Marek's disease virus (MDV) serotypes interferes with expression of the major histocompatibility complex (MHC or B complex) class I (BF) glycoproteins. BF surface expression is blocked after infection of OU2 cells with MDV serotypes 1, 2, and 3. MDV-induced T-cell tumors suffer a nearly complete loss of cell surface BF upon virus reactivation with 5-bromo-2'-deoxyuridine (BUdR). The recombinant virus (RB1BUS2gfpDelta) transforming the MDCC-UA04 cell line expresses green fluorescent protein (GFP) during the immediate early phase of viral gene expression. Of the UA04 cells induced to express the immediate early GFP, approximately 60% have reduced levels of BF expression. All of the reactivated UA04 and MSB1 tumor cells expressing the major early viral protein pp38 display reduced levels of BF. Thus, BF down-regulation begins in the immediate early phase and is complete by the early phase of viral gene expression. The intracellular pool of BF is not appreciably affected, indicating that the likely mechanism is a block in BF transport and not the result of transcriptional or translational regulation.

A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge.

Coccidiosis is caused by several distinct intestinal protozoa of Eimeria sp., and is responsible for intestinal lesions and severe body weight loss in chickens. To develop a DNA vaccination strategy for coccidiosis, an expression vector pMP13 encoding a conserved antigen of Eimeria was constructed by subcloning 3-1E cDNA into pBK-CMV and used to elicit protective immunity against E. acervulina. One-day-old chickens were immunized intramuscularly (IM) or subcutaneously (SC) with various doses of pMP13 expression vector ranging from 5 to 100 ug two weeks apart and were challenged with 5x10(3) E. acervulina. Chickens immunized with 5, 10, 50 or 100 ug of pMP13 plasmid, but not control plasmid, pBK-CMV, showed significantly reduced oocysts following challenge infection with E. acervulina. Two injections were in general more effective than one injection with higher dose of DNA eliciting better protection. At 10 days post challenge infection, maximum levels of circulating antibodies were detected regardless of the routes of injection, although IM injection provided higher levels of serum antibodies compared to SC injection. Serum antibody levels demonstrated a dose-dependent response showing higher antibody production at higher DNA dose. DNA immunization with pMP13 also induced significant changes in T-cell subpopulations in the spleen and duodenum intraepithelial lymphocytes. At 4 days post DNA immunization, pMP13-immunized chickens showed lower CD8, and higher CD4(+) and gammadelta T(+) cells in the duodenum compared to the pBK-CMV-immunized chickens. Following challenge infection with E. acervulina, pMP13-immunized chickens showed lower CD8(+) and alphabeta T(+) cells, and higher CD4(+) cells than pBK-CMV-immunized chickens in the duodenum. These findings demonstrate that DNA immunization with pMP13 induce local and systemic host immune responses against Eimeria.

Recombinant Marek's disease virus (MDV)-derived lymphoblastoid cell lines: regulation of a marker gene within the context of the MDV genome.

Marek's disease is a herpesvirus (Marek's disease virus [MDV])-induced pathology of chickens characterized by paralysis and the rapid appearance of T-cell lymphomas. Lymphoblastoid cell lines (LBCLs) derived from MDV-induced tumors have served as models of MDV latency and transformation. We have recently reported the construction of mutant MDVs having a deletion (M. S. Parcells et al., J. Virol. 69:7888-7898, 1995) and an insertion (A. S. Anderson et al., J. Virol. 72:2548-2553, 1998) within the unique short region of the virus genome. These mutant MDVs retained oncogenicity, and LBCLs have been established from the mutant-induced tumors. We report the characterization of these cell lines with respect to (i) virus structure within and reactivated from the cell lines, (ii) surface antigen expression, (iii) kinetics of MDV and marker gene induction, (iv) localization and colocalization of induced MDV antigens and beta-galactosidase (beta-Gal), and (v) methylation status of the region of lacZ insertion in recombinant- and non-recombinant-derived cell lines. Our results indicate that (i) recombinant-derived cell lines contain no parental virus, (ii) the established cell lines are predominantly CD4(+) CD8(-), (iii) the percentage of Lac-expressing cells is low (1 to 3%) but increases dramatically upon 5'-iododeoxyuridine (IUdR) treatment, (iv) lacZ expression is induced with the same kinetics as several MDV lytic-phase genes (pp38, US1, gB, gI, and US10), and (v) the regulation of lacZ expression is not mediated by methylation. Furthermore, the MDV-encoded oncoprotein, Meq, could be detected in cells expressing beta-Gal and various lytic antigens but did not appear to be induced by IUdR treatment. Our results indicate that regulation of the lacZ marker gene can serve as sensitive measure of virus lytic-phase induction and the reactivation from latency.

Establishment of a lymphoblastoid cell line using a mutant MDV containing a green fluorescent protein expression cassette.

We have previously described the construction and characterization of mutant Marek's disease viruses (MDVs) having mutations within the unique-short (US) region of the genome that have retained oncogenicity (Anderson et al., 1998; Parcells et al., 1995). We have also reported the characterization of lymphoblastoid cell lines (LBCLs) derived using these mutant viruses (Parcells et al., 1998). These mutant MDVs were constructed using a lacZ expression cassette. Expression of lacZ was found to be constitutive during lytic infection but was found to be tightly repressed in tumors and the derived LBCLs. The construction of these viruses and the analysis of lacZ induction required the use of toxic substrates or antibody staining to detect lacZ expression. We now report the establishment of an MDV lymphoblastoid cell line, MDCC-UA04, that was derived from a tumor induced by an MDV having an insertion of a green fluorescent protein expression cassette into the US2 gene. Like previous mutant-derived LBCLs, expression of the marker cassette is constitutive in lytic infection, but repressed in tumors and in the UA04 cells. UA04 cells express CD3low, CD4, TCR-2low, MHC class II, and CD28 antigens on their surface. The percentage of UA04 cells expressing GFP is generally low (5-7%), but increases markedly within 48 hrs of 5'-iododeoxyuridine (IUdR) treatment (20-30%) in a manner similar to many MDV lytic antigens. Thus, induction of GFP expression in UA04 cells can serve as a non-invasive marker for MDV reactivation from latency.

The glycoprotein D (US6) homolog is not essential for oncogenicity or horizontal transmission of Marek's disease virus.

RB1BUS6lacgpt, a Marek's disease virus (MDV) mutant having a disrupted glycoprotein D (gD) homolog gene, established infection and induced tumors in chickens exposed to it by inoculation or by contact. Lymphoblastoid cell lines derived from RB1BUS6lacgpt-induced tumors harbored only the mutant virus. These results provide strong evidence that an intact gD homolog gene is not essential for oncogenicity or horizontal transmission of MDV.

Marek's disease virus latency-associated transcripts belong to a family of spliced RNAs that are antisense to the ICP4 homolog gene.

Marek's disease virus (MDV) latency-associated transcripts include at least two MDV small RNAs (MSRs) and a 10-kb RNA which map antisense to the ICP4 homolog gene and are relatively abundant in MDV-transformed lymphoblastoid cells. This report further describes the biological and structural properties of these RNAs. First, these RNAs were detected in primary lymphomas isolated from chickens infected with several oncogenic MDV strains. Second, the MSRs are nonpolyadenylated, whereas, the 10-kb RNA is predominantly polyadenylated. Third, MSRs localize to the nuclei of both lymphoblastoid cells and cytolytically infected chicken embryo fibroblasts. Fourth, the 3'-region splice junctions of the MSRs during latent and productive infection were determined by sequencing RNA-PCR products generated with primers that flank the 3' splice region. The MSRs contain at least three introns, the largest of which overlaps the ICP4 putative translational start site. Fifth, the 5' end of the MSRs initiates approximately 5 kb upstream from the main body of the RNA. The extreme 5' exon is approximately 251 nucleotides (nt) long and is joined to the main body of the transcript upon removal of a 4,852-nt intron. Finally, the 10-kb RNA lies entirely within the repeats flanking the unique short region of the genome. We believe that the MSRs and 10-kb RNA belong to a family of spliced RNAs that map antisense to the ICP4 gene and comprise a complex transcriptional unit expressed during MDV-induced T-cell transformation.

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.

Characterization of a Marek's disease virus mutant containing a lacZ insertion in the US6 (gD) homologue gene.

We report the construction of a Marek's disease virus (MDV) mutant containing the lacZ gene of Escherichia coli inserted into a homologue of the US6 (glycoprotein D, gD) gene of herpes simplex virus. The mutant was constructed using the high-passage GAatt85 MDV strain as the parent virus, since that strain grows readily in chicken embryo fibroblasts using culture conditions conducive to mutant virus construction. The lacZ insertion site was positioned one third of the way into the US6 (gD) open reading frame. Insertion of the lacZ gene disrupted a major 6.2 kb transcript that initiated approximately 2.5 kb upstream of the gD homologue gene in the vicinity of the US3 homologue and sorf4 genes, and extended into the US7 (gI) homologue gene. The mutant virus (US6lac) and the parent virus had similar growth kinetics in cell culture at 37 degrees C and 41 degrees C. Furthermore, the US6lac mutant could be reisolated from the spleens and peripheral blood of infected chickens with a frequency comparable to that of the parent virus. Our results indicate that the gene encoding the gD homologue is nonessential for growth in cell culture or for infection of chickens following intra-abdominal inoculation with an attenuated serotype-1 MDV.