Marek's Disease Tumor Cells Research Articles

Role of gga-miR-29b-3p in suppressing the proliferation, invasion and migration of MSB1 Marek’s disease tumor cells by the targeting of the DNMT3B gene

BackgroundMarek’s disease (MD), a class II infectious, lymphoproliferative disease that mainly afflicts poultry, has been shown to cause wasting, limb paralysis, and often acute death. It is a neoplastic disease caused by a cell-binding herpesvirus that leads to the formation of tumors in various organs and tissues. Our previous reports have found that the microRNA, gga-miR-29b-3p, showed abnormal expression in MD lymphoma. However, it remains unknown whether gga-miR-29b-3p affects MD tumorigenesis.MethodsThe MD tumor cell line MSB1 was chosen to analyze the characteristics of gga-miR-29b-3p in tumors. Cell proliferation and migration were assessed by Cell Counting Kit-8 (CCK-8) and Transwell, respectively, and cell apoptosis and cycle were analyzed via fluorescent staining and flow cytometry, respectively. The regulation between gga-miR-29b-3p and its potential target genes was verified by dual luciferase results and loss-of-function assays. The effect of target genes was verified by examining the degree of RNA interference on MSB1 cells.ResultsAnalysis revealed that gga-miR-29b-3p impaired the proliferation of the MSB1 MD tumor cell line, induced apoptosis without obvious effects on the cell cycle, and suppressed the expression of the invasion-associated MMP2 and MMP9 genes. It was concluded that DNMT3B is the direct target of gga-miR-29b-3p. As expected, the effects of DNMT3B knockdown with small interfering RNA (siRNA) on MSB1 cell proliferation, apoptosis, and cycle were associated with gga-miR-29b-3p overexpression. Moreover, BCL2 and BCL2L1 were downregulated and TNFSF10 was upregulated in both the gga-miR-29b-3p overexpression and DNMT3B knockdown groups. The expression levels of invasion-related genes were decreased post-DNMT3B knockdown. In both the gga-miR-29b-3p overexpression and DNMT3B knockdown conditions, a decrease in MEQ oncogene expression in MD virus was observed.ConclusionsOverall, gga-miR-29b-3p was demonstrated to have a suppressive effect in MD lymphoma progression via the targeting of the DNMT3B gene. Gga-miR-29b-3p overexpression and DNMT3B knockdown inhibited MSB1 cell proliferation through suppressing the pro-apoptotic gene expression and elevating the anti-apoptotic gene expression in the apoptosis pathway. Our study provides a theoretical basis for targeted treatment of MD.

Development of monoclonal antibodies specific to Marek disease virus-EcoRI-Q (Meq) for the immunohistochemical diagnosis of Marek disease using formalin-fixed, paraffin-embedded samples.

Marek disease (MD) is a viral disease characterized by the development of lymphoma in poultry. Although morphologic confirmation of lymphoma is used to diagnose MD, immunohistochemical detection of MD virus-EcoRI-Q (Meq), which is a viral protein that is expressed exclusively in MD tumor cells, would further improve the accuracy of diagnosis. We developed monoclonal antibodies (mAbs) that specifically detect Meq by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) sections. We evaluated the sensitivity and specificity of 14 mAbs that we produced, using FFPE samples of MDCC-MSB1 cells, MD tumor tissues, and tissues of uninfected chickens. Four different antigen retrieval conditions were investigated. Thirteen mAbs reacted with Meq in FFPE sections, but immunohistochemical reactivity and specificity varied depending on the mAb and antigen retrieval condition; heat-induced antigen retrieval (HIAR) was more effective at detecting Meq than the other tested conditions. HIAR pH 9 tended to increase immunoreactivity and decrease specificity. Of the 5 mAbs that immunoreacted strongly with Meq without nonspecific reactions under the optimal antigen retrieval conditions, 3 mAbs (1C1-121, 3A3-112, 5F7-82) did not produce background staining of tumor or non-tumor tissues; 2 mAbs (2C5-11, 4A5-54) produced background staining. The mAb 6B5-128 reacted moderately with Meq without nonspecific reactions and background staining. The remaining mAbs showed weak immunoreactivity or problematic nonspecific reactions. Our results suggest that some of our developed mAbs can be used in IHC to detect Meq in FFPE sections with high specificity, and that the use of IHC may greatly improve the diagnosis of MD.

Knockdown of the Meq gene in Marek's disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek's disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek's disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken. The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines. An experiment was conducted to elucidate the role of Meq in MD tumor transformation. RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1. A small interfering RNA with an interference efficiency of 70% (P<0.01) was transfected into MSB1 cells to knock down the expression of Meq gene. The cell proliferation, cycle and apoptosis were detected post-Meq knockdown. The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h (P<0.01), 60 h (P<0.05) and 72 h (P<0.01) post-Meq knockdown. The cell cycle was unaffected (P>0.05). B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway. The activity of caspase-6 was upregulated (P<0.05) significantly and BCL2 gene expression was downregulated (P<0.05) significantly post-Meq knockdown, suggesting cell apoptosis might be induced. MSB1 cell migration did not exhibit any obvious change (P>0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P<0.05) remarkably post-Meq knockdown. The Meq knockdown might affect the main features of tumorous cells, including proliferation, apoptosis, and invasion, suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.

Adenovirus-mediated co-expression of the TRAIL and HN genes inhibits growth and induces apoptosis in Marek's disease tumor cell line MSB-1.

BackgroundThe objective of this study was to determine the in vitro tumor-inhibitory effect of a recombinant adenovirus expressing a fusion protein of tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) and hemagglutinin-neuraminidase (HN) genes on the MSB-1 Marek’s disease tumor cell line.MethodsTRAIL and HN genes were amplified from lymphocytes in the peripheral blood of chickens and the LaSota strain of Newcastle disease virus (NDV), respectively, using RT-PCR. The two genes were connected with a 2A connecting peptide by site-directed mutagenesis and gene splicing by overlap extension (SOE). The target gene TRAIL-2A-HN was cloned into the shuttle vector pShuttle-CMV. Homologous recombination was carried out with the vector pAdeasy-1 in the bacterium BJ5183 to construct the recombinant adenovirus plasmid pAd-TRAIL-2A-HN. After linearization, the plasmid was transfected into AD293 cells and packaged. Real-time quantitative PCR (RT-PCR) and fluorescence microscopy confirmed the introduction of the recombinant adenovirus into AD293 cells. The TCID50 method (50% tissue culture infectious dose) was employed to determine viral titers for the exprimental and control viruses, which met criteria for use. The Marek’s disease tumor cell line MSB-1 was transfected with the constructed recombinant adenovirus. The infectivity of the recombinant adenovirus and the expression levels of exogenous genes were detected with RT-PCR and western blotting. The effects of the recombinant adenovirus on the growth of MSB-1 cells and cellular apoptosis were determined using flow cytometry.ResultsThe recombinant adenovirus infected the cultured cells in vitro, and replicated and expressed exogenous genes in the cells. The recombinant adenovirus Ad-TRAIL-2A-HN inhibited the growth of MSB-1 cells and induced apoptosis by expressing exogenous genes. The rate of induced MSB-1 cell apoptosis reached 11.61%, which indicated that TRAIL and HN produced synergistic tumor-inhibiting effects.ConclusionThe constructed TRAIL-2A-HN fusion gene combined the apoptosis-inducing function of TRAIL and the adsorptive capacity of HN from NDV for tumor cells, and the capacity of the recombinant adenovirus expressing this fusion gene to induce tumor cell apoptosis was reported. These results provide a basis for future in vivo tumor suppression studies using recombinant adenoviruses.

The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

BackgroundMarek's disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Marek's disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction.ResultsWe confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ∆Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ∆Meq was found to act as a negative regulator of Meq.ConclusionsThe Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis.

The presence of the p53 transcripts with truncated open reading frames in Marek's disease tumor-derived cell lines

Several kinds of the p53 transcripts in which their open reading frames (ORFs) were truncated (ranging from 101 to 765 bp) were identified in Marek's disease (MD)-derived tumor cell lines as well as avian leukosis- and reticuloendotheliosis-derived ones, detected by nested RT-PCR and subsequent nucleotide sequence analysis. In these ORFs, regions encoding the proline-rich and DNA-binding domains of the p53 protein were frequently deleted, and many of these deletions were found to cause frame shift. Western blot analysis using anti-p53 monoclonal antibodies revealed that multiple p53 isoform proteins with various molecular weights including 45–46, 35 and 28 kDa were expressed in these tumor cell lines, though the p53 protein with a molecular weight of 49 kDa was detected in chicken embryo fibroblasts transformed by the SV40 T antigen as a control. Since no deletions were found in the p53 gene of these MD tumor cell lines, truncations in the p53 ORFs observed in this study might result from alternative splicing of the p53 gene.

Anti-Human Class I α3 Domain-Specific Monoclonal Antibody Induces Programmed Cell Death in Murine Cells Expressing Human Class I MHC Transgenes

In vivo and in vitro methods were used to determine whether JMV Marek's disease (MD) tumor cells have B-cell or T-cell characteristics. JMV tumor cells were treated with anti-T-cell serum (ATS) and complement (C) or anti-B serum (ABS) and C and then inoculated into 1-day-old chickens. Within 2 weeks of inoculation, 95% of chickens inoculated with ATS- and C-treated cells died of JMV-induced leukemia, whereas only 10% of chickens inoculated with ABS- and C-treated cells succumbed. This in vivo result indicated that the malignant cells may be B cells. In vitro studies corroborated this conclusion. ABS and C killed 99 to 100% of cultivated JMV cells, whereas little or no killing of JMV cells was achieved by ATS and C, as demonstrated by trypan blue dye exclusion or by release of 51Cr from labeled JMV cells. Furthermore, after staining of JMV cells with fluorescein-conjugated rabbit anti-chicken immunoglobulin, all cells fluoresced, indicating that JMV cells have surface immunoglobulin. With support of these lines of evidence, we conclude that JMV Marek's disease tumor cells have B-cell characteristics.

Marek's Disease Virus Type 1-Specific Phosphorylated Proteins pp38 and pp24 with Common Amino Acid Termini Are Encoded from the Opposite Junction Regions between the Long Unique and Inverted Repeat Sequences of Viral Genome

The nucleotide sequence of the junction region between the long unique (U L) and terminal inverted repeat (TR L) sequences of Marek's disease (MD) virus type 1 (MDV1) DNA revealed the presence of a rightward open reading frame of 155 amino acids. The ORF inserted into an eukaryotic expression vector transiently expressed an antigan in the cytoplasm of COS7 cells which reacted with the monoclonal antibody M21 against an MDVl-specific phosphorylated protein complex consisting of at least the proteins pp38 and pp24. In addition, RNA synthesized in vitro from the ORF under the control of the T7 promoter was translated in vitro using rabbit reticulocyte lysates. A polypeptide of about 24 kDa was immuneprecipitated with M21 antibody. Thus, the MDV1-specific phosphorylated proteins pp38 and pp24 with common amino termini are encoded in the opposite junction regions between the U L and IR L and between the U L and TR L, respectively, of the MDV1 genome. The pp38 gene is transcribed leftward from the viral genome, while the pp24 gene was shown here to be transcribed rightward in a MD tumor cell line as well as in cells productively infected With MDV1.

Association of Delayed Hypersensitivity Wattle Reaction to BCG with Marek's Disease Resistance in Chickens

The influence of cell-mediated immunity on Marek's disease (MD) resistance was investigated, using two White Leghorn lines selected for high (DWR-H) and low (DWR-L) delayed hypersensitivity wattle reaction to BCG antigen. MD resistance was measured by inoculation of MD virus or transplantable MD tumor cells into day-old chicks.Association of cell-mediated immunocompetence with resistance to MD virus was observed, since the DWR-L line was more resistant to MD virus than the DWR-H line. The line differences in MD incidence and in MD mortality were highly significant statistically. On the other hand, resistance to MD tumor cells was nearly identical in both lines. No line differences were observed for both tumor incidence and mortality. The data suggest that resistance of chickens to MD tumor transplant is controlled by a mechanism differing from that for the resistance to MD virus.

Differentiation of Oncogenic and Nononcogenic Strains of Marek's Disease Virus Type 1 by Using Polymerase Chain Reaction DNA Amplification

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.

Use of a soluble tetrazolium/formazan assay for chicken cells.

We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.

Antibody directed against Marek's disease-associated tumor surface antigen can be eluted from Marek's disease tumor cells

Antibody directed against Marek's disease-associated tumor surface antigen (MATSA) was eluted from tumor cells of lymphomas and peripheral blood lymphocytes that were isolated from Marek's disease virus-infected chickens. Feather follicular Marek's disease virus (MDV) antigen could not be demonstrated with this antibody by indirect immunofluorescent (IF) staining. Monoclonal antibody directed against MATSA could completely block the activity of eluted antibody and vice versa. By indirect IF staining using eluted antibody and fluorescein isothiocyanate (FITC) labelled antichicken globulin conjugate, MATSA-bearing cells were detected in MDV infected and herpes virus of turkey (HVT) vaccinated birds. Blocking of immunoglobulin molecules present on B-cells by anti-chicken globulin is critical in this test.

Infectious Anemia Caused by a Parvovirus-Like Virus in Georgia Broilers

Pale chicks with necrotic dermatitis, small bursas of Fabricius (BFs), small thymuses, pale bone marrow, and watery blood were suspected of having parvovirus-like virus- (PVLV) associated disease. Histologic lesions included atrophy or hypoplasia of thymuses and BFs, and septic necrotizing clostridial dermatitis and hepatitis. Clostridium perfringens was cultured from skin and liver. A PVLV was isolated in a Marek's disease tumor cell line (MDCC-MSB1) culture and was identified by physicochemical, immunofluorescent, and morphologic features. This isolate was named GA-1 PVLV. Specific-antibody-negative chicks and embryos infected with heat- or chloroform-treated GA-1 PVLV developed anemia at the same rate. Control chicks never were anemic. This is the first isolation of PVLV from clinically ill chickens in the United States and the first report of PVLV-induced anemia in chickens in the Western Hemisphere.

Expression of a Putative Tumor-Associated Surface Antigen on Normal Versus Marek's Disease Virus-Transformed Lymphocytes&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Various avian tumor cell lines and normal spleen cells from 3 genetic strains of specific-pathogen-free (SPF) chickens were examined for expression of Marek's disease (MD) tumor-associated surface antigen (MATSA). Two anti-MATSA monoclonal antibodies (RPH 6 and EB 29) and a rabbit anti-MATSA antiserum were used in indirect fluorescent antibody tests, and cells were examined by fluorescence microscopy and with a fluorescence-activated cell sorter (FACS). Less than 5% MATSA-positive cells were observed in 2 non-MD tumor cell lines (LSCC-RP 9 and RECC-CU 60) with RPH 6, but 7-82% positive cells were observed with EB 29 or the rabbit antiserum. Five MD tumor cell lines (MDCC-CU 2, -CU 14, -CU 25, -CU 32, and -CU 41) had 12-72% positive cells detected with one or both monoclonals and 31-99% positive cells detected with the rabbit antiserum. Over 90% of cells in all MD lines were la and T3 positive, while values for the same parameters in LSCC-RP 9 were 100 and 3% and for RECC-CU 60, 48 and 51%, respectively. Evidence for cell-cycle-dependent expression of MATSA on MDCC-CU 2 was obtained from cell sorting experiments with the FACS and from examination of the MATSA-staining characteristics of 3 clones derived from the parent culture. Less than 5% MATSA-positive cells were observed in uncultured spleen cells from SPF chickens or in spleen cells stimulated for 48 hours with concanavalin A or phytohemagglutinin-M. However, with one exception, 10-53% of normal spleen cells were MATSA positive with RPH 6, after stimulation by mitogen for 24 or 48 hours followed by maintenance in conditioned medium (CM) for various times or after culture directly in CM for 3 days. More limited experiments with rabbit anti-MATSA antiserum yielded 55-85% MATSA-positive cells. From 60 to 97% of these MD virus-free, MATSA-positive cells were la-positive; and, in 2 cases, 89 and 90% were T3 positive.

Detection of a New Antigen Associated With Chicken Thrombocytes on Marek's Disease Lymphoblastoid Cell Line&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

A monoclonal antibody, 2C12, produced against MDCC-MSB1 cells, reacted with several cell lines derived from Marek's disease (MD), including MDCC-MSB1 cells, normal chicken thrombocytes, and MD tumor cells. It did not react with cell lines derived from avian leukosis or reticuloendotheliosis, cells from normal chicken thymus and bursa, chicken kidney cells and quail fibroblast cultures inoculated with MD virus, or with erythrocytes from 1-day-old and adult chickens, cow, sheep, and horse. Anti-thrombocyte serum prepared in a rabbit reacted with thrombocytes and MDCC-MSB1 cells. The specificities of antibody 2C12 and anti-thrombocyte serum against MDCC-MSB1 cells and thrombocytes were confirmed by cross-absorption, blocking, and double-membrane fluorescent antibody tests. The existence of a polypeptide with an apparent molecular weight of 103,000, common to both MDCC-MSB1 cells and thrombocytes, was demonstrated by Western blotting analyses.

Enhancement by interferon of chicken splenocyte natural killer cell activity against Marek's disease tumor cells

A non-immune natural killer-type cell population (NK) from 6-to 12-week old chickens was able to kill MSB-1 Marek's disease (MD) tumor cells in vitro ; as measured by the 51Cr-release cycotoxicity assay. Removal of T cells, B cells, adherent cells, or any combination of the three populations of cells did not result in diminished levels of cytotoxicity of the remaining spleen cells against MSB-1 cells. The cytotoxicity of chicken NK cells could be rapidly aumgented by polyinosinic-polycytidylic acid (poly I:C) and by the Cal 11914 strain of Newcastle disease virus (NDV), but not by the TCND strain of NDV which is not an interferon (IFN) inducer, indicating that IFN play a role in augmentation of the NK activity in chickens.

Surface Antigens on Marek's Disease Lymphoblastoid Tumor Cell Lines&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;

A total of 32 Marek's disease virus (MDV)-induced cell lines, 1 avian leukosis virus-induced cell line, and 1 reticuloendotheliosis virus (REV)-induced lymphoblastoid cell line were investigated for the presence of Fc receptors, surface IgM, and Ia-like antigen. Surface IgM and Fc receptors could not be demonstrated on any of the cell lines. Ia-like antigen was detected on all MDV-induced cell lines and on the 1 REV-induced cell line. Ia-like antigen was unrelated to other antigens reported to be present on Marek's disease tumor cells. It was also unimportant as a target antigen for MDV-related, allogeneic cell-mediated cytotoxicity. The densities of the cells of 5 MDV-induced cell lines were established on continuous Percoll gradients. Most cells had densities between 1.05 and 1.06 g/ml.

JMV Marek's disease tumor cells have B-cell characteristics

In vivo and in vitro methods were used to determine whether JMV Marek's disease (MD) tumor cells have B-cell or T-cell characteristics. JMV tumor cells were treated with anti-T-cell serum (ATS) and complement (C) or anti-B serum (ABS) and C and then inoculated into 1-day-old chickens. Within 2 weeks of inoculation, 95% of chickens inoculated with ATS- and C-treated cells died of JMV-induced leukemia, whereas only 10% of chickens inoculated with ABS- and C-treated cells succumbed. This in vivo result indicated that the malignant cells may be B cells. In vitro studies corroborated this conclusion. ABS and C killed 99 to 100% of cultivated JMV cells, whereas little or no killing of JMV cells was achieved by ATS and C, as demonstrated by trypan blue dye exclusion or by release of 51Cr from labeled JMV cells. Furthermore, after staining of JMV cells with fluorescein-conjugated rabbit anti-chicken immunoglobulin, all cells fluoresced, indicating that JMV cells have surface immunoglobulin. With support of these lines of evidence, we conclude that JMV Marek's disease tumor cells have B-cell characteristics.

Spontaneous and induced herpesvirus genome expression in Marek's disease tumor cell lines.

We incubated 31 newly established Marek's disease tumor cell lines at 41 degrees C for 48 h after subculturing and then examined them to determine the spontaneous rates of expression of viral internal antigen(s), viral membrane antigen(s), and virus isolation. All but two of the lines were isolated from tumors induced by clone-purified Marek's disease virus strain JM-10, GA-5, RB-1B, and BC-1A in nine different genetic strains of chickens with defined histocompatibility antigens. The line-to-line variations in the rates of spontaneous expression for the antigens or virus rescue were great, but the levels of expression were very low in most cases. The median rates of expression for viral internal antigen, viral membrane antigen, and virus isolation were 32, 8, and 2 positive cells per 10(5) cells, respectively (ranges, 0 to 20,280, 0 to 22,990, and 0 to 220 positive cells per 10(5) cells, respectively). The ratio of viral internal antigen expression to virus isolation was extremely variable and often high, whereas the ratio of viral internal antigen to viral membrane antigen expression was more consistent and generally low. The virus strain which induced the cell line influenced the level of virus genome expression, but the cell genotype did not. Cell lines transformed by JM-10 virus, which exhibited low oncogenicity, had significantly (p less than 0.01) higher rates of expression than cell lines transformed by CA-5 and RB-1B viruses, which exhibited high oncogenicity. Treatment with iododeoxyuridine or incubation at 37 degrees C induced increased rates of expression in most lines but not in all lines. The degree of enhanced expression was inversely proportional to the rate of spontaneous expression.

Mice inoculated with marek's disease tumor cells: Increased number of lymphomas

Germfree derived CFW wd mice were inoculated with tumor cells from chickens with Marek's disease and observed for any long term effect. Nineteen per cent of these animals developed tumors whereas the incidence in control animals was 0.2%. Of those animals which developed tumors, 62% of the animals were observed to have ataxia before tumor development. Marek's disease herpesvirus was never observed in any of the murine tumors.