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In order to investigate the replication timing properties of PCDH11X and PCDH11Y, a pair of protocadherin genes located in the hominid-specific non-pseudoautosomal homologous region Xq21.3/Yp11.2, we conducted a FISH-based comparative study in different human and non-human primate (Gorilla gorilla) cell types. The replication profiles of three genes from different regions of chromosome X (ZFX, XIST and ATRX) were used as terms of reference. Particular emphasis was given to the evaluation of allelic replication asynchrony in relation to the inactivation status of each gene. The human cell types analysed include neuronal cells and ICF syndrome cells, considered to be a model system for the study of X inactivation. PCDH11 appeared to be generally characterized by replication asynchrony in both male and female cells, and no significant differences were observed between human and gorilla, in which this gene lacks X-Y homologous status. However, in differentiated human neuroblastoma and cerebral cortical cells PCDH11X replication profile showed a significant shift towards allelic synchrony. Our data are relevant to the complex relationship between X-inactivation, as a chromosome-wide phenomenon, and asynchrony of replication and expression status of single genes on chromosome X.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek's disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek's disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Marek's disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

Novel approaches to vaccination against very virulent (vv) strains of Marek's disease virus (MDV) are discussed. Fowlpox virus (FPV) and herpesvirus of turkeys (HVT) recombinants expressing MDV antigens have been constructed. It has been shown that glycoprotein B of MDV serotype 1 (gB1) is an effective immunogen which is particularly important for conferring protective immunity in genetically susceptible chickens. However, maternal antibodies against MDV diminished the efficacy of vaccination with recombinant FPV‐gB1. HVT recombinants expressing antigens of MDV and Newcastle disease virus have been constructed and have been shown to be effective in preventing Marek's disease as well as systemic Newcastle disease. Maternal antibodies against MDV and Newcastle disease virus did not affect significantly the efficacy of vaccination with cell‐associated HVT recombinants. The potential of retroviral insertion mutagenesis and other means of delivering MDV antigens for immunization are discussed.

Protocadherin X (PCDHX) and Protocadherin Y (PCDHY) are cell-surface adhesion molecules expressed predominantly in brain. The human PCDH11X/Y gene pair is located in the non-pseudoautosomal X-Y homologous region (Xq21.3/Yp11.2). The possible existence of PCDH11 gene dosage differences between human and non-human primates is of evolutionary significance with respect to species differences and escape from X inactivation, and has been repeatedly debated. Previous investigations on the X/Y homologous status of PCDH11 and adjacent sequences in non-human primates have highlighted the complexity of the molecular pattern and evolutionary history of this genomic region. This paper provides for the first time direct evidence for the absence of the PCDH11 genefrom the Y chromosome of chimpanzee (Pan troglodytes) as well as gorilla (Gorilla gorilla). By confirmingthe suspected lack of X-Y homologous status for PCDH11 in non-human primates, our results reinforce the hypothesis of a hominid-specific role for this gene in brain development.

The deduced amino acid sequences of the open reading frames (ORFs) mapping in the short unique segment (US) of Marek's disease virus (MDV) reported in the accompanying paper have been analysed using computer programs to determine their relationships to herpesvirus proteins. Analysis of the catalytic domains of protein kinases showed that the MDV kinase (MDV PK) was closely related to the alphaherpesvirus protein kinase mapping in US. The results also showed that the MDV PK was more closely related to the cellular kinases that control cell division than to the proto-oncogenes c-src and c-mos and it was predicted that the MDV PK would phosphorylate serine/threonine. The MDV homologue of herpes simplex virus (HSV) glycoprotein D (gD) contained several residues that were conserved in mammalian herpesviruses. In particular, six cysteines were perfectly aligned in all the gDs and there were numerous conservative substitutions. Although only approximately 65% of the MDV homologue of glycoprotein I (gI) of HSV has been sequenced, it was clear that a significant number of amino acid residues including four cysteines were conserved in the gI homologues of MDV and mammalian herpesviruses. Further analysis suggested that MDV gD was more closely related to the gDs of pseudorabies virus (PRV) and equine herpesvirus 1 than to the gD of HSV-1 and HSV-2. It was noted that HSV-2 glycoprotein G (gG), PRV gX and MDV gD were related and that MDV ORF4 was related to MDV gD and probably to HSV-1 gG. The results have shown a clear relationship between the genes of MDV and their counterparts in mammalian alphaherpesviruses and are consistent with the idea that MDV glycoprotein genes in US might have arisen by a process of gene duplication and independent evolution.

Protocadherin X (PCDHX) and Protocadherin Y (PCDHY) are cell-surface adhesion molecules expressed predominantly in brain. The human PCDH11X/Y gene pair is located in the non-pseudoautosomal X-Y homologous region (Xq21.3/Yp11.2). The possible existence of PCDH11 gene dosage differences between human and non-human primates is of evolutionary significance with respect to species differences and escape from X inactivation, and has been repeatedly debated. Previous investigations on the X/Y homologous status of PCDH11 and adjacent sequences in non-human primates have highlighted the complexity of the molecular pattern and evolutionary history of this genomic region. This paper provides for the first time direct evidence for the absence of the PCDH11 genefrom the Y chromosome of chimpanzee (Pan troglodytes) as well as gorilla (Gorilla gorilla). By confirmingthe suspected lack of X-Y homologous status for PCDH11 in non-human primates, our results reinforce the hypothesis of a hominid-specific role for this gene in brain development.

Identification of Marek's disease virus-infected lymphocytes is essential for a full understanding of the pathogenesis of Marek's disease. This paper describes the development of a simple, quantitative and reproducible flow cytometric technique for the identification of the phenotype of Marek's disease virus-infected lymphocytes. The method is based upon the detection of the Marek's disease virus-specific phosphoprotein pp38, in saponin-permeabilized lymphocytes, using the monoclonal antibody BDI, and the identification of the phenotype of pp38+ lymphocytes using monoclonal antibodies against lymphocyte surface markers. pp38 expression in the spleen was demonstrated in the cytolytic phase of infection. The mean proportion of pp38+ lymphocytes at 4 days post-infection was 0.43%. Of these, 95% were B lymphocytes, while only 4% were T lymphocytes (both CD4+ and CD8+). The potential of the technique for the investigation of the cytolytic phase of Marek's disease is discussed.

Summary Precipitating antigens present in extracts of chick embryo cells infected with the HPRS-16 attenuated strain of Marek's disease virus (att-MDV) were separated by gel filtration on Sephadex G200 and some of their properties determined. The two main antigens detected with convalescent MD serum, referred to as ‘B’ and ‘C’ antigens, had mobilities of 0.55 and 0.25 respectively relative to phenol red on electrophoresis in 7.5% acrylamide gel. The B antigen was relatively stable and of low mol. wt. in comparison with the C antigen. B and C antigens were in some instances also detected in culture medium of infected cells, but were distinguishable from the A antigen, a major glycoprotein antigen released into the culture medium of cells infected with HPRS-16. The results of immunodiffusion studies suggest that B antigen is common to MDV and strains of herpes virus of turkeys (HVT) and that at least 2 antigens (including C) are MDV specific. The A antigen was also common to MDV and HVT strains. It was noted however that the capacity of HPRS-16/att to synthesize A antigen was considerably reduced in comparison with HPRS-16 and HVT strains, and in some preparations the A antigen could not be detected. Evidence was also obtained for the presence of HVT-specific antigens associated mainly with the cell fraction.

The enhanced glucose uptake by chick embryo cells as early as 72 h after infection with Rous sarcoma virus (RSV) was confirmed in this study to be an early indicator of cellular transformation. The glucose uptake of C/E BrL cells infected by various doses of BS-RSV of subgroup A showed that the relationship between the log-dose of virus and log-uptake was linear (slope, b1 = 1.30 +/- 0.14) when the ratio of the number of infectious virus particles to the number of cells in the culture was above 1:200. But infection of cultures with a relatively high dose of virus, for instance 10(3.5) focus forming units (f.f.u.) was ineffective for the measurement of cellular transformation using the criterion of glucose uptake, whereas a much lower dose such as 10(1.7) f.f.u. was sufficient to induce foci of transformed cells. We concluded therefore that the statistic of glucose uptake assay (GUA) measured at 72 p.i. is less sensitive than that of the focus count assay (FCA) measured after 10 days as a measure of assessing cellular transformation by RSV. Nevertheless, when the cultures were infected with a higher dose of virus (10(4.3) f.f.u. or more), the GUA could discriminate between the transformed (T) and non-transformed (NT) cultures. This was demonstrated in the two genetic crosses, line 7-2 X WC(F1) and line 7-2 C line. Embryo cultures of these two test-crosses were infected with viruses of subgroups A, B, C and D, and the T and NT phenotypes were ascertained. Also, on the basis of focus counts in the cultures the genetically resistant (R) and susceptible (S) phenotypes in response to various infections were determined. The T and NT phenotypes based on the GUA were compared with the S and R phenotypes, respectively, based on the FCA. It was found that in 47 of the 51 cultures, the phenotypic agreement was perfect, suggesting that glucose uptake by cells of embryo cultures exposed to RSV is a biochemical indicator of genetic susceptibility. The discordant results in 4 cultures are discussed in the light of present knowledge of cellular transformation by RSV.

Summary The restriction endonuclease patterns of viral DNA obtained from serotypes 1 and 2 strains of Marek's disease virus have been compared and homology between the strains examined by hybridization. The results have shown that HPRS 16 (serotype 1) DNA has a structure similar to its attenuated variant HPRS 16/att except for a few fragments that are present only in the virulent strain. Evidence was obtained which suggested that these restriction fragments contained repeat sequences and that insertion of heterogeneous DNA occurred at these sites during attenuation of HPRS 16. The restriction enzyme patterns of HPRS 24 (serotype 2) differed substantially from those of HPRS 16 and HPRS 16/att and reassociation experiments showed that HPRS 24 shares less than 10% homology with either HPRS 16 or herpesvirus of turkeys (serotype 3).

Immunity against Marek's disease was conferred by the use of non-infectious materials extracted with non-ionic detergents from cells infected with the attenuated strain of Marek's disease virus (MDV). Antibody-free Rhode Island Red chicks were inoculated at 1 week of age with cell extracts emulsified in Freund's complete adjuvant and were given a second inoculation 1 week later without adjuvant. Protection against natural infection was obtained in groups inoculated with both soluble (not sedimented at 100,000 times g/2h) and insoluble antigens present in Nonidet P40 (NP40) extract, but only with the insoluble fraction of deoxycholate extract. The results suggest that the immunizing antigens can be partially solubilized with 0.5% NP40 and that the growth and spread of MDV are reduced in immunized chickens.

The susceptibility of 3 lymphoblastoid cell lines (MDCC-MSB1, MDCC-HP1 and MDCC-RP1) derived from Marek's disease tumours to infection with influenza (AoPR8) and pseudorabies viruses was studied. MSB1 and HP1 were more susceptible to infection with influenza virus than was RP1. It was shown in the case of HP1, which was studied in greater detail, that although the majority of the cells synthesized influenza virus haemagglutinin when infected at high multiplicity, only a small proportion produced mature infectious particles. The results obtained from both high and low multiplicities of infection suggested that infection with AoPRs was abortive in the majority of the cells of all 3 cell lines. Infected cultures remained viable when subcultured over several days. All 3 cell lines were resistant to pseudorabies virus. The block in pseudorabies virus replication probably occurred after adsorption. Immunization of chickens with HP1 lymphoblastoid cells superinfected with influenza virus AoPR8 and inactivated with glutaraldehyde gave better protection against Marek's disease than immunization with uninfected HP1 cells.

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.

Summary The production, purification and properties of the ‘A’ antigen induced by Marek's disease virus in tissue culture were investigated. It was found that although virus multiplied on passage in chick kidney, chick embryo and duck embryo cells the antigen was produced only in chick kidney and duck embryo cultures. The results suggested that the ‘A’ antigen produced by duck embryo cells was a glycoprotein having a mol. wt. in the range 70 000 to 90 000 and was heterogeneous in charge (pI 4.5 to 5.5). A 20-fold purification was achieved by electrophoresis in 5% acrylamide gel with a recovery of 45%. An equally good purification was also possible by chromatography on DEAE-Sephadex A50 and Sephadex G200, although the recovery was only 20% in this case.

MAREK'S disease (MD) is a herpesvirus induced lymphoma of chickens characterised by transient virus replication in lymphoid cells and transformation of T cells1. Several mechanisms probably operate in resistance to Marek's disease, but there is indirect evidence that protective immunity is predominantly T-cell dependent2 and comprises both antiviral3 and anti-tumour4 immune responses. Thus, a notable feature which distinguishes immunity to Marek's disease from immunity to other herpesvirus infections studied so far is that both effector and target cells are probably T cells and that both virus and tumour-specific antigens may act as targets for immunological attack. Using a plaque inhibition test as a measure of cell-mediated immunity, I show here that T cells obtained from immunised birds are sensitised to virus-specific antigens present at the surface of infected cells of both lymphoid and non-lymphoid origin. The results provide direct evidence for the role of T cells in eliminating infected leukocytes and in reducing the spread of Marek's disease virus (MDV) in vivo.

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.

The small-plaque effect occurs with a wide range of herpesviruses following irradiation with ultraviolet light. The 37 per cent survival (D37) values, or dose required for one lethal hit (e−1), for herpes simplex, pseudorabies and pigeon herpesviruses in different cells indicate a broad spectrum of host-cell repair capacity. Other DNA-containing viruses such as SV40 and adenoviruses, which also replicate in the cell nucleus, show the small-plaque effect. Ionizing irradiation of herpes simplex virus type 1 (HSV-1) showed but little reduction in plaque-size.

Marek’s disease is a serious lymphoproliferative disease of the fowl which affects most organs and tissues but has an unusual predilection for peripheral nerves. The disease varies in severity ranging in extremes from the chronic disease, in which peripheral nerve involvement and paralytic signs are the most conspicuous features, to an acute fulminating disease in which multiple and diffuse lympho-mata are present. Prior to the development and wide use of vaccines for the control of the disease, it was of great economic importance to the poultry industry throughout the world. In the acute form mortality of 30% of a flock was common, and in some cases mortality reached over 50%.

Summary A Marek's disease lymphoblastoid cell line (MSB-1) has been analysed by immunoprecipitation for expression of tumour-associated antigen, Marek's disease virus (MDV)-specific antigens and antigens specific to avian leukosis-sarcoma viruses. Rabbit antisera raised against two independently derived cell lines after extensive absorption with normal chick cells reacted with a polypeptide of mol. wt. 40000 (40K) in extracts of MSB-1 cells. The 40K polypeptide was not present in myeloblasts or in chick embryo fibroblasts (CEF) infected with MDV and did not react with antiserum raised against normal chicken thymus antigens. The possibility that the 40K polypeptide is a tumour-associated antigen is discussed. Seven MDV-specific antigens were noted in infected CEF (mol. wt. 110K, 100K, 80K, 70K, 50K, 35K and 32K) but none of these was detected in MSB-1 cells. The avian leukosis-sarcoma group-specific antigen P27gag and its precursor Pr76gag were not found in MSB-1 cells, confirming that expression of mature gag protein is not required for transformation by MDV. However, two polypeptides of unknown origin and function (mol. wt. 180K and 110K) were precipitated from MSB-1 cells with a rabbit anti-Rous sarcoma (Schmidt-Rupin, subgroup D) antiserum.

Summary Surface changes were demonstrated in duck embryo cells following infection with both Marek's disease virus (MDV) and a herpes virus of turkeys (HVT) by agglutination with concanavalin A (con A). Materials labelled with fucose were extracted from the surface of cells with Nonidet. Antigens and con A receptors were isolated from these extracts by affinity chromatography and were compared using double labelling and electrophoresis in polyacrylamide gels. Eight or nine glycoproteins containing fucose were detected in cell surface extracts. Major con A receptors (average mol. wt. 95000 and 130000) detected in MDV and in HVT infected preparations were also present in uninfected cells. Antigens isolated from MDV and HVT infected preparations by immunoadsorption using MDV antiserum were different from the major con A receptors. The relationship between con A binding glycoproteins and antigenic glycoproteins is discussed. Two antigens (average mol. wt. 30000 and 43000) associated with the surface of MDV infected cells were MDV specific but the antigens precipitated from culture fluids of MDV and HVT infected cells with MDV antiserum appear to be identical.

Immunization with inactivated viral antigens protected chickens against Marek's disease. Non-immunized chickens could be protected by injections of spleen cells but not of serum from immunized, histocompatible donors. Chickens rendered agamma-globulinaemic by bursectomy and irradiation could also be immunized against Marek's disease by inoculation with viral antigens, but spleen cells from these immunized, bursectomized and irradiated donors did not confer protection on the recipients into which they were injected. It was concluded that, although in the bursectomized, immunized donors cell-mediated immunity alone was able to provide a fair degree of protection against Marek's disease, the protection afforded against the disease by spleen-cell transfer was at least partly attributable to the transfer of antibody-producing cells, and that humoral immunity, while not being an absolute requirement for resistance, is normally an important component of the resistance mechanism.

The study of the molecular biology of Marek’s disease virus (MDV) started more than a decade ago when Lee et al. (1,2) characterized its DNA. However, progress in the analysis of virus structural proteins has been hampered by the unusual cell-associated properties of MDV and the lack of enveloped infectious particles in cultured cells. For these reasons and because of the shortage of conditional lethal mutants, genetic analysis of virus functions by classical approaches has not been possible. The recent progress in molecular cloning of MDV (3,4) and the development of monoclonal antibody to virus-specific epitopes (5,6) have provided alternative approaches which hold great promise for furthering our understanding of oncogenesis and for defining virus structural components and their functions.

Marek's disease herpesvirus (MDV) can cause either a productive-restrictive or lytic infection, a latent infection or can transform thymus-derived lymphocytes. RNA was extracted from infected chicken embryo fibroblasts (CEF) or from lymphoblastoid tumour cell lines. Some of the infected CEF were treated with 200 micrograms/ml cycloheximide to identify immediate early (IE) transcripts, and others with 1 microM 1-(2-fluoro-2-deoxy-B-D-arabinofuranosyl)-5-methyluracil (FMAU), an inhibitor of herpesvirus DNA synthesis to identify early transcripts. An extensive Northern blot analysis was carried out using DNA probes spanning almost the complete MDV genome. In the lytically infected CEF at least 66 discrete transcripts were detected, ranging in size from 9.1 kb to 0.6 kb. Eleven IE transcripts were identified, of which 8 were mapped in the genome segment consisting of the IRL, IRS, US and TRS. Six transcripts were identified as early genes. In the MD lymphoblastoid cell lines MDCC-HPI, a non-producer cell line, and MDCC-CU41, a non-expression cell line, 4 and 7 transcripts were detected, respectively. These RNAs were transcribed from IE genes located mainly in the repeat sequences flanking UL and US and in US. Treatment of the lymphoblastoid cell lines with 20 micrograms/ml 5-iodo-2-deoxyuridine resulted in the additional transcription of 1 RNA species in HPI and 9 in CU41. Most of the transcripts present in lytically infected cells were also detected in MDCC-CU36, a cell line with a high percentage of antigen-positive cells (expression cell line).

Summary The small-plaque effect induced by exposure of herpes simplex virus to u.v. light was investigated. New results are presented showing that the effect was due to a delay of multiplication of virus in the cells initiating plaques. The delay occurred after entry of virus into the cell and before the replication of virus. The ultimate cause of the delay was probably the formation of thymine dimers, since the small-plaque effect was reversed by photoreactivation in avian cells. Evidence was obtained for co-operative reactivation and host-cell reactivation in infected BHK cells but neither of these phenomena could be related to the small-plaque effect.

Summary Baby hamster kidney cells were infected with herpes simplex virus under one-step growth conditions. Virus-specific antigens were traced by ‘direct’ and ‘indirect’ immunofluorescence and their times of appearance estimated by agar-gel diffusion using an antiserum made by immunization of rabbits with herpes-virus-infected rabbit cells. Twelve distinct precipitin lines were detected by agar-gel diffusion and a succession of fluorescent patterns was distinguished. Quantitative estimations indicated progressive accumulation of antigen in various patterns within the nucleus, in the cytoplasm and at the cell membrane. These patterns were correlated with the cellular distortions seen with a conventional cytological technique.