Marek's Disease Herpesvirus Research Articles

Transformation of T-lymphocyte subsets by Marek's disease herpesvirus

Marek's disease herpesvirus (MDV)-transformed lymphoblastoid tumor cell lines were characterized for the presence of the surface markers. Monoclonal antibodies were used for CD3 (T-cell receptor [TCR] complex), TCR1, TCR2, and TCR3, CD4, CD8, and Ia antigen by indirect fluorescence staining followed by microscopic examination or flow cytometry. The lymphoblastoid cell lines were obtained from tumors from chickens infected with MDV (n = 44) or from local lesions induced by inoculation of allogeneic, MDV-infected chick kidney cells (n = 56). Lymphocytes were harvested from these lesions between 4 and 16 days postinoculation and cultured in vitro to establish cell lines. All cell lines expressed Ia antigen and CD3 and/or TCR and thus are activated T cells. Most of the cell lines developed from tumors were CD4+ CD8-; only one cell line was negative for both markers. Sixteen percent of the cell lines were TCR3+, while the remainder were TCR2+. The cell lines developed from local lesions were much more heterogeneous: 45% were CD4- CD8+, 34% were CD4- CD8-, and only 21% were CD4+ CD8-. The number of TCR3+ cell lines was larger than expected for the CD4- CD8+ and CD4- CD8- cell lines, as judged from the presence of these cells in the blood. These results indicate that several subsets of T lymphocytes can be transformed by MDV, depending on the pathogenesis of infection. Activation of T cells as a consequence of the normal pathogenesis or by allogeneic stimulation seem to be a first important step in the process of transformation.

Monospecific antibodies to Marek's disease virus antigen B dimer (200 kDa) and monomer (130 and 60 kDa) glycoproteins neutralize virus infectivity and detect the antigen B proteins in infected cell membranes.

Monospecific antibodies were prepared by nitrocellulose blot immunoaffinity to 3 polypeptide components of the host-membrane associated B antigen of Marek's disease herpesvirus (MDV) and to its soluble A antigen. The B antigen comprised a 200 kDa dimer which is 2-mercaptoethanol (2-ME) labile, a monomer of 130 kDa and a 60 kDa protein, both of which are 2-ME resistant. Cross-immunoblotting studies showed that the anti-dimer antibody recognized the dimer protein as well as the 130 and 60 kDa components. In contrast, the anti-130 kDa antibody gave the strongest signal on blots of reducing gels indicating that the monomer is largely formed by in vitro reduction with 2-ME. All four antibodies recognized membrane antigens on chicken embryo fibroblasts infected with MDV vaccine viruses representative of the three serotypes and in addition, neutralized the homologous MDV isolate. The anti-dimer antibody was greatest, the anti-monomer antibody was the weakest and the anti-60 kDa antibody intermediate in neutralizing efficacy to all four viruses. We conclude from these studies that the B antigen presents at least two classes of neutralizing epitopes: one is discontinuous and of broad specificity on the intact dimer molecule and the other, on the 130 and 60 kDa proteins, is continuous and of lower avidity.

Latent transcripts of Marek's disease virus are clustered in the short and long repeat regions

Marek's disease herpesvirus (MDV) induces tumors in chickens, and lymphoblastoid cells derived from such tumors contain the viral genome in a latent state and do not produce infectious virus. Poly(A)+ RNAs extracted from MDV-induced kidney lymphoma cells and from MKT-1 cells, a nonproducing lymphoblastoid cell line derived from a tumor induced by MDV, were electrophoretically separated under denaturing conditions, transferred to a solid substrate, and hybridized with labeled DNA probes representing approximately 95% of the virus genome. These analyses revealed 29 viral RNA transcripts in the kidney lymphoma and 32 viral RNAs in MKT-1 cells. In both instances, the transcripts hybridized to a restricted region comprising approximately 20% of the MDV genome located in the repeats flanking the long and short unique sequences and in the adjacent unique sequences. The sizes of the transcripts derived from kidney lymphoma and MKT-1 cells and the distributions of the homologous regions in the viral genome were very similar. The most abundant transcripts were homologous to the BamHI I2 fragment. The results suggest that expression of the MDV genome in MKT-1 cells closely reflects the expression of the MDV genome in tumors. The gene expression over extended regions of the genome is in concordance with that observed in latent gammaherpesviruses rather than that observed in latent alphaherpesviruses.

Herpesviruses of birds ‐ a review

The family of herpesviridae is divided into the subfamilies alpha-, beta- and gammaherpesvirinae. Members of the alphaherpesvirinae (infectious laryngo-tracheitis in gallinaceous birds and duck viral enteritis in waterfowl) may cause substantial economic and ecological losses. Marek's disease and turkey herpesviruses are the only members of the gammaherpesvirinae. Most herpesviruses from free-living and pet birds are tentatively classified as bctaherpesvirinae. These may induce diseases, with variably non-specific clinical signs but including respiratory distress, ocular lesions, enteritis and liver involvement. Prominent pathological lesions consist either of haemorrhages on respiratory or intestinal epithelium or of multi-focal necrotic lesions in livers, spleen and bone marrow. At least four different plaque types of betaherpersvirinae can be differentiated in infected chick embryo fibroblasts or chick kidney cell cultures. Infectivity may be cell-associated or detectable in supernatant fluids and transformed cultured cells. All avian herpesviruses are presently grouped on the basis of results of cross neutralisation tests into 11 different serotypes.

Purification of genomic sized herpesvirus DNA using pulse-field electrophoresis

A new method for the separation of genomic sized herpesvirus DNA was developed. This method utilizes the improved separation of extremely high molecular weight DNA molecules by pulse-field electrophoresis. This method has allowed us to separate and purify herpesvirus DNA (> 100 kbp) free of cellular DNA, directly from infected cells. In the case of cell associated herpesviruses (such as Marek's disease herpesvirus), this method is more efficient than present isolation procedures. The DNA obtained is able to be restriction enzyme digested and used in cloning experiments.

Transplantable Marek's disease lymphomas. III. Induction of MHC‐restricted tumor immunity by lymphoblastoid cells in F1 hosts

F1 chickens, which were a cross between birds of the related inbred lines G-B1 and G-B2, were immunized with in vitro cultured virus-non-producer lymphoblastoid cells that were either syngeneic or allogeneic with the challenge tumor cells. The lymphoblastoid cells were derived from Marek's disease herpesvirus (MDV)-induced transplantable tumors. Previous findings showed that such immunization of G-B1 and G-B2 chickens prevented early mortality caused by the tumors. Lymphoblastoid cells syngeneic with the tumors were more effective than allogeneic cells, suggesting that an MHC-restricted immune response was induced, or, alternatively, that immune elimination of allogeneic cells prevented them from initiating strong immunity to MDV-associated tumor antigens. Immune elimination of the immunizing cells should not occur in F1 birds heterozygous for the 2 different parental MHC haplotypes (B6 and B13). Early mortality among F1 chickens immunized with lymphoblastoid cells that were syngeneic with the challenge tumor cells was significantly lower than for non-immunized control chickens or for birds immunized with lymphoblastoid cells that were allogeneic with the challenge tumor cells. Our results suggest that MDV-induced tumor antigens may be recognized by the host as altered-self MHC antigens.

Partial transcription map of Marek's disease herpesvirus in lytically infected cells and lymphoblastoid cell lines

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).

Discovery of Noninfectious Viral Genes Complementary to Marek's Disease Herpes Virus in Quail Susceptible to Cholesterol-Induced Atherosclerosis

Japanese quail genetically selected on the basis of atherosclerosis susceptibility were tested for infection by Marek's disease herpesvirus (MDV). Viral DNA was detected in the atherosclerotic aortas of susceptible (SUS) quail by the technique of DNA hybridization. Southern blot analysis demonstrated that restriction mapping of aortic DNA was specific and different from that of MDV. Screening of quail embryos by dot-blot hybridization detected that MDV DNA existed in 100% of SUS quail tested. Resistant (RES) quail were a mixed population, with 16% of embryos resembling the SUS group. Functional MDV was not found by a number of methods including virus isolation, serological test, and exposure of sentinel chicks to SUS quail. These results suggest that the SUS quail possess a portion of the MDV genome in the germline, and the viral genes have been coselected by their susceptibility to cholesterol-induced atherosclerosis.

Augmentation of retrovirus-induced lymphoid leukosis by Marek's disease herpesviruses in White Leghorn chickens

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.

Synthesis and processing of the Marek's disease herpesvirus B antigen glycoprotein complex.

The Marek's disease herpesvirus B antigen (MDHV-B) complex was previously immunologically identified and molecularly characterized as a set of three glycoproteins designated gp100, gp60, and gp49 on the basis of apparent molecular weight and immunoprecipitation with both polyclonal and monoclonal antibodies. Immunoprecipitation analysis, previously with polyclonal and more recently with monoclonal antibodies, of infected cell lysates labeled with [35S]methionine in the presence of tunicamycin, an inhibitor of N-linked glycosylation, revealed two putative precursor molecules of 88,000 daltons (pr88) and 44,000 daltons (pr44). High-resolution pulse-chase studies revealed that gp100 was a glycosylated intermediate which was processed to yield gp60 and gp49. This cleavage was inhibited by monensin, an inhibitor of glycoprotein processing. Endo-beta-N-acetylglucosaminidases F and H (endo-F, endo-H) reduced gp100 to pr88, indicating that the latter is an intermediate in the biosynthetic pathway. These same enzymes reduced gp49, and to a lesser extent gp60, to pr44, suggesting that pr44 is their polypeptide backbone. Significant support for this concept is the fact that the same monoclonal antibody recognized all three molecules, gp60, gp49, and pr44. In the presence of monensin, terminal addition of complex sugars was also prevented, since gp60 was replaced by a slightly faster migrating component which was insensitive to both endo-F and endo-H. Cell-free translation of infected-cell mRNA, followed by immunoprecipitation analysis with either polyclonal or monoclonal antibody, resulted in detection of a putative unglycosylated precursor polypeptide of 44,000 daltons. Since pr88 was not the initial precursor polypeptide of the MDHV-B complex, its existence may have resulted from dimerization of pr44. Again, detection of both pr88 and pr44 with the same monoclonal antibody is consistent with this interpretation. These collective data obtained from the cell-free and in vivo studies with polyclonal and monoclonal antibodies reactive with MDHV-B are consistent with the concept that pr44, the initial gene product, dimerizes to form pr88 and demonstrate that pr88 is actually a processing intermediate glycosylated to gp100, another processing intermediate, which is then processed to gp60 and gp49.

Maintenance of Marek's Disease Herpesvirus Latency in vitro by a Factor Found in Conditioned Medium

Chicken spleen cells latently infected with Marek's disease virus were cultured with and without conditioned medium (CM) obtained from concanavalin A-stimulated chicken spleen cell cultures. The expression of viral internal antigen(s) (VIA), which is usually associated with cultivation, was prevented or markedly reduced by the CM. This effect required the continued presence of CM, since its removal after 48 h resulted in the subsequent appearance of VIA. Although CM contains both gamma interferon (IFN-gamma) and interleukin 2, our studies suggest that the 'latency-maintaining activity' (LMF) may not be associated with either of these products of stimulated lymphocytes. However, IFN-gamma may also have had some suppressive effect. LMF appears to have an Mr greater than 10,000 and to be inactivated by heating to 90 degrees C for 5 min.

Structure and complete nucleotide sequence of the Marek's disease herpesvirus gp57-65 gene

The gene encoding the precursor polypeptide of the Marek's disease herpesvirus (MDHV) secretory glycoprotein gp57-65 (formerly identified as A antigen) has been sequenced. Previous results had localized the gene to a 4.6-kilobase (kb) segment of the BamHI B fragment in the unique long region of the MDHV genome. S1 nuclease protection experiments were used to more precisely locate the 5' initiation and approximate 3' termination points of the approximately 1.8-kb MDHV gp57-65 mRNA within this segment. These results indicated that the entire MDHV gp57-65 coding sequence is contained within a 2.35-kb PvuII-EcoRI fragment, with the direction of transcription from PvuII to EcoRI (5' to 3'). Nucleotide sequence analysis of this region revealed a single open reading frame of 1,515 base pairs. The MDHV gp57-65 coding sequence has an overall guanosine-plus-cytosine content of 41%. Translation of the single open reading frame would produce a polypeptide of 505 amino acids, with a calculated molecular weight of 56,805. The putative gp57-65 precursor polypeptide contains features common to many glycoproteins. These include a hydrophobic amino-terminal region (amino acids 1 to 27) that may function as a signal peptide and nine potential N-linked glycosylation sites (Asn-X-Ser/Thr). These two features, predicted from nucleotide sequence data, are consistent with the published data showing that gp57-65 has a signal peptide and N-linked glycosylation (R. J. Isfort, R. A. Stringer, H.-J. Kung, and L. F. Velicer, J. Virol. 57:464-474, 1986). The predicted sequence indicates that overall the polypeptide is relatively hydrophobic, with a possible 18-residue carboxyl-terminal membrane anchor sequence. This sequence appears to be less prominent than those commonly found in integral membrane glycoproteins. The lack of a strong hydrophobic anchor sequence may help to explain the predominantly secretory nature of MDHV gp57-65.

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Marek's disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek's disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

Detection of specific DNA segments of Marek's disease herpes virus in Japanese quail susceptible to atherosclerosis

Genetically selected lines of Japanese quail, highly susceptible (SUS) and resistant (RES) to atherosclerosis, were used to study the possible involvement of Marek's disease herpes virus (MDV). An EcoRl gene library of MDV cloned in pBR328 was used to prepare the 32P-DNA probe in dot-blot and Southern blot hybridizations to detect the presence of MDV DNA sequence in the aorta, embryo and other tissue specimens. The viral DNA was found present in the aorta of SUS quail and it increased with the severity of the aortic lesion. For the DNA isolated from the atherosclerotic aorta, the endonuclease restriction map is specific but not identical to MDV genome. When screening the embryos of SUS and RES quail, it was found that all the SUS were positive with approximately 10 or more viral genome equivalents or virus copies per cell. The RES embryos were heterogeneous, 41% negative (< 0.1 copy per cell), 43% intermediate (1–10 copies per cell) and 16% positive (10 or more copies per cell). The vaccination of SUS quail with the herpes virus of turkey vaccine did not prevent the disease. These results indicated that a part of MDV genome or another related herpes virus genome was integrated into the host DNA of SUS quail. The integrated viral gene or genes are believed to be important in atherogenesis, because they are genetically co-selected with the atherosclerosis-susceptibility.

Identification of the gene encoding Marek's disease herpesvirus A antigen.

The gene encoding the glycoprotein Marek's disease herpesvirus A antigen (MDHV-A) precursor polypeptide pr47 was delineated by using Northern blot (RNA blot) analysis and hybrid selection of its mRNA with cloned MDHV DNA, cell-free translation of the mRNA, and immunoprecipitation of the polypeptide. The resulting piece of DNA with strongly positive hybrid selection results was a 2.2-kilobase-pair (kbp) PvuII-EcoRI restriction fragment localized to the center of the 18.3-kbp MDHV BamHI B fragment of the total virus genome. The localization was specific since no other small restriction subfragment of the larger BamHI B fragment was able to hybrid select significant MDHV-A mRNA and the gene mapped only in the BamHI B fragment of the total virus genome. Northern blot analysis confirmed the localization of the MDHV-A gene on the 2.2-kbp fragment and detected its mRNA as a 1.8-kilobase species, a size consistent with encoding a 47-kilodalton polypeptide. This is the first report of an MDHV gene being mapped to the MDHV viral genome. This opens the way for the use of recombinant DNA technology to study the nature of the gene encoding a secreted virus-specific glycoprotein that could possibly be involved in immunoprevention, immunosuppression, or immunoevasion, immune phenomena known or speculated to be involved in this oncogenic herpesvirus system.

Molecular characterization of Marek's disease herpesvirus B antigen

The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B-antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O-linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed R alpha PM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of very virulent pathotypes of Marek's disease virus from vaccinated chickens in Europe.

Two very virulent strains of Marek's disease virus were isolated from two separate farms in northern Italy that had experienced outbreaks of Marek's disease in vaccinated flocks. The isolates were similar to very virulent strains reported in the USA in terms of their enhanced pathogenicity, both for chickens vaccinated with the herpesvirus of turkeys, and for genetically resistant chickens. This first description of very virulent strains of Marek's disease virus from outside the USA suggests that at least some of the increasingly frequent disease outbreaks reported in Europe may be associated with such strains and that the adoption of bivalent or polyvalent vaccines containing, for example, attenuated Marek's disease virus plus herpesvirus of turkeys may be beneficial in the field.

Herpesvirus infection prevents activation of cytoplasmic cholesteryl esterase in arterial smooth muscle cells.

Herpesvirus infection has been shown to alter the cholesteryl ester cycle in avian arterial smooth muscle cells, resulting in cytoplasmic cholesteryl ester accumulation (Hajjar, D. P., Falcone, D. J., Fabricant, C. G., and Fabricant, J. (1985) J. Biol. Chem. 260, 6124-6128). In this study, we attempted to define some of the regulatory mechanisms associated with the control of cytoplasmic cholesteryl esterase in Marek's disease herpesvirus (MDV)-infected cells. We found that cholesteryl esterase activity in MDV-infected cells could not be activated by dibutyryl cyclic AMP, dibutyryl cyclic AMP added together with protein kinase, or agonists of adenylate cyclase. Activation of cytoplasmic cholesteryl esterase activity occurred in uninfected cells and in cells infected with a control virus, turkey herpesvirus. Furthermore, the rate of cholesterol efflux from arterial smooth muscle cells challenged with dibutyryl cyclic AMP was unchanged in MDV-infected cells as compared to uninfected or turkey herpesvirus-infected cells in which efflux was increased. We propose that the reduced cytoplasmic cholesteryl esterase activity in lipid-laden, herpesvirus-infected cells is due partly to its inability to be activated by the cyclic AMP-protein kinase mechanism. This may contribute to the pathologic changes seen in MDV-infected arterial cells, including accumulation of intracellular cholesteryl esters.

Synthesis, processing, and secretion of the Marek's disease herpesvirus A antigen glycoprotein.

The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.

Preparation of monoclonal antibodies against Marek's disease virus and herpesvirus of turkeys.

Fifty-two monoclonal antibodies specific for Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) were established. Twenty-two of these antibodies were specific for three different serotypes. Of the remaining antibodies, 22 cross-reacted with all combinations of the three serotypes (1·2·3, 1·2, 1·3 and 2·3). Using the serotype-specific antibodies, 10 presumable serotyped MDV and HVT and 7 field isolates could be serotyped by indirect immunofluorescence test. One of 7 field isolates was identified to a serotype 2 MDV which has been isolated for the first time in Japan.