Lyophilization of Cell-Free Marek's Disease Herpesvirus and a Herpesvirus from Turkeys

Cell extracts of the JM and GA strains of Marek''s disease herpesvirus and the FC 126 strain of turkey herpesvirus were lyophilized with various stabilizers. Much higher virus titers were obtained with stabilizer than without stabilizer. Titers increased even further in the case of the Marek''s disease virus strains by the addition of a chelating agent, disodium ethylenediaminetetraacetate.     

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.     

Genomic difference of herpesvirus of turkeys at low and high passage levels in culture of O1 and FC126 strains

Serial passages in culture of herpesvirus of turkeys resulted in structural genomic changes at the regions common to two virus strains with loss of their protective ability against Marek's disease.     

Growth Characteristics of a Herpesvirus of Turkeys

The herpesvirus of turkeys remains associated with cells attached to the culture vessel throughout its growth cycle. The level of infectious centers is essentially constant between 24 and 48 hr when the culture medium is unchanged. Thereafter, the number of infectious centers decreases. The cessation of virus synthesis is not due to insufficient numbers of uninfected cells or interferon production. Herpesvirus of turkeys does not replicate at 22 C and is similar to other herpesviruses in its sensitivity to interferon. The results define conditions to be considered in the preparation of infected cell suspensions commonly used as a vaccine against Marek's disease virus.     

Identification with monoclonal antibodies of glycoproteins of Marek''s disease virus and herpesvirus of turkeys related to virus neutralization.

By use of monoclonal antibodies cross-reactive with Marek's disease virus and herpesvirus of turkeys, three glycoproteins (for Marek's disease virus, gp115/110, gp63, and gp50; for herpesvirus of turkeys, gp115, gp62 and gp52) related to virus neutralization were identified. Immunization of chickens or rabbits with these glycoproteins purified by affinity chromatography resulted in production of neutralizing antibodies.     

Map location of homologous regions between Marek''s disease virus and herpesvirus of turkey and the absence of detectable homology in the putative tumor-inducing gene.

The DNA region having homology between Marek's disease virus and herpesvirus of turkey was assigned to the restriction map of Marek's disease virus by Southern blot hybridization. Under moderate conditions at the level of 15% mismatching, homology was found to be distributed throughout the Marek's disease virus genome. The long inverted-repeat regions (TRL and IRL), which are considered to play a significant role in tumorigenicity, did not show any homology to herpesvirus of turkey DNA.     

Neutralization Studies with Marek''s Disease Virus and Turkey Herpesvirus

Use of Marek's disease virus (MDV) in a neutralization test presents several problems, which are described, making this potentially useful test difficult. To obviate these difficulties, a plaque reduction test has been designed based on cross-neutralization of turkey herpesvirus (HVT) by serum-neutralizing MDV. The technique for such a neutralization test is outlined. Kinetics of development of neutralizing antibodies in chickens inoculated with HVT and MDV are described. The neutralization test can be used to evaluate viability of HVT vaccines and the possible role of neutralizing antibodies in the protection afforded by vaccination against MDV-induced tumors.     

A repeat sequence, GGGTTA, is shared by DNA of human herpesvirus 6 and Marek''s disease virus.

Some regions of the genomes of human B-lymphotrophic virus (HBLV), also designated as human herpesvirus 6, and Marek's disease virus were found to hybridize to each other under moderate to stringent conditions, scoring from 10 to 30% base-pair mismatch. Nucleotide sequence analysis showed that a 6-base-pair repetitive sequence, GGGTTA (DR2), present in the IRS-IRL junction region of the Marek's disease virus genome, was also reiterated in the HBLV genome. The function(s) of such a sequence is unknown, but this is the first report of homology between HBLV and a nonhuman herpesvirus.     

DNA Configuration in the Core of Marek''s Disease Virus

Marek's disease virus DNA appears to be wound around a central structure connecting the two inner poles of the capsid. Based on electron micrographs of Marek's disease virions, a diagram of spatial configuration of virus DNA is presented. This diagram may explain the pleomorphic character of the herpesvirus core observed with the electron microscope.     

Marek's Disease Herpesviruses II. Purification and Further Characterization of Marek's Disease Herpesvirus A Antigen

Marek's disease herpesvirus A antigen was purified greater than 200-fold with a 24% recovery by ion exchange column chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The antigen had an isoelectric point of 6.68 ± 0.03 in the presence of 1 M urea and 0.05% Brij 35, a nonionic detergent, and approximately 6.5 in the absence of dissociating agents. When analyzed by electrophoresis on analytical polyacrylamide gels, the purified antigen migrated as a single broad band which stained for both protein and carbohydrate, suggesting that it was a highly purified heterogeneous glycoprotein. However, the antigen was not purified to homogeneity as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and by immunodiffusion analysis. Antibody to Marek's disease herpesvirus A antigen was prepared in a rabbit, and antibody to two contaminating antigens was removed by adsorption to yield monospecific antisera.     

Marek's Disease Herpesviruses I. Production and Preliminary Characterization of Marek's Disease Herpesvirus A Antigen

A method was developed for the large-scale production of Marek's disease herpesvirus A antigen in duck embryo fibroblast roller bottle cultures in quantities sufficient to permit its purification and characterization. Maximum yield was obtained in serum-free culture medium harvested daily. The Marek's disease herpesvirus A antigen was stable at pH 2.0 and was a glycoprotein based on its sensitivity to trypsin, specific immune co-precipitation of radioactive amino acids and glucosamine, and detection of radioactive glucosamine by immunodiffusion and autoradiography. The antigen aggregated and lost titer upon storage but dissociated readily and regained titer in 1 or 2 M urea and 0.05% Brij 35. Fresh unaggregated antigen or antigen dissociated with urea and Brij 35 sedimented at 3.7S on sucrose gradients. The apparent molecular weight of the glycoprotein antigen was estimated to be 44,800 by gel filtration on Sephadex G-200 in the presence of 2 M urea and 0.05% Brij 35.     

Protection against Marek''s disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek''s disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.     

Use of Japanese Quail Embryo Fibroblast Cells for Propagation and Assay of Turkey Herpesvirus FC-126

Japanese quail (Coturnix coturnix japonica) fibroblast cell culture monolayers were found to provide a very satisfactory system in which to propagate and assay turkey herpesvirus FC-126, which is used for production of Marek's disease vaccine. Japanese quail cells were more sensitive than duck cells and of approximately equal sensitivity to chicken cells. Foci of infection developed rapidly and uniformly, were of larger size, and were more easily discernible in quail cells than in chicken cells.     

表达新城疫病毒HN基因的重组火鸡疱疹病毒的构建

应用聚合酶链反应技术,用设计带有限制性酶切位点的引物,特异地扩增从起始密码子开始的１ ．８ ｋｂ 新城疫病毒（ Ｎ Ｄ Ｖ） 血凝素神经氨酸酶（ Ｈ Ｎ） 基因开放式阅读框,然后插入ｐ Ｔ Ｋ２ Ｂ 的 Ｎｈｅ Ⅰ位点,构建了含 Ｎ Ｄ Ｖ Ｈ Ｎ 基因的插入载体ｐ Ｔ Ｋ Ｈ Ｎ１ 和ｐ Ｔ Ｋ Ｈ Ｎ２ ,再与感染火鸡疱疹病毒（ Ｈ Ｖ Ｔ） 细胞的总 Ｄ Ｎ Ａ 共转染鸡胚成纤维细胞（ Ｃ Ｅ Ｆ） ,经有限稀释法和 Ｄｏｔｂｌｏｔ 筛选,得到含有 Ｈ Ｎ 基因的重组体ｒ Ｈ Ｖ Ｔ１ 和ｒ Ｈ Ｖ Ｔ２ 。经组织培养传代和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 分析,表明重组体在感染细胞中表达了 Ｈ Ｎ 蛋白。重组体在 Ｃ Ｅ Ｆ 上的生长特性与亲本病毒相同,且在连续传代过程中保持稳定。为国内新城疫基因工程疫苗研制奠定了基础。     

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.     

Roles of avian herpesvirus microRNAs in infection, latency, and oncogenesis

MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation.     

Monoclonal antibodies with specificity for three different serotypes of Marek's disease viruses in chickens

A total of 50 antibody-secreting hybridoma cells against Marek's disease virus (MDV) and turkey herpesvirus (HVT) have been produced. Eleven hybridomas were used for serotyping a panel of 15 pathogenic and nonpathogenic strains of MDV and HVT, representing three serotypes. The antibodies from the culture medium have fluorescence antibody (FA) titers of up to 100 and those from mouse ascitic fluid have titers ranging from 10(4) to 10(6). Monoclonal antibody T81 is type-common, i.e., it reacts at equal titer with all MDV and HVT tested. Of the remaining 10 antibodies, eight react only with pathogenic and attenuated strains of MDV (presumably serotype 1), one reacts only with nonpathogenic MDV (presumably) serotype 2), and one reacts only with strains of HVT (presumably serotype 3). Two hybridomas belong to IgG2a and IgG2b subclasses, respectively, and the remaining nine belong to IgG1 subclass. None of the antibodies specific for MDV strains reacted with homologous viruses in serum neutralization (SN), agar gel precipitin (AGP), or membrane immunofluorescence tests. Antibody L78, which is specific for HVT, was reactive with its homologous virus in the SN test; antibody from the culture medium showed an SN titer of 10 and that from mouse ascites a titer of 10,000. None of the antibodies specific for MDV or HVT reacted with other avian or mammalian herpesviruses, avian leukosis viruses (ALV), reticuloendotheliosis viruses (REV), or Marek's disease tumor-associated surface antigen (MATSA) expressed in a lymphoblastoid cell line, MDCC-MSB-1.     

Induction of Deoxyribonucleic Acid Synthesis and the Oncogenicity of Marek''s Disease Virus

Leukocytes recovered from birds inoculated with an oncogenic strain of Marek's disease virus synthesized deoxyribonucleic acid in vitro at a rate 7.9 times that of leukocytes from normal birds or birds inoculated with the nononcogenic herpesvirus of turkeys. A close relationship was observed between the level of in vitro deoxyribonucleic acid synthesis and in vivo tumor formation.