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.     

Amplification of a tandem direct repeat within inverted repeats of Marek''s disease virus DNA during serial in vitro passage.

DNA of the oncogenic strain BC-1 of Marek's disease virus contains three units of tandem direct repeats with 132 base pairs in the terminal repeat and internal repeat, respectively, of the long region of the Marek's disease virus genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of the tandem direct repeats.     

Replicatin of the resident marek''s disease virus genome in synchronized nonproducer MKT-1 cells

MKT-1, a virus nonproducer lymphoblastoid cell line established from a Marek's disease tumor, was synchronized by double thymidine block to determine the sequence of events in the synthesis of cellular and latent marek's disease virus DNA. Cellular DNA synthesis was measured by incorporation of [3H]thymidine, whereas viral DNA synthesis was determined by DNA-DNA reassociation kinetics. The results of these studies indicate that the resident Marek's disease viral DNA in MKT-1 cells replicates during the early S phase of the cell cycle, before the onset of active cellular DNA synthesis. This observation is similar to that seen in the replication of resident Epstein-Barr virus DNA in synchronized Raji cells.     

Genomic expansion of Marek''s disease virus DNA is associated with serial in vitro passage.

An EcoRI restriction endonuclease pattern of Md11 virus DNA, a very virulent strain of Marek's disease virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we constructed a partial EcoRI map of a series of MDV clones (gift from H. J. Kung). The clones were used to identify a region of the Md11 genome which is altered as the oncogenic virus is passaged in vitro. This region was mapped into a 1.8-kilobase segment in the inverted-repeat sequences flanking the long unique region of the virus genome. The alteration appeared to result from multiple DNA insertions that produced an increase of 0.6 to 5.4 kilobases. Although the expansion of this region did not diminish the ability of MDV to replicate in vitro, it may be associated with the loss of Marek's disease oncogenicity.     

Deoxyribonucleic Acid of Marek''s Disease Virus in Virus-Induced Tumors

DNA was extracted from [(3)H]thymidine-labeled Marek's disease virus (MDV) and purified by two cycles of CsCl gradient centrifugation in a fixed-angle rotor. The DNA was transcribed in vitro into (32)P-labeled complementary RNA (cRNA). MDV cRNA did not hybridize with DNA from chicken embryo fibroblast cultures or from chicken spleen, but hybridized efficiently with DNA from MDV particles or MDV-infected cell cultures. Five Marek's disease tumors from different chickens and different organs (ovary, liver, testis) were all found to contain MDV DNA sequences. The relative amount of MDV DNA varied from tumor to tumor and was between 3 and 15 virus genome equivalents per cell. The content of virus DNA per cell in spleens from tumor-bearing chickens was much lower than in tumors from the same animals. MDV-infected cell cultures contained a large proportion (28-59%) of virus antigen-positive cells, as measured by immunofluorescence, but tumor cells were negative in this respect (<0.02% positive cells). These data indicate that MDV is present in a provirus form in tumor cells.     

鸡端粒酶RNA基因的克隆

本研究采用扩增条件优化的PCR扩增技术,以MDCC-MSBl细胞基因组DNA为模板扩增出鸡端粒酶RNA(chicken telomerase RNA,chTR)全长基因,克隆到pMD18-T载体中,经酶切鉴定和PCR鉴定后测定序列.序列分析表明所克隆的鸡端粒酶RNA基因全长465 bp,其中模板区的11个核苷(5'-CUAACCCUAAU-3')合成端粒亚单位(TTAGGG)n.chTR基因的克隆为进一步研究chTR在马立克氏病发病过程中的作用以及马立克氏病的发病机制提供可能的序列基础.     

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.     

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.     

Status of Marek's disease virus in established lymphoma cell lines: herpesvirus integration is common.

Six cell lines derived from Marek's disease lymphomas of chickens and turkeys were investigated for the status of Marek's disease virus (MDV) DNA. In the transformed T- and B-cell lines, viral DNA could be detected by conventional Southern blot hybridization, by Gardella gel electrophoresis, and by in situ hybridization of metaphase and interphase chromosomes. Integration of viral DNA into the host cell chromosome was observed in all cell lines. Two to 12 integration sites of viral DNA could be detected in metaphase chromosome spreads. The integration sites were characteristic for the individual cell lines and were preferentially located at the telomers of large- and mid-sized chromosomes or on minichromosomes. In four of six cell lines, a minor population of latently infected cells supported the lytic cycle of MDV, giving rise to linear virion DNAs. In one of these cell lines, a third species of MDV DNA could be detected with properties reminiscent of covalently closed circular DNA. The finding that MDV integrates regularly into the genomes of latently infected cells is crucial to understanding the molecular biology of herpesvirus-induced tumors in the natural host.     

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.     

火鸡疱疹病毒细菌人工染色体的构建

火鸡疱疹病毒(HVT)为一种-疱疹病毒,因其与马立克氏病病毒(MDV)抗原相关性而被广泛用作预防马立克氏病(MD)的活疫苗.[目的]本研究的目的是构建HVT全基因组感染性细菌人工染色体(BAC).[方法]利用Eco-gpt(黄嘌呤鸟嘌呤磷酸核糖转移酶)基因和BAC载体pBeloBAC11的基本功能序列,构建重组病毒转移载体Pgab-gpt-BAC11.通过将Pgab-gpt-BAC11与HVT感染细胞总DNA共转染原代鸡胚成纤维细胞(CEF),待出现病毒噬斑后,利用霉酚酸(MPA)阻断核酸代谢途径,经过筛选获得纯化的重组病毒purified-Rhvt.提取purified-Rhvt感染细胞总DNA电转化大肠杆菌DH10B感受态细胞,在氯霉素抗性平板上筛选阳性克隆,并用酶切和PCR方法对其进行鉴定.随机选取BAC克隆提取BAC DNA转染次代CEF,完成HVT重组病毒的拯救.[结果]经过6轮筛选后获得纯化的重组病毒,并筛选到25个BAC分子克隆化病毒.其中BAC6、BAC8和BAC10再次启动病毒感染,产生与野生型HVT感染CEF相似的病毒噬斑形态,说明已经获得拯救出的HVT重组病毒.[结论]本研究构建了HVT全基因组感染性细菌人工染色体,建立了HVT反向遗传操作技术平台.     

The pp38 gene of Marek's disease virus (MDV) is necessary for cytolytic infection of B cells and maintenance of the transformed state but not for cytolytic infection of the feather follicle epithelium and horizontal spread of MDV

Marek's disease virus has a unique phosphoprotein, pp38, which is suspected to play an important role in Marek's disease pathogenesis. The objective of the present study was to utilize a mutant virus lacking the pp38 gene (rMd5Deltapp38) to better characterize the biological function of pp38. This work shows that the pp38 gene is necessary to establish cytolytic infection in B cells but not in feather follicle epithelium, to produce an adequate level of latently infected T cells, and to maintain the transformed status in vivo.     

Macrophage restriction of Marek's disease virus replication and lymphoma cell proliferation.

Macrophages are shown to restrict the replication of Marek's disease virus (MDV) and isotope uptake by spleen cells from chickens bearing Marek's disease (MD) tumors. The titer of virus from duck embryo fibroblasts (DEF) co-cultivation with MDV-spleen cells pretreated to deplete marcophages was 4- to 18-fold higher than with untreated cells. Treated MDV-spleen cells increased isotope uptake by 2-fold. These restrictive activities are attributable to macrophage regulation of cell proliferation.     

Evaluation of DNA homology of Marek's disease virus, herpesvirus of turkeys and Epstein-Barr virus under varied stringent hybridization conditions

Marek's disease virus (MDV) showed only 0.3-0.6% homology in DNA sequence with herpesvirus of turkeys (HVT) at Tm-24.4 degrees C in spite of its antigenic similarity to the latter. Southern blot hybridization under stringent conditions showed that the homology between MDV and HVT is located in the restricted portion of these viral genomes. At Tm-49.6 degrees C, which permits the detection of homology with one base mismatch in three between the MDV and HVT DNAs, sequences with weak homology were found to be distributed over most of these viral genomes. No homology was detected between Epstein-Barr virus and either MDV or HVT DNA.     

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.     

马立克氏病病毒强毒GA株基因组BamH Ⅰ-L片段的序列分析

马立克氏病病毒（Ｍａｒｅｋ’ｓｄｉｓｅａｓｅｖｉｒｕｓ,ＭＤＶ）是一种能诱导鸡淋巴组织增生或淋巴肿瘤的细胞结合性疱疹病毒,由此而引起的马立克氏病（Ｍａｒｅｋ’ｓｄｉｓｅａｓｅ,ＭＤ）也是迄今为止唯一可以利用疫苗进行有效控制的肿瘤性疾病。鉴于此,作为研...     

Isolation of a reticuloendotheliosis virus from chickens inoculated with Marek's disease vaccine.

Over a period from spring to fall in 1974, a disease with delayed growth, anemia, abnormal feathers, and leg paralysis as main symptoms broke out in flocks of chickens inoculated with Marek's disease vaccine. A virus was isolated from affected birds in the field and the same lot of Marek's disease vaccine as inoculated into these birds. It had a common antigenicity to the T strain of reticuloendotheliosis virus (REV) and could not be discriminated from this strain on the basis of morphology or property. When chicks were inoculated with it, they presented essentially the same symptoms as the birds affected in the field. Since the disease was reproduced in this manner, it was presumed to have been caused by REV contained in the vaccine as contaminant. The virus persisted in the body for long time and also induced horizontal infection.     

Structure of Marek's disease virus DNA: detailed restriction enzyme map.

Purified virion DNA (120 X 10(6) molecular weight [MW]) of Marek's disease virus strain GA was cleaved with BamHI restriction endonuclease, and 27 out of the 29 fragments were cloned into bacterial plasmids. Restriction maps for BamHI, BglI, and SmaI endonucleases were constructed. The genomic structure of Marek's disease virus DNA was found to be similar to that of herpes simplex virus types 1 and 2. A long unique region (75 X 10(6) MW, located at 10 X 10(6) to 85 X 10(6) MW [10-85] from the left end of the genome), which was subdivided into segment 1 (22 X 10(6) MW, located at 10-32) and segment 2 (51 X 10(6) MW, located at 34-85) by direct repeats (32-34), was flanked by a long terminal region (10 X 10(6) MW, located at 0-10) and a long inverted region (10 X 10(6) MW, located at 85-95). A short unique region (8 X 10(6) MW, located at 103-111) was flanked by a short terminal region (8 X 10(6) MW, located at 111-119) and a short inverted region (8 X 10(6) MW, located at 95-103). The direct repeat fragments (0.9 X 10(6) could be isolated by cleavage with SmaI. The right terminal end was found to be heterogenous .