Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.     

Adaptation of Marek's disease virus to the Vero continuous cell line

Marek's disease virus (MDV) is a highly infectious, cell-associated oncogenic herpesvirus. Production of MD vaccines has been limited to primary chicken and duck embryo fibroblast (CEF and DEF) cultures. These have a limited life span and cannot be readily stored in liquid nitrogen. Moreover, the need to prepare CEF and DEF cells on a regular basis from 10 to 11 day-old embryos derived from a flock that must be tested continuously for the presence of avian pathogens adds to the cost of vaccine production. A continuous cell line that would support MDV replication could have significant advantages for the rapid large-scale preparation of MD vaccines. In this report, we describe the adaptation to growth of CEF-grown preparations of serotype 1 and serotype 3 (herpesvirus of turkeys; HVT) strains of MDV in cells of the Vero continuous cell line. Although both viruses produced typical CPE, higher levels of infectious progeny and more extensive virus-specific immunofluorescence were obtained for HVT than for the serotype 1 virus. PCR and pulsed field electrophoresis (PFE) analysis of the DNA from Vero cells infected with either virus confirmed the presence of virus-specific DNA.     

Cell-mediated cytolysis of lymphoblastoid cells expressing Marek's disease virus-specific phosphorylated polypeptides.

Cell-mediated immune responses against Marek's disease virus (MDV)-antigens were examined using reticuloendotheliosis virus (REV)-transformed lymphoblastoid cell line CU91 and three cell lines derived from CU91. CU210 was established by establishing a latent MDV infection in CU91. Transfection of CU210 with pNL1, a selectable plasmid or with pNL1 and the cloned BamHI A fragment of MDV DNA resulted in the establishment of CU212 and CU211, respectively. CU211 expressed a MDV-specific phosphorylated polypeptide, while CU210 and CU212 were negative for MDV antigens. Only CU211 was lysed by MDV-specific effector cells. All cell lines were lysed by syngeneic REV-specific effector cells, although high levels of expression of the phosphorylated protein reduced the level of REV-specific lysis.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Pathogenicity studies with plaque-purified preparations of Marek's disease virus strain CVI-988

The pathogenicity of Marek's disease (MD) strain CVI-988 vaccine, eight plaque-purified preparations originating from this strain, and the vaccine HVT FC126 (based on herpesvirus of turkeys) was determined by intramuscular administration of high virus doses to day-old specific-pathogen-free Rhode Island Red (RIR) chickens, which are extremely MD-susceptible. Paralysis and neuritis were observed in 88% of RIR chickens inoculated with MDV CVI-988 at the cell-passage level of the commercial vaccine. HVT FC126 caused paralysis in two of 39 RIR chickens tested, of which one had an endoneural lymphoma, and another three had endoneural inflammation. Five plaque-purified MDV CVI-988 virus preparations at various cell-culture-passage levels caused no lesions. Of another three clones, two caused inflammatory B-type lesions in the nerves of 1/10 chickens, and the third clone caused inflammatory nonneoplastic MD lesions in the liver of 1/11 chickens.     

In vivo characteristics of a transplantable Marek's disease lymphoblastoid cell line, MDCC-MSB1-41C

A Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, was highly transplantable and lethal for chickens. Autopsies showed extensive metastasis in various organs. The transplantabilities of the parent cell line, MDCC-MSB1, and another derivative line, MDCC-MSB1-33C, were transient. MD virus (MDV) could be isolated from the kidneys but not from the peripheral blood leukocytes of chickens inoculated with the MSB1-41C cell line. In addition, anti-MDV antibodies were produced both in chickens inoculated with this cell line and in controls raised with inoculated chickens, but several attempts to isolate MDV from this cell line in vitro failed.     

Cytotoxic T lymphocyte responses to Marek's disease herpesvirus-encoded glycoproteins

Cell-mediated immune responses are important for protective immunity to Marek’s disease (MD), especially because MD herpesvirus (MDV) infection is strictly cell-associated in chickens with the exception of the feather follicle epithelium. A system previously developed using reticuloendotheliosis (REV)-transformed cell lines stably expressing individual MDV genes allows the determination of relevant MDV proteins for the induction of cytotoxic T lymphocyte (CTL) responses. To examine the importance of glycoproteins for the induction of CTL, the MDV genes coding for glycoproteins (g) C, D, E, H, I, K, L, and M were stably transfected into the REV-transformed chicken cell lines RECC-CU205 (major histocompatibility complex (MHC): B²¹B²¹) and RECC-CU91 (MHC: B¹⁹B¹⁹). All transfected cell lines were lysed by REV-sensitized, syngeneic splenocytes obtained from MD-resistant N2a (MHC: B²¹B²¹) and MD-susceptible P2a (MHC: B¹⁹B¹⁹) chickens, indicating that the expression of individual MDV glycoproteins did not interfere with antigen processing pathways. Only cell lines expressing gI were recognized by CTL from both N2a and P2a MDV-infected chickens. Cell lines expressing glycoproteins gC and gK, and to a lesser extent, gH, gL, and gM were lysed by syngeneic MDV-sensitized splenocytes from N2a birds but not P2a birds. In contrast, gE was recognized by MDV-sensitized effector cells from the P2a line and not the N2a line. Glycoprotein D was not recognized by either line, with the exception of one marginally significant P2a assay. These results indicate that late viral glycoproteins are relevant for the induction of cell-mediated immunity during MDV infection.     

Resistance to Marek's disease herpesvirus-induced lymphoma is multiphasic and dependent on host genotype

Genotype-dependent differences in Marek's disease (MD) susceptibility were identified using 14-day-old line N and 6(1) (resistant) and 151 and 7(2) (susceptible) inbred chickens infected with HPRS-16 MD virus (MDV). All line 72 chickens developed progressive MD. Line 15I had fluctuating MD-specific clinical signs and individuals recovered. A novel histologic scoring system enabled indices to be calculated for lymphocyte infiltration into nonlymphoid organs. All genotypes had increased mean lesion scores (MLSs) and mean total lesion scores after MDV infection. These differed quantitatively and qualitatively between the genotypes. Lines 6(1) and 7(2) had a similar MLS distribution in the cytolytic phase, although scores were greater in line 7(2). At the time lymphomas were visible in line 7(2), histologic lesions in line 6(1) were regressing. AV37+ cells were present in similar numbers in all genotypes in the cytolytic phase, suggesting that neoplastically transformed cells were present in all genotypes regardless of MD susceptibility. After the cytolytic phase, AV37+ cell numbers increased in lines 7(2) and 15I but decreased in lines 6(1) and N. In the cytolytic and latent phases, in all genotypes, most infiltrating cells were CD4+. After this time, line 7(2) and 15I lesions increased in size and most cells were CD4+; line 6(1) and N lesions decreased in size and most cells were CD8+. In all genotypes, AV37 immunostaining was weak in lesions with many CD8+ cells, suggesting that AV37 antigen expression or AV37+ cells were controlled by CD8+ cells. The rank order, determined by clinical signs and pathology, for MD susceptibility (highest to lowest) was 7(2) > 15I > 6(1) > N.     

Autosomal trisomy 20 (61,XX,+20) in a malformed bovine fetus.

A 240-day-gestation female bovine fetus with severe anasarca, palatoschisis, cheiloschisis, mild cranioschisis, and a flattened facies was collected at a slaughterhouse, and a fibroblast line was established from the fetal skin. Chromosome preparations were Q-banded, and chromosome counts were taken that indicated the presence of 61 chromosomes in cells of the fetus (the normal diploid number for domestic cattle is 60). Q-band karyotypes were constructed, and Q-band analysis revealed the presence of three copies of chromosome 20. Trisomy 20 (61,XX,+20) was confirmed through the use of two-color fluorescence in situ hybridization of bovine bacterial artificial chromosome clones that were specific to chromosome 20 and the X chromosome.     

Comparison of blood and feather pulp samples for the diagnosis of Marek's disease and for monitoring Marek's disease vaccination by real time-PCR

Comparison of blood and feather pulp (FP) samples for the diagnosis of Marek's disease (MD) and for monitoring Marek's diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Marek's disease virus (MDV) DNA load was evaluated in 21 chickens suffering from MD. For each chicken, samples of blood and FP were collected and MDV DNA load was quantified. Solid tumors are the sample of choice for MD diagnosis by real time-PCR and, hence, 14 solid tumors were included in the study as positive controls. Load of MDV DNA in FP was equivalent to that detected in solid tumors (threshold cycle [Ct] ratio above 1.7). MDV DNA load in blood samples was lower than in solid tumors and FP samples. Nonetheless, there was a statistically significant correlation of the results obtained from FP and blood (r = 0.92). Results of the Pearson correlation test showed that Ct ratio values of 1.7 in FP correspond to Ct ratio values of 1.2 in peripheral blood. For monitoring vaccines, serotypes 2 and 3 MDV DNA load was evaluated in blood and FP samples of vaccinated chickens. Serotype 2 MDV DNA load was evaluated in samples of blood and FP from 34 chickens vaccinated with SB-1 strain. Serotype 3 MDV DNA load was evaluated in blood and FP samples from 53 chickens vaccinated with HVT strain. For both serotypes, frequency of positive samples and load of vaccine DNA was higher in FP than in blood samples. There was not a statistically significant correlation between the load of SB-1 DNA (r = 0.17) or HVT DNA (r = -0.04) in FP and blood. Our results show that the load of serotypes 1, 2, and 3 DNA is higher in FP than in blood. Diagnosis of MD could be done using both FP and blood samples. Monitoring of MD vaccination by real time-PCR required the use of FP samples. There were a high percentage of false negative samples when using blood to detect serotypes 2 and 3 MDV by real time-PCR.     

Effects of vaccine dose, virus challenge dose and interval from vaccination to challenge on protection of broiler chickens against Marek's disease virus challenge

OBJECTIVES: To examine the effects of varying the doses of turkey herpesvirus (HVT) vaccine and Marek's disease virus (MDV) challenge at two intervals after vaccination on the protection of chickens against challenge with MDV. DESIGN AND PROCEDURE: Experiment 1, a dose response study, consisted of 11 doses of HVT vaccine administered at hatch followed by challenge with 100 plaque forming units (pfu) of MDV 5 days post vaccination. Experiment 2, a 2 x 6 x 2 factorial design, included two HVT vaccine types, six different doses of HVT vaccine and 50 pfu and 200 pfu of MDV challenge 2 days post vaccination. All chickens were reared up to day 56 post challenge when all survivors were killed humanely. Dead and killed chickens were examined for gross MD tumours. RESULTS: Experiment 1 showed a significant positive linear relationship between dose of HVT vaccine and protective index in chickens challenged 5 days post vaccination. However the range of protective index observed was limited. In Experiment 2 neither HVT vaccine provided significant protection at any dose. There was no significant effect of vaccine type or MDV challenge dose on overall protection against challenge. Chickens challenged with 200 pfu of MDV had significantly higher mortality and MD incidence than those with 50 pfu. CONCLUSIONS: HVT vaccine dose had a significant impact on protective index, but vaccination to challenge interval appeared to have greater impact on the protective efficacy of vaccination. A fourfold increase in challenge dose increased mortality rate and incidence of MD.     

Identification of the cross-reactive antigens between Marek's disease virus and pseudorabies virus by monoclonal antibodies

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.     

细菌人工染色体及其在马立克氏病病毒研究中的应用

简要介绍了细菌人工染色体（Bacterial artificial chromosome,BAC）载体及其修饰技术,重点综述了BAC在马立克氏病病毒基因功能、疫苗研究中的应用。细菌人工染色体是近十几年发展起来的一种新型DNA克隆载体系统,具有操作简单、遗传稳定、容量大等明显的优势,主要用于构建基因组文库、转基因动物模型、分子克隆化病毒等。     

Study on Oocyte Nuclear Factor Promotes the Early Development of Tetraploid Somatic Cell Nuclear Transfer Embryos

The study was aimed to investigate the role of porcine oocyte nuclear factors during reprogramming. Somatic cell nuclei was introduced into intact MⅡ oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. And then the influence of the oocyte nucleus on tetraploid SCNT embryo development was examined by assessing characteristics including cleavage rate and blastocyst rate. The results showed that the cleavage rate of tetraploid SCNT embryos,diploid parthenogenetic embryos and haploid parthenogenetic embryos was extremely significantly higher than that of standard diploid SCNT embryos (P<0.01). The blastocyst rate and the total number of cells in tetraploid SCNT embryos were extremely significantly higher than that of standard diploid SCNT embryos (P<0.01).Overall,tetraploid SCNT embryos had a higher developmental competence than standard diploid SCNT embryos. In conclusion, the embryonic model was established in which a fetal fibroblast nucleus and an oocyte M Ⅱ plate coexist. Tetraploid SCNT represented a new research platform that was potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.     

Expression of Marek's disease virus phosphorylated polypeptide pp38 produces splice variants and enhances metabolic activity

The phosphorylated polypeptide (pp)38 of oncogenic Marek's disease (MD) herpesvirus (MDV) is expressed during lytic infections in vivo and in vitro, but its functions have not been fully elucidated. The quail cell line QT-35, latently infected with MDV, was used to generate QTP32 in which pp38 is expressed under control of a tetracycline controlled promoter to examine possible functions of pp38. Induction of pp38 did not influence late MDV genes expression, but it enhanced mitochondrial dehydrogenase activity significantly. Two new pp38 splice variants were found in induced QTP32 cells, in additional in vitro systems and MDV-infected chickens. Differential expression of full-length pp38 and splice variants suggests that the splice variants are important during latency and perhaps transformation. Polypeptides of 40 and 20kDa were detected by Western blot using monoclonal antibody H19. These polypeptides were also produced in DF-1 cells transfected with a pp38 construct in which the splice acceptor sites had been mutated. Our results add important new information to the role of pp38 in the pathogenesis of MD. The data suggest that pp38 and the two newly described splice variants may influence metabolic activity, which may have important consequences for the understanding of latency and tumor development.     

Effect of diluting Marek's disease vaccines on the outcomes of Marek's disease virus infection when challenged with highly virulent Marek's disease viruses

Dilution of Marek's disease (MD) vaccines is a common practice in the field to reduce the cost associated with vaccination. In this study we have evaluated the effect of diluting MD vaccines on the protection against MD, vaccine and challenge MD virus (MDV) kinetics, and body weight when challenged with strains Md5 (very virulent MDV) and 648A (very virulent plus MDV) by contact at day of age. The following four vaccination protocols were evaluated in meat-type chickens: turkey herpesvirus (HVT) at manufacturer-recommended full dose; HVT diluted 1:10; HVT + SB-1 at the manufacturer-recommended full dose; and HVT + SB-1 diluted 1:10 for HVT and 1:5 for SB-1. Vaccine was administered at hatch subcutaneously. One-day-old chickens were placed in floor pens and housed together with ten 15-day-old chickens that had been previously inoculated with 500 PFU of either Md5 or 648A MDV strains. Chickens were individually identified with wing bands, and for each chicken samples of feather pulp and blood were collected at 1, 3, and 8 wk posthatch. Body weights were recorded at 8 wk for every chicken. Viral DNA load of wild-type MDV, SB-1, and HVT were evaluated by real time-PCR. Our results showed that dilution of MD vaccines can lead to reduced MD protection, reduced relative body weights, reduced vaccine DNA during the first 3 wk, and increased MDV DNA load. The detrimental effect of vaccine dilution was more evident in females than in males and was more evident when the challenge virus was 648A. However, lower relative body weights and higher MDV DNA load could be detected in chickens challenged with strain Md5, even in the absence of obvious differences in protection.     

卵母细胞核因子促进核移植四倍体胚胎早期发育研究

研究旨在探讨猪卵母细胞核因子在重编程过程中发挥的作用。将体细胞引入未去核的MⅡ期卵母细胞中,构建体细胞核与卵母细胞核共存的核移植四倍体胚胎。通过分析核移植四倍体胚胎的早期发育情况探讨卵母细胞核因子对核移植四倍体胚胎早期发育的影响。结果显示,核移植四倍体胚胎、孤雌二倍体胚胎及孤雌单倍体胚胎这3组胚胎的卵裂率极显著高于核移植二倍体胚胎（P<0.01）,且核移植四倍体囊胚率及总细胞数也极显著高于核移植二倍体囊胚（P<0.01）。与通过标准核移植程序构建的核移植二倍体胚胎相比,核移植四倍体胚胎具有更强的发育能力。本研究建立了一个体细胞核与完整卵母细胞核因子物质共存的四倍体胚胎模型,有助于研究供体核与卵母细胞核之间的联系,为研究核因子在重编程过程中发挥的作用提供了平台。     

Load of challenge Marek's disease virus DNA in blood as a criterion for early diagnosis of Marek's disease tumors

Outbreaks of Marek's disease (MD) in vaccinated flocks still occur sporadically and lead to economic losses. Unfortunately, adequate methods to predict MD outbreaks are lacking. In the present study, we have evaluated whether high load of challenge MD virus (MDV) DNA in peripheral blood could aid in the early diagnosis of MD and in monitoring efficacy of vaccines against MD. One experiment was conducted to simulate field conditions by combining various vaccines (turkey herpesvirus [HVT] and HVT + MDV serotype 2 [SB1]) and challenge viruses (GA, Md5, and 648A). Vaccine efficacy among our experimental groups ranged from 13.3% to 94.2%. Each chicken was sampled three times during the length of the experiment (3, 5, and 15 wk postchallenge [wpc]), and gross lesions were evaluated in chickens that died and at termination of the experiment. DNA was extracted from whole blood and buffy coats from each sample, and the load of challenge MDV DNA and HVT DNA were quantified by real-time polymerase chain reaction. Chickens that developed MD by the end of the experiment had higher load of challenge MDV DNA (threshold cycle [Ct] glyceraldehyde-3-phosphate dehydrogenase [GAPDH]/Ct glycoprotein B [gB] ratios of 1.0, 1.04, and 1.05 at 3, 5, and 15 wpc, respectively) than those that did not develop MD (Ct GAPDH/Ct gB ratios of 0.7, 0.69, and 0.46 at 3, 5, and 15 wpc, respectively). However, load of HVT DNA in blood was not correlated with the development of tumors (Ct GAPDH/Ct HVT ratios from 0.04 to 0.10 in both groups). Vaccinated groups with >75% protection had statistically significant less challenge DNA virus (Ct GAPDH/Ct gB ratios of 0.76, 0.70, and 0.45 at 3, 5, and 15 wpc, respectively) than less protected groups (Ct GAPDH/Ct gB ratios of 0.92, 0.97, and 0.85 at 3, 5, and 15 wpc, respectively). No differences in the load of HVT DNA could be found between protected and nonprotected groups at any time point of the study (Ct GAPDH/Ct HVT from 0.05 to 0.09 in both groups). Our results showed that load of challenge MDV DNA but not load of HVT DNA in blood can be used as criterion for early diagnosis of MD.     

Effects of IgY antibody on the development of Marek's disease.

The effects of passive immunization with immunoglobulin Y (IgY) on the pathogenesis of Marek's disease (MD) were examined in an experimental line of White Leghorn chickens highly susceptible to MD. Purified IgY with anti-MDV antibody activity, when injected into chicks, delayed the development of MDV viremia and lesions until 9 days postinoculation (PI) with Marek's disease virus (MDV). The blastogenic response of spleen cells to concanavallin-A was depressed at 6 days PI in the birds without passive immunization, whereas it was not totally depressed until 17 days in birds passively immunized with IgY anti-MDV antibody.     

Identification of virus-specific antigens in cultured cells infected with Marek's disease virus serotype 2 and CVI-988 strain

For the identification of serotype-specific antigens of Marek's disease virus (MDV) serotype 1 (MDV1) or serotype 2 (MDV2), a total of 24 hybridoma clones, secreting monoclonal antibodies (MAbs) against CVI-988 (MDV1) or HPRS-24 (MDV2) strain, were established and characterized by immunofluorescence assay, virus neutralization and immunoprecipitation analysis. Based upon the molecular weights (mol. wt.) of the immunoprecipitated polypeptides, the MAbs were subdivided into 7 groups. Among them, two groups of MAbs reacted with antigens that have not been reported, were identified. MAbs belonging to the first group reacted with CVI-988- and MDV2-specific antigens with mol. wt. ranging from 29 K to 34 K (29/34 K). This antigen was not found in cells infected with Md/5 and JM strains of MDV1, and the results of kinetic analysis of antigen expression showed this antigen appeared to be related to late membrane antigens. MAbs belonging to the second group immunoprecipitated MDV2-specific antigens with mol. wt. of 37 K, 33 K and 31 K from HPRS-24-infected cells or with those of 37 K, 34 K and 31 K from SB-1(MDV2)-infected cells, and these antigens appeared to be related to early antigens. MAbs belonging to the other 5 groups included those which recognized similar antigens reported previously or the antigens characterized insufficiently in this study.