Restriction enzyme map of herpesvirus of turkey DNA and its collinear relationship with Marek's disease virus DNA

The genome of herpesvirus of turkey (HVT) was shown to consist of long and short unique regions flanked by inverted repeats (J. Cebrian, Kaschka-Dietrich, C., Berthelot, N., and Sheldrick, P., 1982, Proc. Natl. Acad. Sci. USA 79, 555-558). In this paper we report the construction of the linkage map of HVT DNA for BamHI, HindIII, and PstI restriction endonucleases. The maps were constructed by hybridization of 19 cloned BamHI fragments of HVT DNA to electrophoretically separated digests of genomic DNA. Our results indicate that the terminal and internal inverted repeats (TRL and IRL) flanking the long unique sequences (UL) are spanned by BamHI-F fragment and a -F-related terminal fragment, respectively, whereas the terminal and internal inverted repeats (TRS and IRS) flanking the short unique sequences (US) are mostly contained in BamHI-A fragment. Both BamHI-A and -F showed a heterogeneity in size, suggesting the presence of amplification of certain sequences in the inverted repeats. We also report that the HVT genome is collinear with the genetically related Marek's disease virus (MDV) genome, as determined by hybridization of labeled cloned HVT DNA fragments with electrophoretically separated MDV DNA fragments.     

Comparative studies on Marek's disease virus and herpesvirus of turkey DNAs.

DNA of Marek's disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between the two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek's disease, appeared similar to DNA of the vaccine strain in the size buoyant density and in its restriction endonuclease cleavage pattern.     

Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.

We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Marek's disease virus in feather tips of chickens, and this has been used clinically to monitor a flock's response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.     

Differentiation between strains of Marek's disease virus and turkey herpesvirus by immunofluorescence assays

Two serological types of Marek's disease virus and a herpesvirus of turkeys have been differentiated by indirect immunofluorescence tests as (1) pathogenic strains of Marek's disease virus (MDV) and their attenuated variants: HPRS-16, HPRS-16/att, HPRS-B14, JM, JM/att, GA, VC and 'Oldenburg', a recent field isolate; (2) apathogenic strains HPRS-24 and HPRS-27 of MDV; (3) herpesvirus of turkeys strain FC126 and its HVT(A-) variant. Virus strains could not be distinguished on the basis of qualitative differences in immunofluorescent staining of intracellular virus-induced antigens. Results were similar whether chicken kidney, chicken embryo fibroblast or duck embryo fibroblast cell cultures were used. Fluorescence of virus-induced antigens was stronger with homologous than with heterologous antisera. Using the direct immunofluorescence technique Marek's disease virus and turkey herpesvirus infections could be distinguished. There were never any significant differences in the appearance and distribution of antigen in infected cells treated with homologous or heterologous antisera at dilutions of comparable activity using the indirect immunofluorescence technique. Antibody titres of antisera were 4 to 8-fold higher in the indirect immunofluorescence test against the homologous virus-induced antigens than against heterologous antigens. Cross-reactions between the 3 serological types could be prevented by absorption of antisera with the appropriate antigens. Cross-reactions could also be prevented by the appropriate dilution of antisera before use in the indirect immunofluorescence test.     

A method for partial purification of Marek's disease herpesvirus A-antigen from feather follicles of infected chicken

A method for partial purification of Marek's disease herpesvirus A antigen from feather follicles of infected chickens is described. The A antigen partially purified by pH 4 treatment and affinity chromatography when analyzed in polyacrylamide gel electrophoresis was found to be a mixture of 7 Coomassie blue stained fractions.     

Isolation of chicken anaemia agent and Marek's disease virus from chickens vaccinated with turkey herpesvirus and lesions induced in chicks by inoculating both agents

A disease that was characterised by high mortality, necrotic lesions, severe thymic and bursal atrophy, as well as Marek's disease (MD) lesions, occurred in two young layer chicken flocks vaccinated with turkey herpesvirus (HVT) at hatching. An agent similar to the chicken anaemia agent, designated CAA82-2, and a virulent MD virus strain, designated Md82-2, were isolated from the kidneys of affected chickens. Dual inoculation of young chicks with both agents resulted in early mortality associated with severe necrosis and depletion of lymphocytes in the lymphoid organs and aplasia of the bone marrow. The similarity of this disease induced in chicks to an early mortality syndrome caused by variant MD virus strains was discussed.     

Further study on the three-dimensional structure of the core of Marek's disease virus and herpesvirus of turkey

Summary Three-dimensional structures of the core of Marek's disease virus and herpesvirus of turkey were examined by the tilting apparatus of an electron microscope. Various types of the core found in the infected cells were considered to represent developmental stages of the viruses. The basic structure of the core consisted of a toroid surrounding a cylindrical mass, which was clearly demonstrated by tilting the core in two directions. A cylindrical mass spooled by more than two toroids, which seemed to constitute a spiral band of 10 to 20 nm, was demonstrated. The maturation process of the cores of the viruses was also discussed.With 7 Figures     

Detection of Marek's disease virus DNA in chicken but not in human plasma

Marek's disease virus (MDV) causes a common lymphomatous and neuropathic disease in domestic chickens and, less commonly, turkeys and quail. It is a member of the alpha-herpesviruses and until now was considered to be strongly cell associated. In 1991, MDV was suggested to be the causative infectious agent of multiple sclerosis (MS) in humans. In a previous study, we investigated the leukocytes of 107 well-defined MS patients for the presence of MDV DNA but were unable to confirm a role for MDV in the pathogenesis of MS. A recent report (S. Laurent, E. Esnault, G. Dambrine, A. Goudeau, D. Choudat, and D. Rasschaert, J. Gen. Virol. 82:233-240, 2001) described the detection of MDV DNA in 20% of 202 human serum samples, regardless of whether the individuals were exposed to poultry. The detection of MDV DNA in chicken serum samples was reported as well. The aim of the present study was to investigate whether we can confirm the presence of MDV DNA in chickens and humans if we use plasma as the source for nucleic acid isolation. Leukocytes and plasma specimens from 16 chickens experimentally infected with MDV serotype 1 and plasma specimens from 300 volunteer blood donors were tested for MDV DNA by two different TaqMan PCR assays. MDV DNA was repeatedly found in the leukocytes as well as in the plasma specimens of all 16 animals. All human samples analyzed, however, tested negative by both assays. Accordingly, Marek's disease in chickens can be diagnosed by detection of MDV DNA in plasma as well as in leukocytes. Once again, we found no evidence for the spread of MDV to humans.     

The feather tips of commercial chickens are a favorable source of DNA for the amplification of Marek's disease virus and avian leukosis virus,subgroup J

Marek's disease virus (MDV), a herpesvirus, and avian leukosis virus, subgroup J (ALV-J), a retrovirus, are oncogenic viruses of poultry. The present report describes a case-report study aimed at examining the efficacy of amplifying MDV and/or ALV-J from feather-tip DNA as compared with DNA purified from liver and spleen. We show that the polymerase chain reaction for MDV and ALV-J env using DNA from feather tips was more effective for diagnosis of naturally infected commercial chickens than using the liver and spleen.     

Relation between common antigen and membrane antigens associated with Marek's disease herpesvirus and turkey herpesvirus infections

Summary The appearance of two kinds of membrane antigen (MA) in Marek's disease herpesvirus (MDHV) or herpesvirus of turkey (HVT) infected cells was examined using antiserum to the common antigen (common-Ag). No early-appearing MA (EMA) was detected in these cultures until 16 hours post-inoculation, after which a number of cells in these cultures had late-appearing membrane antigen (LMA) as detected by immunofluorescence (IF). Fluids from cultures infected with these viruses were examined for the presence of the common-Ag by agar-gel precipitation (AGP) test. No common-Ag was detected until 16 hours, but after 24 hours, it was detected by AGP test.These results suggest that the common-Ag is related to LMA but not to EMA.With 2 Figures     

Use of lectins to detect and differentiate subtypes of Marek's disease virus and turkey herpesvirus glycoproteins in tissue culture

Biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex were used for the first time to reveal glycoproteins in chicken and duck embryo fibroblasts infected with three prototype members of the avian herpesvirus group, Marek's disease virus serotypes 1 and 2 and turkey herpesvirus. By using a panel of 10 lectins, a pattern of reactivity emerged which was both group- and type-specific. Morphological details of the lectin-stained cells include cytoplasmic granulation, capping and bleb-like protrusions of the cell membrane. Although no antibody is necessary for the reaction, this novel approach allows specific detection of the glycan moieties of viral glycoproteins as they are synthesized during infection.     

Kinetics of Marek's disease virus (MDV) infection in broiler chickens 1: effect of varying vaccination to challenge interval on vaccinal protection and load of MDV and herpesvirus of turkey in the spleen and feather dander over time

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Marek's disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Marek's disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Marek's disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from ～40% to ～80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log₁₀MDV load in the spleen (vaccinated, 2.99±0.20/10⁶ spleen cells; unvaccinated, 4.60±0.23/10⁶ spleen cells) and dander (vaccinated, 5.28±0.13/mg; unvaccinated, 6.00±0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.     

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.     

Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek's disease virus but fails to increase protection against virulent challenge.

As Marek's disease virus continues to evolve towards greater virulence, more efficacious vaccines will be required in the future. We expressed chicken interleukin-2 (IL-2) from a turkey herpesvirus (HVT) in an attempt to increase the efficacy of HVT as a vaccine against Marek's disease. The recombinant IL-2/HVT was safe for in ovo vaccination, although it replicated less in the birds compared with the parent HVT strain. Expression of IL-2 increased the neutralizing antibody response against HVT but did not increase the protection against virulent Marek's disease virus challenge.     

Replication of herpesvirus DNA. VI. Virions containing either isomer of pseudorabies virus DNA are infectious

Pseudorabies virus DNA exists in two isomeric forms in which the short unique sequence is present in two orientations with respect to the long unique sequence. The viral DNA present in the virions of 21 individual plaques was analyzed. In all cases, equimolar amounts of the two isomeric forms of the DNA were present, indicating that isomerization of the DNA is a rapid process which is complete by the time a small plaque (<2 mm) has developed. Virions containing either isomeric form of the DNA adsorb equally well to cells and either isomeric form of the DNA has the same likelihood of becoming associated with the cell nucleus and to form circles (or concatemers) before initiation of DNA synthesis. The two isomeric forms of viral DNA are also equally represented in the genomes that mature first from concatemeric replicating DNA in cells infected at low multiplicities (0.01 PFU/cell). Furthermore, during the first round of DNA replication in cells infected at low multiplicity, equimolar amounts of the two isomeric forms of the DNA replicate. Since, in this experiment, each cell was infected with a maximum of one viral particle per cell, we conclude that virions containing either isomeric form of the DNA can initiate infection. Previous data (J. M. DeMarchi, T. Ben-Porat, and A. S. Kaplan, 1979, Virology97,457–463) have indicated that each cell, in which infection is initiated, is able to produce a plaque. We therefore conclude that virions containing either isomeric form of the DNA are infectious.     

Depression of vaccinal immunity to Marek's disease by infection with reticuloendotheliosis virus.

The effect of infection with low-virulence, tissue culture-propagated strains of reticuloendotheliosis virus (REV) on protective vaccinal immunity against Marek's disease (MD) lymphomas was investigated. Vaccinated chickens inoculated at hatching with greater than 10(4) focus-forming units of REV and challenged with MD virus were poorly protected against MD lesion development as indicated by protective indices of 53 to 79% for strain CS (P less than 0.05) and 42 to 49% for strain T (P less than 0.01) compared to 78 to 100% for REV-free controls. Furthermore, the response of blood lymphocytes to mitogen stimulation and the antibody response to sheep erythrocytes and Brucella abortus were less in REV-inoculated chickens than in controls. The REV-induced depression of immune responses was more severe in chickens infected with mildly pathogenic strain T than in chickens infected with the apathogenic strain CS and was generally transient with both virus strains. Little or no depression of immune responses was observed in chickens inoculated with less than 10(3) focus-forming units of REV. These studies extend knowledge on the immunodepressive ability of low-virulence REV strains and establish that infection with these viruses depresses certain parameters of MD vaccinal immunity, an important model for cellular immunity against virus-induced neoplasia in the chicken.     

Effect of reticuloendotheliosis virus and infectious bursal disease virus on Marek's disease herpesvirus latency

Birds infected with reticuloendotheliosis virus (REV) were exposed to Marek's disease virus (MDV) to determine if the establishment of MDV latency was affected by REV-induced immunosuppression, while other chickens, already latently infected with MDV, were challenged with REV or infectious bursal disease virus (IBDV) to determine if the consequent immunosuppression caused a return to cytolytic infection. Immunosuppression was assessed by in vitro mitogen stimulation assays with spleen cells. Latently MDV-infected cells were free of viral internal antigen(s) (VIA) but could be identified by their ability to produce VIA after in vitro cultivation. The results were unexpected: chickens infected with either of these viruses had very low, and often undetectable, levels of MDV infection when compared with appropriate controls. REV infection interfered with early cytolytic MDV infection, and IBDV and REV both failed to activate latent MDV infection in the face of inferred (IBDV) or demonstrated (REV) immunosuppression by these viruses. Apparently, both viruses reduced the number of MDV infected cells since neither cytolytic nor latent infection could be demonstrated. This was based on an absence of cells with VIA either before or after cultivation and, in the case of REV infection, on failure to detect MDV-DNA using a dot-blot hybridisation technique.     

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.