Use of recombinant pp38 antigen of Marek''s disease virus to identify serotype 1-specific antibodies in chicken sera by Western blotting

A fowlpox recombinant expressing the pp38 antigen of Marek's disease virus has been constructed. Production of pp38 in chick embryo fibroblasts (CEF) infected at a m.o.i. of 1 pfu/cell occurred over a period of 5 days and reached a peak at 72 h after infection. The pp38 antigen could be released from infected cells by freezing and thawing. Western blot analysis showed that denatured pp38 antigen reacted with antisera from chickens inoculated with serotype 1 MDV but failed to react with antisera from chickens inoculated with MDV serotype 2 or HVT. The results suggest that MDV pp38 contains a serotype 1-specific epitope which becomes available upon denaturation of the antigen and that this could be exploited to identify MDV-specific antibodies in epidemiological studies. The relationship between pp38 and the related polypeptides pp24 and pp41 in MDV-infected cells was also examined. The results suggest that pp24 and pp38 are synthesised independently and that MDV coded proteins (probably a protein kinase) might be required to convert pp38 to pp41.     

A mass spectrometry-based proteomic approach to study Marek's Disease Virus gene expression

Marek's Disease Virus (MDV) is an avian herpesvirus that causes a lymphoproliferative disorder in chickens. MDV transitions between a lytic phase in which new viruses are produced and a latent phase in which the virus lays dormant. The mechanism controlling this lytic-to-latent switch remains unclear. To better understand the lytic phase of MDV infection, a mass spectrometry-based strategy was developed to identify viral proteins and to qualitatively examine their abundance in lytically infected chicken embryo fibroblast (CEF) cells. A combination of strong cation exchange chromatography (SCXC) and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (murpLC/MS/MS) was used to resolve peptides from tryptic digests of MDV-infected CEF cell lysates. Peptides were identified by searching the tandem mass spectra against a protein database containing both MDV proteins and all currently available Gallus gallus proteins using the SEQUEST algorithm. A total of 427 MDV peptides, corresponding to 82 unique proteins, were identified, with 56 of them detected with at least two unique peptides. Overall, nearly 80% of all putative MDV proteins expressed in infected CEF cells were identified. We anticipate that this approach will be a viable method for determining how viral and host proteome changes occurring in Marek's Disease pathogenesis regulate the switch between the lytic and latent phases of the MDV life cycle.     

Antibodies against a novel nucleolar and cytoplasmic antigen (p105-p42) present in the sera of patients with a subset of rheumatoid arthritis (RA) with signs of scleroderma

We identified three patients (two of them relatives) with RA and signs of scleroderma whose sera contained a high titre of IgG class antibodies against the nucleoli and the nucleoplasm of cells of different mammalian origins. Sera from these patients uniformly immunoprecipitated four polypeptides, from a ³⁵S-methionine-labelled HeLa cell extract, whose mol. wts were 120, 105, 95 and 42 kD. Of these, the 95-kD protein was highly phosphorylated. By immunoblotting, these sera reacted with 105-, 95- and 42-kD proteins and affinity-purified antibodies from these, demonstrating that 105- and 95-kD proteins shared cross-reactive epitopes. Moreover, affinity-purified antibodies from each of these proteins immunoprecipitated the whole complex. Localization studies using immunoelectron microscopy and in vivo actinomycin-D-treated cells demonstrated that the 105-, 95- and 42-kD proteins were present in the granular component of the nucleolus and the nucleoplasm. In addition, the 105- and 95-kD were present in free polyribosomes as well as ribosomes attached to endoplasmic reticulum. Pulse/chase experiments strongly suggested that the complex was accomplished shortly after a 10-min pulse. It was preferentially present in the nucleus after a 2 h chase and in both nucleus and cytoplasm after a 5 h chase. We conclude that a protein complex with a main nucleolar distribution is a new autoantigen (p105-p42) recognized by autoantibodies present in the serum of a subgroup of patients with RA and scleroderma signs. These antibodies could be useful as diagnostic markers and as tools for further studies involving the biology of the nucleolus.     

Marek's disease virus serotype-1 antigens A and B and their unglycosylated precursors detected by western blot analysis of infected cells

The antigenic profile of cell cultures infected with Marek's disease virus (MDV) was determined by the immunoblotting method using convalescent immune serum obtained from chickens that survived infection with MDV strain GA5. The MDV antigen profile in infected cell lysates could be accurately determined since this method has advantages over the immunoprecipitation method used in other studies. We studied six very virulent MDV isolates and the prototype of serotype 1 MDV, the GA5 strain. Immunoblots of NaDodSO₄-polyacrylamide gel electrophoresis (PAGE) performed under reducing conditions revealed a main viral antigen (B) of 120–130 kD, which was present in all cell lysates infected with MDV isolates. Analysis of infected cell proteins by NaDodSO₄-PAGE performed under nonreducing conditions, revealed a 205 kD major MDV antigen, which, under reducing conditions, was identical to the 130 kD major antigen. The unglycosylated precursors of the major MDV antigens were elucidated. Two polypeptides of 43 and 45 kD were found to be the unglycosylated precursors of MDV antigen A (the glycosylated form of which appears in 4 distinct bands). The unglycosylated precursors of the MDV major antigen B were found to be three polypeptides of 80, 110, and 125 kD.     

Antigen B of the vaccine strains of Marek's disease virus and herpesvirus of turkeys presents heat-labile group and serotype specific epitopes

Summary Antigen B of Marek's disease virus (MDV) vaccine strains CVI988 and SB1 (serotypes 1 and 2) and herpesvirus of turkeys (HVT) (serotype 3) is formed of oligomeric molecules that are detergent-stable and heat-labile. Immunoblots of native membranal extracts of HVT- and MDV-infected chick embryo fibroblasts (CEF) probed with avian monoserotypic antisera, murine monoclonal antibodies (mAb) to the three serotypes and mAb to antigen B showed two distinct patterns of high molecular weight oligomeric antigens. Serotypes 1 and 3 vaccine viruses formed one set and serotype 2, the other. Avian monotypic sera to serotypes 1 and 3 viruses detected two high molecular weight bands of 230 and 300 kDa in MDV-1 and HVT-infected CEF but only a weak diffuse zone ranging from 130 to 230 kDa in extracts of SB1-infected CEF. No 300 kDa band was discernible in the SB1 extract when blotted with avian monotypic 1 and 3 antisera. MAbs to MDV serotypes 1 and 3 and to antigen B also detected the 230 and 300 kDa antigens, while the mAb to SB1 detected a 50 kDa antigen in the SB1-infected extract only. Furthermore, the antigen B mAb did not reveal high mol. wt. oligomers in SB1-infected CEF extracts. Antigen B oligomers were rapidly destroyed by heating at 95°C and the rate of denaturation of the 230 and 300 kDa oligomers differed for each of the three vaccine viruses. We propose that antigen B of MDV1 and HVT has a complex conformation created by juxtaposition of dimers (230–250 kDa) and trimers (300 kDa), and is inserted in the infected cell membrane so that conformational, discontinuous epitopes are formed in addition to continuous epitopes. It appears that HVT protects chickens against oncogenic strains of MDV1 by virtue of the cross reactivity of the conformational determinants located on these oligomers. Serotype 2 vaccine shares some of its antigenic determinants with serotypes 1 and 3, while its unique immunogenic features form the basis of the protective synergism achieved when serotypes 2 and 3 vaccines are combined together.     

The expression of Marek's disease tumour-associated surface antigen in various avian species

Marek's disease tumour-associated surface antigen (MATSA) was detected on lymphoid cells from chickens infected with Marek's disease virus (MDV) or the herpesvirus of turkeys (HVT), from a chicken-quail hybrid infected with MDV, and from turkeys infected with the GA strain of MDV, but not on cells from turkeys infected with HVT or the HPRS-16 strain of MDV or from MDV-infected Japanese quails or bobwhite quails, despite the presence of lymphomas in some Japanese quails. Two complementary explanations are offered for these observations/Firstly, certain combinations of virus strain and host species may not result in malignant transformation although infection has occurred; secondly, if, as seems likely, MATSA is a modified host antigen, antigens on malignant cells arising in a foreign host may not be recognised by an antiserum prepared against MATSA of chicken origin.     

Host responses are induced in feathers of chickens infected with Marek's disease virus

Control measures are ineffective in curtailing Marek's disease virus (MDV) infection and replication in the feather follicle epithelium (FFE). Therefore, vaccinated birds which subsequently become infected with MDV, shed the virulent virus although they remain protected against disease. The present study investigated host responses generated against MDV infection in the feather. We observed that in parallel with an increase in viral genome load and viral replication in the feather, there was a gradual but progressive increase in infiltration of CD4+ and CD8+ T cells into the feather pulp of MDV-infected chickens, starting on day 4 and peaking by day 10 post-infection. Concomitant with infiltration of T cells, the expression of interleukin (IL)-18, IL-6, interferon (IFN)-gamma and major histocompatibility complex class I genes was significantly enhanced in the feather pulp of MDV-infected chickens. The finding that host responses are generated in the feather may be exploited for developing strategies to control MDV infection in the FFE, thus preventing horizontal virus transmission.     

Development of a cell line system susceptible to infection with vaccine strains of MDV

Despite reliance on the need to continually prepare fresh cultures of chick embryo fibroblasts (CEFs) to make Marek's disease (MD) vaccines, MD vaccines are the most widely used vaccines in the poultry industry. Preparation of CEF's accounts for approximately 40% of the costs associated with producing MD vaccines. A significant reduction in MD vaccine production costs could be realized if a continuous cell lines were available for MD vaccine production. Recently, we reported development and characterization of a cell line system (OCL) that supports growth and replication of oncogenic serotype 1 Marek's disease virus (MDV). Here we report development of three cell line systems for production of MD vaccine. These cell lines support the growth and replication of attenuated serotype 1 MDV (CVI-OCL), serotype 2 MDV (SB1-OCL) and serotype 3 MDV (HVT-OCL). MDV is maintained in a stable state in the OCL cells and the infected cells can be continuously grown. The vaccines made from these cell lines are safe and protect White Leghorn chickens against challenge with very virulent serotype 1 MDV, similar to traditional vaccines made from CEF cells. These cell line systems can significantly reduce the costs associated with MD vaccine production. Furthermore, the increased stability of MDV and the potential for positive selection of recombinant MDV suggest that OCL will be ideal for production of more effective MDV vaccines using recombinant DNA technology.     

Molecular-biological characterization of Marek's disease virus. II. Differentiation of various MDV and HVT strains

Immunological cross-reactions were found between each of the four major virus-specific polypeptides of Marek's disease virus (MDV) strains CVI 988, K and HPRS-16/att, and herpesvirus of turkey (HVT) Fc126 by one-dimensional (1D) gel analysis of immunoprecipitates from the lysate or culture medium of infected cells. The results, however, did not allow a serotype classification of MDV and HVT strains. Comparison of the two-dimensional (2D) gel patterns of virus-specific polypeptides of nine MDV and HVT strains with different biological properties revealed many similarities. Strain CVI 988 provided the reference pattern, consisting of 35 polypeptides, 18 of which were glycosylated. Based on their similarities in migration characteristics (size, charge, and heterogeneity of spots) during 2D gel electrophoresis, 11 virus-specific polypeptides or polypeptide complexes were identified in the patterns of nearly all virus strains analyzed. One of these polypeptides was glycoprotein gp5, the putative A antigen of MDV and HVT, which was also detected in the medium of cells infected with HPRS-16/att, a strain which was reported to have lost its capacity for production of A antigen. In addition to the similarities mentioned above, differences were found in migration behavior of virus-specific polypeptides (complexes) p4/p5/p6, gp3, gp5, and gp8/gp9, which were confirmed by coelectrophoresis experiments. The most conspicuous difference was found between three patterns of gp5 which seem to be characteristic for three groups of viruses: (I) high- and low-virulent MDV strains and their attenuated variants; (II) avirulent and apparently nononcogenic MDV strains; (III) herpesviruses of turkey. The minor differences found for the other polypeptides mentioned above further substantiated this molecular-biological classification of MDV and HVT strains and, in addition, enabled the differentiation of two HVT strains within molecular-biological group III.     

Demonstration of a marek's disease virus-specific antigen in tumour lesions of chickens with marek's disease using monoclonal antibody against a virus phosphorylated protein

By use of monoclonal antibody against a Marek's disease virus (MDV) serotype 1-specific phosphorylated protein, MDV antigen-positive cells were demonstrated in tumour lesions of various visceral organs of chickens with Marek's disease. However, these tumour lesions did not appear to have the MDV glycoproteins gA and gB, which are considered to be late gene products of the virus genome gA and gB as well as the phosphorylated protein were detected in the feather follicle epithelium, which is a permissive site for MDV replication.     

Extracellular export of Shiga toxin B-subunit/ haemolysin A (C-terminus) fusion protein expressed in Salmonella typhimurium aroA-mutant and stimulation of B-subunit specific antibody responses in mice

The Shiga toxin B-subunit has been fused to the 23-kD C-terminus of Escherichia coli haemolysin A (HlyA) and exported from attenuated antigen carrier strain of Salmonella typhimurium aro A (SL3261). The expression of the gene fusion under the control of a synthetic modified β-lactamase promoter (constitutive expression) and under the iron-regulated aerobactin promoter showed that the fusion protein could be stably expressed and exported out of the bacterial cell in significant amounts so long as high copy number plasmids were not used. Oral and i.p. immunization of mice with the hybrid salmonellae resulted in significant B-subunit specific mucosal and serum antibody responses. A comparative analysis of the location of hybrid proteins in the antigen carrier bacterial cell (i.e. cytoplasmic expression and extracellular export) has shown that both modes of expression result in antigen-specific immune responses. This is the first report demonstrating that foreign polypeptides fused to the 23-kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella vaccine strains and that such exported polypeptides can result in antigen-specific immune responses.     

An in vivo system for analysis of stable complex formation between Src and AFAP-110

The actin filament-associated protein, AFAP-110, forms a stable complex with activated pp60src (Src) as both an SH2 and SH3 binding partner. AFAP-110 contains two putative SH3 binding motifs and six putative SH2 binding motifs, indicating that interactions with Src may be complex. In order to evaluate the mechanism by which AFAP-110 and Src form a stable complex, an in vivo expression system was utilized that permits co-expression of each protein from the same plasmid construct subsequent to transient transfection of Cos-1 cells. Sixty hours post-transfection, western blot analysis indicated that both Src and AFAP-110 were expressed efficiently. Furthermore, Src phosphorylated AFAP-110 on tyrosine and was in stable complex with this protein. These results reflect observations detected in chick embryo fibroblast (CEF) cells. This system has several advantages in that (a) it is rapid, (b) high levels of protein can be overexpressed, stable complex formation occurs, and all transfected cells have the potential of expressing both protein products, (c) proper protein folding and post-translational modifications are likely to occur, (d) mAbs directed against Src and AFAP-110 are specific for these avian cDNA gene products, and (e) the plasmid constructs are amenable to site-directed mutagenesis, allowing for rapid analysis of the mechanism of stable complex formation.Abbreviations CEF chick embryo fibroblasts - Src activated pp60^527F - cSrc repressed pp60^c-src - AFAP-110 actin filament-associated protein, 110 kDa - CMV cytomegalovirus - SV40 simian virus 40 - PVDF polyvinylidene fluoride - Ab F1 anti-AFAP-110 polyclonal rabbit - mAb 4C3 anti-AFAP-110 monoclonal antibody - EC10 anti-Src monoclonal antibody     

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein

The synthesis and processing of the nucleocapsid protein (pp60) of the JHM strain of murine coronaviruses were examined. Pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). Immunoprecipitation using mouse anti-JHMV antiserum indicated that p57 was virus specific. Immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. The p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein.     

Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens

Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek's disease virus (MDV) and were maintained under pathogen-free conditions from the day of hatch until 75 weeks of age. Spontaneous ALV-like bursal lymphomas, also termed lymphoid leukosis (LL)-like lymphomas, were detected in 7% of the ALVA6 breeders. There was no evidence of infection with exogenous and endogenous ALV as determined by virus isolation tests of plasma and tumour tissue homogenates. For the next three generations, serotype 2 MDV was eliminated from the trivalent MD vaccine used. Results show, for the first time, that removal of serotype 2 MDV from MD vaccines eliminated spontaneous LL-like lymphomas within 50 to 72 weeks of age for at least three consecutive generations. Two experiments were also conducted to determine the influence of in ovo vaccination with serotype 2 MD vaccines on enhancement of spontaneous LL-like lymphomas in ALVA6 chickens. Chickens from the 2012 generation were each inoculated in ovo or at hatch with 5000 plaque-forming units of serotype 2 MDV. Results indicate that by 50 weeks of age the incidence of spontaneous LL-like lymphomas in chickens inoculated in ovo with serotype 2 MDV was comparable with that in chickens inoculated with virus at hatch, suggesting that the augmentation effect of serotype 2 MDV is independent of age of vaccination.     

Molecular and biological characterization of a Marek’s disease virus field strain with reticuloendotheliosis virus LTR insert

A Marek’s disease virus (MDV) field strain designated GX0101 was isolated from a layer flock and confirmed to be a recombinant virus with an insert of a long terminal repeat (LTR) from the reticuloendotheliosis virus (REV). A chimeric molecule containing an REV-LTR insert of 539 bp and its flanking sequences from MDV was amplified and sequenced. An REV-LTR downstream from the Internal Repeat Short (IRS) region has 77.4–98.6% homology to seven REV field strains isolated from different avian species in different parts of the world. The insertion site is located downstream of SORF 1 and upstream of SORF2 in the IRS region near the junction with the Unique Short (US) region in the MDV serotype 1 genome. Chicken experiments were conducted to determine the oncogenicity of the recombinant GX0101 virus and its transmissibility to contact chickens. Dot blot hybridization was used to detect the presence of the pp38 gene in feather tips from GX0101 or Md5 infected and contact birds. The pp38 was detected in GX0101 contact birds about 1–2 weeks earlier than in Md5 birds when both groups were vaccinated with HVT vaccine. Long term pathogenicity tests in specific pathogen free (SPF) chickens reveal that the recombinant GX0101 has a higher virulence than GA, but less virulence than Md5, the very virulent pathotype of MDV. This is the first report on an oncogenic serotype 1 MDV field strain with LTR insert and its pathogenicity.