Electronic Library of Scientific Literature
Volume 43 / April 1999 / number 2-3
FEMS SUPPORTED WORKSHOP
August 8–11, 1998, Smolenice Castle, Slovakia
The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, 910 East 58th Street, Chicago, IL 60637, USA
Summary. – Herpes simplex virus 1 (HSV-1) encodes at least 84 polypeptides to perform two functions: to enable viral replication and to create the environment in which the entry of the virus into host cells, synthesis of virion components, assembly and egress are optimized. Whereas the former are indispensable for viral replication, the latter, numbering 47, can be deleted without a major effect on viral replication in cells in culture. Of particular interest are gene products whose function is either to modify cellular proteins (set 1) or to block entirely their function (set 2). An example of set 1 is the infected cell protein No. 0 (ICP0), a promiscuous transactivator of genes introduced into cells by infection or transfection. In its nuclear phase this protein binds to cyclin D3, extends its life by many hours, and sequesters it in nuclear structures known as ND10. In its cytoplasmic phase, ICP0 binds the translation elongation factor EF-1delta. Another viral protein, the UL13 protein kinase, hyper-phosphorylates EF-1 delta. ICP0 and the protein kinase stimulate protein synthesis and cause the cell to induce the synthesis of pre-S phase cellular proteins the virus needs for its replication. The gamma134.5 protein, a prototype of set 2, also has multiple functions. One, mapped at its carboxyl terminus, blocks the effects of double-stranded RNA-dependent protein kinase R (PKR) that is activated by all wild-type and mutant viruses examined to date. PKR phosphorylates eIF-2alpha and shuts off protein synthesis. gamma134.5 protein binds protein phosphatase 1 and redirects it to dephosphorylate eIF-2alpha. Although PKR is activated in wild-type-infected cells, protein synthesis is unaffected. HSV-1 encodes in addition at least two proteins, ORF O and ORF P that are repressed during productive infection. The ORF P protein localizes in spliceosomes and blocks the synthesis of viral proteins derived from spliced mRNA. The ORF O protein binds ICP4, the major regulatory protein, and prevents it from binding to DNA. The role of ORF O and ORF P proteins in the establishment of latency is uncertain. A significant discovery that has emerged from these studies is that viral proteins can perform several functions that may be totally unrelated.
Acta virologica 43: 75 – 80, 1999
BECKER, Y. ASHER, S. BUJANOVER, G. DARAI
Department of Molecular Virology, Faculty of Medicine, The Hebrew University of
Jerusalem, P.O. Box 12272, Jerusalem 91120, Israel;
Institut für Medizinische Virologie, Ruprecht Karls Universität Heidelberg, Heidelberg, Germany
Summary. – Herpesviruses evolved from an ancestral viral genome that contained five blocks of genes which provide the members of this family of viruses with structural and enzymatic properties. These genes allow the herpesviruses to infect a host by entering into the nuclei of the cells, the site of replication and transcription of the viral DNA. The viral mRNAs are released into the cell cytoplasm where synthesis of enzymatic and structural proteins occurs. The latter proteins are responsible for the formation of the infectious virions. Herpesviruses that were able to adapt to different hosts during the evolution of the species (speciation) had acquired additional genes from transposons or retrotransposons that allowed them to successfully maintain their hold in the specific vertebrate host. The present overview deals with molecular differences between Marek’s disease virus type 1 (MDV-1) and herpes simplex virus type 1 (HSV-1) and the specialized genes that differentiate MDV-1 from HSV-1, the promoters of the viral genes that control gene expression and the nuclear localization signals. Dynamic changes in the viral genomes that may occur during viral DNA replication and recombination and their effects on virus pathogenicity and genome evolution will be discussed.
Key words: MDV-1; HSV-1; DNA genome; evolution of herpesviruses; DNA
replication; DNA recombination
Acta virologica 43: 81 – 89, 1999
A. BARROW, K. VENUGOPAL
BBSRC Institute for Animal Health, Compton, Berkshire, RG20 7NN, United Kingdom
Summary. – Marek’s disease virus (MDV) strains with increasing virulence have been reported from many parts of the world. Many of these recent MDV isolates produce an acute early cytolytic disease with high mortality and severe atrophy of the lymphoid organs, thymus and the bursa of Fabricius. Although the degree of the atrophic changes and the virulence of the virus are correlated, the molecular basis of the increased virulence is not known. We examined the characteristics of the disease induced by 3 such MDV isolates, C12/130, MR36 and MR48, isolated from Europe. All the three viruses produce high early mortality and atrophy of the lymphoid organs. As a first step in understanding the determinants of the increased virulence of these isolates, we have compared the sequences of MEQ and the ICP4 genes of these three viruses with that of the published sequences. Some of the amino acid changes seen within the Meq and ICP4 proteins were conserved in all the three isolates and could account for the increased virulence characteristics.
Key words: MDV; pathotypes; MEQ; ICP4
Acta virologica 43: 90 – 93, 1999
J.-L. LIU, S.-F. LIN, L. XIA, P. BRUNOVSKIS, D. LI, I. DAVIDSON, L.F. LEE, H.-J. KUNG
Department of Molecular Biology and Microbiology, School of Medicine, Case Western
Reserve University, Cleveland, OH 44106-4960, USA;
Avian Disease and Oncology Laboratory, US Department of Agriculture, Agricultural Research Station, East Lansing, MI, USA;
Department of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel
Summary. – One of the hallmarks of oncogenic viruses is their ability to subvert the growth regulation and evade immune response of the host. There are a number of tricks devised by various virus families. Oncogenic herpesviruses often accomplish this by encoding homologs of cellular genes involved in these functions. These viral homologs sometimes are hyperactive forms of their cellular counterparts, which function to overtake the cellular pathways, other times serve as decoys to mask the cellular functions. Marek’s disease virus (MDV) carries at least two genes in that category. We have previously described Meq protein (MEQ gene product), a transcriptional factor with homology to proto-oncogenes Jun and Fos in the bZIP domain. Meq dimerizes with Jun or Fos and the Meq/Jun heterodimer is able to transactivate promoters with AP-1 site. We show here that Meq and Jun colocalize in living cells, adding to the physiological significance of the dimer formation. In addition, we present data to show that Meq and Jun can functionally complement each other in cis and in trans, using transformation and transactivation assays. Finally we describe the discovery of an IL8 chemokine homolog, designated as v-IL8 (viral IL8) in the MDV genome and discuss its possible function in MDV infection.
Key words: MDV; MEQ; IL-8; bZIP; chemokine; herpesviral oncogene
Acta virologica 43: 94 – 101, 1999
A.M. MILES, A.S. ANDERSON, E.L. BERNBERG, J. KENT, J.K. ROSENBERGER, C.R. POPE, R.W. MORGAN
Delaware Agricultural Experiment Station, Department of Animal and Food Sciences, College of Agricultural Sciences, University of Delaware, Newark, DE 19717-1303, USA
Summary. – We compared the RB1B and T. King (TK) serotype 1 isolates of Marek’s disease virus (MDV) in vivo. Body and organ weights, mortality, and lesions indicated that the TK inoculum established early infection more efficiently than RB1B and did greater damage to the bursa of Fabricius and thymus. Subsequent studies showed that the TK inoculum that we used contained chicken infectious anemia virus (CIAV). Therefore, pathogenicity profiles shown here should be interpreted with the presence of CIAV contamination in the TK stock in mind.
Key words: Marek’s disease; MDV; herpesvirus of turkeys; herpesvirus
Acta virologica 43: 102 – 105, 1999
R.L. DIENGLEWICZ, M.S. PARCELLS
Center of Excellence for Poultry Science, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA
Summary. – We have previously described the construction and characterization of mutant Marek’s disease viruses (MDVs) having mutations within the unique-short (US) region of the genome that have retained oncogenicity (Anderson et al., 1998; Parcells et al., 1995). We have also reported the characterization of lymphoblastoid cell lines (LBCLs) derived using these mutant viruses (Parcells et al., 1998). These mutant MDVs were constructed using a lacZ expression cassette. Expression of lacZ was found to be constitutive during lytic infection but was found to be tightly repressed in tumors and the derived LBCLs. The construction of these viruses and the analysis of lacZ induction required the use of toxic substrates or antibody staining to detect lacZ expression. We now report the establishment of an MDV lymphoblastoid cell line, MDCC-UA04, that was derived from a tumor induced by an MDV having an insertion of a green fluorescent protein expression cassette into the US2 gene. Like previous mutant-derived LBCLs, expression of the marker cassette is constitutive in lytic infection, but repressed in tumors and in the UA04 cells. UA04 cells express CD3low, CD4, TCR-2 low, MHC class II, and CD28 antigens on their surface. The percentage of UA04 cells expressing GFP is generally low (5–7%), but increases markedly within 48 hrs of 5'-iododeoxyuridine (IUdR) treatment (20–30%) in a manner similar to many MDV lytic antigens. Thus, induction of GFP expression in UA04 cells can serve as a non-invasive marker for MDV reactivation from latency.
Key words: MDV; mutants; cell lines; latency; mutagenesis
Acta virologica 43: 106 – 112, 1999
Z. XING, Q. XIE, R.W. MORGAN, K.A. SCHAT
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell
University, Ithaca, NY 14853, USA;
Department of Animal and Food Sciences, University of Delaware, Newark, DE, USA
Summary. – Monoclonal antibodies (MAbs) were prepared against ICP4 of Marek’s disease virus (MDV). Mice were inoculated with ICP4 obtained from High-Five insect cells infected with a recombinant baculovirus expressing ICP4. MAbs were selected by enzyme-linked immunosorbent assay (ELISA) using MDV-infected and control chick kidney cells as antigens. One of the MAbs, 5H8, recognized an epitope toward the carboxyl terminus of ICP4 based on staining of reticuloendotheliosis virus-transformed cells transfected with full-length and truncated ICP4 constructs. This MAb recognized ICP4 in chicken embryo fibroblasts (CEFs) infected with MDV strains JM16 and HVT but not with SB-1 strain. Using Western blot analysis a protein of 155 kDa was detected in CEFs infected with JM16 and HVT strains.
Key words: MDV; ICP4; monoclonal antibody
Acta virologica 43: 113 – 120, 1999
A.M. LEVY, E.D. HELLER, G. LEITNER, I. DAVIDSON
Department of Animal Science, Faculty of Agriculture, Hebrew University of Jerusalem,
P.O. Box 12, Rehovot 76100, Israel;
Kimron Veterinary Institute, Beit Dagan, Israel
Summary. – Marek’s disease virus (MDV) is an oncogenic alphaherpesvirus. Its specific phosphorylated protein, pp38 has been implicated in MDV oncogenesis. In order to check whether the known anti-viral or anti-proliferative actions of interferon (IFN) are of importance in Marek’s disease (MD), chicken embryo fibroblasts (CEFs) were infected with attenuated serotype-1 MDV, strain CVI988, or with herpesvirus of turkeys (HVT). Different concentrations of native chicken IFN were added to the cell cultures, prior to their infection. After incubation, MDV plaques were counted. Analysis by flow cytometry for pp38 expression was performed by using three monoclonal antibodies (MAbs) and for HVT by using an anti-glycoprotein B (gB) MAb. Increasing IFN quantities caused a reduction in a stepwise manner of plaque numbers as well as a suppression of pp38 and gB expression in the CVI988- and HVT-infected cells, respectively.
Key words: IFN; MDV; oncogenesis; herpesvirus
Acta virologica 43: 121 – 127, 1999
K. Ohashi, T. Morimura, M. Takagi, S.-I. Lee, K.-O. Cho, H. Takahashi, Y. Maeda, C. Sugimoto, M. Onuma
Laboratory of Infectious Diseases, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Summary. – To characterize the molecular events involved in both apoptosis and transformation process induced by Marek’s disease virus (MDV), the expressions of the bcl-2 and bcl-x genes, ones of the dominant apoptosis-regulating genes, in Marek’s disease (MD) tumor cell lines and cells prepared from MDV-infected chickens were analyzed. The expression of bcl-2 was down-regulated in both CD4+ and CD8+ T cells prepared from MDV-infected chickens at 3 weeks p.i. No bcl-2 transcript was detected in MD tumor-derived MSB1 and MTB1 cell lines, which had been established from primary MD tumors. On the other hand, the bcl-xL transcript whose product can also inhibit apoptosis was expressed in cell lines derived from MD. By the treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin, normal CD4+ T cells were induced to express bcl-xS which can promote apoptosis, while bcl-xL was constitutively expressed in MD cell lines. Our results suggest that bcl-xL rather than bcl-2 might play an important role in the transformation process by MDV.
Key words: apoptosis; bcl-2; bcl-x; MDV
Acta virologica 43: 128 – 132, 1999
D. EWERT , J. DUHADAWAY
The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA
Summary. – Strains of Marek’s disease virus (MDV), a herpesvirus, have been shown to augment the development of lymphoid leukosis induced by retroviruses, the avian leukosis virus (ALV) or the reticuloendotheliosis virus. In this study we explored the possibility that the ability to augment lymphoid leukosis may be correlated with the ability of different strains of MDV to block apoptosis. Subclones of the ALV-transformed B cell line, DT40, which was free of MDV DNA were infected with either R2/23 strain of MDV-1, SB1 strain of MDV-2, or turkey herpes virus (HVT), a MDV-3. We found that most of the normal DT40 cells and DT40 cells infected with the R2/23 became apoptotic when cultured in serum-reduced medium. By contrast, the frequency of apoptotic cells was greatly reduced in the DT40-SB1 and DT40-HVT subclones. These findings suggest that because the SB-1 strain persists in the ALV-infected cells, it may augment lymphoid leukosis by inhibiting apoptosis and providing a survival advantage to the B cells which have a deregulated myc proto-oncogene.
Key words: ALV; Marek’s disease; MDV; leukosis; apoptosis
Acta virologica 43: 133 – 135, 1999
I. DAVIDSON, R. BORENSTEIN
Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, P.O. Box 12, 50250 Israel
Summary. – The avian herpesvirus, Marek’s disease virus (MDV) and several retroviruses, reticuloendotheliosis virus (REV), avian leukosis virus (ALV) (chickens) and lymphoproliferative disease virus (turkeys) are oncogenic and immunosuppressive agents. These viruses were detected either alone, or in various combinations in blood and tumor DNAs of commercial birds using PCR. We present a 5-year retrospective study that included 207 chicken and 52 turkey flocks. Of these, 32 chicken and 18 turkey flocks were negative. Of the positive chicken and turkey flocks 76% and 75%, respectively, had a single, while the rest, 24% and 25%, had a multiple virus infection. In the chickens of the multiple virus-infected flocks, 14% and 17% of the blood and tumor DNAs carry dual MDV and REV and/or ALV sequences, that is about 30% of the PCR-positive, and about 5% of the total DNAs analysed. Multiple virus sequences were detected only in the turkey blood DNAs – 11% of 84 samples. Following that quantitation we aimed to analyse the molecular status of the retrovirus sequences in order to determine whether retrovirus sequences were integrated into the herpesvirus genome. We focused on the MDV BamH1-H 132 bp tandem repeat fragment proximity using a combined PCR (cPCR) to identify chimeric PCR products. That included amplification with heterologous combinations of the MDV and retroviral LTR primers. In 13 of 35 DNAs that had both MDV and retrovirus sequences new products were produced. Of 4 MDV+REV chimeric products that were sequenced, one was homologous to the Chicken Repeat element 1 non-LTR type retrotransposon. No evidence for a retrovirus LTR integration was found in the 132 bp repeat proximity, but in two of these products we detected nucleotide stretches of 20 bp and 21 bp with a 70% and 71% homology to the REV-LTR. Also, the amplification of the chimeric products using a retrovirus primer denoted that at least short nucleotide stretches homologous to retroviral LTR primer were present in these DNAs, and that they might resemble ancient retroviral insertions, as previously demonstrated (Isfort et al., 1992).
Key words: avian oncogenic viruses; PCR; herpesviruses; retroviruses;
molecular interactions; multiple virus infection
Acta virologica 43: 136 – 142, 1999
K. VOELCKEL, E. BERTRAM, I. GIMENO, U. NEUMANN, E.F. KALETA
Institut für Geflügelkrankheiten, Justus-Liebig-Universität, Frankfurter Str.
91, 35392 Giessen, Germany;
Klinik für Geflügel, Tierärztliche Hochschule, Hannover, Germany;
Departamento Patologia Animal II, Universidad Complutense de Madrid, Madrid, Spain
Summary. – Since 1994 tumorous lesions have been monitored in turkeys on three farms in Germany. On one of these farms, chickens also had tumorous lesions. Affected turkeys were retarded in growth, apathic, pale and almost unable to move. The older the animals got, the more indistinct the clinical signs became. Mortality started at an age of 5 weeks and reached between 20% and 60% by the end of the fattening period of about 20 weeks.The aetiological differential diagnosis includes reticuloendotheliosis (RE), lymphoproliferative disease (LPD), lymphoid leukosis (LL) and Marek’s disease (MD). Repeated serological examinations did not establish the presence of antibodies against REV, LLV or MDV-1. Cloacal swabs were negative for LL P27 antigen in ELISA. Solid tumors of various sizes as well as diffuse infiltrations were predominantly seen in the liver, spleen and kidney. Pleomorphic cell infiltration was rarely noted in the plexus brachialis and nervus ischiaticus. Herpesvirus of turkeys (HVT) was the only virus isolated from buffy coat cells derived from affected turkeys in chicken embryo kidney cell (CEK) and chicken embryo fibroblast (CEF) cultures. Use of polymerase chain reaction (PCR) for the amplification of the 132 bp repeat region provided evidence for the presence of MDV-1 DNA in tumor tissue from several diseased turkeys. No evidence was found for the presence of REV.
Key words: MDV; PCR; turkeys
Acta virologica 43: 143 – 147, 1999
M.S. HOLLAND, R.F. SILVA
Department of Pathology, Michigan State University, East Lansing, A529 East Fee Hall,
MI 48823, USA;
USDA-ARS, Avian Disease and Oncology Laboratory, East Lansing, MI, USA
Summary. – Using in situ hybridization, latent turkey herpesvirus (HVT) transcription was examined in lymphoid and/or nonlymphoid tissues. Blood samples were taken for virus isolation from chickens at 7 and 240 days post infection (PI) representing time points for productive and latent turkey herpesvirus infections, respectively. Spleen, thymus, sciatic and brachial nerves from infected chickens were analyzed for latent HVT transcription and HVT glycoprotein B (gB) expression at 240 days PI. Using indirect immunofluorescence, HVT gB expression was not detected in any tissues examined at 240 days PI. HVT genomic fragments from a HVT BamHI library were used as probes in in situ hybridization assays. In the spleen, thymus, sciatic and brachial nerves, latent HVT transcription occurred from the repeat regions flanking the unique long region (TRL and IRL). However, fine-mapping of this region revealed a difference in latent HVT transcriptional pattern. A SmaI map of the HVT BamHI-F fragment was made to further fine-map latent HVT transcription. A 1.6 kbp SmaI subfragment hybridized to cells infected with latent HVT in the spleen and thymus. However, the 1.6 kbp SmaI subfragment did not hybridize to cells of the brachial or sciatic nerves. In addition, a 2.0 kbp SmaI subfragment hybridized to cells in the thymus but not in the spleen, sciatic or brachial nerves. The above results suggest that latent turkey herpesvirus exhibits tissue-specific transcription.
Key words: HVT; latency; transcription; tissue tropism
Acta virologica 43: 148 – 151, 1999
P. WU, W.M. REED, S. YOSHIDA, D. SUI, L.F. LEE
Department of Pathology, Michigan State University, East Lansing, MI, USA;
USDA-ARS Avian Disease and Oncology Laboratory, East Lansing, 3606 East Mount Hope Road, MI 48823, USA;
Jichi Medical School, Tochigi, Japan
Summary. – A 2439 bp open reading frame (ORF) was identified from the DNA sequence of BamHI-F and -K2 fragments of Marek’s disease virus of serotype 1 (MDV-1) GA strain, which predicts an 813 amino acid polypeptide. This peptide is homologous to HSV-1 gH, and has typical glycoprotein features. There are nine potential N-linked glycosylation sites within the extracellular domain. A fragment of the gH ORF was cloned into pGEX vector in frame with glutathione S-transferase (GST) to produce a GST-gH fusion protein in Escherichia coli. The GST-gH fusion protein was used to develop gH monoclonal and polyclonal antibodies. Expression of gH was detected in duck embryo fibroblasts (DEFs) infected with MDV-1 GA strain by immunofluorescence assay (IFA) with these antibodies. Virus neutralization and plaque-forming inhibition analyses were conducted with the gH antiserum. There were no neutralization and plaque-forming inhibition activities of gH antiserum. Comparison of the DNA sequence of gH gene between GA and RB1B strains of MDV-1 revealed major difference in the upstream control elements of gH ORF.
Key words: MDV; HSV; glycoprotein H; DNA; protein sequence
Acta virologica 43: 152 – 158, 1999
M. NIIKURA, R.L. WITTER, H.-K. JANG, M. ONO, T. MIKAMI, R.F. SILVA
Avian Disease and Oncology Laboratory, Agricultural Research Service, USDA, 3606 East
Mount Hope Rd., East Lansing, MI 48823, USA;
Department of Veterinary Microbiology, Faculty of Agriculture, University of Tokyo, Tokyo, Japan
Summary. – Glycoprotein D (gD) and its homologues are essential for many alphaherpesvirus life cycles. A gene encoding a homologue of gD was recently found in the Marek’s disease virus (MDV) genome. Interestingly, gD-negative MDV mutants apparently replicate unimpaired in both cell culture and chickens. In this study, we show the expression of the gD homologue of MDV in feather follicle epithelium in infected birds. The gD homologue was detected in a few feather follicles even when most of the follicles were expressing pp38 or gB, other MDV-specific proteins. These observations indicate that the MDV gD homologue is expressed in a very limited set of cells and may be differently regulated. Since feather follicle epithelium of infected birds are the only place where the infectious cell-free MDV virions can be recovered, analysis of the transcriptional regulation of gD may lead to the understanding of the cell-associated nature of MDV.
Key words: Marek’s disease; glycoprotein; herpesvirus; chicken; nonessential
Acta virologica 43: 159 – 163, 1999
V. ZELNÍK, V. MAJERÈIAK, D. SZABOVÁ, H. GEERLIGS, J. KOPÁÈEK, L.J.N. ROSS, J. PASTOREK
Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 46
Bratislava, Slovak Republic;
Fort Dodge Animal Health Holland, Weesp, The Netherlands;
Institute for Animal Health, BBSRC, Compton nr. Newbury, United Kingdom
Summary. – Glycoprotein D (gD) belongs to family of conserved structural proteins of alpha-herpesviruses. During productive infection of cells by herpes simplex virus 1 (HSV-1) gD has several important functions, is involved in virus penetration to and release from infected cells and is one of main targets of neutralizing antibodies. Similar functions are shared also by other alpha-herpesvirus gD homologues. Surprisingly, in previous studies it was found that MDV gD expression could not be detected during infection in vitro using immunological methods. In this study we have analyzed expression of MDV gD and its biological consequences. In vitro expression using rabbit reticulocyte lysate and/or overexpression in transfected cells showed that the second ATG codon is required for synthesis of mature, glycosylated gD. In addition, it was found that gD overexpression is neither toxic for transfected cells nor is involved in membrane fusion. After MDV infection of a proprietary cell line stably transfected with plasmid overexpressing MDV gD, no viral particles could be found in culture. On the other hand, cells overexpressing the MDV gD were sensitive to MDV infection in similar way as parental, non-transfected cells. From our study and results of other authors we propound the following conclusions: (i) MDV gD expression is blocked during in vitro infection at transcription level; (ii) MDV gD is lacking many important functions characteristic for other alpha-herpesvirus gD homologues; (iii) overexpression of single MDV gD does not result in production of mature infectious MDV particles.
Key words: MDV; cell-associated herpesvirus; gene expression; glycoprotein D
Acta virologica 42: 164 – 168, 1998
Z. CUI, A. QIN, L.F.LEE, P. WU, H.-J. KUNG
Department of Veterinary Medicine, Yangzhou University, Yangzhou 225009, P.R. China;
Avian Disease and Oncology Laboratory, USDA-ARS, East Lansing, MI, USA;
Department of Microbiology and Molecular Biology, Case Western Reverse University, Cleveland, OH, USA
Summary. – A recombinant virus, CVI/rpp38, was developed from the Marek’s disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chich embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by A resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.
Key words: MDV; pp38; monoclonal antibody H19; point mutant
Acta virologica 43: 169 – 173, 1999
S.M. REDDY, D. SUI, P. WU, L. LEE
Avian Disease and Oncology Laboratory, USDA-ARS, 3606 East Mount Hope Road, East Lansing, MI 48823, USA
Summary. – DNA sequence analysis of the BamHI-C fragment of Marek’s Disease Virus (MDV) reveals the presence of a 513 amino acid open reading frame (ORF). This ORF codes for a protein with an estimated Mr of 58,901. Comparison of the amino acid sequence with those available in the Swiss-Prot database indicates extensive homology with a protein kinase (PK) of herpes simplex virus (HSV) and varicella-zoster virus (VZV). In Northern blot hybridization, a transcript of 2.0 kb was detected in MDV (GA strain) infected duck embryo fibroblasts (DEFs). A portion of the ORF was expressed in Escherichia coli as a trpE-fusion protein and used to generate antiserum in New Zealand rabbits. This antiserum specifically detected a protein of 60 kDa in MDV serotype 1, 2 and 3 infected DEFs or chicken embryo fibroblasts (CEFs) by Western blot analysis. This ORF codes for a functional PK.
Key words: MDV; protein kinase; UL13; nucleotide sequence
Acta virologica 43: 174 – 180, 1999
M. BUBLOT, E. LAPLACE, J.-C. AUDONNET
Merial, Biological Research, 254, rue Marcel Mérieux, BP 7009, 69342 Lyon Cedex 07, France
Summary. – The sequence of BamHI-I fragment of the herpesvirus of turkeys (HVT) FC126 strain DNA was analyzed for the presence of potential open reading frames (ORFs). Four complete (ORFs 2 to 5) and 2 partial ORFs (ORFs 1 and 6) were detected. ORFs 2 and 3 were homologous to the HSV-1 UL55 and the EHV-1 gene 3, respectively. The ORF 6 was already partially sequenced by Smith et al. (Virology 207, 205-216, 1995), and was homologous to a Marek’s disease virus (MDV) ORF located in a similar position (ORF 21; Ross et al., Virus Genes 7, 33-51, 1993a). No significant homology was found for the other ORFs. ORF 4 was antisense to ORF 3. Two HVT recombinants having an expression cassette inserted into two intergenic sites were generated and tested for viremia in chickens. Results demonstrated that these 2 intergenic loci are non-essential for in vitro and in vivo HVT replication. A 650 bp deletion in the repeat region flanking UL (TRL and IRL (BamHI-F)) has been identified in some DNA molecules of HVT FC126 strain. This deletion covers the entire truncated pp38 homologous ORF and the N-terminus of a small ORF which has no detectable homology with any known gene. Our results indicate that (1) this genomic region including the HVT pp38 homologue was not essential for in vitro and in vivo growth of HVT, and (2) this deletion had no apparent effect on Marek’s disease (MD) protection induced by HVT.
Key words: HVT; insertion locus; recombinant virus; pp38
Acta virologica 43: 181 – 185, 1999
A.A. ABUJOUB, D.L. WILLIAMS, J.D. REILLY
Origen Inc., Molecular Virology Division, 3900 Collins Road, Lansing, MI 48910, USA
Summary. – 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 (OCLTM) 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-OCLTM), serotype 2 MDV (SB1-OCLTM) and serotype 3 MDV (HVT-OCLTM). MDV is maintained in a stable state in the OCLTM 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 OCLTM will be ideal for production of more effective MDV vaccines using recombinant DNA technology.
Key words: MD vaccines; fibroblast cell lines; safety studies; pathogenesis
Acta virologica 43: 186 – 191, 1999
A.M. CRONENBERG, C.E.H. VAN GEFFEN, J. DORRESTEIN, A.N. VERMEULEN, P.J.A. SONDERMEIJER
Virological Research Department, Intervet International BV, P.O. Box 31, 5830 AA Boxmeer, The Netherlands
Summary. – The sequence encoding part of the 100 kDa refractile body protein (Ea1A) from Eimeria acervulina was cloned into the US10 locus of herpesvirus of turkeys (HVT) downstream of LTR promoter. Expression of the fusion protein was shown in vitro. Recombinant HVT showed a delayed and slightly reduced level of viremia compared to the parent strain in SPF chickens as well as in broilers. Effect on the performance of broilers vaccinated with recombinant or parent HVT was measured by challenge at day 24 with a high dose of E. acervulina and E. maxima oocysts. A significant improvement in weight of animals vaccinated with the recombinant HVT was detected at the end of the challenge period.
Key words: herpesvirus of turkeys; recombinant vaccine; coccidiosis
Acta virologica 43: 192 – 197, 1999
H.J. GEERLIGS, M.W. WESTSTRATE, T.L. PERTILE, J. RODENBERG, M. KUMAR, S. CHU
Fort Dodge Animal Health Holland, Bio R&D Department, van Houtenlaan 36, 1381 CP
Weesp, The Netherlands;
Fort Dodge Laboratories, Fort Dodge, IA, USA
Summary. – With the emergence of very virulent Marek’s disease virus (MDV) strains, vaccines based on herpesvirus of turkeys (HVT) appear to be not powerful enough to confer full protection, whereas in chicken flocks vaccinated with MDV CVI 988 strain protective immunity sometimes is generated not early enough for full protection. For this reason combination vaccines containing HVT as well as CVI 988 have been developed. In this paper the beneficial effect of combining both types of virus strains in one vaccine for early protection is shown in a vaccination challenge experiment, in which one-day-old chickens were vaccinated with suboptimal dosages of the monovalent vaccines and the same dosages in a combination vaccine. After 5 days the chickens were challenged with a very virulent MDV strain and subsequently observed for a period of approx. 50 days. It appeared that the combination vaccine provided better early protection than the monovalent vaccines. In addition, the combination vaccine was tested as vaccine administered in ovo. It appeared that after in ovo vaccination the vaccine conferred adequate protection against challenge with a very virulent MDV strain, 5 days after hatch, and that protection after in ovo vaccination was similar to that obtained after subcutaneous vaccination with the same combination vaccine.
Key words: Marek’s disease; vaccine; subcutaneous vaccination; in ovo
vaccination; protection; very virulent MDV strain
Acta virologica 43: 198 – 200, 1999
X. LIU, D. PENG, X. WU, L. XING, R. ZHANG
Department of Veterinary Science, Yangzhou University, 12 Sunong Road, Yangzhou, Jiangsu 225009, P.R. China
Summary. – Recombinant fowlpox virus (rFPV) was constructed to express glycoprotein B (gB) gene from CVI988/Rispens strain of Marek’s disease virus (MDV). The rFPV-gB/R alone and in combination with herpesvirus of turkey (HVT) preparations were evaluated for their protective efficacy against challenge with very virulent MDV strains Md5 and RB1B in different chickens. The rFPV-gB/R alone induced protection comparable to that by HVT vaccines in both Ab- SPF chickens and Ab+ production chickens. Significant protective synergism was observed in one of these two types of commercial production chickens when rFPV-gB/R was combined with HVT of either cell-associated or cell-free preparations. Immunogenesis studies showed that rFPV-gB/R, just like conventional vaccines, significantly reduced the level of viremia, splenocytes infection and feather follicle shedding of challenge virus in vaccinated chickens.
Key words: recombinant fowlpox virus; vaccine; glycoprotein B; MDV;
Acta virologica 43: 201 – 205, 1999