Disease Virus Gene Research Articles

PCR method for detecting recombinant baculoviruses with the insertion of a large gene

A PCR strategy was developed using primers specific to an infectious bursal disease virus (IBDV) gene as well as primers flanking the polyhedrin region of baculovirus to verify the presence of IBDV gene in the recombinant baculovirus and confirm the absence of wild-type baculovirus contamination. This method can be applied to detect the presence of large genes in the recombinant baculovirus with greater sensitivity and avoid the need of modifying the typical PCR procedure provided by the manufacturer. © Rapid Science Ltd. 1998

Transcriptional analysis of Marek's disease virus (MDV) genes in MDV-transformed lymphoblastoid cell lines without MDV-activated cells.

Spontaneously activated MDV is rarely included in MDV-transformed cells, while it may influence the results of transcriptional analysis. A population consisting of 10(3) MDV-transformed cells probably did not include spontaneously activated MDV, since the estimated frequency of MDV-transformed cells including activated MDV was below 0.01% according to limiting-dilution polymerase chain reaction (PCR) and the presence of the major early antigen pp38 in 6 transformed cell lines. Reverse transcriptase-PCR (RT-PCR) products corresponding to ICP27, pol, TK, US3, A41, gA, gB and UL50 genes were undetectable in 10(3) cells by Southern hybridization of the RT-PCR products. Transcripts of the VP16 and SORF2 genes were detected in the 10(3) cells of MSB-1, and the pp14 gene transcript was found in 10(3) cells of RPL-1 but not in 10(3) cells of HPRS-1, MOGA-2, MSB-1 or MTB-1. A transcript corresponding to the ICP4 sequence was detected as a 0.7 kbp RT-PCR product in 10(3) cells of these MDV cell lines but not in the retrovirus-transformed 1104B1 cell line. The transcript corresponding to the 0.7 kbp RT-PCR product suggested a splice by its size and sequence. Thus, transcriptional analysis of 10(3) MDV-transformed cells revealed that the transcript corresponding to the ICP4 sequence was a common transcript in latently infected MDV-transformed cells, while most of the genes did not transcribe in these cells.

Characterization of Marek's disease herpesvirus-specific cytotoxic T lymphocytes in chickens inoculated with a non-oncogenic vaccine strain of MDV.

Previously we have reported that reticuloendotheliosis virus (REV)-transformed cell lines expressing Marek's disease virus (MDV) genes pp38, meq or gB were lysed by syngeneic MDV-specific splenocytes from major histocompatibility complex (MHC):B9B19 and MHC:B1B21 chickens. In contrast, REV-transformed cell lines expressing the MDV gene ICP4 were only lysed by syngeneic MDV-specific splenocytes from MHC:B21B21 chickens. In this study we report that this syngeneic cell-mediated immune response is induced by cytotoxic T lymphocytes (CTL). Splenocytes from MDV vaccine strain, SB-1 inoculated MHC:B19B19 and MHC:B21B21 chickens, depleted for CD4+, CD8+, TCR gamma delta +, TCR alpha beta 1+ and/or TCR alpha beta 2+ cells, were used as effector cells in chromium-release assays. Effector cells depleted of CD8+ or TCR alpha beta 1+, but not TCR gamma delta + or TCR alpha beta 2+, markedly reduced the MDV-specific release. Depletion of CD4+ effector cells did not influence the specific release significantly. This is the first report on identification of virus-specific CD8+ CTL in chickens inoculated with a non-oncogenic vaccine strain of MDV.

Antigen-specific lymphoproliferative responses to tetanus toxoid: a means for the evaluation of Marek's disease virus-induced immunosuppression in chickens

Antigen-specific lymphoproliferative responses were examined in chickens following immunization with tetanus toxoid (Ttx). The immune competence of chickens was assessed by mitogen assay utilizing phytohemagglutinin (PHA)-stimulation and Ttx-specific antigen proliferation assay (Ttx-APA). Immune spleen cells but not peripheral blood leucocytes demonstrated specific proliferation following stimulation in vitro in a Ttx-APA. In this study, we examined firstly the effects of Marek's disease (MD)-associated immunosuppression on specific immune responses. The humoral and cell-mediated immune responses were monitored by enzyme-linked immunosorbent assay (ELISA) and Ttx-APA, respectively. Secondly, we examined if vaccination against MD using a conventional herpesvirus of turkeys (HVT) vaccine and two recombinant HVT (rHVT) vaccines would affect the development of Ttx-specific immune responses. The rHVT vaccines used in this study included two constructs: one expressing both Newcastle disease virus (NDV) and MD virus (MDV) genes (HVT/NDV/MDV), and another expressing only MDV genes (HVT/MDV). The mitogenic responses of spleen cells of the vaccinated chickens were inconsistent allowing no definitive conclusions about vaccinal immunosuppression. The results of the Ttx-APA indicated that Ttx-specific lymphoproliferative responses provide a meaningful measure of immunosuppression. The MDV-induced immunosuppression resulted in the inhibition of Ttx-specific lymphoproliferation in vitro. Both HVT and rHVT vaccines were not immunosuppressive as indicated by the development of normal Ttx-specific lymphoproliferative responses in chickens. These results indicate that vaccination against MD results not only in the prevention of tumor formation but also protection from possible virus-induced immunosuppression.

Detection of infectious bursal disease virus by reverse transcription-polymerase chain reaction amplification of the virus segment A gene

A reverse transcription-polymerase chain reaction (RT-PCR) amplification assay was developed to detect infectious bursal disease virus (IBDV) gene sequences in clinical samples, infected cell cultures and chicken embryos. Two pairs of primers were designed to amplify the 5'- and 3'-termini of segment A genes that partially code for the IBDV proteins VP2 and VP3, respectively. One primer pair specifies a 309-bp fragment, the other a 520-bp fragment. Direct RT-PCR analysis of 5 bursal samples of chickens derived from a suspected first outbreak of infectious bursal disease in New Zealand yielded the 309-bp and 520-bp by fragments. The identity of both amplified fragments was confirmed by restriction endonuclease analysis, chemiluminescence Southern blot hybridization and direct cycle sequencing. RT-PCR amplification of RNAs extracted from 4 out of 5 IBDV isolates propagated in Vero cells, chicken embryo fibroblasts and specific pathogen-free chicken embryos yielded IBDV-specific fragments of unpredicted small sizes.

Identification and characterization of a Marek's disease virus gene encoding DNA polymerase

DNA sequence analysis revealed a gene encoding the Marek's disease virus (MDV) DNA polymerase (pol) within the BamHI-E fragment of the long unique region of the virus genome. Identification is based on an extensive amino acid homology between the MDV open reading frame and the DNA pol (UL30) of the herpes simplex virus. We describe here a 3540-base-pair fragment of the MDV DNA encoding 1180 amino acids with a Mr of 133,920 daltons as the viral DNA pol gene, with the analysis of transcription and translation. In Northern blot hybridization, a transcript of 4.0 kb was detected in GA-MDV-infected duck embryo fibroblast (DEF) cells. An antiserum was generated in rabbit using TryE-pol fusion protein expressed in E. coli. This antiserum specifically immunoprecipitated a protein of 135 kD from lysates of MDV-GA-infected DEF cells. MDV DNA pol showed extensive homology to five distantly related herpesviruses: equine herpesvirus (EHV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV). Comparison of amino acid sequences among the herpesviruses highlights nine highly conserved regions. Three of the conserved regions are in the N-terminus in the 3′−5′ exonuclease domains and the remaining six are in the C-terminus in the catalytic domains. The predicted structural characters are in good agreement with the published data on a number of human herpesvirus DNA pol. The identification of MDV DNA pol gene may lead to a better understanding of MDV replication.

Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells

Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes ( B 19 B 19 and B 13 B 13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a ( B 19 B 19) and S13 ( B 13 B 13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

Identification and Characterization of Marek's Disease Virus Genes Homologous to ICP27 and Glycoprotein K of Herpes Simplex Virus-1

We have identified two Marek's Disease Virus (MDV) genes within the Eco RI-B fragment of MDV-GA genomic DNA. EcoRI-B is 11.3-kb long and maps within the long unique region of MDV genomes. A 3.2-kb fragment of Eco RI-B has been sequenced and contains two open reading frames, ORF53 and ORF54. ORF53 (MDV gK), a homolog to HSV-1 glycoprotein K (gK), is 1062 nucleotides long and encodes 354 amino acids (395 kDa). ORF54, designated MDV ICP27, based on significant similarity to HSV-1 ICP27, is 1419 nucleotides long and encodes 473 amino acids (54.5 kDa). In Northern blot hybridization, two overlapping transcripts (2.9 and 1.6 kb) were detected in MDV-infected DEF cells treated with cycloheximide, suggesting that both transcripts belong to the immediate-early gene family. Amino acid sequence analysis of MDV gK shows some common glycoprotein features, including a putative N-terminal signal sequence, four N-linked glycosylation sites, and four potential transmembrane domains. Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV-1 ICP27 and VZV ORF4 shows a high degree of conservation within the C-terminus. The C-terminal region of HSV-1 ICP27 has been demonstrated to be critical to its function. A conserved zinc finger metal-binding motif C (442)-X 4-C (447)-X 13-H (461)-C (467) was also found in the C-terminus of MDV ICP27. Furthermore, MDV ICP27 upstream sequences contain four copies of consensus sequence elements similar to the tegument protein target sequence TAATGARAT. TrpE-ICP27 fusion protein was expressed in Escherichia coli, and rabbit antisera were generated using purified fusion protein. A 55-kDa protein has been detected in both MDV-GA- and Md11-infected cells using immunoblot analysis.

Identification and Characterization of a Marek's Disease Virus Gene Homologous to Glycoprotein L of Herpes Simplex Virus

We have identified three Marek's disease virus (MDV) open reading frames (ORFs) within the BamHI D fragment of MDV genome. The predicted polypeptides are homologous to UL1 (glycoprotein L, gL), UL2 (uracil-DNA glycosylase), and UL3 (nuclear localizing phosphoprotein) of herpes simplex virus type 1 (HSV-1). Comparison of the deduced amino acid sequences of these three ORFs with HSV-1 counterpads revealed overall identities of 18, 43, and 49%, respectively. In spite of the low overall amino acid identity with HSV-1 gL, the first open reading frame was identified as a gL homolog of HSV-1 based not only on the gene arrangement but also on a limited amino acid conservation among gL homologs of alpha-herpesviruses. To characterize the expression of the MDV gL gene, an antiserum to a hydrophilic region of the gene expressed in a bacterial expression vector was produced. Immunoprecipitation with this antiserum revealed a 25,000-Da polypeptide in MDV-infected cells. Furthermore, the 25,000-Da polypeptide migrated as a 18,000-Da polypeptide following PNGase F treatment. This result is consistent with the predicted molecular weight of MDV gL, considering the two potential Nglycosylation sites and the predicted N-terminal signal sequence. A recombinant fowlpox virus expressing the MDV gL gene was generated to characterize this glycoprotein. Unlike gL in MDV-infected cells, gL expressed by recFPV-gL was highly sensitive to Endo H, indicating that it was probably retained in the endoplasmic reticulum and was not properly processed to a mature form. Therefore, similar to HSV-1, coexpression and complex formation of MDV gL and gH may be required for proper processing and transport of gL to the cell surface.

Evaluation of swinepox virus as a vaccine vector in pigs using an Aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp50 and gp63.

Pigs were vaccinated by scarification or intramuscular injection with a swinepox virus-Aujeszky's disease (pseudorabies) recombinant (rSPV-AD) constructed by inserting the linked Aujeszky's disease virus genes coding for glycoproteins gp50 and gp63, attached to a vaccinia virus p7.5 promoter, into the thymidine kinase gene of swinepox virus. By 21 days after vaccination, 90 and 100 per cent of the animals vaccinated by scarification or intramuscular injection, respectively, had developed serum neutralising antibodies to Aujeszky's disease virus. Upon challenge with virulent virus, significantly fewer vaccinated pigs developed clinical Aujeszky's disease, nasal shedding of challenge virus was markedly reduced, and the vaccinated groups of pigs maintained or gained weight during the week after challenge whereas the unvaccinated control group lost weight. No transmission of rSPV-AD to in-contact controls was detected during the three weeks before challenge. In a second experiment, serum neutralising antibodies to Aujeszky's disease virus persisted for 150 days after the pigs were vaccinated with rSPV-AD by scarification or intramuscular injection and all the pigs showed an anamnestic response when they were revaccinated.

Complete nucleotide sequence of the Marek's disease virus ICP4 gene

The Marek's disease virus (MDV) gene encoding a homologue to the ICP4 protein of herpes simplex virus has been mapped to BamHI fragment A based on the physical map of the MDV genome (Fukuchi et al., 1984). The gene lies completely within the inverted repeat flanking the unique short region of the genome. The complete nucleotide sequence of the MDV ICP4 gene has been determined. The coding region is 4245 nucleotides long and has an overall G + C content of 52%. The MDV ICP4 protein is predicted to have a structure similar to that of ICP4-like proteins of other herpesviruses in that it has five distinct regions, the second and fourth of which are highly conserved. In addition, the protein contains the characteristic run of serine residues located toward its amino terminus. The MDV ICP4 gene is expressed in MDV-infected chicken embryo fibroblasts.

Recombinant fowlpox viruses expressing the glycoprotein B homolog and the pp38 gene of Marek's disease virus.

Two Marek's disease virus (MDV) genes, one homologous to the glycoprotein B gene of herpes simplex virus and encoding the B antigen complex and the other encoding a 38-kDa phosphorylated protein (pp38), were inserted into the fowlpox virus (FPV) genome under the control of poxvirus promoters. Randomly selected nonessential regions of FPV were used for insertion, and the vaccinia virus 7.5 kDa polypeptide gene promoter or a poxvirus synthetic promoter was used for expression of MDV genes. Gene expression in cells infected with these recombinants was highly influenced by the promoter (the synthetic promoter being more effective) but was only slightly influenced by the insertion site and by the transcription direction of the insert relative to the direction of the flanking FPV sequences. Cells infected with an FPV recombinant expressing the MDV gB gene reacted positively with a monoclonal antibody specific to this glycoprotein in an immunofluorescence assay. Immunoprecipitation of infected cell lysates showed three glycoproteins identical to those associated with the B antigen complex of MDV (100, 60, and 49 kDa). Cells infected with a recombinant expressing the pp38 gene reacted positively with an anti-pp38 monoclonal antibody in an immunofluorescence assay. The generated protein was phosphorylated and had a molecular weight similar to that of the native pp38 protein. Sera from chickens immunized with an FPV recombinant expressing the MDV glycoprotein B gene reacted with MDV-infected cells.

Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells.

The identification of unique Marek's disease (MD) virus (MDV) antigens expressed not only in lytically infected cells but also in latently infected MD lymphoblastoid tumor cell lines is important in understanding the molecular mechanisms of latency and transformation by MDV, an oncogenic lymphotropic herpesvirus of chickens. Through cDNA and nucleotide sequence analysis, an open reading frame (designated the pp38 ORF) which encodes a predicted polypeptide of 290 amino acids was identified in BamHI-H. Demonstration that the pp38 ORF spans the junction of the MDV long unique and long internal repeat regions (MDV has an alphaherpesvirus genome structure) precludes the presence of the gene encoding the B-antigen complex (gp100, gp60, and gp49) in the same region of BamHI-H, where it was originally thought to exist. Duplication of the complete pp38 ORF was not observed in BamHI-D, but part of it (encoding 45 amino acids) was found in the long terminal repeat region of the fragment. By use of trpE-pp38 fusion proteins, antisera against pp38 were prepared. By immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a predominant virus-specific 38,000-dalton polypeptide (designated pp38) and a minor 24,000-dalton polypeptide (designated p24) were found. No precursor-product relationship was found between pp38 and p24 by pulse-chase analysis, and only pp38 was detected by Western blot (immunoblot) analysis with antiserum to pp38. pp38 was found to be phosphorylated and present in oncogenic serotype 1-but not nononcogenic serotype 3-infected cells. Expression of the gene encoding pp38 was relatively insensitive to phosphonoacetic acid inhibition, suggesting that pp38 may belong to one of the early classes of herpesvirus proteins. pp38 was also detected in the latently infected MSB-1 lymphoblastoid tumor cell line. The detection of antibody against pp38 in immune chicken sera indicates that pp38 is an immunogen in birds with MD. Most of the properties described here for a protein detected by methods based on finding the ORF first are identical to those of a 38-kDa phosphoprotein reported by others, suggesting that they are the same. Collectively, the data reported here provide (i) more definitive information on the complete ORF of another MDV gene and the protein that it encodes, (ii) clarification of the gene content within a specific region of the MDV genome, and (iii) the molecular means to conduct further studies to determine whether pp38 plays a role in MDV latency and transformation.

Structural analysis and transcriptional mapping of the Marek's disease virus gene encoding pp38, an antigen associated with transformed cells.

The gene encoding a Marek's disease virus (MDV) pp38 phosphoprotein has been identified, sequenced, and localized to the BamHI H fragment to the left of the putative MDV origin of replication. The open reading frame was defined by sequencing of a lacZ-pp38 fusion protein gene from a lambda gt11 expression library. The entire open reading frame is 290 amino acids long and codes for a protein with a calculated molecular weight of 31,169, compared with the size of 38 kDa of the phosphorylated form estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. S1 nuclease protection analysis showed that the pp38 gene is transcribed leftward as an unspliced mRNA. On the basis of transcriptional mapping studies, the pp38 transcript is predicted to be about 1.8 kb in length without a poly(A) sequence. Its promoter-enhancer region overlaps that of the major rightward BamHI H 1.8-kb transcript implicated in tumor induction. This region contains Oct-1, Sp1, and CCAAT motifs as well as the putative origin of replication. The pp38 protein is the only presently known antigen that is consistently associated with the transformation state. It may play a significant role in MDV transformation.

Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed in E. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region of BamHI-H or EcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8. Immunoprecipitation of 35S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of 32P-phosphate-labeled or 3H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides. Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.

Newcastle disease virus gene clones comprising polynucleotides encoding the HN and/or F protein of Newcastle disease virus RNA; vaccine

Newcastle disease virus gene clones comprising polynucleotides encoding the HN and/or F protein of Newcastle disease virus RNA; vaccine

A second protease of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) genes are expressed as a polyprotein which is rapidly processed into the four primary cleavage products L, P1, P2, and P3. In secondary cleavage reactions, these are further processed into the mature proteins. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. For analysis of its biological function, the L gene was mutated by site-directed mutagenesis of cloned cDNA. In vitro translation of in vitro transcripts of these DNAs and expression studies in Escherichia coli showed that the L mutants affect the processing of the viral polyprotein. The mutants isolated were partially or totally defective in processing the polyprotein at the L/P1 junction. These mutants could, however, be processed in the presence of the wild-type L protein. Furthermore, an antiserum directed against the L protein inhibited processing at the L/P1 cleavage site, so that the release of the L protein from the polyprotein was blocked. These data reveal that the L gene product represents a viral protease which catalyzes its own release from the nascent polyprotein.

Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins.

Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambda PL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in 35S-labeled extracts from foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and-mouth disease virus gene products not previously identified in vivo or in vitro.