PubMed Health⌕ Search

Biomedical subjects

M Malkinson

Publications and source records attributed to M Malkinson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Open reading frames in a 4556 nucleotide sequence within MDV-1 BamHI-D DNA fragment: evidence for splicing of mRNA from a new viral glycoprotein gene.

A DNA segment of the MDV-1 BamHI-D fragment was sequenced, and the open reading frames (ORFs) present in the 4556 nucleotide fragment were analyzed by computer programs. Computer analysis identified 19 putative ORFs in the sequence ranging from a coding capacity of 37 amino acids (aa) (ORF-1a) to 684aa (ORF-1). The special properties of four ORFs (1a, 1, 2, and 3) were investigated. Two adjacent ORFs, ORF-1a and ORF-1, were found by computer analysis to have the properties of two introns encoding a glycoprotein: ORF-1a encodes an aa sequence with the properties of a signal peptide, and ORF-1 encodes a polypeptide with a membrane anchor domain and putative N-glycosylation sites in the aa sequence. ORF-1a and ORF-1 were found to be transcribed in MDV-1-infected cells. Two RNA transcripts were detected: a precursor RNA and its spliced form. Both are transcribed from a promoter located 5' to ORF-1a, and splice donor and acceptor sites are used to splice the mRNA after cleavage of a 71-nucleotide sequence. This finding suggest that ORF-1a and ORF-1 are two introns of a new MDV-1 glycoprotein gene. The DNA sequence containing ORF-1 was transiently expressed in COS-1 cells, and the viral protein produced in these cells was found to react with anti-MDV serotype-1 Antigen B-specific monoclonal antibodies. These studies indicate that the protein encoded by ORF-1 has antigenic properties resembling Antigen B of MDV-1. A gene homologous to ORF-1 was detected in the genome of both MDV-2(SB1) and MDV-3(HVT), which serve as commercial vaccine strains. Two additional ORFs were noted in the 4556 nucleotide sequence: ORF-2, which encodes a 333 aa polypeptide initiating in the UL and terminating in the TRL prior to the putative origin of replication, and ORF-3, which encodes a 155 aa polypeptide that is partly homologous to the phosphoprotein pp38 encoded by the BamHI-H sequence. The 65 N-terminal aa of the two gene products are identical, both being derived from the nucleotide sequences in the TRL and IRL, respectively. Additional homologous aa sequences are the hydrophobic aa domain in the middle of both proteins. The functions of ORF-2, ORF-3, and additional ORFs are under study.

Amino Acid Sequence↗
PubMed1994

Distribution of Ia antigen positive cells in chicken embryos infected with oncogenic Marek's disease virus (MDV) and MD vaccine viruses of serotypes 1, 2 and 3.

Chick embryos infected at Day 13 of embryonic development (ED) with the oncogenic serotype 1 Marek's Disease Virus, isolate B (MDV-B) and three MDV vaccines (CVI988, SB1 and HVT, serotypes 1, 2 and 3, respectively) and uninfected chick embryos were studied for the distribution of Ia antigen positive dendritic cells (DC), B cells and MDV antigen positive (Ag+) cells in the lymphoid organs and chorioallantoic membrane (CAM). The immunofluorescence study was conducted on acetone-fixed organ touch impressions using monoclonal antibodies to Ia antigen, and MDV serotypes 1, 2 and 3 and polyclonal antibodies to bursal Ig-bearing (Ig+) B cells. DC were found mainly in the thymus and spleen and Ig+ cells in the bursa, thymus and spleen of normal embryos. All virus-infected embryos had MDV Ag+ cells in the spleen. MDV-B and SB1 infected embryos also had MDV Ag+ cells in the bursa, MDV-B Ag+ cells in the CAM and SB1-Ag+ cells in the thymus. Infection with MDV altered the distribution pattern of DC in a serotype-specific manner: to a lesser extent, infection with MDV-B and SB1 induced their appearance in the CAM, while HVT and CVI988 depleted the DC population from all organs except the bursa and the thymus, respectively. Infection with MDV-B depleted the Ig+ cells from all organs. These results suggest that virus-specific patterns of change in the distribution of DC and B cells occur in various tissues and organs of the chick embryo as a result of infection with oncogenic and apathogenic strains of MDV.

Animals↗
PubMed1994

Diagnostic test for lymphoproliferative disease virus infection of turkeys, using the polymerase chain reaction.

Lymphoproliferative disease virus, a type-C retrovirus, is the etiologic agent of a naturally acquired lymphoproliferative disorder in turkeys. The disease is characterized by rapid induction of lymphoproliferative lesions with morphologic and histopathologic features resembling those of reticuloendotheliosis. Owing to lack of overt clinical manifestations, early detection of lymphoproliferative disease virus is essential for preventing the rapid horizontal spread of the disease that can decimate flocks. We describe development of a simple, rapid, sensitive, and highly specific assay, using the polymerase chain reaction, capable of providing differential diagnosis of the disease soon after infection.

Animals↗
PubMed1993

PCR detection of amplified 132 bp repeats in Marek's disease virus type 1 (MDV-1) DNA can serve as an indicator for critical genomic rearrangement leading to the attenuation of virus virulence.

A radioactive PCR test was developed that amplified the very virulent Marek's disease virus-1 (vvMDV-1) DNA sequence containing the 132 bp repeats. In apathogenic MDV-1 (CVI 988, Rispens), amplified DNA bands containing multiple copies of 132 bp repeats were identified. In the present study this PCR technique was used to monitor the passage level of vvMDV-1 in chicken embryo fibroblasts (CEF) in which the number of tandem 132 bp repeats was increased. It was found that at passage level 32 of vvMDV-1-B isolate, the 132 bp tandem repeat was already markedly amplified and its pattern resembled that of the MDV-1 (CVI 988, Rispens) vaccine virus DNA. In the vvMDV-1Z strain, amplification of the 132 bp repeat was not detectable at a similar passage level. The PCR test demonstrated that the apathogenic MDV-1 Md11/75c virus developed by extensive in vitro passaging has amplified 132 bp DNA repeats similar to those of the commercial vaccine virus (CVI 988, Rispense). It was also found that the pattern of viral RNA from infected cells detectable by Northern blot hybridization was markedly changed from a 2.4 kb RNA species in cells infected with vvMDV-1 viruses, to four RNA species (ranging from 2.2 to 4.4 kb) in cells infected with passage 32 of MDV-1-B strain, to a very large number of undefined RNA species synthesized in cells infected with attenuated MDV-1 viruses (CVI 988, Rispens and Md 11/75c).

Animals↗
PubMed1992

Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek's disease viruses (MDV) and vaccine viruses of MDV-serotypes 2 and 3.

A polymerase chain reaction (PCR) test based on primers flanking the 132 bp tandem repeat in pathogenic MDV-1 DNA was developed. These primers amplify a dimer or a trimer 132 bp repeat in pathogenic MDV-1 DNA from blood and organs of commercial chickens with Marek's disease (MD) symptoms. Using the same primers in a radioactive PCR test, it was possible to distinguish between vvMDV-1 and the non-pathogenic MDV-1 CVI-988 vaccine in which the 132 bp repeats in the DNA were increased up to 9 repeats. The MDV-1 specific primers did not amplify MDV-2 (SB1) and MDV-3 (HVT) DNA. Primers prepared according to the nucleotide sequence of MDV-1 antigen A gene amplified MDV-1 DNA only. Specific primers prepared according to the nucleotide sequence of MDV-3 (HVT) antigen A gene amplified MDV-3 DNA but not MDV-1 nor MDV-2 DNA. The results of the present study show that the PCR tests can be used for the early identification of vvMDV-1 DNA in pathological samples from diseased commercial chickens and to distinguish between the vvMDV-1 and the three types of virus vaccines used to immunize chickens. The tests are accurate and can be performed in the presence of vaccine virus DNA in the sample.

Animals↗
PubMed1992

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

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 > or = 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 > or = 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 degrees C and the rate of denaturation of the 230 and > or = 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 (> or = 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.

Animals↗
PubMed1991

Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT).

Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR 322. Under stringent conditions of hybridization (low salt concentration, 10% dextran sulfate at 68 degrees C), two of the cloned HVT DNA fragments (13.5 and 11.0 kbp) hybridized to three MDV DNA fragments: BamHI-C, D, and G. The 13.5 kbp fragment detected two transcripts of 1.8 and 3.0 kb in RNA extracted from HVT-infected chicken embryo fibroblasts, while the 11.0 kbp fragment detected three transcripts of 1.6, 2.0, and 3.0 kb in the extracted RNA. Using hybrid selection, specific RNA was isolated by hybridization with the two cloned HVT DNA fragments and then was translated in vitro using rabbit reticulocyte lysate, and the resulting proteins were analyzed by NaDodSO4-PAGE. The RNA selected by the two HVT DNA fragments coded for a 30 kD protein and for several smaller proteins 10-20 kD in size, while the RNA selected by the 11.0 kb HVT DNA fragment was translated into a 40 kD protein. Immunoprecipitation of these in vitro synthesized proteins with hyperimmune anti-HVT chicken serum showed that they were of viral origin.

Animals↗
PubMed1991

Monospecific antibodies to Marek's disease virus antigen B dimer (200 kDa) and monomer (130 and 60 kDa) glycoproteins neutralize virus infectivity and detect the antigen B proteins in infected cell membranes.

Monospecific antibodies were prepared by nitrocellulose blot immunoaffinity to 3 polypeptide components of the host-membrane associated B antigen of Marek's disease herpesvirus (MDV) and to its soluble A antigen. The B antigen comprised a 200 kDa dimer which is 2-mercaptoethanol (2-ME) labile, a monomer of 130 kDa and a 60 kDa protein, both of which are 2-ME resistant. Cross-immunoblotting studies showed that the anti-dimer antibody recognized the dimer protein as well as the 130 and 60 kDa components. In contrast, the anti-130 kDa antibody gave the strongest signal on blots of reducing gels indicating that the monomer is largely formed by in vitro reduction with 2-ME. All four antibodies recognized membrane antigens on chicken embryo fibroblasts infected with MDV vaccine viruses representative of the three serotypes and in addition, neutralized the homologous MDV isolate. The anti-dimer antibody was greatest, the anti-monomer antibody was the weakest and the anti-60 kDa antibody intermediate in neutralizing efficacy to all four viruses. We conclude from these studies that the B antigen presents at least two classes of neutralizing epitopes: one is discontinuous and of broad specificity on the intact dimer molecule and the other, on the 130 and 60 kDa proteins, is continuous and of lower avidity.

Animals↗
PubMed1990

Unique sequences in the env gene of avian hemangioma retrovirus are responsible for cytotoxicity and endothelial cell perturbation.

An avian retrovirus isolated from spontaneous cavernous hemangiomas of layer hens codes for an env protein that induces a cytopathic effect on a wide variety of cultured avian and mammalian cells and also causes thrombogenicity of endothelial cells. Sequence analysis of the avian hemangioma inducing virus revealed unique elements in both its env gene and its LTR. We propose that these elements are responsible for the biological and pathogenic characteristics of the virus.

Amino Acid Sequence↗
PubMed1988

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.

Animals↗
PubMed1988

RNA transcripts of Marek's disease virus (MDV) serotype-1 in infected and transformed cells.

RNA was isolated from two strains of Marek's disease virus (MDV-Z and MDV-B). The virus was grown in duck embryo fibroblasts (DEF) for 96 hr, 72 hr in the presence of phosphonoacetic acid (PAA) and 24 hr in the presence of cycloheximide added at the time of infection. With the use of DNA probes representing about 80% of the MDV genome, an extensive Northern blot analysis of the RNA was carried out. A similar analysis was done with RNA extracted from the MDV-transformed cell line MSB-1. This study revealed 42, 25 and 29 discrete viral RNA transcripts in MDV-Z and MDV-B-infected DEF and in the MSB-1 cell line, respectively, ranging in size from 0.8 to 13 kb. In MDV-Z-infected DEF, there were twelve late RNA species, two early and eight immediate-early viral transcripts. In MDV-B-infected DEF there were eleven late RNA species, two early and seven immediate-early viral transcripts. The RNA species were homologous for all the probes used except the BamHI-G DNA fragment where no RNA transcripts were detected in the MSB-1 cell line. The RNA transcripts were used to produce a preliminary viral RNA map. Comparison of the location and sizes of the viral RNA transcripts in MDV-infected and MDV-transformed cells revealed several differences.

Animals↗