Yael Asher

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 Mareks 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.

Effect of herpes simplex virus type-1 UL41 gene on the stability of mRNA from the cellular genes: β-actin, fibronectin, glucose transporter-1, and docking protein, and on virus intraperitoneal pathogenicity to newborn mice

Infection with HSV-1 is accompanied by the shut-off of cellular gene expression. The virion-associated function is encoded by the viral gene UL41. An HSV-1 mutant, vhs-1, which has a genomic deletion in the UL41 gene, is incapable of inducing the shut-off of cellular gene expression. The effect of HSV-1 infection on the shut-off of the cellular genes (or mRNA degradation) was studied specifically with the cellular genes for β-actin, fibronectin, glucose transporter-1, and the docking protein. The level of these specific mRNAs was measured in cells infected with several HSV-1 strains and was compared to that of vhs-1- and mock-infected cells. It was possible to demonstrate a marked reduction in the level of the specific mRNA from these cellular genes in cells infected with several HSV-1 strains but not with the vhs-1 mutant. The pathogenicity of the HSV-1 vhs-1 mutant to newborn mice was studied. It was found that the mutant is less pathogenic to newborn mice than its parental strain HSV-1 KOS.

Incorporation of lipids into herpes simplex virus particles.

Summary The synthesis of lipids in BSC 1 cells and their association with herpes simplex virus was studied. Radioactive choline was found to be incorporated mainly into lecithin. Infection of cells with herpes simplex virus did not affect the synthesis of cellular lipoproteins. Infected cells, prelabelled with radioactive choline, yielded virus particles which contained radioactively labelled cellular lipids. Treatment of herpes virus particles with sodium deoxycholate caused the dissociation of the particles and the release of capsomeres and soluble proteins.

Defective herpes simplex virus DNA: circular and circular-linear molecules resembling rolling circles.

The formation of defective herpes simplex virus (HSV) in BSC-1 cells and the synthesis of defective virus DNA was studied. The fourth consecutive passage of undiluted virus yielded defective DNA that was 0.008 g/ml more dense than wild type (w.t.) virus DNA. The amount of defective DNA increased at passage 6 concomitantly with the decrease in infectious virus progeny. The synthesis of defective DNA was always accompanied by w.t. virus DNA synthesis. Defective DNA from both infected nuclei and defective virions had a mol. wt. of 100 X 10(6) and was linear as determined by electron microscopy. Electron microscopy of defective virus DNA at passage 6 revealed circular molecules varying in size in addition to linear DNA molecules with the length of intact virion DNA. The circular DNA molecules had contour lengths of 10, 5, 2.5 and less than 2.5 micron. The smallest circular DNA molecules had a contour length of 0.3 micron, possibly one virus gene. In addition, circular-linear DNA molecules were observed in which both the circular and the linear components varied in length. Most of these DNA molecules had circular components of either 2.5 or 5.0 micron, and linear components varying in length from less than 1 to 50 micron. Based on the present study, it is proposed that the S component of w.t. virus DNA is fragmented into small circular molecules that serve as templates for DNA synthesis, possibly by the rolling circle mechanism.

HSV-1 virulence for mice by the intracerebral route is encoded by the BamHI-L DNA fragment containing the cell fusion gene

SummaryThe phenotype of pathogenicity by direct intracerebral inoculation of herpes simplex virus type 1 (HSV-1) was mapped in the viral genome. This phenotype could be rescued by cotransfection of unit length HSV-1 DNA of an avirulent strain with the BamHI fragment L (0.70–0.738 map units) cloned from a virulent strain. The virulence function was localized in the 2.0 Kb NruI-BamHI fragment in the right-hand side of BamHI-L, the same region that encodes a virus cell-fusion gene (3). Transduction of virulence was linked with the phenotype of a larger plaque size. It is concluded that a neurovirulence function resides in the BamHI-L fragment of the HSV-1 genome, closely linked to the viral gene for cell fusion.

Effect of zinc ions on the synthesis of herpes simplex virus DNA in infected BSC-1 cells

Abstract Treatment of herpes simplex virus-infected cells with ZnSO 4 (0.2 m M ) inhibited the synthesis of viral DNA. A similar concentration of ZnSO 4 had no effect on DNA synthesis in uninfected cells. In vitro analyses revealed a marked depression of the DNA-polymerase activity in nuclei obtained from zinc-treated infected cells.

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 Mareks 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).

Irreversible Inhibition of Herpes Simplex Virus Replication in BSC-1 Cells by Zinc Ions

Zinc sulfate added to the medium of herpes simplex virus-infected BSC-1 cells, at a concentration of 0.1 mM, inhibited the synthesis of infectious virus progeny by 95 to 96%. A concentration of 0.2 mM zinc sulfate inhibited herpes simplex virus synthesis by 99.8% as determined by centrifugation in sucrose gradients and by plaque assay on BSC-1 monolayers. The inhibition of herpes simplex virus replication in BSC-1 cells was found to be irreversible.

In vitro synthesis of DNA in nuclei isolated from herpes simplex virus-infected cells, untreated and treated with metabolic inhibitors

Abstract Nuclei isolated from herpes simplex virus (HSV)-infected cells are able to synthesize viral and cellular DNA molecules under in vitro conditions in the presence of the four deoxynucleoside triphosphates, 6 mM Mg2+ and 8% (w/v) sucrose. Under similar conditions, nuclei from uninfected cells do not synthesize DNA. Analysis of the in vitro synthesized DNA molecules which were released from the nuclei revealed heterogenous DNA species, mostly of low molecular weight, which had the density of viral DNA. Nuclei were isolated from infected cells treated in vivo with metabolic inhibitors, and the in vitro DNA synthesis was studied. Nuclei from cells treated with inhibitors which affect DNA (distamycin A and camptothecin) did not synthesize DNA in vitro, while nuclei treated with certain other inhibitors (hydroxyurea, cytosine arabinoside, or cordycepin) showed a low DNA synthesis. Analysis of the DNA products revealed that only cellular DNA was synthesized in nuclei of cells treated with hydroxyurea and cytosine arabinoside.

Phosphonoacetic Acid-Resistant Mutants of Herpes Simplex Virus: Effect of Phosphonoacetic Acid on Virus Replication and In Vitro Deoxyribonucleic Acid Synthesis in Isolated Nuclei

Phosphonoacetic acid (PAA) inhibits the replication of herpes simplex virus in BSC-1 cells and the in vitro synthesis of deoxyribonucleic acid (DNA) in isolated nuclei. Phosphonopropionic acid at a concentration of 100 μg/ml had no effect on herpes simplex virus replication. PAA-resistant mutants were obtained at a rate of 1 in 104 plaque-forming units after 5-bromodeoxyuridine mutagenization of the virus. These mutants replicate in BSC-1 cells in the presence of 100 μg of PAA per ml and induce a PAA-resistant DNA polymerase that synthesizes DNA in vitro in the presence of PAA.