- Volume 64, Issue 2, 1983
Volume 64, Issue 2, 1983
- Review Article
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- Articles
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- Animal
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Cellular DNA Surrounding Integration Sites of an Avian Retrovirus
More LessSUMMARYThe size of the direct repeats of cellular DNA next to a spleen necrosis virus (SNV) provirus from infected rat cells and the nature of the cellular DNA surrounding SNV integration sites in chicken DNA were studied. A five-base pair repeat, ATTTT, was observed at the SNV-rat cell junctions. Five of ten SNV proviruses from chicken cells were flanked by unique DNA and five others were flanked by repetitive DNA. No large rearrangements of cellular DNA at SNV integration sites were observed. A clone of uninfected chicken DNA containing the presumptive unoccupied integration site for one SNV provirus was obtained and part of it was sequenced. A small substitution, approximately 60 nucleotides, was observed at the site of virus DNA insertion. Its significance is not known.
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Inhibition of Rous Sarcoma Virus-induced Transformation by Preinfection with Rhabdoviruses
More LessSUMMARYIn vivo preinfection of chicks with rabies virus (RV) or vesicular stomatitis virus (VSV) ts 1026 inhibits tumour formation after superinfection with Rous sarcoma virus (RSV). The degree of inhibition depends on the titre of the infecting viruses and the interval between rhabdovirus and RSV infection. In vitro, cells preinfected with VSV ts 1026 under non-permissive conditions and superinfected with RSV, are not transformed as judged by cell morphology, serum requirement for growth or the capacity to form colonies in soft agar, all these being the same as in uninfected cells. Doubly infected cells take up less deoxyglucose than cells infected with RSV only and more than cells infected with VSV only. RSV multiplication is inhibited in doubly infected cells: the supernatant fluid of these cells contains fewer focus-forming units and less reverse transcriptase activity than that of cells infected with RSV only. Doubly infected cells contain both VSV and RSV internal antigens 15 days after infection. The supernatant fluid of cells infected with VSV and maintained under non-permissive conditions inhibits transformation by RSV and multiplication of RSV, but not of VSV. Under non-permissive conditions, the rhabdoviruses undergo at least part of the infectious cycle, but no infectious virus is produced. RV antigen can be detected in the brain of parenterally infected chicks and VSV antigen in cells infected 15 days previously. We conclude that the inhibition of RSV multiplication and expression is probably due to one or more processes linked to the persistence of rhabdovirus components and that it cannot be attributed exclusively to interferon.
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Purification of Highly Radioactive Mouse Interferon Produced by Ehrlich Ascites Tumour Cells Induced by Sendai Virus
More LessSUMMARYMouse interferon (IFN) was produced to high titres after induction of Ehrlich ascites tumour cells with Sendai virus by using an improved procedure. The IFN molecules were labelled during their synthesis by the incorporation of [3H]leucine and [3H]lysine. Electrophoretically homogeneous labelled IFN with a molecular weight of 34000 was obtained after a two-step purification procedure using poly(I)-agarose and octyl-Sepharose column chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The specific radioactivity of this IFN was about 10 ct/min/IFN unit.
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Studies on a Temperature-sensitive Mutant of Fowl Plague Virus Having a Mutation in Gene 7 Coding for the M Protein
More LessSUMMARYA fowl plague virus (FPV) temperature-sensitive mutant, ts 303/1 having a ts mutation in gene 7 coding for the matrix (M) protein has been obtained. The mutant induced synthesis of virus-specific RNA and polypeptides as well as ribonuclear protein (RNP) formation in cells under non-permissive conditions; however, haemagglutinin cleavage was reduced, functionally active haemagglutinin and neuraminidase were absent and virions were not formed. In mutant-infected cells at 36 °C haemagglutinin cleavage was also reduced and virions formed had an altered NP:M ratio as well as a decreased haemagglutinin content. A population of virions formed under these conditions was heterogeneous both in morphology and in buoyant density. The data obtained suggest that a mutation in the M proteins of orthomyxoviruses can affect processing of the haemagglutinin and impair final stages of virion morphogenesis.
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In vitro Inhibition of Negative Strand Virus Transcriptase Activity by Proteins Soluble in Acidic Chloroform–Methanol
More LessSUMMARYThe effect of proteins soluble in acidic chloroform-methanol (ACMS proteins) on the transcriptase activity of virus ribonucleoproteins (RNPs) in vitro has been studied. Experiments with ACMS membrane (M) proteins from type A and B orthomyxoviruses, as well as from vesicular stomatitis virus, showed that inhibition of the viral RNP transcriptase activity occurred when they interacted with M proteins isolated from viruses of a different serotype, or even of a different family. The presence of ACMS proteins capable of inhibiting the transcriptase activity of orthomyxovirus RNP in vitro was also detected in human blood plasma and among proteins produced by human leukocytes. Determination of the minimum concentration of M protein inhibiting the RNP transcriptase activity, and analysis of the fowl plague virus M protein-RNP complex formed in the in vitro system, showed that the M protein was capable of inhibiting RNP transcriptase activity at a M:RNP ratio of 0.1 to 0.2:1.
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Gene Coding Assignments for growth Restriction, Neutralization and Subgroup Specificities of the W and DS-1 Strains of Human Rotavirus
More LessSUMMARYGene coding assignments for growth restriction, neutralization and subgroup specificities were determined for two human rotavirus strains, DS-1 and W, which represent two distinct serotypes. The 4th gene segment of both viruses was associated with restriction of growth in cell culture. The 9th gene segment of W virus and 8th segment of DS-1 were associated with serotype specificity, while the 6th gene segment of W virus was associated with subgroup specificity.
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Rapid Detection of Infectious Pancreatic Necrosis Virus (IPNV) by the Enzyme-linked Immunosorbent Assay (ELISA)
More LessSUMMARYThe enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the presence of infectious pancreatic necrosis virus (IPNV) antigen in cell cultures and in fish. Virus antigen could be detected in infected cell cultures before visible cytopathic effect (c.p.e.) was evident and cell cultures showing complete viral c.p.e. produced intense colour reactions. Virus antigen was detected in infected fry during and immediately after an epizootic, but although IPNV-carrier fish could be detected by the ELISA technique, the sensitivity of detection was not as great as that of isolation of the infectious virus in cell culture. The major IPNV serotypes, Sp, Ab and Vr, cross-reacted at only a low level and it was shown that the ELISA technique could be used to serotype IPNV strains rapidly. None of 10 other fish pathogenic viruses reacted with plates sensitized for IPNV detection. The time taken to perform the technique was reduced to 1 h 35 min at room temperature and this still allowed the results to be readily assessed visually as antigen-positive or antigen-negative.
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Studies on the Mechanism of Temperature Sensitivity of Infectious Pancreatic Necrosis Virus Replication
More LessSUMMARYYields of infectious pancreatic necrosis virus from fathead minnow cell cultures were maximal at 20 °C. The virus failed to replicate at 28 °C and neither virus-specific mRNA nor virus-specific polypeptides could be detected when infected cells were maintained at this temperature. Intrinsic thermolability of virus infectivity or inability to adsorb to cells at 28 °C could not account for the temperature-dependent block in virus morphogenesis. Analysis of infectious virus production and virus-specific polypeptide and RNA synthesis following shifts from the permissive (20 °C) to the non-permissive temperature (28 °C) at various times after infection indicated that multiple temperature-sensitive (ts) steps were involved in the inhibition of virus replication.
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Protective Role of Foot-and-Mouth Disease Virus Antibody in vitro and in vivo in Guinea-pigs
More LessSUMMARYPlaque reduction neutralization assays, using foot-and-mouth disease virus (FMDV) type A, strain 119 and immune serum from convalescent guinea-pigs infected with this strain of virus and performed with monolayers of a swine kidney cell line, resulted in biphasic neutralization curves because of the presence of as many as 30 to 50% of non-neutralized virus particles at peak activity. These results were found using gum tragacanth, agar, agar containing DEAE-dextran, and methylcellulose overlays and were also found using monolayers of guinea-pig embryo tongue and guinea-pig embryo heelpad cells. Non-neutralized virus in immune serum-FMDV mixtures was neutralized after the addition of anti-species antibody, demonstrating that the non-neutralized virus fraction consisted of virus in the form of infectious immune complexes. These complexes were not infectious when inoculated intraperitoneally into suckling mice or intracardially into guinea-pigs. They were infectious, however, if inoculated intradermally into the tongue or rear heelpads of guinea-pigs. Low doses of passively transferred immune serum did not protect guinea-pigs against the formation of primary vesicles after intradermal tongue or heelpad challenge with virus but did protect against systemic spread of virus to the remaining uninoculated feet. Higher doses of passively transferred immune serum protected against tongue challenge but even higher doses were required to protect against heelpad challenge. The role of antibody in protection against the systemic spread of FMDV may be due to infectious immune complexes being removed from the blood by the reticuloendothelial system. In the dermis of the tongue and heelpad, the immune complexes remain infectious, resulting in the formation of local vesicles except when these tissues contain very high concentrations of antibody.
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The Genome-linked Protein of Picornaviruses. VIII. Complete Amino Acid Sequence of Poliovirus VPg and Carboxy-terminal Analysis of its Precursor, P3-9
More LessSUMMARYVPg, the genome-linked protein of poliovirus, and its putative precursor P3-9, were radiolabelled and subjected to carboxypeptidase-A digestion. The release of amino acids was followed by identification and quantification on an amino acid analyser. Both proteins were found to be co-terminal with a sequence of -valyl-glutamine-COOH, an observation that provides further evidence that host cell trimming of virus-specific peptides does not play a role in poliovirus protein processing. Radiolabelled VPg was subjected to automated Edman degradation. The combined results complete the structural analysis of VPg, a polypeptide 22 amino acids in length with a molecular weight of 2354. Only one form of VPg has been found linked to virion RNA and it originates by a cleavage at glutaminyl–glycine pairs at both termini. The observation is consistent with other cleavages found in the virus processing scheme.
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A Comparative Analysis of Restriction Enzyme Digests of the DNA of Herpes Simplex Virus Isolated from Genital and Facial Lesions
More LessSUMMARYThe restriction endonuclease (RE) cleavage patterns of the DNA of herpes simplex virus (HSV) genital isolates from two geographically distinct groups and from one group of facial isolates were examined. Eleven of 21 genital isolates from females, 1 of 27 genital isolates from males and all 17 of the facial isolates were HSV type 1 (HSV-1). The groups of isolates of the same serotype could not be distinguished by significant differences in the frequency of variable restriction endonuclease sites or molecular weight of variable length fragments. Simultaneous consideration of two or more variable sites has disclosed some which are apparently correlated in the HSV-1 isolates, and which could provide a useful marker for phylogenetic relationships. However, most pairs showed no correlation, while certain sites appeared more closely correlated in the genital than in the facial isolates. All 39 HSV-2 isolates could be distinguished from each other on the basis of a combination of variable RE sites and variable length fragments. BglII RE sites appeared less variable than other RE sites.
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Replicative Forms of Human Cytomegalovirus DNA with Joined Termini Are Found in Permissively Infected Human Cells But Not in Non-permissive Balb/c-3T3 Mouse Cells
More LessSUMMARYBalb/c-3T3 mouse cells were found to be highly restricted non-permissive hosts for human cytomegalovirus (HCMV) strain Towne. These cells did not produce infectious progeny virions, did not permit virus DNA replication, and allowed expression of only a single, major, virus-specific, immediate-early polypeptide. Virus DNA synthesis was examined by three different experimental approaches. In infected Balb/c-3T3 cells, no 32P-labelled newly synthesized DNA was found at the virus density in CsCl gradients and no virus-specific fragments were detected after cleavage with restriction enzymes. Similarly, hybridization experiments revealed no net increase in total virus DNA over the amount of input virus-specific DNA sequences. In contrast, infected permissive human fibroblast cells synthesized 32P-labelled virus-specific DNA fragments and accumulated greatly increased amounts of total hybridizing virus DNA. Experiments with a cloned BamHI L-S joint fragment probe provided evidence for the formation of either circular or concatemeric replicative forms of HCMV DNA in which all half-molar terminal fragments were missing and the proportion of quarter-molar joint fragments increased. These forms were abundant in the first 48 h after infection of permissive human cells and mature linear monomeric forms accumulated thereafter. No detectable joining of the termini of input virus DNA occurred in either non-permissive Balb/c-3T3 cells or in human fibroblast cells in the presence of phosphonoacetic acid. In the infected Balb/c-3T3 cells a single major protein corresponding to the 68K immediate-early polypeptide could be detected within 2 h after cycloheximide reversal. Few, if any, other virus proteins were synthesized at later times or in the absence of inhibitors. The 68K protein was overproduced in Balb/c-3T3 cells to such an extent that it became a major component of the nuclear fraction and could be readily detected by direct staining procedures in polyacrylamide gels.
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Defective Particles from a Persistent Baculovirus Infection in Trichoplusia ni Tissue Culture Cells
More LessSUMMARYAn HZ-1 baculovirus clone isolated from persistently infected Trichoplusia ni (TN-368) tissue culture cells was homogeneous as determined by particle morphology and restriction enzyme analysis of virus DNA. Re-establishment of a persistent infection of TN-368 cells with this clone (B1) was unsuccessful. There was a 99% decrease in infectious virus titre following 10 passages in tissue culture at a high multiplicity of infection, and the 10th passage virus population was capable of establishing a persistently infected cell line (TN-B1). Based on particle morphology, restriction enzyme analysis, and focus-forming plaque assays, the virus recovered from TN-B1 cells included a high percentage of defective virus particles which did not interfere with the infection of second-passage B1 virus particles. The role of defective HZ-1 virus particles in the establishment of persistent infections is discussed.
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Properties of the Major Nucleocapsid Protein of Heliothis zea Singly Enveloped Nuclear Polyhedrosis Virus
More LessSUMMARYThe major nucleocapsid protein of Heliothis zea single nucleocapsid nuclear polyhedrosis virus is a low mol. wt., basic, DNA-binding protein present in the core of the capsid. It is rich in arginine and helix-destabilizing residues and possesses no lysine nor hydrophobic residues. Circular dichroism analysis showed that the protein undergoes a major conformational change in high salt solutions involving the tyrosine side chains. There is sufficient of this protein in the nucleocapsid for all the genome phosphate to be neutralized by arginine residues. A comparison of the protein with similar basic proteins from Spodoptera litura granulosis virus, Oryctes rhinoceros non-occluded baculovirus, and Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus showed that they are all arginine-rich, lysine-poor, DNA-binding proteins.
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Antiviral Effects of Human Fibroblast Interferon from Escherichia coli against Encephalomyocarditis Virus Infection of Squirrel Monkeys
More LessSUMMARYRecombinant DNA methodology has allowed the production of human fibroblast interferon (IFN-β) from Escherichia coli and this material, in highly purified form, has been shown to reduce viraemia and mortality in encephalomyocarditis (EMC) virus-infected squirrel monkeys. These effects are dose related: six treatments over 4 days at 106 U/kg and 3 × 103 U/kg have comparable efficacy, whereas treatments at 103 U/kg are ineffective. The recombinant DNA-derived IFN-β appears to be as effective as natural fibroblast cell-derived IFN-β and both materials are effective by the intramuscular or intravenous routes. Thus, even though previous studies have shown that low circulating concentrations of IFN-β are observed after intramuscular injections, the present data indicate that this slow release from the muscle can still confer protection.
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Elimination of Endogenous Xenotropic Retroviruses from Peritoneal Macrophages in Virus-induced T Cell Lymphoma
T. Sher, M. Haas and M. FeldmanSUMMARYPeritoneal macrophages did not support the replication of 136.7 and 4SP, T cell lymphoma-inducing viruses, either in vivo or in vitro. Interestingly, endogenous xenotropic viruses, which were detected in more than 50% of the tested samples of peritoneal macrophages of normal C57BL/6 mice, were eliminated from peritoneal macrophages removed from 136.7- or 4SP-inoculated, T cell lymphoma-bearing mice. This elimination occurred about 2 weeks after virus inoculation. X-irradiation (400 rads) seemed to accelerate the elimination of xenotropic viruses from the peritoneal macrophages of mice inoculated with the radiation-dependent variant, the 4SP virus. The significance of this elimination of viruses from macrophages following inoculation with T cell lymphoma-inducing viruses is discussed.
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Host Range Differences among Xenotropic Type C Retroviruses Isolated from Mouse Kidney Cell Cultures
More LessSUMMARYBy co-cultivation procedures, infectious xenotropic type C viruses have been recovered from kidney cells of several strains of mice. They have host-range patterns which place them into separate subgroups. In cells cultivated from one NZB kidney, two biologically different xenotropic type C retroviruses were found. One, X-NZB/K-1, infects and replicates well in human and mink fibroblast cells but does not induce foci in mink S+L – cells with good efficiency. The other, X-NZB/K-2, infects and replicates well in mink but not human fibroblast cells, and induces foci readily in mink S+L – cells. Cross-infection studies indicate that these viruses, classified as xenotropic by host range and envelope properties, are genetically stable.
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Studies on the Methylation of Avian Sarcoma Proviruses in Permissive and Non-permissive Cells
More LessSUMMARYThe extent of methylation of several avian oncogenic proviruses was determined by using the restriction endonucleases HpaII and MspI. The results indicated that the transformation-defective proviruses (RAV-O or B77-td), which are exogenously introduced into avian host cells, were not methylated. However, endogenous proviruses (RAV-O) or ASV proviruses present in non-permissive host cells were found to be partly or completely methylated. The methyl-sensitive restriction endonuclease PvuI, which recognizes a unique site within the long terminal repeat in the ASV genome, failed to cleave proviruses present in several non-permissive host cells. From these results we suggest that modification of the sequence around the PvuI site results in reduced levels of transcription.
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