- Volume 44, Issue 3, 1979
Volume 44, Issue 3, 1979
- Articles
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Inhibition of the Replication of Parvovirus X14 by 5-Iodo-2′-deoxyuridine Pre-treatment of Cell Cultures
More LessSUMMARYPre-treatment of rat embryo cell cultures with 5-iodo-2′-deoxyuridine (IdUrd) inhibits the replication of parvovirus X14. Reduced yields of haemagglutinating and infectious particles were observed. Adsorption of virus to cells was not affected, but both virus protein and DNA synthesis were inhibited. Fewer cells were capable of supporting protein or antigen synthesis as determined by immunofluorescence. Virus-specific DNA was detected in IdUrd pre-treated cells, but the amount synthesized was considerably less than that from control cultures. Cellular DNA synthesis was also inhibited in IdUrd pre-treated cells. Therefore, the replication of parvoviruses appears dependent upon host cell factors involved in cellular DNA synthesis.
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Invasion of Mouse Brain by Mount Elgon Bat Virus
More LessSUMMARYMount Elgon bat virus killed mice up to 13 days of age when given intranasally. Virus reached the brain of these mice via the olfactory nerve route without obvious multiplication in any tissues other than the nasal mucosa of 1- to 6-day-old mice and in the absence of viraemia or circulating virus neutralizing antibody. Large amounts of interferon were, however, synthesized in brain where virus grew to high titres. In mice older than 13 days virus did not multiply in brain but it reached the olfactory bulbs and persisted until virus neutralizing antibody appeared in the nasopharynx. No antibody was detected in blood of the resistant mice, nor was interferon detected in their brains or nasal mucosa. Immunosuppression of the resistant mice with cyclophosphamide resulted in moderate virus growth in mid- and hind-brain accompanied by interferon synthesis and death of the mice. The local immune response prevented invasion of mid- and hind-brain in the resistant mice.
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Characterization of Influenza Virus RNA Transcripts Synthesized in vitro
More LessSUMMARYPolyadenylated transcripts synthesized in vitro by detergent-disrupted influenza virus resemble virus mRNAs in that they possess the complement of the 3′ terminus of the genome RNAs but lack sequences corresponding to the same 5′ terminal region, including the homologous sequence of nucleotides 1 to 22. Transcription is initiated at the 3′ terminus by both ApG and GpG as well as in the absence of added primer.
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Is the DNA of Virus T7 Methylated?
More LessSUMMARYDNA modification of T7 wild type and of T7 M-mutants was studied by determining the percentage of 5-methylcytosine(5MC)/cytosine(C) and N6-aminopurine (6MA)/adenine (A) and by evaluating the plating efficiencies of restriction-sensitive T7 M-mutants on modifying and non-modifying host strains. Only 0.03% adenine and 0.02% cytosine were methylated in the DNA of T7 wild type as well as in T7 M-mutants, which was independent of the host DNA methylation (30- to 50-fold higher). The restriction of T7 M-mutants, determined from the plating efficiencies, was not altered on modifying or non-modifying hosts. These results indicate that host-specific modification is blocked during T7 development and that this is not due to the M-protein.
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A Solid-Phase System (SPACE) for the Detection and Quantification of Rotavirus in Faeces
More LessSUMMARYThis report describes the development of a solid-phase haemadsorption system using chromic chloride-linked, antibody coated erythrocytes. It is proposed to call this technique solid phase aggregation of coupled erythrocytes (SPACE). The system is suitable for the detection of virus antigens, such as from rotavirus infections, which are present in ‘dirty’ or ‘mixed’ preparations such as faeces, urine or exudates. The test uses microtitre U-form plates coated with specific antivirus antibody; faecal suspensions are added and virus or antigen allowed to adsorb. The plates are then washed and adsorbed antigens are detected by the addition of virus-specific IgG-coated erythrocytes. The resultant settling pattern is read in the same manner as a conventional haemagglutination test. The system is compared with electron microscopy and fluorescent antibody techniques.
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Inhibition of Protein Synthesis by Vaccinia Virus. II. Studies on the Role of Virus-induced RNA Synthesis
More LessSUMMARYCytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2 µg/ml but not 20 µg/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20 µg/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated cells, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.
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Genetic Analysis of Adenovirus Type 2. IX. The Physical Locations of Structural Genes
More LessSUMMARYSeveral virus-specified proteins of adenovirus type 2 (Ad2) and type 5 (Ad5) can be distinguished by their differential electrophoretic mobility in SDS-polyacrylamide gels. By comparing the physical maps of the genomes of interserotypic recombinants between ts mutants of these viruses with the pattern of proteins expressed, the physical locations of some genes can be determined on the adenovirus DNA molecule. The proteins of a total of 103 recombinants were analysed; 44 of these are of known DNA structure. Structural genes for the proteins analysed span the following loci on the conventional adenovirus map: V (30 to 43); III (43 to 44); II and 66K (55 to 57); 100K, 36K, 32K (69 to 71); IV (80 to 90).
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Integration of Herpes Simplex Virus Type 1 DNA into the DNA of Growth-arrested BHK-21 Cells
More LessSUMMARYThe ability of HSV-1 DNA to become associated with host cell DNA in an alkaline-stable form has been demonstrated following infection of a ts baby hamster kidney growth mutant (ts BTN-1), at the non-permissive temperature (39.5 °C). After 8 h pre-incubation at 39.5 °C, ts BTN-1 cells infected at this temperature using m.o.i. ranging from 0.5 to 200 p.f.u./cell fail to replicate virus DNA even though transport of input virus genomes to the nucleus is the same at both permissive and non-permissive temperatures.
Virions containing 3H-labelled DNA were used to infect ts BTN-1 cells at 39.5 °C, and the total cellular DNA isolated from these cells was resolved into host and virus material by repeated CsCl equilibrium gradient centrifugation. A significant amount of the input radioactivity was found as a distinct band in the host region in both neutral and alkaline CsCl gradients, strongly suggesting a covalent association between host and virus DNAs. Evidence for this association was strengthened by demonstrating that radioactive material (virus DNA) banding in the host region of CsCl gradients could be driven towards the density expected for virus DNA following degradation of the putative hybrid molecules by shearing.
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In Vivo and In Vitro Phosphorylation of Murine Mammary Tumour Virus Proteins
More LessSUMMARYA comparative study of in vitro and in vivo phosphorylation of murine mammary tumour virus, a type B RNA virus, is reported. The protein kinase activity associated with murine mammary tumour virus catalysed the in vitro phosphorylation of endogenous virus polypeptides. This kinase activity required a divalent metal cation, a non-ionic detergent, and was stimulated in the presence of dithiothreitol. Exogenous cyclic AMP was not required. The 32P-labelled products of the in vitro reaction were completely sensitive to pronase digestion and the phosphate was attached mainly by phosphomonoester linkage to serine residues. As determined by SDS-polyacrylamide gel electrophoresis, heterogeneous labelling of major and minor virus polypeptides was observed under in vitro conditions.
In contrast, the in vivo labelling of type B virus produced by a continuous cell line (MuMT-73), established from pooled mammary adenocarcinomas of Balb/cfC3H mice, demonstrated specific phosphoproteins associated with murine mammary tumour virus. The major phosphorylated proteins were found to have mol. wt. of 18000 and 12000 (p18 and p12) after isolation by molecular sieving chromatography and analysis by gel electrophoresis.
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Analysis of Hepatitis B Surface Antigen Components Solubilized with Triton X-100
More LessSUMMARYThree glycoproteins of intact hepatitis B surface antigen (HBsAg) with mol. wt. of 32000, 30000 and 28000 respectively were identified by reaction with 125I-concanavalin A (Con A) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen was effectively disrupted with Triton X-100 to produce a structure with a sedimentation coefficient of 3.9S. Affinity chromatography of disrupted HBsAg using concanavalin A-Sepharose 4B (Con A-Sepharose) resulted in two fractions. The first contained material which did not bind to the lectin and consisted of a single polypeptide of mol. wt. 64000. Further studies revealed this component to be serologically identical to serum albumin and to lack any affinity for antibody to HBsAg. A comparison of the tryptic peptide map of this polypeptide with that of purified serum albumin demonstrated identical amino-acid sequences. The second fraction contained material which bound to Con A and contained two polypeptides with mol. wt. of 28000 and 23000 respectively. HBsAg reactivity was associated with this fraction. This procedure allows the preparation of HBsAg sub-units in milligram quantities for further immunological studies.
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Some Effects of Buffers on the Infectivity and Appearance of Virus Inocula used for Tobacco Protoplasts
More LessSUMMARYInocula of either tobacco rattle (TRV) or raspberry ringspot (RRV) viruses prepared in tris chloride, pH 8.0, were as infective for tobacco mesophyll protoplasts as those prepared in potassium phosphate, pH 6.0. Both types were more infective than inocula prepared in potassium citrate, pH 6.0. However, at sub-optimal concentrations of poly-l-ornithine (< 0.8 µg/ml), tris-chloride buffered inocula of TRV were much more infective than phosphate buffered inocula.
Electron microscopy showed that all inocula contained aggregated virus particles. Aggregates of TRV formed in phosphate or citrate buffers contained 2 to 30 particles whereas those formed in tris chloride were much larger, containing hundreds of virus particles. Such a difference was not evident in TMV inocula, in which virus particles were aggregated into strands of up to 25 µm in length. In contrast aggregates of RRV were approx. spherical, about 150 to 300 nm in diam., with those formed in tris-HCl being more irregular than those formed in citrate or phosphate. These results indicate that the infectivity of an inoculum is not related simply to the type or size of aggregate formed.
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Transcription of the Genome of Adenovirus Type 12. V. Kinetic and Size-class Analyses of Nuclear and Messenger RNA in Productive Infection
More LessSUMMARYIn human KB cells productively infected with adenovirus type 12 (Ad12) the kinetics of synthesis of Ad12-specific RNA were investigated. Using 2 h labelling periods, striking differences were observed in the time course and the rate of synthesis of virus RNA comparing the nuclear, cytoplasmic and polysomal RNA fractions. A protracted onset of virus RNA synthesis was followed by an exponential increase between 15 and 40 h p.i.; reaching a maximum at 40 to 45 h p.i., the fraction of the newly synthesized Ad12-specific RNA remained constant up to 66 h in the nucleus, but decreased in the cytoplasm. Characteristic patterns were found in the size distribution of Ad12-specific messenger RNA synthesized early and late in the infection cycle. By using 10 min labelling periods, the size distribution and the degree of polyadenylation of the primary transcripts were determined. Early in infection, the size distribution of the virus-specific nuclear RNA resembled that of the mRNA, whereas late after infection most of the label was found in high mol. wt. RNA sedimenting between 30 and 55S. Eighty percent of the nuclear RNA was polyadenylated and belonged to the high mol. wt. RNA class. During the early phase of infection, approx. 20% of the virus genome was found to be transcribed symmetrically, and later after infection the entire Ad12 genome. Symmetrical (self-complementary) transcripts were preferentially derived from the terminal parts of Ad12 DNA.
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Mechanism of Acquired Resistance to Herpes Simplex Virus Infection as Studied in Nude Mice
More LessSUMMARYThe role of antibody and cell-mediated immunity in the resistance of adult mice to intracutaneous infection with herpes simplex virus type 1 (HSV-1) was studied in nu/nu and nu/+ mice. In nu/+ mice, local skin lesions began to appear at the site of inoculation on the 4th day after intracutaneous challenge with the virulent Hayashida strain of HSV-1. Zosteriform skin lesions were observed in some animals. Almost complete regression of the lesions had occurred by the 16th day p.i. In contrast, all of the nu/nu mice that developed local skin lesions died after development of severe zosteriform skin lesions. After repeated intraperitoneal inoculations with the avirulent SKa strain of HSV-1, nu/nu mice did not produce detectable amounts of neutralizing antibody and succumbed to infection, indicating no development of resistance.
Passively transferred neutralizing antibody prevented nu/nu mice from developing zosteriform skin lesions by the Hayashida strain of HSV-1, as long as the minimum concentration of serum antibody was maintained and prolonged their survival time. Adoptive transfer of 1.0 × 107 immune nu/+ spleen cells to nu/nu mice provided almost complete recovery from infection with production of sporadic low levels of anti-HSV antibody. The protective action of the immune spleen cells was lost after pre-treatment with anti-θ serum and fresh guinea pig serum prior to transfer of the cells. These data indicate that T cell-mediated cellular immunity plays a major role in recovery from intracutaneous HSV infection in mice, while antibody-mediated protection due to passive administration of HSV antibody is effective only in limiting the spread of virus.
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Biochemical and Antigenic Comparisons of the Envelope Glycoproteins of Venezuelan Equine Encephalomyelitis Virus Strains
More LessSUMMARYPulse-chase experiments after synchronous initiation of translation indicate that the larger Venezuelan equine encephalomyelitis (VEE) virus membrane glycoprotein, E2, is derived by proteolytic cleavage of the precursor, PE2. The structural proteins of VEE virus strains representing each of the antigenic subtypes and varieties have been compared by discontinuous SDS-polyacrylamide gel electrophoresis. Nucleocapsid proteins of all isolates were similar in size (mol. wt. 35 to 36 × 103). The mol. wt. of E1 varied from 48 to 51 × 103 and the mol. wt. of E2 glycoproteins ranged from 53 to 59 × 103. Pixuna virus contained a third envelope glycoprotein of 59 × 103 mol. wt. in addition to the two major glycoproteins of mol. wt. 53 × 103 and 48 × 103 respectively. The isoelectric points (pI) of E1 and E2 for all VEE strains studied were approx. 7 and 9 respectively. Both glycoproteins of TC-83 virus induced precipitating antibodies which reacted only with the homologous purified E1 and E2 glycoproteins. Antibodies to E2 protein of each virus neutralized virus infectivity and inhibited the agglutination of goose erythrocytes by virions. Haemagglutination-inhibition tests using antisera to E2 glycoproteins of prototype viruses, representing each of the antigenic subtypes and varieties, differentiated the viruses into subtypes I, II, III and IV with subtype I divided into variants 1AB, 1C, 1D and 1E.
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An Enzyme-linked Immunosorbent Assay (ELISA) for PBS Z1, a Defective Phage of Bacillus subtilis
More LessSUMMARYThe sensitivity, reproducibility and specificity of an enzyme-linked immunosorbent assay (ELISA) for the defective phage PBS Z1 of Bacillus subtilis have been investigated. It was shown that phages in concentrations between 108 and 2.5 × 1010 particles/ml could be assayed with this method. The coefficient of variation for concentrations between 5 × 108 and 5 × 109 particles/ml was approx. 10%. From some other Bacillus phages tested, only the defective phages resembling PBS Z1 in morphology were detected efficiently with the ELISA for PBS Z1. A comparison is made between ELISA and other assays for PBS Z1.
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Host-cell Response to Herpes Virus Infection in Central and Peripheral Nervous Tissue in Vitro
More LessSUMMARYIn an organotypic nerve cell culture system, all cells in both the central and the peripheral nervous system (CNS, PNS) components supported replication of herpes simplex virus types 1 and 2 (HSV 1, HSV 2). In HSV 1 infection, cellular response was particularly characterized by the formation of small syncytia (which involved neurons) and by the presence of bundles of interwoven fine filaments within the nuclei of infected cells. In HSV 2 infection, groups of parallel tubules characteristically formed in the nuclei of infected cells. All cells in the CNS or PNS succumbed to virus infection, some within 24 h (e.g. oligodendrocytes) and others after 48 h (e.g. neurons), with the exception of astrocytes. Although among the first cells to develop virus nucleocapsids in their nuclei, astrocytes became swollen and filled with increased numbers of bundles of glial filaments within 24 h after infection; by 48 h the actual number of astrocytes was increased by as much as three- to fourfold over the number in controls. The results suggest that astrocytes may have a unique mechanism which modifies virus infection and the cells not only survive, but can also become reactive.
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Sindbis Virus RNA Replication. I. Properties of the 38S RNA Species
More LessSUMMARYFour species of single-stranded virus RNA (49S, 38S, 33S and 26S) were detected in chick embryo fibroblasts infected with Sindbis virus. The relative amounts of these RNAs were unaffected by the m.o.i. There was also no significant difference in the molar proportions of the four RNA species when purified virion RNA was used as the inoculum. These findings suggest that the 38S and 33S species represent products of the transcription of non-defective virion RNAs. Kinetic analyses of RNA synthesis indicated that during a 1 min pulse more radioactivity was associated with the 38S than with the 49S RNA and as the length of the pulse increased, the ratio of 38S/49S decreased, with the 49S appearing as the predominant species. Furthermore, addition of cycloheximide within the first 3 h p.i. resulted in detection of only the 49S species. Synthesis of all four species was unaffected when the drug was added after this time period. These data suggest that the 38S species may represent newly synthesized 49S molecules and some protein(s) synthesized within the first 3 h p.i. is necessary for maintaining the 38S conformational form.
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Physico-chemical Characterization and Partial Purification of Mouse Immune Interferon
SUMMARYMouse immune (type T) interferon was produced from suspensions of spleen cells (1 × 107 cells/ml) treated with 3 µg/ml of phytohaemagglutinin. The crude interferon was chromatographed on four sorbents with varying affinities, namely concanavalin A-Sepharose, Affi-Gel 202, Blue Sepharose CL-6B and Phenyl-Sepharose CL-4B. With each of these the interferon activity was observed to have considerable heterogeneity. By means of affinity chromatography, mouse immune interferon was purified 100 to 200 times with concomitant complete recovery of activity.
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Characterization of Adenovirus Protein IX
More LessSUMMARYProtein IX from adenovirus type 2 was purified by two methods, one from groups of nine hexons obtained by disrupting purified virus by heating in the presence of deoxycholate, and the other by a previously published method. The purified protein was used to obtain a monospecific antiserum. Protein IX was found to possess both sub-group- and type-specific antigenic determinants which were apparently accessible within the groups of nine hexons. Approximately 15 molecules of IX were found per group of nine hexons and from considerations of symmetry it seemed possible that IX was located at the ‘corner to edge’ contacts between hexons in the icosahedron.
The protein in infected cells was found to possess approximately neutral charge as determined by immunoelectrophoresis. This was consistent with the amino acid composition, which showed it to be rich in serine, alanine and leucine with approximately half of its glutamic and aspartic acid residues amidified, and the isoelectric point of 6.0, as determined by two dimensional gel analysis. No free N-terminal amino acid was detectable. It is suggested that a unique tryptophan residue is located at around position 70 from the blocked N-terminus, on the basis of chemical cleavage by BNPS-skatole. Based on one tryptophan residue a total of 107 amino acids and a mol. wt. of 11200 was deduced. Analysis of 35S-methionine-labelled infected cell extracts in a two-dimensional gel system showed that the synthesis of polypeptide IX could be detected early in infection, i.e. in the presence of an inhibitor of DNA synthesis.
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Standardization in Inoculation Procedure and Effect of a Resistance Gene on Infection of Tomato Protoplasts with Tobacco Mosaic Virus RNA
More LessSUMMARYInoculation at 0 °C improved the efficiency of infecting tomato protoplasts with RNA of tobacco mosaic virus (TMV). Up to 41% infection was obtained when inoculation was at 0 °C with 10 µg/ml RNA in the presence of 1 µg/ml poly-d-lysine in 0.01 m-potassium citrate buffered 0.7 m-mannitol (pH 5.2). The homozygous gene for resistance Tm-1 was expressed in tomato protoplasts inoculated with RNA of TMV-L, a common tomato strain of TMV; no virus progeny were detected by fluorescent antibody staining or infectivity assay. Virus multiplied rapidly in protoplasts from susceptible homozygotes. Protoplasts homozygous for Tm-1 were infected by the RNA of TMV-CH2, a tomato strain which can overcome this resistance in plants. These results resemble those previously reported for inocula using intact virus, and suggest that Tm-1 blocks virus growth after the uncoating stage.
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