- Volume 42, Issue 3, 1979
Volume 42, Issue 3, 1979
- Articles
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Late Transcription and Simultaneous Replication of Simian Adenovirus 7 DNA as Revealed by Spreading Lytically Infected Cell Cultures
More LessSUMMARYMiller’s technique of spreading DNA was applied to monkey cells productively infected with simian adenovirus 7. This permitted the visualization of cellular DNA transcription, both nucleolar and non-nucleolar, and of late transcription and replication of virus. Virus double-stranded DNA, thin fibres with very few nucleosome-like particles, were observed carrying either transcription or replication complexes. In addition, both RNP transcripts and replication forks were found on some virus duplex DNA. Virus single-stranded DNA replicative intermediates were identified on the basis of their increased thickness and contrast which results from the presence of a DNA binding protein.
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Relationship Between Virus Neutralization and Serum Protection Bioassays for IgG and IgM Antibodies to Foot-and-Mouth Disease Virus
More LessSUMMARYThe time interval between administering the serum and the virus was found to influence the results of the in vivo mouse protection test for foot-and-mouth disease antibodies. In particular, for both IgG and IgM antibodies to strain A12 virus, the mouse protection index increased from zero to a maximum at about 6 h and remained high for at least five days.
Variations in the antiserum concentration, on a log scale, had a proportional effect on the mouse protection index, if between 1 and 3. The constant of proportionality was unity for IgM and 2 for IgG antibody. Comparison with in vitro neutralization tests revealed essentially parallel neutralization curves. The lower serum titre in the protection test, if computed for less than 103 LD50/dose, was accounted for by the simple dilution of the inoculated serum into the volume of the mouse. Consequently, in the low titre range, the same virus-antibody reaction and its effect are operable in each of the two tests. Analysis of literature data in which both the in vivo protection test and the in vitro neutralization test results were available on the same sera showed consistency with the above conclusions for both cattle and swine sera.
The protection test had a highly atypical survival pattern occurring at antibody concentrations expected to neutralize more than 103 LD50/dose. The resulting in vivo dampening effect on virus titre is postulated to be caused by the excess antibody of the passive immunity test interfering with the spread of infection. The effect is analogous to an anomaly caused by not removing the inoculum in quantal tissue culture assays and it prevents quantification of antibody levels in strong sera.
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Effect of Interferon on Murine Leukaemia Virus Infection. IV. Formation of Non-infectious Virus in Chronically Infected Cells
More LessSUMMARYInterferon (150 units/ml) was used to treat SC-1 and AKR-2B cells which were chronically infected with murine leukaemia virus (MuLV). This led to a 100-fold decrease in the amount of infectious virus released into the medium and a 10-fold decrease in the number of virus particles measured by the virion-associated reverse transcriptase assay. However, there was little change in the amount of cell-associated infectious virus, though nearly twice as many cell-associated virions were counted in electron micrographs. With both types of cells, interferon blocked MuLV replication at the post-budding stage, but it did not change the morphology of the particles produced or their content of virion 70S RNA.
Infectious virus assembled on the cell membranes of interferon-treated cells was less stable at 37°C than that grown in the absence of interferon. Release of infectious virus from interferon-treated cells was not inhibited by actinomycin D or cycloheximide, though both agents inhibited virus production in controls.
These results show that interferon inhibits MuLV replication through effects on virion assembly; these lead both to the formation of non-infectious particles and of fewer virions. Kinetic analysis further shows that interferon affects MuLV assembly rapidly and induction of an antiviral protein may not be required.
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Infectious Lymphocytes in Lymphocytic Choriomeningitis Virus Carrier Mice
More LessSummaryThe infectivity of blood and lymphoid organs of mice persistently infected with lymphocytic choriomeningitis virus was found to be predominantly associated with lymphocytes and both T and B cells were infectious. A hypothesis is presented in which it is assumed that lymphocytes in carrier mice are infected via their LCM virus-specific antigen receptors, thereby leading to their antigen-triggered clonal expansion followed by infection and functional inactivation.
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Radioimmunoassay of Hepatitis B e-antigen (HBeAg): identification of HBeAg not associated with Immunoglobulins
More LessSUMMARYA radioimmunoassay for hepatitis B e-antigen (HBeAg) is described. Polystyrene beads coated with IgG prepared from a human serum containing antibodies to HBeAg (anti-HBe) and anti-HBe IgG labelled with 125I-p-hydroxyphenylpropionic acid N-hydroxysuccinimide ester were used in the test. The radioimmunoassay was approximately 1000-fold more sensitive than immunodiffusion. At least a transient presence of HBeAg in serum appears to be a common feature of infections by hepatitis B virus. The radioimmunoassay was instrumental in establishing conditions for identification of apparently free monomeric HBeAg. The HBeAg has an approximate mol. wt. of 35000 and was recovered after isoelectric focusing in fractions with a pH between 4.25 and 4.8. Polyacrylamide gel electrophoresis revealed the presence in HBeAg of a polypeptide with an apparent mol. wt. of 17000.
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Rapid Diagnosis of Tick-borne Encephalitis by means of Enzyme Linked Immunosorbent Assay
More LessSUMMARYAn enzyme-linked immunosorbent assay was applied for determining separately IgM and IgG antibodies against tick-borne encephalitis virus. A micro-modification in microtitre plates proved to be at least as sensitive as the HI test. However, more precise information could be achieved by a macrotest using antigen coated polystyrene balls. False positive results in IgM antibody determinations could be caused by a rheumatoid factor. A high content of IgM antibodies in a serum could impair the determination of its IgG antibodies but not vice versa. Titres were expressed in comparison to a positive control serum.
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Demonstration of Hepatitis B e Antigen in Hepatitis B Core Particles Obtained from the Nucleus of Hepatocytes Infected with Hepatitis B Virus
More LessSUMMARYLiver tissue infected with hepatitis B virus was homogenized and nuclei were separated by centrifugation. Hepatitis B core particles were obtained from the nucleus by the digestion with pronase followed by ultracentrifugation in a sucrose density gradient. Hepatitis B core particles were then treated with sodium dodecyl sulphate and 2 mercaptoethanol and tested for hepatitis B e antigen (HBeAg) by the haemagglutination method. The antigenicity of HBeAg was clearly demonstrated in hepatitis B core particles so treated, although untreated core particles did not reveal any detectable HBeAg activity. The localization of HBeAg in hepatitis B core particles was further supported by the results of a fluorescent antibody technique. When a frozen section of the liver infected with hepatitis B virus was stained with the specific rabbit antibody against HBeAg labelled with fluorescent isothiocyanate, only nuclei of hepatocytes were stained, in a similar distribution to hepatitis B core antigen visualized by fluorescent antibody against hepatitis B core antigen in a nearby section.
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Demonstration of an unusual DNA polymerase activity associated with the L cell virion
More LessSUMMARYPurified preparations of L cell virions (LCV) were found to possess an associated DNA polymerase activity. This enzyme was active with poly(C). oligo(dG) and poly(Cm). oligo(dG) and was able to transcribe poly(A). oligo(dT). Endogenous DNA synthesis was also demonstrable in disrupted virion preparations but this reaction was enhanced, rather than inhibited, by RNase pre-treatment. The effects of variations in a number of the assay parameters on these activities were examined in an attempt to determine the class of DNA polymerase involved.
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Two Molecular Species of Mouse L Cell Interferon Differing in Lectin Binding
More LessSUMMARYBinding of L cell interferon to lectins, Wistaria floribunda agglutinin (WFA) and concanavalin A (Con A) was studied by affinity chromatography. Of the two molecular species of L cell interferon, F (mol. wt. 24000) and S (mol. wt. 36000), only the latter was bound efficiently to WFA-Sepharose and eluted quantitatively with d-galactose followed by a pH 3 buffer, suggesting a substantial difference between the two interferon species in their carbohydrate structure. Both were partially bound to Con A-Sepharose and eluted with α-methyl-d-glucoside, indicating that at least some of the F species are also glycoprotein, and that both F and S interferons are heterogeneous as regards their affinity to this lectin.
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The Measurement of Haemagglutinin and Matrix Protein Present on the Surface of Influenza Virus Infected P815 Mastocytoma Cells
More LessSUMMARYA thermodynamic approach has been used to measure the amount of haemagglutinin and matrix protein expressed at the surface of P815 cells infected for periods between 4.5 and 11 h with either WSN (HoN1) or JAP (H2N2) strains of type A influenza virus. This involved measuring the interaction of different concentrations of labelled (Fab)2 preparations of specific antibody with normal and infected cells. Assuming that one molecule of (Fab)2 bound to one molecule of antigen, values for the number of molecules of antigen/infected cell ranged from 7.6 × 105 to 1.7 × 107 for haemagglutinin and 1.3 × 105 to 1.1 × 106 for matrix protein. The ratio of haemagglutinin/matrix protein was lower for WSN-infected cells (1.7) than for JAP-infected cells (10). The same reagents were reacted with three purified A type virions, WSN, JAP and Port Chalmers (H3N2). Each preparation bound anti-matrix protein (Fab)2 though the value for haemagglutinin/matrix protein was much higher (66) than for infected cells and suggested that a virion may have a small number (about 12) of matrix protein molecules exposed though it was not excluded that the matrix protein detected was exposed only on damaged virions. Pre-treatment of infected cells with unlabelled reagent (anti-haemagglutinin) reduced the subsequent binding of the same labelled reagent but not the binding of the labelled matrix protein reagent and vice versa, suggesting that the haemagglutinin and matrix protein were not very close to each other on the cell surface.
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The Responses of Nude-Athymic Mice to Nominally Avirulent Togavirus Infections
More LessSUMMARYFollowing intraperitoneal infection by an avirulent strain of Semliki Forest virus, athymic nude mice showed almost normal clearance of viraemia and a transitory peak of antibody activity at 5 to 9 days which fell to less than about 0.1% of the normal antibody activity from the 14th day. When nude mice received a transfer of normal spleen cells from sex-matched litter mates at 1 day before infection, a pattern of high and continous antibody synthesis was established for at least the following 7 weeks.
This clear T-cell dependence of the regulation of serum antibody synthesis was unrelated to the development of regulatory (pre-challenge) or protective (post-challenge) immunity since, particularly for female nude mice, up to 60% were benignly and protectively infected in the absence of detectable antibody activity. The brains of such nude mice showed persistence of infectivity for at least 7 weeks at 10 to 104 p.f.u./brain after avirulent infection and at about 103 to 104 p.f.u./brain after virulent challenge. The prior transfer of normal spleen cells to nude mice enabled them to clear brain infectivity as efficiently as normal mice.
These results are discussed in terms of the evident interplay of both T-lymphocyte dependent and T-lymphocyte independent functions in the control of brain infectivity, in the expression of virulence and in the stimulation of regulatory and protective immunity.
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Isolation and Characterization of BK Virus-Transformed Rat and Mouse Cells
More LessSUMMARYThe isolation and characterization of four groups of BK virus (BKV)-transformed rat embryo fibroblast (RE) and mouse kidney (MK) cells are described. They consist of (1) seven RE lines transformed with a BKV pool containing a high proportion of defective virions, and (2) 16 RE, (3) 14 Balb/c-MK and (4) 2 Swiss ICR-MK lines, all transformed, at different input multiplicities, with a pool of BKV free of defective virions.
None of the lines produces BKV, all contain BKV T antigen and all grow to higher saturation densities and have higher plating efficiencies than do the corresponding control cells. Cells of the RE lines, transformed with the BKV pool containing defective virions, form colonies in soft agar and produce tumours in irradiated weanling rats, while those of the RE lines transformed with the defective virion-free pool do neither. Cells of the Balb/c-MK, but not of the ICR-MK lines are tumorigenic, although cells of both groups form colonies in soft agar. In general, those lines transformed at higher multiplicities express the biological properties associated with transformation more strongly than do those transformed at lower multiplicities.
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Productive Influenza Virus Infection of Synchronized Chick Embryo Fibroblast Cells
More LessSUMMARYThe effects of cell metabolic activity on the outcome of influenza virus infection were studied in partially synchronized chick embryo fibroblast cultures. There was no evidence to show that the time in the cell cycle at which cells were infected had any significant effect on the final virus yield. However, some differences were detected in the length of the latent period between infections established in synchronized or in stationary cells. Influenza virus could replicate in synchronized or normal cell cultures in which DNA synthesis was inhibited with 9-β-d-arabinofuranosyladenine (ara-A).
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Effects of Leukocyte and Fibroblast Interferon on Events in the Fibroblast Cell Cycle
More LessSUMMARYSerum-depleted human foetal skin fibroblasts were stimulated by addition of 10% foetal calf serum to proliferate synchronously for at least one cell cycle. This proliferation was suppressed by leukocyte or fibroblast interferon (IF), which prolonged the G1 phase and diminished the rate of DNA synthesis during the S phase in a dose-dependent manner. When used in identical concentration, as judged in terms of units of antiviral activity, fibroblast IF had more pronounced effects on cell cycle events than leukocyte IF. Interferon exerted its effect in early G1, before the cells were irreversibly committed to DNA synthesis.
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Detection and Quantification of Foot and Mouth Disease Virus by Enzyme Labelled Immunosorbent Assay Techniques
More LessSUMMARYEnzyme labelled immunosorbent assays (ELISA) have been developed to detect and quantify foot and mouth disease (FMD) virus using flexible plastic microtitre plates. The methods were successful for the specific detection of FMD virus and were 50 to 100 times more sensitive than the complement fixation test. The application of the ELISA techniques to FMD virus typing and subtyping, and to the assay of antigen concentration during manufacture of vaccines is discussed.
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A Comparison of Polypeptides in Measles and SSPE Virus Strains
More LessSUMMARYThe polypeptide patterns of twelve subacute sclerosing panencephalitis (SSPE) and measles viruses have been compared by slab gel electrophoresis. The polypeptide patterns of nine strains of SSPE and measles virus were identical. Differences in the NP protein and the HA protein of the Oddo strain, the P protein of the large plaque variant of the Lec strain of SSPE and in the M protein of the Hu 2 measles strain could not be correlated with biological characteristics such as plaque morphology, origin or haemagglutination properties.
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Virus Carrier State Suppresses Tumorigenicity of Tumor Cells in Athymic (Nude) Mice
More LessSUMMARYNude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.
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Impairment of Hormone Dependent Signal Transfer by Chronic SSPE Virus Infection
M. Halbach and K. KoschelSUMMARYIn a CNS-derived cell line, the cellular response to hormonal stimulation, represented by the rise of intracellular cAMP levels, is impaired under the influence of a persisting neurotropic virus infection. This dysfunction is caused by the decrease in adenylate cyclase activity, most probably due to the virus-induced loss of active catalytic units.
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