- Volume 44, Issue 1, 1979
Volume 44, Issue 1, 1979
- Review Article
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- Articles
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A Fluoroimmunoassay for Measurement and Visualization of Antibody Bound to Surface Antigens of HSV-Infected Cells
More LessSUMMARYAn assay based on quantitative spectrofluorometry of surface immunofluorescence was applied to the study of reactions between antibody and surface antigens of viable HSV-infected cells in suspension. Fluorescence was expressed in terms of photon counts per second. Both the direct and indirect fluorescent antibody techniques proved acceptable for assay of surface reactions, yielding values of specific fluorescence as high as eight times those of controls. Fluorescence microscopy of cell populations assayed by spectrofluorometry allowed for simultaneous visual examination of surface antigens.
Comparison of the fluoroimmunoassay with the 51Cr-release test for cytolytic antibody to HSV-induced surface antigens revealed the latter to be the more sensitive, with antibody titres ranging up to four times those detected by fluoroimmunoassay. General correlation between the two assays was found using both rabbit and human sources of antisera.
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Characterization of Virus DNA Synthesized in KB Cells Infected with Two Temperature-sensitive Mutants of Adenovirus Type 5
More LessSUMMARYKB cells were infected with H5ts36 or H5ts125, two adenovirus type 5 (Ad5) mutants with a temperature-sensitive synthesis of virus DNA. Infection was started at the nonpermissive temperature and at 16 h p.i. the temperature was shifted down to the permissive temperature. Shortly after the shift-down H5ts125-infected cells showed an accumulation of purely single-stranded DNA of virus origin, which was not observed in H5ts36-infected cells. This single-stranded DNA has been characterized by hybridization and sedimentation analysis. It was found that the single-stranded DNA was derived from both complementary strands and consisted of short fragments. The observation that the single-stranded DNA accumulates in H5ts125-infected cells under conditions in which the amount of DNA binding protein is reduced, suggests that the DNA-binding protein is not only involved in initiation, but also in elongation of nascent strands.
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Comparisons of Virulence of Influenza Virus Recombinants in Ferrets in Relation to their Behaviour in Man and their Genetic Constitution
D. Campbell, C. Sweet and H. SmithSUMMARYTwo parent viruses, A/Finland/4/74 (H3N2) and A/Okuda/57 (H2N2), virulent and attenuated respectively for man, showed similar differences of virulence in ferrets as judged by estimations of 50% minimal infectious doses (MID50), the level and persistence of nasal infection, the height and duration of pyrexia and the level of lung infection. In ferrets, two recombinant clones, WRL 94 (H3N2) and WRL 105 (H3N2), were almost as virulent as A/Finland and indistinguishable from one another, a result which agreed well with genetic analysis (Hay et al., 1977); the RNA pieces of these recombinants appeared identical and largely derived from the virulent parent (A/Finland).
The results in ferrets did not agree with tests on clone WRL 94 in small numbers of human volunteers but they were not inconsistent with those on clone WRL 105 in larger numbers. It is possible therefore that careful tests in ferrets may yield more accurate information on the virulence of strains than limited tests in human volunteers.
A rapid test for virulence in ferrets is described. It could be used to screen many additional recombinants thereby yielding information on the genetical basis of virulence and indicating possible vaccine strains for more thorough testing in ferrets and in man.
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Enhanced Proliferation of Endogenous Virus in Chinese Hamster Cells Associated with Microtubules and the Mitotic Apparatus of the Host Cell
More LessSUMMARYChinese hamster ovary cells harbour intracytoplasmic virus-like particles of type A which are closely associated with sites of microtubule formation. We report here the enhanced proliferation of these particles and their release at the cell membrane by using either 5-bromodeoxyuridine or dibutyryl cyclic AMP. The extracellular mature particles are similar in morphology to retroviruses of type B. Close association of the type A virus precursors with microtubule organizing centres, i.e. kinetochores, centrioles and basal bodies, and with microtubules per se, is confirmed by studying the effects of the microtubule inhibitors Colcemid and vincristine sulphate. The role of microtubules in the activation and transport of the intracytoplasmic type A particles is discussed.
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Production of Tubular Structures in Vero Cells Infected with Herpes Simplex Virus Type 2: Effects of Ultraviolet Light Irradiation and Antimetabolites
More LessSUMMARYIn order to investigate the nature of tubular structures specifically found in herpes simplex virus type 2 (HSV-2)-infected cells, the multiplication of HSV-2 was studied in Vero cells cultured in the presence of varying concentrations of cytosine arabinoside (Ara-C) and cycloheximide (CH), inhibitors of DNA synthesis and protein synthesis respectively. Ara-C, at a concentration of 60 µg/ml, inhibited the multiplication of HSV-2 by more than 99% and also prevented the appearance of tubular structures and virus particles in the nuclei of infected cells. Nevertheless, the synthesis of virus specific surface antigens of HSV-2-infected Vero cells was not reduced, as revealed by the fluorescent antibody technique. On the other hand, 10 µg/ml of CH inhibited both the appearance of tubular structures and virus particles and the synthesis of virus specific surface antigens by more than 99%. These observations strongly suggest that the appearance of tubular structures is one of the late events in the process of virus multiplication.
To measure the comparative genome size needed to produce membrane antigens, tubular structures and infectious centres, the effect of u.v.-inactivation of HSV-2 on these processes was studied. After u.v.-irradiation, the capacity to induce tubular structures was inactivated at a slower rate than the capacity to form infectious centres, but at a faster rate than the induction of surface antigens. Furthermore, more tubular structures could be induced by u.v.-inactivated virus than by the nonirradiated virus which was diluted to the same infectivity as the u.v.-irradiated virus. These results indicate that expression of the entire genome is not required for the production of tubular structures.
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Physicochemical Properties and Restriction Maps of Simian Adenovirus Type 38 DNA
More LessSUMMARYThe sedimentation constant of simian virus type 38 (SV-38) DNA was estimated to be 31.6S. The intrinsic viscosity of DNA was on average 86.5 dl/g and the length of the molecule determined by electron microscopy was 10.6 µm. The average mol. wt., as determined by sedimentation and viscometry, was 21.5 × 106, which agreed well with the value derived from the length of the molecule (21.4 × 106) and with the value of 21.2 × 106 determined by the relative electrophoretic mobility of the DNA fragments produced by restriction endonucleases EcoR1, SalI and BglII.
The buoyant density of the DNA in caesium chloride and caesium sulphate was 1.7185 and 1.4295 g/ml respectively. The melting temperature of the DNA in 1 × SSC was 93.5 °C. The GC content calculated from ρ and Tm values was 59.3%.
BglII cleaves SV-38 DNA at three sites producing four fragments with mol. wt.: A, 9.3 × 106; B, 5.6 × 106; C, 3.3 × 106; and D, 2.9 × 106. After treatment with EcoR1 and SalI, SV-38 DNA is cleaved into five and six fragments respectively, with mol. wt. for EcoR1 fragments: A, 8.2 × 106; B, 6.5 × 106; C, 4.0 × 106; D, 1.27 × 106; and E, 1.07 × 106, and for SalI fragments: A, 6.5 × 106; B, 5.4 × 106; C, 4.2 × 106; D, 2.8 × 106; E, 2.5 × 106 and F, 0.25 × 106. The sequence of fragments within the SV-38 DNA molecules for BglII was deduced to be BDCA, and for EcoR1 - BCEAD.
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Mouse Hybrid Cell Lines produce Antibodies to Herpes Simplex Virus Type 1
More LessSUMMARYA solid-phase radioimmunoassay procedure has been devised for the assay of antibodies produced in the mouse to herpes simplex virus type 1 (HSV-1). It is based on the adsorption of virus to flexible micro-well plates and uses radio-iodine-labelled rabbit antibody against mouse immunoglobulin to assess antibody binding. Using this assay for screening, cell hybrids have been obtained which yield monoclonal antibody to HSV-1. The hybrids are between spleen cells from hyperimmune mice and an immunoglobulin-non-secreting, azaguanine resistant myeloma cell line (NS-1). From 480 hybrid cell lines initially examined, five stable cell lines were obtained which released HSV-1-specific antibody in vitro and in vivo. Mice carrying transplants of these cell lines yield binding titres in serum of up to 1/25000. Both IgG and IgM antibodies were obtained in this way.
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The Replication of Frog Virus 3 in an Amphibian Cell Line (XTC-2) Derived from Xenopus laevis
More LessSUMMARYFrog virus 3 (FV3) has been demonstrated to replicate in a Xenopus laevis cell line, XTC-2. The virus has been titrated in XTC-2 cells by plaque assay, but the efficiency of plaquing is lower than in minnow or hamster cells. The rate of replication is greater in XTC-2 cells than in BHK cells and this has been correlated with the appearance of virus specific polypeptides in infected cells. Electron microscope observations have shown that FV3 replicates in the cytoplasm of XTC-2 cells and that the virus may leave the cell by budding at the plasma membrane.
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The Physico-chemical Characterization of Bovine Ephemeral Fever Virus as a Member of the Family Rhabdoviridae
More LessSUMMARYThis study of the physico-chemical properties of bovine ephemeral fever virus was initiated to establish whether or not it should be classified as a rhabdovirus. In contrast to the regular bullet-shaped morphology of some rhabdoviruses the virus particles are often cone-shaped or slight variants from bullet-shaped. The virion contains single-stranded RNA sedimenting at 42S and six proteins with mol. wt. of 164, 101, 64, 53, 43 and 29 × 103. The protein P101 is located on the surface of the virus and is glycosylated. It is removed by treatment of the virus particles with trypsin. Protein P64, the nucleoprotein, was found to be a phosphoprotein, like the N protein of rabies virus, whereas in vesicular stomatitis virus NS is the phosphorylated protein.
Virus harvests contain defective-interfering particles. The particles are short cone-shaped forms about one-third the length of the infectious virion and similar in morphology to defective-interfering particles of vesicular stomatitis virus. These particles interfere with the replication of bovine ephemeral fever virus but not with the Indiana serotype of vesicular stomatitis virus. They contain single-stranded RNA sedimenting at 18 to 20S. The particles appear to have a protein composition identical to that found in the virus particle.
The physico-chemical properties of bovine ephemeral fever virus justify its inclusion in the family Rhabdoviridae. The protein composition differs in detail from that found for vesicular stomatitis and rabies viruses, but is similar to that found for Obodhiang and kotonkan, two rabies serogroup viruses isolated from insects in Africa.
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Determinants of Spontaneous Recovery and Persistence in MDCK Cells Infected with Lymphocytic Choriomeningitis Virus
More LessSUMMARYMDCK cells that normally would have been killed by standard lymphocytic choriomeningitis (LCM) virus were saved either by pre- or co-infection with defective interfering (DI) virus. The ability of these spared cells to produce virus-specific antigen (as well as infectious virus) and resist being killed by standard virus challenge was followed for at least 35 days. During this period both types of cultures displayed unique cycling patterns for the above characteristics. The most striking difference was the longevity of the infections. Cultures exposed to DI particles prior to standard virus became persistently infected, while co-infection with both virus types led to spontaneous curing with no trace of the previous infection. The basis for these dissimilar outcomes was traced to a hitherto undetected non-defective LCM virus (called SP) in the DI virus stocks used to preinfect MDCK cells. SP virus was not present in standard virus stocks but arose in long-term persistently infected L cells that had been initially infected with standard virus. Cloned SP virus shared species-specific antigens with standard virus, was resistant to inhibition by DI virus and was capable of turning self-curing cultures into cultures persistently synthesizing both DI and SP virus.
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Protein Synthesis in Bunyamwera Virus-infected Cells
More LessSUMMARYIn Vero cells infected with Bunyamwera virus there is a rapid inhibition of cell RNA and protein synthesis to levels of 30 and 3% respectively of the control rate, both the rate of inhibition and the time lag before its initiation being multiplicity dependent. Using u.v.-irradiated virus, investigation of the mechanism of inhibition of host cell protein synthesis indicates that synthesis of new virus components is required for inhibition to occur. Quantitative comparison of the proteins synthesized in infected cells shows that at higher m.o.i. synthesis of virus, as well as cellular proteins, is inhibited. Bunyamwera virus-infected Vero cells synthesized three virus-specific proteins identified as the structural virion proteins. Nucleoprotein is synthesized predominantly early in infection while the major envelope glycoprotein and the minor glycoprotein are synthesized predominantly late in the infection cycle.
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Effect of Undiluted Passage on the Polypeptides of Measles Virus
More LessSUMMARYMeasles virus induces a large polypeptide (L; mol. wt. 180 K), a large glycopolypeptide (H; mol. wt. 80 K), a nucleocapsid associated polypeptide (P; mol. wt. 70 K), a nucleocapsid polypeptide (N; mol. wt. 60 K), a second glycopolypeptide (F0; mol. wt. 60 K), a matrix or membrane polypeptide (M; mol. wt. 37 K) and a small polypeptide (S; mol. wt. 15 K). The second glycopolypeptide (F0) appears to be cleaved in purified measles virus. Defective interfering particles accumulate during passage of measles virus leading to a decrease in the amounts of virus-specific protein synthesized in infected cells. Even in the best preparations of purified measles virus, host proteins are always detected and these become more predominant in preparations with low infectivity.
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Feline Syncytium-forming Virus: DNA Provirus Size and Structure
More LessSUMMARYAn infectious DNA assay has been used to investigate the size and structure of the genome of feline syncytium-forming virus (FSFV). The dose response between DNA extracted from FSFV-infected cells and plaque number on feline embryo cells followed two-hit kinetics and the mol. wt. of the proviral DNA was estimated as approx. 6 × 106.
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Enzyme-assisted Purification of two Phloem-limited Plant Viruses: Tobacco Necrotic Dwarf and Potato Leafroll
Y. Takanami and S. KuboSUMMARYTobacco necrotic dwarf (TNDV) and potato leafroll (PLRV), viruses which are normally phloem-limited and are transmitted by aphids in the persistent manner, were successfully purified using Driselase, an enzyme that macerates plant tissue. Frozen tissue of Physalis floridana infected with TNDV or PLRV was homogenized in buffer containing Driselase and incubated with shaking. The virus particles were then purified by clarifying extracts with chloroform and n-butanol, precipitation of virus with polyethylene glycol, differential centrifugation and density-gradient sedimentation. The average yield of TNDV purified without the enzyme was 0.5 mg/kg of fresh tissue and of that purified with the enzyme was 4.7 mg. Yield of PLRV purified using the enzyme was 1.3 mg/kg. The sedimentation coefficients of both TNDV and PLRV were s 20, w = 115S. Enzyme treatment had no influence on the infectivity of the viruses, assessed either using tobacco mesophyll protoplasts or using aphids fed through membranes. The enzyme method described here may be useful for purification of other phloem-limited viruses.
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Initial Interaction of Human Fibroblast and Leukocyte Interferons with FS-4 Fibroblasts
More LessSUMMARYHuman FS-4 cells were exposed to human fibroblast interferon for various times and further incubated in the absence of interferon until challenged with vesicular stomatitis virus. Addition of antibody to fibroblast interferon at the time of removal of interferon did not alter the development of the antiviral state. If cells were exposed to interferon for 45 min at either 0 or 37 °C, they developed resistance upon subsequent incubation at 37 °C. However, less resistance developed if the cells were initially incubated at 0 °C. Our results indicate that a single interaction of fibroblast interferon with susceptible cells, either at 0 or 37 °C, is sufficient for the subsequent development of an antiviral state, at least in the short term experiment.
The kinetics of development of the antiviral state were compared with fibroblast and leukocyte interferon. The rise in the degree of antiviral resistance was steeper and maximal levels of resistance were reached sooner when FS-4 cells were incubated with increasing concentrations of fibroblast interferon than with leukocyte interferon. This suggests a greater affinity of fibroblast interferon for these cells.
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Interferon Induction by Viruses. II. Sindbis Virus: Interferon Induction Requires One-Quarter of the Genome – Genes G and A
More LessSUMMARYWe have measured the amounts of interferon formed by chick cells ‘aged’ in vitro in response to different amounts of infectious wild-type Sindbis virus. Our results suggest that one plaque-forming unit is enough to induce maximum interferon formation. With higher m.o.i. the yield of interferon is less.
To inactivate the interferon-inducing activity of Sindbis virus, four times more u.v.-radiation was needed than to inactivate the infectivity of the virus. This suggests that only 25% of the virus genome need be intact in order to induce interferon. Temperature-sensitive Sindbis virus mutants from the three RNA+ complementation groups, C, D and E, gave rise to interferon in chick cells incubated at a non-permissive temperature. Similarly, mutants from two of the RNA− groups, B and F, gave rise to interferon, but not mutants from groups G and A.
We conclude that no pre-formed inducer of interferon is present in Sindbis virus. It appears, however, that genes G and A represent a special one-quarter of the genome which must be functional in order to synthesize an interferon-inducing moiety. We suggest that this moiety is a double-stranded RNA molecule formed after synthesis of a segment of RNA complementary to the genome.
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