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Volume 44,
Issue 2,
1979
Volume 44, Issue 2, 1979
- Articles
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Intracellular State of Marek’s Disease Virus DNA in Two Tumour-derived Chicken Cell Lines
More LessSUMMARYThe intracellular state of Marek’s disease virus (MDV) DNA was investigated in two permanent chicken cell lines: HPRS-1 and MSB-1. The HPRS-1 line was established from an ovarian lymphoma of a chicken with Marek’s disease and is a virus-non-productive line, while the MSB-1 line originates from another animal with a Marek’s disease splenic lymphoma and is a low producer line. By repeated isopycnic centrifugation in CsCl, MDV DNA in the HPRS-1 line showed properties of integrated DNA, whereas in cells of the productive MSB-1 line both integrated and free virus DNA appeared to be present. Under denaturing conditions (0.1 m-NaOH) the virus DNA remained associated with the cellular DNA as revealed by equilibrium centrifugation in CsCl and hybridization of the DNA in each single fraction with 32P-labelled complementary RNA transcribed from the DNA of the GA strain of MDV. Shearing of HPRS-1 DNA to a mol. wt. of about 8 × 106 released only part of the virus DNA to the density of free virus DNA, while a large proportion of MDV DNA could still be localized at the density of cellular DNA. Sedimentation velocity experiments with HPRS-1 and MSB-1 DNA originally fractionated on CsCl gradients revealed integrated virus DNA sequences in both cell lines and an additional peak of virus DNA at the position of free linear MDV DNA in the MSB-1 line. No fast-sedimenting virus DNA molecules with properties of covalently closed circular structures could be detected. Further evidence for the presence of integrated virus DNA sequences in MDV-transformed cells was provided by the Hirt (1967) precipitation procedure.
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A Solid-phase Immunosorbent Technique for the Rapid Detection of Rubella IgM by Haemagglutination Inhibition
More LessSUMMARYThe rubella haemagglutination inhibition (HAI) test has been modified for the detection of rubella-specific IgM. The rubella HAI test is performed in microtitre plate wells in which IgM from patients’ sera has been selectively retained by anti-human IgM bound to the polystyrene surface. The test requires only anti-human IgM-coated microtitre plates, in addition to the standard rubella HAI reagents. The results obtained in this test, a solid-phase immunosorbent technique (SPIT), are in good agreement with results obtained by the sucrose density gradient centrifugation technique (DGCT). Advantages include the essentially unrestricted number of sera which can be tested daily, the availability of results within 24 h and the lack of interference by rheumatoid factor and by rubella-specific IgG.
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Induction of Simian Virus 40-specific Tumour Rejection by the Ad2+ND2 Hybrid Virus
More LessSUMMARYImmunization of BALB/c mice with Ad2+ND2, a non-defective hybrid virus containing about half of the early region of simian virus 40 (SV40) DNA covalently integrated into the human adenovirus 2 (Ad2) genome, can confer protection against subsequent challenge by syngeneic SV40 tumour cells. Analysis of subcellular fractions from Ad2+ND2-infected cells shows a close correlation between the tumour rejection activity and the presence of the two SV40-specific proteins induced by this hybrid virus. These two proteins, with mol. wt. of 56000 (56K) and 42000 (42K), can be specifically immunoprecipitated using sera obtained from hamsters bearing SV40-induced tumours. Such immunoprecipitates, which contain no detectable contaminating components as determined by polyacrylamide gel electrophoresis, can efficiently immunize mice against SV40 tumour challenge, suggesting that the 56K and 42K proteins are directly responsible for the induction of tumour rejection. Moreover, we have found, by immunoprecipitation, a novel antigen in SV40-transformed BALB/c cells, also of 56000 mol. wt.; possibly, this 56K protein is responsible for induction of transplantation immunity in SV40-transformed cells.
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Modulation of Herpes Simplex Virus Replication in Adenovirus Transformed Cells
More LessSUMMARYThe ability of herpes simplex virus type 1 to replicate in cells transformed by adenovirus type 5 is strongly dependent on the origin of the cells. Studies show that adenovirus transformed rat cells lose their permissiveness while cells of hamster or human origin retain their ability to replicate HSV although at a reduced level when compared to the untransformed parent cells. One line of adenovirus transformed rat cells, 107, demonstrates thermosensitive events, allowing HSV to replicate at 34 °C but not at 37 °C. Analysis of the biochemical events taking place at 37 °C showed that virus-specific DNA synthesis was greatly reduced but that all of the late virus structural proteins could be observed after SDS-polyacrylamide gel electrophoresis. It was also demonstrated that shut-off of host macromolecular synthesis appeared to be less efficient after HSV infection of 107 cells than after infection of more permissive cells such as the non-transformed REF line. Collectively the data show that interactions between HSV and the host cell are perturbed when the cell is transformed by type 5 adenovirus. The degree of perturbation ranges from a slight reduction in number of progeny to a completely abortive infection.
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Sequence Studies of Poliovirus RNA. IV. Nucleotide Sequence Complexities of Poliovirus Type 1, Type 2 and Two Type 1 Defective Interfering Particles RNAs, and Fingerprint of the Poliovirus Type 3 Genome
More LessSUMMARYThe 32P-labelled genomes of poliovirus type 1, 2 and 3 have been digested with RNase T1 and the products separated by two-dimensional gel electrophoresis. All three fingerprints differ in the separation pattern of the large oligonucleotides. The molar yields of the large RNase T1-resistant oligonucleotides of type 1 and type 2 RNA of poliovirus RNA are close to one. By comparing the yields of these oligonucleotides to the amount of RNA from which they originated, the chain length of type 1 poliovirus RNA was found to be 7851 ± 567 nucleotides (mol. wt. 2.66 ± 0.19 × 106) and that of poliovirus type 2, 8181 ± 578 nucleotides (mol. wt. 2.77 ± 0.19 × 106). The chain length of two defective interfering particle (DI) RNAs of poliovirus type 1 were determined to be 7042 ± 999 nucleotides for DI(1) and 6639 ± 674 nucleotides for DI(2).
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Studies of the Effect of Chloramphenicol, Ethidium Bromide and Camptothecin on the Reproduction of Rous Sarcoma virus in Infected Chick Embryo Cells
More LessSUMMARYA function for mitochondria in the reproduction of Rous sarcoma virus (RSV) in chronically and newly infected chick embryo cells was studied by using chloramphenicol and ethidium bromide. Chloramphenicol (CAM) and ethidium bromide (EB) were both shown to decrease the rate of growth of infected chick embryo cells and to inhibit the synthesis of mitochondrial macromolecules. Both drugs however had little or no effect on the incorporation of labelled leucine, thymidine and uridine into total cellular macromolecules. Neither CAM (80 µg/ml) nor EB (0.4 µg/ml) inhibited the production of infectious virus. In contrast, camptothecin, an inhibitor of cellular but not mitochondrial macromolecular synthesis, was shown to depress the production of infectious virus. The results indicate that the mitochondrial macromolecular synthesis machinery of RSV-infected chick embryo cells does not contribute to virus production.
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Characterization of Autographa californica Nuclear Polyhedrosis Virus Deoxyribonucleic Acid
More LessSUMMARYThe DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) was isolated from extracellular non-occluded virions. The guanosine plus cytosine content of AcNPV DNA was estimated from its buoyant density in CsCl and its Tm value to be about 43 mol %. In ethidium bromide-CsCl gradients double-stranded covalently closed DNA molecules (form I) were detected, while relaxed circular (form II) and linear (form III) DNA molecules were demonstrated by velocity sedimentation in sucrose. The mol. wt. of AcNPV DNA was calculated to be about 78 × 106 from the sedimentation value of form III DNA. Digestion of AcNPV DNA with restriction endonucleases EcoRI and BamI resulted in 21 and 7 fragments, respectively, after analysis on agarose gels. The mol. wt. of the fragments and their molar ratio were determined. By this method the mol. wt. of AcNPV DNA was calculated to be about 83 × 106.
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Pathogenesis of Murine Cytomegalovirus Infection: the Macrophage as a Permissive Cell for Cytomegalovirus Infection, Replication and Latency
More LessSUMMARYMacrophages harvested from the peritoneal cavities of mice of several strains were permissive to infection with murine cytomegalovirus (MCMV). Macrophages from six mouse strains released equivalent amounts of plaque-forming virus into the culture fluids and cells from three mouse strains scored similarly in numbers of infectious centres. Twenty to 50% of the infected macrophages obtained after thioglycollate activation formed infectious centres. When studied by in situ hybridization, more than 82% of infected macrophages (with or without thioglycollate activation) contained MCMV DNA.
Macrophages obtained from latently infected mice were examined for their content of MCMV. Using co-cultivation assays, MCMV was frequently recovered from thioglycollate activated macrophages harvested from latently infected mice but only rarely recovered from non-activated macrophages. MCMV DNA-mouse DNA hybridization assays revealed four to seven virus genome DNA copies per 100 cells. These studies indicate that macrophages harvested from mice susceptible (BALB/cSt) or resistant (C3H) to MCMV infection replicated virus equivalently and that macrophages are a reservoir of MCMV during latent and chronic infections. Activation of macrophages may be one of the important steps leading to the exacerbation of in vivo latent infections.
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Studies on the Induction of IgG-Fc Receptors and Synthesis of IgM in Primary and Chronically-Infected Lymphoid (Raji) Cells by Herpes Simplex Virus
More LessSUMMARYThe present paper reports on the induction of two cell surface markers on human lymphoid cells following herpes simplex virus (HSV) infection. While both primary and chronic infections of human lymphoid cells led to the induction of receptors for the Fc region of 7S IgG, chronic HSV infection was also characterized by the induction of surface-bound IgM. Surface and intracellular Fc receptors were detected in the human lymphoid cell line, Raji, infected with HSV types 1 and 2. Under optimal conditions with a multiplicity of infection (m.o.i.) of 50 to 100 p.f.u. per cell, this marker was inducible in only about 53% of the infected cells. Kinetic studies revealed the appearance of these receptors at around 5 h following HSV infection and they reached a plateau 16 to 18 h p.i. Interestingly, this Fc receptor expression (i.e. percentage of positive cells) was found to be similar in primary and chronically HSV-infected Raji cells. Both human leukocyte interferon and phosphonoacetic acid (PAA), an inhibitor of herpesvirus DNA polymerase activity, effectively inhibited Fc receptor synthesis during primary HSV-infection and these two agents suppressed its induction in chronically HSV-infected Raji (Raji-HSV) cells. This inhibitory or suppressive effect, particularly of PAA, suggests that this HSV-induced Fc receptor may represent a late virus function in the infected cell. Unlike primary HSV infection, about 80% of the chronically HSV-infected Raji cells were found to express surface-bound IgM. This IgM induction was suppressed by long-term interferon treatment but not with PAA-treatment. Superinfection studies of interferon and PAA-treated Raji-HSV cells indicated that only the former would develop Fc receptors suggesting a protective role of this IgM against superinfection by HSV.
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Multi-factorial Specification of Virus-Host Interactions: Studies with Strains of Venezuelan Equine Encephalomyelitis Virus in Mice
More LessSUMMARYMice of different ages were infected i.p. or i.c. by 23 different strains of VEE virus. The course of the virus host interaction was specified in terms of the efficiency of infection, the outcome of infection as lethality or protection and the survival time. These separately quantifiable features all showed several host-maturation events that combine to provide a multifactorial specification of virus-strains and host-responses. This base-line for correlations with the responses of principal hosts (equidae and man) may be expanded to test correlations with the antigenic or in vitro characteristics of virus-strains.
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Simian Foamy Virus-induced Immunosuppression in Rabbits
More LessSUMMARYA persistent foamy virus infection was established in rabbits and its effect on the cell-mediated (CMI) and humoral immune response was studied. Virus was consistently isolated from viable, peripheral blood mononuclear cells collected 7 to 164 days after inoculation, by co-cultivation, with cell monolayers. The response of leukocytes from infected rabbits to in vitro stimulation with phytohaemagglutinin was studied by two techniques: incorporation of 3H-thymidine and production of the lymphokine, immune interferon. Both parameters of the cell-mediated immune response were depressed in leukocytes collected from foamy virus infected rabbits during the first 2 weeks of the infection. This depression in the cell-mediated immune response was not observed after 14 days p.i. Since primary or reactivation infections with herpes viruses are common following immunosuppression, it was interesting to note that one of the rabbits persistently infected with foamy virus developed a herpes virus infection. The humoral response in infected rabbits appears to be unaffected since the virus infection did not alter the development of antibodies to sheep erythrocytes. Transient depression of the cell-mediated immune response in foamy virus infections may be important in initiating persistence and demonstrates for the first time that foamy viruses can, indeed, have adverse effects.
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The Isolation of Mengo Virus Stable Non-capsid Polypeptides from Infected L Cells and Preliminary Characterization of an RNA Polymerase Activity Associated with Polypeptide E
More LessSUMMARYIsolation of the Mengo virus stable non-capsid virus polypeptides E, F, G and I from infected L cells has been achieved. Unstable precursors were eliminated by incubation in the presence of pactamycin and capsid polypeptides were removed by ultracentrifugation and affinity chromatography. Subsequent sodium dodecyl sulphate (SDS)-hydroxylapatite chromatography resolved the non-capsid proteins into two major peaks which comprised F plus G and E plus I, respectively. The individual polypeptide species were separated by gel filtration on Sephadex G-100 in the presence of SDS.
Polypeptide E was isolated in an undenatured form by gel filtration of infected cell extracts (from which precursor and capsid polypeptides had been removed) on Bio-Gel A-5m agarose beads. Purified polypeptide E was found to co-sediment with Mengo virion RNA during centrifugation in a sucrose density gradient and it was also capable of binding to poly(A)-Sepharose. Assay mixtures containing polypeptide E exhibited an RNA polymerase activity which was dependent upon exogenous virus RNA template and oligo(U) primer and which was not affected by the addition of virus capsid polypeptides or extracts from uninfected cells.
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Fer de Lance virus (FDLV): a Probable Paramyxovirus Isolated from a Reptile
More LessSUMMARYA new virus has been isolated by inoculation of lung tissues of diseased snakes into snake embryos. Homogenates of infected embryo tissues caused c.p.e. in cell cultures incubated at 30 °C. The virus replicates in a wide variety of reptilian or mammalian cell types incubated at 30 °C, inducing either syncytium formation or minimal or no cytopathic changes. Efficient replication occurs in embryonated hens′ eggs at 27 to 30 °C. The virus haemagglutinates guinea pig and chick erythrocytes; it possesses a neuraminidase similar to the receptor-destroying enzyme of Vibrio cholera. Electron microscopic observations of infected cells examined in thin section revealed pleomorphic viruses 146 to 321 nm in diam. resembling known myxoviruses. Internal nucleocapsid strands are 15 to 16 nm in diam.; nucleocapsid observed in negatively stained preparations measures 14 nm in diam. The virus was determined to possess a nucleoprotein core containing a 50S single-stranded unsegmented RNA genome. All characters of the virus are similar to those of the paramyxovirus group except that the nucleocapsid diam. is intermediate between that of paramyxoviruses and pneumoviruses. The virus is antigenically distinct from known myxoviruses and is unique among myxoviruses in its restriction to growth at temperature below 37 °C.
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Morphogenesis of Nuclear Inclusions and Virus Capsids in HEL Cells Infected with Temperature-sensitive Mutants of Human Cytomegalovirus
More LessSUMMARYThe morphogenesis of nuclear inclusions and virus capsids in human embryonic lung cells infected with ts mutants of human cytomegalovirus at permissive (34 °C) and non-permissive (39 °C) temperatures was studied by indirect immunofluorescence (IF) and electron microscopic analyses and compared with the morphogenesis of these structures in wild-type virus infection with or without phosphonoacetate. Mutants tested belonged to five different complementation groups: two groups were DNA− (those unable to synthesize virus DNA at 39 °C) and the others were DNA+. Based on the previous finding that the electron-dense, reticular nuclear inclusions (EM-NI) observed by the thin-section analysis correspond with nuclear inclusions (IF-NI) detected by the indirect IF staining (i.e. they occupy the same space in the nucleus), the following conclusions were obtained in ts mutant infection at 39 °C: (i) the formation of EM-NI, IF-NI and virus capsids requires replication of virus DNA. (ii) The formation of EM-NI is not necessarily accompanied by the formation of IF-NI; EM-NI itself is not IF-positive unless it acquires virus-specific late antigens. (iii) The assembly of virus capsids occurs only in those cells in which EM-NI is formed; however, it can occur without the formation of IF-NI. (iv) Virus capsids assembled are not the major antigens responsible for the fluorescence of nuclear inclusions.
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Differences in the Morphology of Herpes Simplex Virus Infected Cells: I. Comparative Scanning and Transmission Electron Microscopic Studies on HSV-1 Infected HEp-2 and Chick Embryo Fibroblast Cells
More LessSUMMARYInfection with herpes simplex virus type 1 (HSV-1) induces different morphological changes in different cell lines. This is demonstrated by comparative scanning (SEM and transmission (TEM) electron microscopic investigations of cell cultures prepared under identical conditions. SEM of HSV-1 infected HEp-2 cells reveals a slightly altered cell surface: only the number of the microvilli is reduced. Large amounts of released virions are detectable adhering to the outer plasma membrane. Ultra-thin sections show typical virus maturation steps in the nuclei (formation of nucleocapsids and virus budding from the inner lamella of the nuclear membrane) and in the cytoplasm (egress of enveloped nucleocapsids through membranous structures). HSV-infected primary chick embryo fibroblast (CEF) cells are characterized by crumpled and rough surfaces without virus particles adhering to the membrane. Ultra-thin sections exhibit atypical virus maturation with many unenveloped nucleocapsids within the cytoplasm. The distribution of HSV-induced antigen(s) on the surface of the infected cells is identical in the two cell systems as determined by the peroxidase labelling technique. The c.p.e. (as seen by phase contrast light microscopy) is similar in both HEp-2 and CEF cells: both fusion and rounding up is induced in the infected cells.
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Comparative Studies of Wild-type and Cold-mutant (Temperature-sensitive) Influenza Viruses: Independent Segregation of Temperature-sensitivity of Virus Replication from Temperature-sensitivity of Virion Transcriptase Activity during Recombination of Mutant A/Ann Arbor/6/60 with Wild-type H3N2 Strains
More LessSUMMARYRNA 1* (see end of Summary) of a cold-adapted and temperature-sensitive (ts) influenza virus mutant A/Ann Arbor/6/60 has a different mobility from RNA 1 of wild-type (wt) A/Ann Arbor/6/60 when subjected to electrophoresis through acrylamide/agarose gels in the absence of denaturing agents. Detection of this lesion in RNA 1 of the mutant virus was dependent on the temperature of the gel during electrophoresis. Because RNA 1 is believed to code for a protein involved in virus-specific RNA synthesis we compared phenotypes of virion transcriptases in the wt and mutant viruses. The enzyme of the mutant virus was found to be about 40% less active at 40 °C than the enzyme of the wt virus when related to their activities at 31 °C. Two cold-adapted ts recombinants which derive their RNA 1 from the mutant A/Ann Arbor/6/60 have virion transcriptases with a phenotype similar to that of their mutant parent. Three different cold-adapted ts recombinants, however, which also derive their RNA 1 from the mutant A/Ann Arbor/6/60, have virion transcriptases with a phenotype similar to that of wt virus. We conclude, therefore, that the conditional-lethal ts property of A/Ann Arbor/6/60 mutant and its recombinants is independent of the phenotypic marker observed for the A/Ann Arbor/6/60 mutant virion transcriptase, and that the lesion in RNA 1 of the mutant may also be unrelated to the observed difference between virion transcriptases of the mutant and wt A/Ann Arbor/6/60 viruses. The phenotypes of the virion transcriptases in recombinants did, however, correlate with the derivation of their RNA 2. This suggests that the increased temperature-sensitivity of virion transcriptase of the A/Ann Arbor/6/60 mutant is caused by either (1) a lesion (not necessarily conditionally lethal) that occurred in its RNA 2 during the course of cold-adaptation, or (2) a lesion in another gene whose product is a component of the virion transcriptase complex, but which lesion is only expressed phenotypically when there is a synergistic interaction in the transcriptase complex with the product of A/Ann Arbor/6/60 RNA 2.
* Our nomenclature for RNAs 1, 2 and 3 relates to results of electrophoresis under reference conditions where urea is omitted from gels and water at a temperature of 38 to 39 °C is circulated through the heat exchanger of the electrophoresis apparatus. Application of this nomenclature to describe in the text results obtained with other conditions of electrophoresis has only been done where control experiments have been performed which verify the validity of such an extrapolation.
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Characterization of Infection and Replication of Mason-Pfizer Monkey Virus in Human Cell Cultures
More LessSUMMARYHuman cells derived from both normal and neoplastic tissues can be infected by Mason-Pfizer monkey virus (MPMV) without accompanying cytopathology. Infection of cell cultures such as human rhabdomyosarcoma (A204) results in a persistently infected cell line which can be subcultured over 30 sequential culture passages without significant change in phenotypic properties according to reverse, transcriptase (RT), MPMV p27 antigen content, virus particle count and infectivity titre. Productive virus infections were established at relatively low virus particle (VP) input multiplicities (p.i.m.; about 0.06 VP/cell) in A204 cell cultures. At higher p.i.m. (about 600 to 6000 VP/cell) newly synthesized virus was detected within 4 days post infection. Although virus production was cumulative following primary infection, after subculture of infected cultures MPMV production was greater during active cell division. Using synchronization techniques, MPMV replication in persistently infected cultures was found to be cell cycle-dependent. The major internal antigen, p27, was synthesized in G2 and newly synthesized virus particles were released predominantly during mitosis and early G1. Colcemid arrest of cells during mitosis inhibited subsequent MPMV release. Consequently, production of extracellular virus depends upon the progression of cells through the mitotic stage. These data, which provided a basic understanding of the virus-host relationship that occurs in primate cells productively infected with MPMV, were used as a guideline for isolating MPMV-like viruses from experimentally and naturally infected Rhesus monkeys.
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Correlation of Pathogenicity and Gene Constellation of Influenza A Viruses. III. Non-pathogenic Recombinants Derived from Highly Pathogenic Parent Strains
More LessSUMMARYWe have demonstrated by recombination of two highly pathogenic avian influenza viruses [A/FPV/Rostock (Hav1N1) × A/turkey/England/63 (Hav1-Nav3)] that recombinants can be isolated which are pathogenic as well as nonpathogenic for chickens. They carried the glycoproteins of either parent strains, and all are produced in infectious form in chick embryo cells. Genetic analysis revealed that the non-pathogenic recombinants possess a mixed RNA polymerase complex, consisting of pol 1, pol 2, ptra and NP gene products, while, with one exception, the pathogenic recombinants have the genes coding for the polymerase activity from one or other parent virus. The biological properties of the recombinant viruses did not correlate with their pathogenicity for chickens.
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Pneumoviruses: the Cell Surface of Lytically and Persistently Infected Cells
More LessSUMMARYHuman embryonic lung (MRC-5), feline embryo (FEA), mink lung (Mv1Lu) and monkey kidney (BSC-1) cells infected by respiratory syncytial virus showed characteristic morphological changes when viewed by scanning electron microscopy. The surfaces of respiratory synctial virus-infected cells developed a profusion of slender filaments after 48 h incubation at 31 °C. Similar changes in surface morphology were observed in BSC-1 cells infected by murine pneumonia virus. Filament production therefore appears to be a common property of pneumo-viruses. Filaments were not observed in cells infected with either syncytial and non-syncytial herpes simplex virus, the cytocidal vesicular stomatitis and Batai (Bunyaviridae) viruses, or the focus-inducing rabbit fibroma virus.
Filament production was not observed in cells infected with ts mutants of respiratory syncytial (RS) virus during incubation at the restrictive temperature, or in a persistently infected culture of BSC-1 cells at 37 °C. The persistently infected cells (the RS ts 1/BSC-1 line) had some of the characteristics of cells transformed by oncogenic viruses, namely ability to overlap adjacent cells and agglutination by a low concentration of concanavalin A. The pseudo-transformed phenotype was temperature-dependent, however, and suppressed by raising the temperature of incubation to 39 °C. The presence of virus antigen at the cell surface was similarly temperature-dependent in these cells, diminished at high temperature (39 °C) and enhanced at low temperature (31 °C), suggesting that the changes in the host cell were the result of insertion of virus protein into the cell membrane. Evidently, persistent infection by a cytoplasmic virus can produce alterations in the host cell usually associated with transformation by nuclear viruses.
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Resistance to Bacteriophage ϕ6 by Pseudomonas phaseolicola
More LessSUMMARYResistance to bacteriophage ϕ6 by Pseudomonas phaseolicola HB10Y occurred at a frequency of about 1 in 120000 cells. Statistical analysis by the fluctuation and re-spreading tests indicated that the majority of ϕ6r bacteria resulted from random mutation rather than from contact with the phage. However, exposing the bacteria to heated ϕ6 (50 °C for 1 min) or ϕ6 nucleocapsid (virus minus the lipid envelope) prior to testing for resistance increased the frequency two- to threefold; ϕ6 dsRNA, unheated ϕ6, or heated ϕ91 did not have this effect. Nine resistant variants were selected for further characterization. When DNA isolated from the variants was subjected to agarose gel electrophoresis, seven of the nine strains had two plasmid DNA bands typical of HB10Y; the remaining two strains had three plasmid bands. ϕ6 attached to the pili of six of the nine variants; four of the variants actually had higher adsorption rate constants than HB10Y. Culture supernatants from the ϕ6r strains did not inactivate ϕ6. One of the resistant strains, D5, produced large amounts of infectious ϕ6 particles during its exponential phase of growth without a significant effect on its growth rate. Treatment of D5 with ϕ6-antiserum resulted in the loss of ϕ6 production; however, unlike typical carrier-state cells, D5 retained its resistance to ϕ6.
The results indicate that resistance to ϕ6, although occurring at a relatively high frequency, is not due to one common event, such as loss of a plasmid and/or attachment sites.
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