- Volume 66, Issue 7, 1985
Volume 66, Issue 7, 1985
- Review Article
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- Animal
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Examination of the Immunological Relationships between Flaviviruses Using Yellow Fever Virus Monoclonal Antibodies
More LessSUMMARYMonoclonal antibodies prepared against vaccine strains of yellow fever (YF) virus were initially characterized by fluorescence microscopy of Vero cells infected with YF virus strain 17D. When similarly tested against representatives of all flavivirus subgroups, the antibodies produced a wide spectrum of reactions ranging from the monospecific to the broadly cross-reactive; at least five antigenic domains in the YF virus envelope glycoprotein were identified. Monoclonal antibodies differentiated between YF virus vaccine strains (17D, 17DD, FNV), wild-type viruses and plaque variants selected from a 17D pool. One isolate from a patient with YF was antigenically similar to the Brazilian vaccine strain 17DD. Several of the antibodies reacting with the YF viral envelope glycoprotein in biological tests identified the 54K envelope glycoprotein; 45K and 26K polypeptides in YF 17D virus-infected cells were also identified by radioimmunoprecipitation and polyacrylamide gel electrophoresis. Neither of these polypeptides was found in uninfected cells. They may represent short-lived precursors of the 54K protein, post-translational cleavage or breakdown products. Other antibodies reacted with a 48K polypeptide in virus-infected cell lysates. This may be the non-structural NV3 protein described for YF virus. Its appearance on the surface of unfixed infected cells, but not on released virions, was demonstrated by fluorescence microscopy.
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Properties and Characterization of Monoclonal Antibodies to Tacaribe Virus
More LessSUMMARYMonoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies.
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Characterization of Major Structural Proteins of Measles Virus with Monoclonal Antibodies
More LessSUMMARYWe have prepared and characterized monoclonal antibodies against five major structural proteins, i.e. the HA, P, NP, F and M proteins, of measles virus. At least three non-overlapping antigenic sites were delineated on the HA protein, three on the P, four on the NP, four on the F and five on the M proteins by competitive binding assays. Antigenic sites on the HA and F proteins roughly represented functional domains defined by serological tests. The reactivity of monoclonal antibodies with various measles virus strains including those from subacute sclerosing panencephalitis (SSPE) and other members of the morbillivirus family was studied by immunofluorescence. A monoclonal antibody or set of monoclonal antibodies to each of the antigenic sites showed a characteristic pattern of cross-reactivity with heterologous strains. The HA and NP proteins were antigenically the most variable, followed by the F and M proteins, while the P protein was relatively stable. None of the 14 anti-M monoclonal antibodies reacted with non-virus-producing SSPE cells, strongly suggesting the absence of M protein in these cells.
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Effect of Measles Virus Antibodies on a Measles SSPE Virus Persistently Infected C6 Rat Glioma Cell Line
More LessSUMMARYMaintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.
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Relationships between the Multiplication of Chronic Bee-Paralysis Virus and its Associate Particle
More LessSUMMARYThe amounts of chronic bee-paralysis virus associate (CPVA) produced in bees injected with purified chronic bee-paralysis virus (CPV) were negatively associated with the amounts, lengths of particles and maximum sedimentation coefficients of CPV. The ratio of the amounts of CPV/CPVA varied widely between individuals, but more CPVA was produced and CPV/CPVA ratios were smaller when CPVA was injected with the CPV preparations than when the latter were injected alone. The multiplication of the RNAs of CPV and of CPVA were also negatively correlated. The nature of the relationship between CPV and CPVA and the packaging arrangement of CPV RNA are discussed.
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Propagation and Preliminary Characterization of a Chicken Candidate Calicivirus
More LessSUMMARYThe present paper reports the propagation and partial characterization of calici-like virus particles previously observed in gut homogenates obtained from stunted broiler chicks. Radiolabelling experiments with [3H]uridine showed that the virus possesses an RNA genome and that it can replicate in primary chick embryo fibroblasts in the presence of actinomycin D. In vivo studies showed that the virus can be serially passaged in specific pathogen-free chicks and virus particles can be recovered from the gut and excreta of infected birds. The morphological appearance and biophysical properties of the virus were similar to those of feline calicivirus, which supports the view that it should be tentatively classified as a member of the Caliciviridae.
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Phosphorylation of Recombinant Interferon-gamma by Kinases Released from Various Cells
More LessSUMMARYRecombinant interferon-gamma (IFN-γ) in contact with human embryonic fibroblasts or with a great variety of cells from different animal species was phosphorylated in the presence of [γ-32P]ATP and magnesium ions by a protein kinase released in the culture medium. Using SDS-polyacrylamide gel electrophoresis, we found that both the monomeric (17000 to 18000) and dimeric (34000 to 35000) molecular weight forms of IFN-γ became intensely radioactive. Serine, but not threonine or tyrosine, was phosphorylated. It is of interest that the kinase released from reputedly insensitive cells also phosphorylated IFN-γ. The process did not noticeably degrade the antiviral functions of the molecule nor did it affect, at least in a detectable manner, its anti-proliferative effect on WISH or Daudi cells. Furthermore, the antigenic structure and its capacity to react with monoclonal antibodies were also unaltered. It is presently not known which biological function is regulated by the phosphorylating process.
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Purification and Characterization of the Major Envelope Glycoprotein of Simian Foamy Virus Type 1
SUMMARYSimian foamy virus type 1 (SFV-1), the prototype of the Spumavirinae, was subjected to disruption and serial purification procedures. Separation of SFV-1 envelope components from viral cores was verified by electron microscopy, density gradient centrifugation and polyacrylamide gel electrophoresis. After affinity chromatography of the envelope polypeptides on a concanavalin A-Sepharose column, a highly purified 70000 mol. wt. protein was recovered. Glycosylation of this gp70 was confirmed by glucosamine labelling. Immunological studies with anti-SFV antisera confirmed the type-specificity of this envelope gp70.
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Sindbis Virus Glycoproteins Are Abnormally Glycosylated in Chinese Hamster Ovary Cells Deprived of Glucose
More LessSUMMARYWe have previously demonstrated that Sindbis virus infection of Chinese hamster ovary (CHO) cells altered the protein glycosylation machinery of the cell, so that both normal, full-size (nine mannose-containing) oligosaccharides and abnormal, ‘truncated’ (five mannose-containing) oligosaccharides are transferred from lipid-linked precursors to newly synthesized viral membrane glycoproteins. In the present studies, we have examined the precursor oligosaccharides on viral glycoproteins that were pulse-labelled with [3H]mannose in the presence or absence of glucose, since glucose starvation of uninfected CHO cells has been reported to induce synthesis of truncated precursor oligosaccharides. Pulse-labelling in the absence of glucose led to a greater than 10-fold increase in the relative amount of the truncated precursor oligosaccharides being transferred to the newly synthesized viral glycoproteins and to an apparent underglycosylation of some precursor viral polypeptides, with some asparaginyl sites not acquiring covalently linked oligosaccharides. The mature virion glycoproteins from CHO cells which were pulse-labelled in the absence of glucose and then ‘chased’ in the presence of glucose contained proportionately more unusual Man3GlcNAc2-size oligosaccharides. These small neutral-type oligosaccharides were apparently not as good a substrate for further processing into complex acidic-type oligosaccharides as the normal Man5GlcNAc2 intermediate that results from the full-size precursor oligosaccharides.
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Ia Antigens and Fc Receptors of Mouse Peritoneal Macrophages as Determinants of Susceptibility to Lactic Dehydrogenase Virus
T. Inada and C. A. MimsSUMMARYThe relationship between susceptibility of mouse peritoneal macrophages to lactic dehydrogenase-elevating virus (LDV) infection and expression of I region-coded antigens (Ia) on these cells was investigated. The proportion of Ia-positive cells in resident peritoneal macrophages from adult and suckling mice were 4 to 10% and 50 to 70% respectively. Approximately the same percentage of the cells were susceptible to LDV, as detected by fluorescent antibody staining. In adult mice, double-labelling experiments showed that most of the Ia-positive cells were LDV-infected. When the cells were cultured for more than 24 h in vitro, Ia-positive cells rapidly disappeared and the culture became resistant to LDV. Removal of Ia-positive cells by treatment with anti-Ia plus complement or enrichment using an anti-Ia-coated Petri dish simultaneously removed or enriched for LDV-susceptible cells. Treatment of cells with trypsin (1 mg/ml) removed their I-A and I-E antigens and simultaneously abolished susceptibility for LDV. When LDV was preincubated with subneutralizing amounts of antibody, infectivity for macrophages was enhanced and the proportion of LDV-infected cells was higher than that of Ia-positive cells. This suggests that Fc receptors on macrophages can act as receptors for LDV coated with antiviral IgG.
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Characterization by Western Blotting of the Immunogens of Infectious Bursal Disease Virus
More LessSUMMARYThe Australian isolate of infectious bursal disease (IBD) virus (002/73) was purified from infected bursae by rate-zonal and density-equilibrium centrifugation and characterized by polyacrylamide gel electrophoresis. Two major polypeptides having approximate mol. wt. of 32000 (32K) and 37K and three other polypeptides of approximate mol. wt. 29K, 41.5K and 91.5K were present in all preparations of virus having a buoyant density of 1.33 g/ml. Western blotting of the polypeptides of IBD virus showed that the initial antibody response of chickens infected with live virus or injected with an inactivated oil-emulsion vaccine was directed primarily towards the 32K polypeptide. Only sera obtained late in the response to live virus or following hyperimmunization contained antibodies recognizing the 29K, 37K and 41.5K polypeptides. An antibody response to the 91.5K polypeptide was not detected routinely by this technique. It was concluded that the 32K polypeptide is a major immunogen of IBD virus.
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Chronic Shedding of Bovine Enteric Coronavirus Antigen-Antibody Complexes by Clinically Normal Cows
More LessSUMMARYUsing an ELISA for the detection of virus-specific immune complexes, ten cows were found to be shedding bovine enteric coronavirus. The shedding patterns from five of these animals were followed for a period of 12 weeks, and all were found to be chronically shedding virus. Despite the presence of both faecal and serum antibody the infection was not cleared; therefore, the role of cell-mediated immunity (CMI) was investigated by immunosuppressing the chronically shedding cows with dexamethasone. No major role for CMI in maintaining the chronic infection could be determined, although immunosuppression did result in a temporary reduction in the shedding of virus-specific immune complexes.
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Cell Cycle Position and Expression of Encephalomyocarditis Virus in Mouse Embryo Fibroblasts
L. Mallucci, V. Wells and D. BeareSUMMARYInfection of mouse embryo fibroblasts in G1 or S phase with encephalomyocarditis virus gave different kinetics of viral RNA synthesis. In S phase cells, RNA synthesis was faster and reached higher levels than in G1 cells. Virus-specified proteins were fewer in G1 cells than in S cells during the early stage of the infection and c.p.e. in G1 cells appeared about 4 h later than in S cells. Addition of a cellular factor with ability to affect cell conformation had an inhibitory effect on viral RNA synthesis.
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Temperature Sensitivity of the Transcriptase of Mutants tsB1 and tsF1 of Vesicular Stomatitis Virus New Jersey Is a Consequence of Mutation Affecting Polypeptide L
More LessSUMMARYTwo conditional transcriptase-negative mutants of vesicular stomatitis virus (VSV) serotype New Jersey, tsB1 and tsF1, their revertants tsB1/R1 and tsF1/R1 and the wild-type virus were dissociated into pellet, NS and L fractions and, after reconstitution of these in various combinations, the transcriptase activities were assayed in vitro at the permissive (31 °C) and restrictive (39 °C) temperatures. The pellet fractions contained the virion RNA-polypeptide N complexes, while the NS and L fractions were essentially pure preparations of these polypeptides. The synthesis of RNA by the reconstituted pellet and L fractions was inhibited at 39 °C only when the L fractions of tsB1 or tsF1 were used. Addition of the NS fractions to the reconstituted pellet and L fractions did not alter the rates of RNA synthesis. These results demonstrate that polypeptide L is the temperature-sensitive polypeptide of both mutants tsB1 and tsF1 and support previous observations that polypeptide L is the transcriptase itself. The fact that a second mutant of complementation group F, tsF2, is transcriptase-positive but replicase-negative suggests that polypeptide L is involved both in transcription and replication. Intracistronic complementations may account for the observation that the temperature-sensitive mutations affect polypeptide L in complementation groups B and F.
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The Physical State of Human Papillomavirus Type 16 DNA in Benign and Malignant Genital Tumours
More LessSUMMARYCloned DNA from human papillomavirus (HPV) type 16 was subjected to restriction enzyme analysis. A genome size of 7.8 ± 0.1 kb was determined and restriction maps were prepared. Fragments of HPV 16 DNA were nick-translated and hybridized with fragments of HPV 6b DNA. The two genomes appeared to be colinear. The physical state of HPV 16 DNA in genital tumours was analysed. In each of six benign tumours the viral DNA was detected exclusively as 8 kb circles. In four malignant tumours the viral DNA appeared to be integrated within the host genome but one cervical carcinoma and one case of Bowen’s disease also contained oligomeric episomal molecules of viral DNA. One cervical carcinoma (WV 2965), containing only integrated viral DNA, was examined in detail. HPV 16 DNA was integrated as head-to-tail tandem repeats at more than one site. Three virus/cell junction fragments from this tumour were cloned. Two contained lengths of repetitive cellular DNA and one a length of apparently single copy cellular DNA.
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The Ribonucleotide Reductase Induced by Herpes Simplex Virus Type 1 Involves Minimally a Complex of Two Polypeptides (136K and 38K)
More LessSUMMARYHerpes simplex virus type 1 (HSV-1) encodes a polypeptide of apparent mol. wt. 136000 (Vmw136) known to be a component of the virus-specified ribonucleotide reductase. Monoclonal antibodies that precipitate this polypeptide also precipitate a polypeptide of mol. wt. 38000 (Vmw38) from extracts of HSV-1-infected cells. The basis for this co-precipitation has been investigated using a monoclonal antibody directed against Vmw136 and an oligopeptide-induced antiserum directed against the carboxy terminus of Vmw38. We have also made use of a temperature-sensitive (ts) mutant of HSV-1 which maps within the sequences encoding Vmw136 and which induces a thermolabile ribonucleotide reductase. Our experiments show (i) Vmw136 and Vmw38 form a complex in infected cells and (ii) the mutation in the ts mutant results in the two polypeptides being unable to form the complex at the non-permissive temperature. We speculate that association of the two polypeptides is necessary for ribonucleotide reductase activity. No evidence was found for involvement of host proteins in the proposed virus-induced ribonucleotide reductase complex. The terms RR1 and RR2 are suggested for the large and small subunits of the HSV-induced enzyme.
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A Herpes Simplex Virus Type 1 Mutant with a Deletion in the Polypeptide-coding Sequences of the ICP4 Gene
More LessSUMMARYA deletion mutant derived from herpes simplex virus type 1 (HSV-1) strain ANG was analysed. The deletion mapped within the polypeptide-coding region of the immediate-early ICP4 gene. Based on DNA sequence data the deletion was shown to comprise 84 base pairs. In the wild-type genome of strain ANG these sequences were almost completely homologous to the known sequences of HSV-1 strain 17. The ICP4 polypeptide induced by the mutant was similar in size to the wild-type ICP4 protein and was recognized by a monoclonal antibody against ICP4. The data presented suggest that the deletion corresponds to a region on the ICP4 polypeptide that is nonessential for the replication of the virus in vitro.
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Epstein-Barr Virus Nuclear Antigen (EBNA): Size Polymorphism of EBNA 1
More LessSUMMARYThe mol. wt. of the polymorphic Epstein-Barr virus (EBV) nuclear antigen (EBNA) molecule (EBNA 1) encoded by the BamHI K fragment of the EBV DNA has been determined in 14 EBV-carrying lymphoblastoid and Burkitt’s lymphoma cell lines. There is no obvious correlation between the size of this polypeptide and any properties of the cells from which it is derived, other than those related to the strain of transforming virus. We confirm that the polymorphic region of this molecule is the glycine-alanine copolymer encoded by the third internal repeat of the EBV genome (IR3) and we consider the significance of this domain.
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Inhibition of Herpes Simplex Virus Type 1 Penetration by Cytochalasins B and D
More LessSUMMARYInternalization of herpes simplex virus type 1 (HSV) KOS strain by HEp-2 cells was reversibly inhibited by pretreatment of cells with cytochalasins B and D. Internalization of virus following preincubation at 4 °C and temperature shift to 37 °C was normally preceded by a 5 to 8 min lag period and was complete within 20 to 30 min. A similar lag period followed HSV addition at 37 °C. Cytochalasin D was fivefold more active on HSV entry than cytochalasin B, with 50% inhibition at 2 µm and 10 µm respectively. Inhibition was completely reversible, such that all cell-bound infectious virus was recovered upon removal of cytochalasin. In conjunction with previous reports, the activity of cytochalasin on HSV entry suggests that a change in cytoskeletal structure following virus attachment triggers a microfilament activity important for internalization of HSV by HEp-2 cells.
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