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Volume 65,
Issue 5,
1984
Volume 65, Issue 5, 1984
- Bacterial
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A Phage-resistant Mutant of Lactobacillus casei which Permits Phage Adsorption but Not Genome Injection
More LessSUMMARYA phage-resistant mutant of Lactobacillus casei (strain YIT 9021) was capable of adsorbing both PL-1 and J-1 phages, but did not yield phage-infected cells. The mutant and wild-type strains were identical in morphology and sugar composition of the cell walls. Attempts to induce prophages from strain YIT 9021 were unsuccessful. Electron microscopic examination of negatively stained mixtures of phage PL-1 and YIT 9021 bacteria revealed that the phages were adsorbed to the cells in a tail-first orientation. All the adsorbed phages had DNA-filled heads. It was concluded that PL-1 adsorbed normally but was blocked in the injection of the phage genome into the cell.
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- Animal
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Determination of the Sequence Alteration in the DNA of the Herpes Simplex Virus Type 1 Temperature-sensitive Mutant ts K
More LessSUMMARYThe alteration in the DNA sequence responsible for the mutant phenotype of the herpes simplex virus type 1 temperature-sensitive mutant ts K has been determined. A single C:G base pair present in the wild-type Vmw 175 immediate-early polypeptide-coding sequence has been replaced by T:A in the mutant gene. This results in the substitution of an alanine by a valine codon. In two revertants the mutant T:A base pair has been resubstituted by C:G.
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Successful Use of Oligopeptides as Immunogens in the Preparation of Antisera to Immediate-Early Gene Products of Herpes Simplex Virus Type 1
More LessSUMMARYAntisera to two herpes simplex virus-type 1 (HSV-1) immediate-early gene products (IE12 and IE175) have been produced using oligopeptides, constructed on the basis of proposed DNA coding sequences, as immunogens. In both cases the synthetic peptides were linked to bovine serum albumin via an N-terminal tyrosine prior to immunization. Both the IE12 and the IE175 antisera reacted with their respective HSV-1 antigens and with antigens produced by the HSV-1 immediate-early mutant tsK but not with functionally equivalent antigens induced by HSV-2. IE12 induced by tsK was found to have an altered mobility with respect to the wild-type IE12 and its precipitation was accompanied by a second, minor, component of lower mobility. Revertants of tsK gave similar results. Labelling IE12 with a variety of amino acids indicated that the first of three possible initiation codons was used in its translation from mRNA. The results imply that the other initiation codons were not used. Wild-type IE12 is produced for at least 3 h after release of cycloheximide block and appears to be turned over rapidly.
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The Genome Structure of Cowpox Virus White Pock Variants
More LessSUMMARYA previous report described restriction endonuclease analysis of white pock variants of red cowpox virus and their characterization as deletion mutants lacking certain sequences including the repetition from one specific terminus of the wild-type genome. Further analysis has confirmed the terminal deletion but demonstrated that this is compensated at the site of deletion by the presence of an inverted duplication of a variable amount of sequence from the opposite terminus, with the effect of restoring a terminal repetition and the covalent, terminal crosslink. Nine of 11 white pock variants showed a similar deletion of about 21 Mdal mapping contiguously from the right-hand terminus and extending into a 2.4 Mdal restriction fragment. Two white variants showed larger deletions of about 24 and 27 Mdal respectively. These deletions were compensated by a copy of sequences from the opposite terminus which ranged in size from 3 to 27 Mdal. No terminal deletions smaller than 21 Mdal were observed in cowpox white variants or in clones retaining the red phenotype. In contrast with other orthopoxviruses, no deletions involving the left-hand terminus were found. Some independent white isolates had similar sizes of sequence copied from the opposite terminus, but some sibling clones from a single, pock-purified white isolate with the same size of deletion had different sizes of duplicated sequence. Other siblings isolated from an independent, three times pock-purified white clone, itself derived from a single parental red pock, differed from each other in the size of both the deletion and the duplicated sequence. These observations suggest preference for deletion in a particular region, conjunction of the genome termini during DNA replication and a requirement for the preservation of symmetrical termini in orthopoxvirus genome function.
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Human Teratocarcinomas Cultured in vitro Produce Unique Retrovirus-like Viruses
More LessSUMMARYWe have previously reported that among a series of human tumours investigated, only human teratocarcinoma cell lines derived from testicular tumours or pulmonary metastases of patients in Germany and the U.S.A. produced retrovirus-like particles spontaneously, albeit in low amounts. In a recent publication electron microscopical data suggested that the human teratocarcinoma-derived (HTD) particles were morphologically closely related, but not identical, to the type C retroviruses of animals. In this communication, the explantation of three human teratocarcinoma cell lines is briefly described. Evidence is presented that HTD particles (i) are synthesized only in a fraction of the epithelioid and differentiating cells; (ii) can be induced biochemically in a manner characteristic of retroviruses; (iii) either are not infectious or possess a peculiar host range; (iv) are immunologically unrelated to animal retrovirus strains; (v) possess an endogenous RNA-dependent DNA polymerase activity that can be banded at 1.16 g/ml in linear sucrose gradients. These results may be taken as suggestive evidence that HTD particles represent a novel group of unique retroviruses.
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Monoclonal Antibodies to Hepatitis Be Antigen (HBeAg) Derived from Hepatitis B Core Antigen (HBcAg): Their Use in Characterization and Detection of HBeAg
More LessSUMMARYA panel of mouse hybridomas secreting monoclonal antibody to serum hepatitis Be antigen (HBeAg) was produced from mice immunized with denatured hepatitis B core antigen (HBcAg). This panel could be divided broadly into two groups. Within each group, the monoclonal antibodies recognized a single antigenic site, designated either e-α or e-β, and generally exhibited a high degree of cross-inhibition. In contrast, between the two groups of antibodies there was little or no cross-inhibition. The antigens of serum HBeAg and denatured HBcAg appeared to be very similar. Both behaved as molecules carrying only a single e-α or e-β site, in spite of native serum HBeAg having an apparent molecular weight of 300000. It is inferred that e-α and e-β sites may be involved in the polymerization of HBeAg into HBcAg and that during this process mutual masking of antigenic sites may occur.
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Comparison of Atypical Rotaviruses from Calves, Piglets, Lambs and Man
More LessSUMMARYSome rotaviruses from calves, piglets, and lambs were detected by electron microscopic examination of faeces but not by an enzyme-linked immunosorbent assay which relies on detection of group antigen. On further examination by polyacrylamide gel electrophoresis, these viruses had 11 segments of dsRNA, as had typical rotaviruses, but arranged in atypical patterns. From humans, three rotaviruses with atypical electrophoretypes were also detected. Gnotobiotic animals were infected with atypical calf, piglet and lamb rotaviruses, and used to provide antigen and antiserum for an immunofluorescent comparison of these rotaviruses with conventional rotaviruses and other previously described atypical rotaviruses from piglets and chickens. Two atypical rotaviruses from humans failed to infect gnotobiotic piglets. The atypical rotaviruses could be tentatively categorized into two groups serologically distinct from each other and from conventional rotaviruses, and these distinctions were consistent with electrophoretypes. The atypical chicken rotavirus may form a fourth distinct group. These findings are consistent with the hypothesis that rotaviruses belong to at least four separate groups definable by serology and electrophoretype.
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Adaptation of Coronavirus JHM to Persistent Infection of Murine Sac(-) Cells
More LessSUMMARYCoronaviruses can establish persistent infections in the central nervous system of rodents, and these are associated with demyelinating encephalomyelitis. The effects of persistence on the virus are difficult to study in vivo but may have a crucial influence on the course of infection. We therefore produced a persistent infection in vitro using the neurotropic coronavirus JHM, in order to investigate the events underlying the establishment of such an infection and the adaptation of the virus to persistence. The persistent infection was maintained for over 115 passages and continued to release high levels of infectious virus. During the 18 months of culture the number of cells expressing virus antigen detected by indirect immune fluorescence decreased to 40%. Analysis showed that the carried virus contained a significant proportion of heterogeneous temperature-sensitive mutants. All virus clones isolated possessed the capacity to induce a more productive growth cycle, a less pronounced cytopathic effect and showed a much reduced neurovirulence when inoculated into newborn and weanling rats. Evidence for structural changes involving the surface peplomer protein (E2) was obtained using hybridoma antibodies, which neutralized the parental JHM virus but not the JHM-Pi virus. Defective interfering particles and interferon activities have been excluded as possible agents instrumental in the establishment and maintenance of the chronic infection, and we suggest that the emergence of virus variants of lowered virulence is central to these processes.
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Monoclonal Antibodies for Study of Antigenic Variation in Coxsackievirus Type B4: Association of Antigenic Determinants with Myocarditic Properties of the Virus
More LessSUMMARYMonoclonal antibodies were produced to a field strain (Mil) of group B coxsackievirus type 4 (CBV-4), and to the prototype JVB strain. Nine were neutralizing antibodies and four were non-neutralizing antibodies with virus-specific activity in indirect immunofluorescence (IF) staining. On the basis of reactivity with the panel of monoclonal antibodies, nine different strains of CBV-4 were found to fall into five distinct antigenic groups. Antigenic variants were produced by using the neutralizing monoclonal antibodies to select variants from the Mil virus stock which were no longer susceptible to the selecting antibody. A high frequency of antigenic variation was seen. By using the variants in cross-neutralization and IF tests with the monoclonal antibodies, it was possible to identify five tentative antigenic sites functional in neutralization; one site appeared to be complex and possibly to consist of overlapping epitopes. Reactivity of the monoclonal antibodies was similar, but not necessarily identical, by neutralization and by IF staining. The antigenic variants were found to differ from the parent Mil strain, and from one another, in their myocarditic and cardiotropic properties in a murine model. Two of the variants produced more extensive cardiac pathology, and two produced higher virus titres in the heart than was produced by the parent strain. One variant was notable for extensive production of necrotic lesions in the myocardium. Four of the variants showed less histopathology and three produced less virus in the heart than was produced by the parent strain.
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Rubella Virus: Structural and Non-structural Proteins
More LessSUMMARYRubella virus was rapidly concentrated and purified using polyethylene glycol 6000 as the precipitating agent. Electrophoresis in slab gels defined three structural proteins present in equimolar amounts, with mol. wt. of 59000 (E1), 43000 to 48000 (heterogeneous E2) and 34000 (core protein, C). E1 and E2 were glycosylated; different distributions of labelled carbohydrates within the broad band of E2 indicated that the slower migrating region was enriched in complex oligosaccharides. In infected cells, the counterparts to E1 and E2 were labelled with [3H]mannose and both migrated in gels as sharp bands, indicating that the heterogeneity observed in virion E2 was produced during virus maturation. After radioimmunoprecipitation of infected cell extracts with convalescent rubella serum, the intracellular equivalents of E1, E2 and C were readily defined in gels, as well as several putative non-structural proteins. Four of these were defined more clearly and without resort to immunoprecipitation by labelling with [35S]methionine during hypertonic treatment of infected cells at 24 h; their mol. wt. were 200000, 150000, 87000 and 75000. Pulse-chase experiments under these conditions showed that the largest (ns200) was apparently cleaved to ns150.
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Properties of Sindbis Virus Variants from Infected Culex tarsalis Mosquitoes
More LessSUMMARYPlaque-purified Sindbis virus was passed three times in Culex tarsalis mosquitoes and progeny viruses were isolated by plaque purification on a cloned line of Aedes albopictus cells. Nine of ten clones examined differed from wild-type (wt) virus with respect to their plaque morphology characteristics in chick embryo fibroblast (CEF) and/or A. albopictus cells. Seven clones were temperature-sensitive and failed to replicate or synthesize viral RNA in CEF cells at 41 °C. At 35 °C in CEF cells the majority of isolates synthesized less viral RNA than wt virus. In contrast, all cloned isolates synthesized viral RNA more rapidly than wild-type virus in A. albopictus cells.
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Gamma Interferon Production and Cytotoxicity of Spleen Cells from Mice Infected with Semliki Forest Virus
More LessSUMMARYInterferon (IFN) production by spleen cells from normal mice, mice acutely infected with Semliki Forest virus (SFV) or mice immune to SFV was measured after stimulation in vitro with either infectious or inactivated SFV. All three classes of spleen cells made IFN-αβ in response to infectious SFV. Spleen cells taken from mice late, but not early, after infection, or from immune mice, made IFN-γ in response to inactivated SFV. Amounts of INF-γ and IFN-αβ were similar. Normal spleen cells made no IFN (of any type) in response to inactivated SFV. The cell type producing IFN-αβ appeared to be the macrophage, whilst both T-lymphocytes and macrophages were necessary for IFN-γ production. During the acute infection, the ability of spleen cells to lyse both virus-infected and uninfected target cells arose earlier than the ability to produce IFN-γ. However, cytotoxicity towards uninfected cells fell to near background levels by day 7, whilst cytotoxicity towards infected targets remained high at that time, when IFN-γ production was at its peak. IFN-γ production is therefore temporally associated with cytotoxicity specifically directed against virus-infected targets, and the ability to produce IFN-γ is a late response to SFV infection.
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Monoclonal Antibodies to Human Respiratory Syncytial Virus and Their Use in Comparison of Different Virus Isolates
More LessSUMMARYMonoclonal antibodies to the RSN-2 isolate of human respiratory syncytial (RS) virus have been characterized with regard to their specificity for viral polypeptides and for different RS virus isolates. Five hybridoma antibodies recognized the phosphoprotein VPP32 and the other recognized the matrix protein VPM27. Evidence was obtained to support the view that VPP32 was associated with the nucleoprotein VPN41. Three of the antibodies to VPP32 showed cytoplasmic immunofluorescent staining while the other two showed only surface staining of virus-infected cells. The immunoblot technique was used to determine the immunoreactivity of four of the hybridoma antibodies against human RS isolates other than RSN-2. One of these antibodies reacted exclusively with RSN-2 virus isolate whereas the others detected determinants shared by all human RS isolates tested. Extension of this approach may offer the possibility of typing RS isolates using monoclonal antibodies.
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Abortive Infection of Mumps Virus in Murine Cell Lines
More LessSUMMARYInfection of L929 or BW5147 cells with mumps virus was shown to result in an abortive type of infection in that little or no progeny virus was produced. Both cell lines could adsorb mumps virus, indicating that restricted viral growth in these cells was not attributable to the absence of mumps virus receptors. Using [35S]methionine-labelled virus, it was demonstrated that restriction of virus growth in BW5147 cells was due in part to inefficient virus penetration into the cells. Virus-specific polypeptides were synthesized in mumps virus-infected L929 cells but were not detected in infected BW5147 cells. After addition of actinomycin D or anti-interferon serum to the cultures, mumps virus was able to replicate in L929 cells, whereas no virus growth was apparent in BW5147 cells.
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Distribution of Rabies Virus, Interferon an dInterferon-mediated Enzymes in the Brains of Virus-infected Rats
More LessSUMMARYThe distribution of rabies virus, interferon and interferon-mediated enzymes, pppA(2′p5′A)n synthetase (2–5A synthetase) and poly(rI).poly(rC)-Sepharose-bound protein kinase, was studied in different regions of the brains of rabies virus-infected rats. A broad range of virus infectivity was found in the brain stem, cerebellum, cortex, hippocampus and striatum. Similarly, the levels of interferon were variable (2500 to 60000 units/mg protein in tissue extracts) in the different brain regions studied but were unrelated to the corresponding infectivities. The level of 2–5A synthetase and protein kinase was enhanced several-fold in the individual brain regions and the degree of such enhancement was correlated with the level of interferon.
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Effect of N-Acetyl-muramyl-l-alanyl-d-isoglutamine on Interferon Production in Mice by Newcastle Disease Virus
More LessSUMMARYThe activity (carbon clearance) of the reticuloendothelial system (RES) of mice inoculated intraperitoneally with N-acetyl-muramyl-l-alanyl-d-isoglutamine (muramyl dipeptide, MDP) was greatly stimulated 1 day, but not 7 days after MDP treatment. No enhancement of resistance to ectromelia virus infection and influenza virus infection in mice treated with MDP was observed. In mice splenectomized 1 week after MDP pretreatment, normal levels of circulating interferon were produced in response to Newcastle disease virus (NDV), whereas in the mice treated with MDP after splenectomy, circulating interferon levels were reduced to the same level as produced in the MDP-untreated and splenectomized mice. Interferon production in response to NDV was augmented in non-adherent peritoneal and spleen cell cultures derived from MDP-pretreated mice, whereas it was reduced in peritoneal and splenic macrophage cultures. These results suggest that the non-adherent spleen cells activated with MDP were disseminated from the spleen to other organs, that the lack of enhancement of interferon production in mice pretreated with MDP might be du eto reduced interferon production in macrophages, and that the activation of the RES of the whole body by MDP did not correlate with the enhancement of interferon production in spleen cells or with the reduction of interferon production in macrophages
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Non-A, Non-B Epidemic Hepatitis: Visualization of Virus-like Particles in the Stool by Immune Electron Microscopy
More LessSUMMARYAcute-phase stool samples collected from hepatitis cases during outbreaks of water-borne epidemic hepatitis were examined by immune electron microscopy (IEM). Spherical virus-like particles (27nm in diameter) were visualized in the stool of a hepatitis patient with serological evidence of non-A, non-B (NANB) hepatitis. The IEM demonstrated serological specificity of the antigen with the patient’s own convalescent serum as well as a known pool of NANB hepatitis convalescent sera. It is suggested that these virus-like particles may be the aetiological agent of faeco-orally transmitted NANB epidemic hepatitis in India.
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Antibodies to Hepatitis B Surface Antigen (HBsAg) Elicited by Immunization with a Synthetic Peptide Covalently Linked to Liposomes
More LessSUMMARYPeptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins. The possible use of several distinct synthetic vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens. A synthetic peptide corresponding to residues 135 to 155 (P135–155) of hepatitis B surface antigen (HBsAg) failed to elicit in free form anti-peptide antibodies or anti-HBs. However, polymers of P135–155 (prepared by linking to diaminoalkanes) and synthetic conjugates prepared by binding P135–155 to liposomes or polylysine were immunogenic. A poor correlation was observed between anti-peptide and anti-HBs responses elicited by these conjugates. Glutaraldehyde-fixed liposomes appeared to be the carriers of choice for inducing anti-HBs.
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A New Double-stranded RNA Virus from Gaeumannomyces graminis
More LessSUMMARYAn isometric virus, obtained from an isolate of Gaeumannomyces graminis, had the following properties: diameter 29 nm; sedimentation coefficient 127S; buoyant density in CsCl 1.40 g/ml; one double-stranded RNA component of 1.80 kilobase pairs; one capsid polypeptide species of mol. wt. 66000; A 260/A 280 1.7. The virus was unrelated serologically to three other viruses present in the same fungal isolate and to 12 previously described viruses belonging to G. graminis virus groups I, II and III. On the basis of its distinctive properties the virus is assigned to a new G. graminis virus group IV.
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Comparison of Structural Proteins from Two Potato Yellow Dwarf Viruses
More LessSUMMARYThe structural proteins of the two serotypes of potato yellow dwarf virus, SYDV and CYDV, were isolated after labelling in vitro with either dansyl chloride or 125I. Five different structural proteins were detected for each virus by SDS-polyacrylamide gel electrophoresis. The molecular weights of the G, M1 and M2 proteins differed between SYDV and CYDV. Four structural proteins. G, N, M1 and M2, of thetwo viruses were also compared by peptide mapping after partial proteolysis. The order of similarity between the homologous proteins of each strain was M2 (least similar) < G < N < M1.
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