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Volume 68,
Issue 7,
1987
Volume 68, Issue 7, 1987
- Bacterial
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Altered Phage P1 Attachment to Strains of Escherichia Coli Carrying the Plasmid ColV,I-K94
More LessSummaryPhages P1 vir and P1cmclr100 failed to form plaques on or multiply in Escherichia coli strains carrying the ColV,I-K94 plasmid; with P1cmclr100, the effect occurred both with phage from the lytic cycle and with that induced from a lysogen. The effect was on attachment, these P1 phages attaching poorly to ColV,I-K94+ strains. This receptor defect appeared to result mainly from the presence of ColV-encoded transfer and colicin components in the cells carrying ColV,I-K94 and it was specific to this plasmid. Phage Mu (which uses an attachment mechanism similar to that of phage PI) in the G(+) form attached to both Col− and ColV,I-K94+ strains but the G( −) form attached to neither type.
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Further Characterization of Phage T1 DNA Clones
More LessSUMMARYA series of phage T1 DNA clones containing Sau3A fragments of 1 kb or more were tested for their ability to rescue the defects of amber mutations representing all known T1 genes. Rescue was detected by either complementation or recombination. These data have extended the correlation of the T1 genetic and physical maps and provided more conclusive proof that the large map distances at the left end of the genetic map reflect both increased physical separation of the known genetic markers and enhanced recombination and that the right and left chromosomal ends are recombinogenic to the same extent.
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- Animal
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The Complete Nucleotide Sequence of Coxsackievirus B4 and Its Comparison to Other Members of the Picornaviridae
More LessSUMMARYThe genome of the prototype strain of coxsackievirus B4 (J.V.B. Benschoten) has been cloned in Escherichia coli and its complete nucleotide sequence determined. Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids. The predicted amino acid sequence of the polyprotein shows close homology (88%) to that of the previously sequenced coxsackievirus B3 and to certain regions of the polyproteins of the polioviruses and human rhinovirus 14. This allows identification of putative polyprotein cleavage signals, antigenic domains and other structural features likely to be important to the biological integrity of the virus.
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Analysis of the Bond between Encephalomyocarditis Virus and Its Human Erythrocyte Receptor by Affinity Chromatography on Virus-sepharose Columns
More LessSUMMARYAffinity chromatography was used to analyse the bond between encephalomyocarditis (EMC) virus and glycophorin, the receptor for EMC virus on human erythrocytes. Between 60 and 80% of glycophorin added to virus-Sepharose columns was retained compared with 10 to 20% retention on glycine-Sepharose columns. Elution with 0· m- NaCl released about 80 to 90% of the retained glycophorin from virus-Sepharose columns but little from glycine-Sepharose. Glycophorin remaining on either the virus or glycine columns after 0·2 m-NaCl treatment was released with Triton X-100, suggesting that this material was aggregated and trapped non-specifically. The sensitivity of the EMC virus-glycophorin bond to 0·2 m-NaCl was confirmed by showing that the radiolabelled EMC virus that bound to human erythrocyte membranes in 0·02 m-phosphate buffer was released when washed with this buffer containing 0·2 m-NaCl. It was concluded that weak ionic interactions were involved in this virus-receptor bond. Treatment of glycophorin with neuraminidase prevented it binding to EMC virus-Sepharose indicating the requirement for sialic acid for receptor activity. However, other sialylated molecules did not bind. Only one chymotryptic peptide of glycophorin (CH0) bound to EMC virus-Sepharose, confirming that this peptide contains the virus-binding site as had been previously suggested using other techniques. The effects of two non-ionic detergents and two anionic detergents on the virus-glycophorin bond were examined. In each case a very low concentration of detergent disrupted the bond and the concentration required was in parallel with that which dissolved erythrocyte membranes. It appears that multivalent glycophorin aggregates are required for stable bond formation.
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Conservation in vivo of Protease Cleavage Sites in Antigenic Sites of Poliovirus
More LessSummaryPolioviruses possess three major antigenic sites which have been located chemically and structurally on the particle. One of these sites, designated site 1, is strongly immunodominant for serotype 3, but highly immunorecessive for type 1. We report that monoclonal antibodies directed against site 1 of type 1 poliovirus may be isolated by an altered route of immunization of the donor mice. Site 1 is shown to be highly variable for type 1, but highly conserved for type 3 poliovirus, although the converse would be predicted from their immunodominance. The evidence presented suggests that the antigenic conservation is associated with a strong selective pressure for a proteolytic cleavage site within site 1 of type 3. As proteolytic cleavage results in the loss of the antigenicity of site 1 the presence of the cleavage site in a virus replicating in the gut in the presence of proteases would protect the virus from neutralizing antibodies directed against uncleaved site 1. The conservation of the site in type 3 is thus consistent with the view that site 1 is a significant target of a human as well as a murine immune response against type 3.
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Lymphocytic Choriomeningitis Virus Can Persistently Infect Thyroid Epithelial Cells and Perturb Thyroid Hormone Production
More LessSUMMARYAlthough viral infection has been suspected as the cause of some thyroid disorders, there has been limited data to support this contention seriously. Now we report the first evidence that lymphocytic choriomeningitis virus can persist in the thyroid gland, particularly thyroid epithelial cells in which thyroglobulin (Tg) the precursor of thyroid hormone, is synthesized. Concomitant with the infection of these cells is a significant reduction in Tg mRNA and in the level of circulating thyroid hormones. Another virus (lactate dehydrogenase virus) that causes persistent infection but does not replicate in the thyroid gland failed to alter levels of circulating thyroid hormones. These observations in an experimental model support the hypothesis that viruses may account for some thyroid disorders in man.
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Temporary and Permanent Modifications to a Single Strain of Mouse Scrapie on Transmission to Rats and Hamsters
More LessSUMMARYThe interspecies transmission of scrapie is frequently associated with exceptionally long incubation periods at first passage in the new host compared to later passages (the species barrier effect). The basis of this was investigated using the 139A strain of scrapie which had been cloned by three serial passages in mice at limiting infectious doses. Cloned scrapie was passaged through hamsters (twice) or rats (thrice) and then reisolated in mice. Large species barrier effects were encountered on mouse-to-hamster and hamster-to-mouse passage resulting in the isolation of a mutant strain, 139-H/M, with properties very different from 139A. In contrast, the strain reisolated from rats was indistinguishable from 139A. However, a large species barrier was encountered at the mouse-to-rat passage but not at the rat-to-mouse passage. It is suggested that the transmission of scrapie between species may be associated with (i) no change in properties or (ii) a permanent change in the scrapie genome due to the selection of mutants. A third possibility, the donor species effect, is a temporary change occurring only at first passage in the new host species which is largely or entirely caused by the introduction of material from the previous host. We speculate that the donor species effect could be explained if some host protein forms a functional part of the infectious agent.
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The Predicted Primary Structure of the Peplomer Protein E2 of the Porcine Coronavirus Transmissible Gastroenteritis Virus
More LessSummaryThe complete nucleotide sequence of cloned cDNAs containing the E2 glycoproteinencoding region of the genome of transmissible gastroenteritis virus (TGEV) has been determined. A single large translatable frame of 4·3 kb starting at 8·2 kb from the 3′ end of the genome was identified. Its deduced amino acid sequence contains the characteristic features of a coronavirus peplomer protein: (i) the precursor polypeptide of TGEV E2 is 1447 residues long (i.e. 285 longer than the avian infectious bronchitis coronavirus spike protein); (ii) partial N-terminal sequencing demonstrated that a putative secretory signal sequence of 16 amino acids is absent in the virion-associated protein; (iii) the predicted mol. wt. of the apoprotein is 158K; most of the 32 potential N-glycosylation sites available in the sequence are presumed to be functional to account for the difference between this and the experimentally determined value (200K to 220K); (iv) a typical hydrophobic sequence near the C terminus is likely to be responsible for anchoring the peplomer to the virion envelope.
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Purification of Papillomavirus Structural Polypeptides from Papillomas by Immunoaffinity Chromatography
More LessSummaryA broadly cross-reactive monoclonal antibody directed against papillomavirus, coupled to immunoaffinity columns, was used to isolate bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) structural polypeptides from homogenates of productively infected cells. One of the polypeptides isolated from bovine fibropapillomas appeared to be the BPV-1 major capsid protein since it had a mol. wt. of 54K and was reactive by Western blots with papillomavirus genus- and BPV-1 type-specific rabbit antibodies as well as monoclonal antibodies cross-reactive with BPV-l/BPV-2 and BPV-l/deer PV. A polypeptide from human plantar warts similarly appeared to be the major capsid component since it also had a mass of 54K to 55K and reacted with papillomavirus genus- and HPV type-specific rabbit antibodies. By using this technique structural viral polypeptides from papillomavirus-induced lesions containing readily detectable viral structural antigens but relatively few virus particles, such as seen with mucosotropic HP Vs can now be isolated for mapping of virus-specific epitopes.
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Structural and Immunological Characterization of the Intracellular Forms of an Abundant 68000 M r Human Cytomegalovirus Protein
More LessSUMMARYMurine monoclonal antibodies and polyvalent monospecific antisera reactive with an abundant 68000 M r (p68) human cytomegalovirus (CMV) virion protein were used to characterize this protein within CMV-infected cells. The protein was found to partition within the nucleus and cytoplasm of infected cells. Pulse-chase analysis indicated the p68 was degraded into three proteins of 52000, 51000 and 50000 M r which were found only within infected cells. Both cellular forms as well as the virion p68 were phosphorylated but non-glycosylated. The p68 was synthesized shortly after infection and in the presence of cytosine arabinoside, an inhibitor of viral DNA replication. Studies with monospecific antisera and a panel of monoclonal antibodies specific for the p68 suggested that this protein was not expressed on the surface of infectious virions or infected cells.
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Replacement of Glycoprotein B Gene Sequences in Herpes Simplex Virus Type 1 Strain ANG by Corresponding Sequences of the Strain KOS Causes Changes of Plaque Morphology and Neuropathogenicity
More LessSummaryDNA sequences encoding glycoprotein B (gB) derived from herpes simplex virus type 1 (HSV-1) strain KOS321 were transferred to HSV-1 ANG. In cotransfection experiments the cloned HSV-1 KOS BamHI G fragment served as donor, and genomic DNA of two ANG variants as recipients. One of these variants, HSV-1 ANG path, expresses gC and the other, C18, was a spontaneous gC-negative mutant. Both ANG strains are of the syncytial (syn) phenotype whereas HSV-1 KOS321 is non-syncytial (syn+). Recombinants were identified by means of a monoclonal antibody which selectively recognizes gBKOS. Among the HSV-1 ANG path/gBKOS recombinants, the majority displayed an altered plaque morphology, i.e. they were of the syn+ phenotype. In contrast all of the C18/gBKOS recombinants were of the syn phenotype. The possibility that the mutant C18 carries a syn mutation not present in the parental strain could be excluded. Marker transfer experiments involving subfragments of the gB gene mapped the syn mutation of HSV-1 ANG path to a locus within the gene that has been previously termed syn 3. Subclones of HSV-1 ANG path were established either directly or after intermittent transfection or cotransfection with the KOS BamHI G fragment. The pathogenicity in mice of these clones was compared. The data obtained indicated that at high frequency, the BamHI G fragment confers apathogenicity.
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The Products of Gene US11 of Herpes Simplex Virus Type 1 Are DNA-binding and Localize to the Nucleoli of Infected Cells
More LessSUMMARYWe have used antisera raised against synthetic oligopeptides to characterize the protein products from herpes simplex virus type 1 gene Us11. These antisera recognized predominantly polypeptides of apparent molecular weight 21000 and 22000, but also polypeptides of apparent molecular weight 17500, 15000, 14000 and 11000. Tryptic peptide fingerprint analysis confirmed that these polypeptides were all closely related. The 21000 and 22000 molecular weight polypeptides were shown to be DNA-binding proteins, and immune electron microscopy demonstrated their strong localization within nucleoli of infected cells.
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Herpes Simplex Virus Type 2 Glycoprotein Biogenesis: Effect of Monensin on Glycoprotein Maturation, Intracellular Transport and Virus Infectivity
More LessSUMMARYThe ionophore monensin inhibited the formation of herpes simplex virus type 2 (HSV-2) particles by about 30% but the yields of infectious particles were reduced to 5 % and 1 % for cell-associated and extracellular virus, respectively. The presence of monensin did not affect the processing of the two viral glycoproteins gB-2 and gG-2. However, two other glycoproteins, gC-2 and gD-2, were not processed to their fully mature forms in the monensin-treated cells and only the faster moving pgC-2 and pgD-2 were detected. The cell-associated virus particles contained the glycoproteins gB-2, gC-2, gD-2 and gG-2, whereas the extracellular virus particles contained only gG-2 glycoprotein. These results suggest that HSV-2 particles containing all the viral glycoproteins are transported via the Golgi apparatus to the cell surface but that virus particles containing only gG-2 may follow a different pathway for transport and release.
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Virus-specific IgM and IgG Antibody Production by B Cells during Herpes Simplex Virus Type 2-induced Immunosuppression as Analysed by an Immunospot Assay
S. Nick, B. Metzger, S. Muller and D. FalkeSUMMARYThe mechanism of herpes simplex virus (HSV)-2-induced immunosuppression was analysed by determination of the number of IgM and IgG antibody-secreting B cells in female BALB/c mice using an immunospot assay. Primary HSV-1 or -2 as well as homologous or heterologous booster infections at different times were performed. In accordance with earlier results on humoral antibody generation, in contrast to HSV-1, HSV-2 induced only very low numbers of antibody-producing B cells in dose-response experiments. They appeared late after infection compared to HSV-1. Despite a homologous humoral booster reaction against HSV-1 at day 8 no IgM- or IgG-secreting cells in the spleen could be detected. This non-reactivity of the spleen had vanished 10 days later, when secondary reactions of B cells could be observed. Secondary infections with a high homologous dose of HSV-2 after a low primary dose produced only a low booster response of IgG-secreting B cells. Suppression of humoral antibody production induced by HSV-2 (high dose) waned after more than 50 days, indicating that the HSV- 2-induced suppression did not impair antigen presentation or memory cell generation.
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Role of T Lymphocyte Subsets in Protection and Recovery from Hantaan Virus Infection in Mice
SUMMARYAdult athymic nude (BALB/c background) mice or inbred BALB/c mice were inoculated intraperitoneally with Hantaan virus (HV), and attempts were made to isolate the virus from brain, lung and spleen. Virus was isolated from the organs of BALB/c mice for only a short time after infection but was isolated from various organs of nude mice consistently for at least 84 days after infection. Viral antigen was also detected in various organs of nude mice for a long time after infection. The effects of adoptive transfer of immune serum or immune T cells from BALB/c to nude mice before or after virus inoculation were examined. Before transfer, the T cell fraction was treated with complement (C′) (group 1), anti-L3T4 + C′ (group 2), anti-Lyt1 .2 + C′ (group 3) or anti-Lyt2.2 + C′ (group 4). When transferred before virus inoculation to test the effects on protection against infection, immune serum and T cells of groups 1,2 and 4 were effective. When transferred after HV inoculation to test the effects on clearance of virus, group 1 was the most effective followed by group 2. These results suggest that humoral and cellular immunity both have roles in protection against HV infection, and that T cells possessing L3T4− Lyt2+ markers on the cell-surface are especially important for elimination of infectious virus in vivo.
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Demonstration of Bovine Viral Diarrhoea Virus in Peripheral Blood Mononuclear Cells of Persistently Infected, Clinically Normal Cattle
More LessSummaryPeripheral blood mononuclear cells (PBL) from cattle known to be persistently viraemic with bovine viral diarrhoea virus (BVDV) following a foetal infection, were examined for the presence of viral antigens and cell-associated infectious virus. Using immunocytochemical techniques, physical separations of PBL subsets and virus isolation techniques (directly and by cocultivation) it was found that infection occurred in B and T lymphocytes, monocytes, and a group of cells designated null cells for lack of more specific classification. The latter three groups also supported viral replication, as infectious virus could be isolated from enriched cell populations. BVDV-like particles in cytoplasmic vesicles of PBL subsets were detected by electron microscopy.
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Lactate Dehydrogenase-elevating Virus Induces Anti-Golgi Apparatus Antibodies
More LessSummaryA recently described autoantibody-inducing agent in mice was further characterized. Tentatively designated AGIA (anti-Golgi apparatus-inducing agent), this agent has previously been shown to cause antibody production against Golgi apparatus (GA) antigen of cells from different vertebrate species as well as against tumour surface antigen of the Moloney murine sarcoma virus-non-producer transformant Sac. It was shown to possess the properties characteristic of lactate dehydrogenase-elevating virus (LDV). It induced elevation of lactate dehydrogenase levels in the blood, persistent lifelong viraemia in mice and serum titres of up to 1011 infectious doses (ID50) per ml in the acute phase of infection. Its replication in vitro was limited to subpopulations of murine peritoneal macrophages. Electron microscopy of AGIA-infected macrophages and of serum of infected mice revealed virus-like particles with a morphology resembling LDV. The buoyant density of AGIA was approximately 1·14 g/ml. Both the enzyme-elevating activity and the autoantibody-inducing activity were shown to belong to LDV. Infection of STU mice with two established strains of LDV (LDVROW and LDVPLA) was also found to induce both autoantibody groups. In both cases, after infection with AGIA as well as after infection with the two known LDV isolates, anti- Sac cell antibodies occurred at comparable titres. However, anti-GA antibody titres were rather low after infection with LDVROW and LDVPLA compared with AGIA infection. Serological cross-reactivity was demonstrated between AGIA-, LDVROW-and LDVPLA-infected macrophages. AGIA induced anti-GA antibodies in all six mouse strains tested (STU, DBA/2, BALB/c, C3H/He, NMRI, C57BL/6); however, anti-Sac cell antibodies did not develop in C57BL/6 mice.
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Immune Response to Rotavirus Polypeptides after Vaccination with Heterologous Rotavirus Vaccines (RIT 4237, RRV-1)
More LessSUMMARYThe antibody response to individual rotavirus polypeptides after vaccination with live attenuated heterologous rotavirus vaccines was studied with a radioimmunopreci-pitation assay (RIPA). Following vaccination with bovine rotavirus strain RIT 4237 and rhesus monkey rotavirus strain RRV-1, an antibody response was demonstrated mainly against the inner capsid polypeptides VP2 and VP6 and to a lesser extent against VP3/VP4. There was no qualitative difference between the vaccines in the antibody response; both vaccines induced antibodies against the same polypeptides. Quantitatively the antibody response to RRV-1 was somewhat stronger. All prevaccination sera contained by RIPA low levels of rotavirus antibodies. The effect of these antibodies on the ‘take’ of the vaccines is discussed.
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The Primary in vivo Murine Cytotoxic T Cell Response to the Flavivirus, West Nile
More LessSUMMARYA protocol for obtaining cytotoxic T cell responses to the flavivirus West Nile (WNV) has been developed in vivo. CBA/H (H-2k) mice were immunized with 106 p.f.u. WNV intravenously and their spleen cells used directly in cytotoxic assays. This method reliably produced WNV-immune Tc cells which showed WNV-specific cytotoxic activity on infected L929 (H-2k) target cells. There was inadequate lysis of infected targets by WNV-immune spleen cells when the m.o.i. was less than 100 p.f.u. WNV, or when tertiary mouse embryo fibroblasts, resident peritoneal macrophages or thioglycollate-induced peritoneal macrophages were used as targets. Only L929 cells infected for 16 h with WNV at a m.o.i. of 100 were suitable targets. Cytotoxic activity against WNV-infected target cells was first detected 4 days after immunization, peaked on day 5 and declined rapidly after day 7. An immunizing dose of 103 p.f.u. of WNV was adequate for significant cytotoxicity to be detected; however, the cytotoxic response increased with increasing immunizing doses to plateau levels when 106 p.f.u. WNV were used. The cells responsible for lytic activity were H-2-restricted, Thy-1+, Lyt-2+, L3T4- and virus-specific with respect to WNV and influenza virus.
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Synergistic Neutralization of Rubella Virus by Monoclonal Antibodies to Viral Haemagglutinin
SUMMARYUsing murine monoclonal antibodies (MAbs) to rubella virus haemagglutinin, five epitopes were identified in competitive ELISA binding assays: A, B, D and E by haemagglutination-inhibiting (HI) MAbs with no neutralizing (Nt) activity, and C by a MAb with neither activity. However, when HI and Nt activities were determined in the presence of anti-mouse immunoglobulins, epitopes A, B and D were defined by both HI and Nt MAbs, whereas epitopes C and E were identified by HI MAbs without Nt activity. A synergistic Nt activity, in the absence of anti-mouse immunoglobulins, was displayed by mixtures of antibodies of different epitope groups. Analysis of mixtures of MAb pairs each belonging to a different epitope class, showed that synergistic Nt activity was elicited primarily by the group A epitope, secondarily by groups B and D and only minimally by groups C and E.
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