- Volume 70, Issue 6, 1989
Volume 70, Issue 6, 1989
- Bacterial
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Further Characterization of a Bacteriophage Recovered from an Avian Strain of Chlamydia psittaci
More LessSUMMARYThe genome of a 22 nm icosahedral phage which infects some avian Chlamydia psittaci strains recovered from domestic ducks has been characterized as a ss circular DNA molecule of about 4850 bases. The replicative form of this genome was isolated from purified chlamydial organisms. A restriction endonuclease cleavage site map of the genome was constructed from dsDNA synthesized in vitro from ss phage DNA and EcoRI fragments were then cloned into pUC9. The phage genome was detected only by Southern blot hybridization in C. psittaci which was productively infected with phage; no evidence was found for the integration of phage DNA into the chlamydial chromosome. Three viral polypeptides, of approximate M r values 75K, 30K and 16.5K were identified when phage was analysed by SDS–PAGE. This virus, which we have designated Chpl, is either an aberrant member of the Microviridae or the first member of a new bacteriophage family.
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- Animal
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Structure of Rearranged Genome Segment 11 in Two Different Rotavirus Strains Generated by a Similar Mechanism
More LessSUMMARYThe structures of the rearranged genomic segment 11 of two spontaneous swine rotavirus strains were determined. We found that the rearrangements involved the duplication of normal segment 11 in a head-to-tail orientation, and partial deletions in both monomers. The open reading frame for VP11, the protein encoded by normal segment 11, was maintained. We also showed that the two rearranged genes were transcribed into RNA molecules of the same length as their corresponding genomic segments.
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The Transcription Termination Region of the Adenovirus 2 Major Late Transcript Contains Multiple Functional Elements
More LessSUMMARYIn order to understand the process of transcription termination by eukaryotic RNA polymerase II, the transcription termination region of the advenovirus 2 major late transcription unit was analysed in a transient transfection system. Previously, it had been demonstrated that the entire sequence from map units (m.u.) 97·1 to 100 of the adenovirus 2 genome terminates transcription when inserted into the 5′ or 3′ untranslated sequences of the chloramphenicol acetyltransferase gene. Using subclones and Bal 31 deletion mutants of the termination region, we have shown that the termination region consists of multiple elements each capable of inhibiting gene expression independently. A DNA sequence analysis reveals the presence of a highly repetitive A-rich sequence motif throughout the entire termination region. The data suggest that the A-rich motif may mediate the transcription termination process.
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Herpes Simplex Virus Causes Amplification of Recombinant Plasmids Containing Simian Virus 40 Sequences
More LessSUMMARYSimian virus 40 (SV40) DNA, inserted into a plasmid vector, does not replicate when transfected into baby hamster kidney cells. However, when the recipient cells are superinfected with herpes simplex virus type 1 (HSV-1), extensive amplification of the introduced plasmid occurs. Deletion of the late SV40 region or part of the coding sequences of the small tumour (t) antigen has no effect on the efficiency of amplification, whereas manipulations affecting either the SV40 origin of replication or the integrity of large tumour (T) antigen substantially decrease HSV-induced amplification. Phosphonoacetic acid, an inhibitor of HSV DNA polymerase, strongly inhibits plasmid replication. Also, an HSV-1 mutant with a temperature-sensitive defect in the DNA polymerase gene (tsH) is unable to carry out amplification of test plasmids at the non-permissive temperature. On the other hand, a further mutant (tsS) causes SV40–plasmid amplification independent of the temperature, but this mutant fails to amplify a plasmid with an HSV origin at the non-permissive temperature. Thus, HSV-induced amplification of heterologous DNA is possible in the absence of HSV DNA replication. Since tsS putatively has a defect in the gene coding for an HSV origin-binding protein (UL9), this observation appears plausible. The implications for interaction between herpesviral replication functions and heterologous (possibly cellular) DNA sequences are discussed.
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Evaluation of Antiviral Immunity Using Vaccinia Virus Recombinants Expressing Cloned Genes for Herpes Simplex Virus Type 1 Glycoproteins
More LessSUMMARYImmunization of mice with vaccinia virus recombinants expressing the glycoproteins B or D of herpes simplex virus type 1 (HSV-1) induced humoral antibody as well as multiple aspects of HSV-1-specific T lymphocyte-mediated responses. However, vaccinated mice were not completely resistant to HSV-1 challenge and were unable to eliminate an epithelial infection rapidly. Evidence is presented which indicates that immunization with either vaccinia virus recombinant, while inducing the necessary protective populations of CD4+ T lymphocytes, fails to induce the complementing CD8+ cytotoxic T lymphocytes necessary for high levels of protection against a primary HSV-1 infection. These findings are discussed with relevance to the future development of anti-HSV vaccines.
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Herpes Simplex Virus Type 2 Primes Mouse Macrophages for an Early and Genetically Determined Respiratory Burst Mediated by Interferon-α/β
More LessSUMMARYThe influence of infection by herpes simplex virus type 2 (HSV-2) on the respiratory burst capacity of mouse macrophages was studied by luminol-dependent chemilu-minescence with phorbol myristate acetate (PMA) as trigger. Peritoneal cells from virus-infected mice were strongly primed for a respiratory burst during the acute phase of the infection. By 12 h after infection the response had increased 40-fold over control values. Most of the response was elicited by mononuclear phagocytes. When resting peritoneal macrophages were infected with HSV-2 in vitro a maximal priming effect was seen with 2 × 106 p.f.u./ml of virus after 8 h, but a significant response was obtained after 4 h of infection; after 12 h incubation with virus the response declined to reach background levels at 24 h. Peritoneal cells from C57BL/6 mice which are relatively resistant to HSV-2 showed a higher respiratory burst capacity after infection than cells from more susceptible BALB/c mice. Incubation of macrophages with crude murine interferon (IFN)-α/β produced by macrophages or purified murine IFN-α, in concentrations comparable to those obtained early (2 to 5 h) after infection of macrophage cultures with HSV-2 also augmented the respiratory burst. Addition of an IFN-α/β-specific antiserum to HSV-2-infected cultures almost completely removed the response. We therefore conclude that HSV-2 induces an early and genetically determined activation of macrophages, mediated in an autocrine manner by IFN-α/β secreted by the macrophages early during infection.
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Isolation and Characterization of a Functional Murine Interferon Alpha Gene which Is Not Expressed in Fibroblasts upon Virus Induction
More LessSUMMARYA mouse genomic segment containing three new members of the murine interferon alpha (MuIFN-α) gene family was isolated from a fibroblastic cosmid library. A 4 kb EcoRI fragment contained a new MuIFN-α gene named MuIFN-α8. The nucleotide sequence of the coding and flanking regions of this gene showed a high level of homology to those of known members of the MuIFN-α family. Transient expression of the MuIFN-α8 gene in COS cells and oocyte translation of in vitro transcripts both led to a biologically active protein. The antiviral activity was neutralized by monoclonal and polyclonal MuIFN-α antibodies. Although the 5′ flanking sequence shows features characteristic of an IFN regulatory region, the MuIFN-α8 gene is not expressed in murine fibroblasts treated with Newcastle disease virus or poly(I)·poly(C).
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Host Genetic Control of Incubation Periods of Creutzfeldt-Jakob Disease in Mice
More LessSUMMARYHost genetic control of the incubation period of Creutzfeldt-Jakob disease (CJD) was studied using various inbred strains of mice, including B10 congenic strains. Various incubation periods were found in mice injected either intracerebrally or intraperitoneally with the Fukuoka 1 strain of the CJD agent; NZW/Sea and A/JJms had the shortest, and B10.AKM/Ola and C57BL/6J the longest, incubation periods. Length of the CJD incubation period did not correlate with the genetic markers tested, i.c. the murine major histocompatibility (H-2) complex (which has previously been reported to be linked to a gene influencing CJD incubation period in mice), coat colour or sex genes. In NZW/Sea × C57BL/6J F1 hybrid mice the CJD incubation periods were similar to that of the parent with the longest incubation period. Incubation periods of the backcross progeny from F1 and NZW/Sea were intermediate between those of the parental mice and had a unimodal distribution pattern. A similar observation was made on the progeny of the A/JJms × C57BL/6J mating. On the other hand, the length of incubation period for the NZW/Sea × B10.AKM/Ola F1 hybrid fell between those for the two parents and the NZW/Sea × A/JJms F1 hybrid had a significantly longer incubation period than those of the two parents. These results suggest that polygenes probably control the length of the CJD incubation period in mice.
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Production of Antibodies Directed against Microtubular Aggregates in Hepatocytes of Chimpanzees with Non-A, Non-B Hepatitis
SUMMARYWe have previously used Epstein–Barr virus transformation to establish two clonal lymphoblastoid cell lines (48-1 and S-1) producing monoclonal antibodies against microtubular aggregates that appear in the hepatocytes of chimpanzees with non-A, non-B hepatitis (NANBH). To obtain additional antibodies directed against the same structure, the mouse hybridoma method was employed. Partially purified microtubular aggregates were prepared from liver homogenates of a chimpanzee with NANBH and used as the immunogen. Hybridoma cultures were first screened by radioimmunoassay against the partially purified antigen and secondly by immunofluorescence (IF) using liver sections from a chimpanzee with NANBH. Twenty-seven cultures exhibited positive IF reactions similar to those observed with the original antibodies, 48-1 and S-1, and were cloned by limiting dilution. The specificities of the monoclonal antibodies were tested by IF on liver biopsy specimens from chimpanzees with hepatitis A, B, D or NANBH and from normal chimpanzees. All the antibodies proved to be IgG. Immunoelectron microscopy revealed that all 27 antibodies bound to the same structure, the microtubular aggregates, in hepatocytes of chimpanzees with NANBH. To determine the size of the antigen polypeptide recognized by these antibodies, polyacrylamide gel electrophoresis and Western blot assays were performed. Nine of the 27 antibodies specifically reacted with a single polypeptide of M r 44K (p44). The remaining 18 antibodies detected no antigen polypeptide on the filters. The anti-p44 antibodies were then tested using cross-competition assays with 125I-labelled antibodies, and were found to be classifiable into three groups. In addition, the results indicate that at least three distinct epitopes are located on p44: epitope A recognized by group 1, epitope B recognized by group 2 and epitope C recognized by group 3.
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Detection of Dengue 4 Virus Core Protein in the Nucleus. I. A Monoclonal Antibody to Dengue 4 Virus Reacts with the Antigen in the Nucleus and Cytoplasm
More LessSUMMARYA mouse monoclonal antibody (MAb) to dengue 4 (DEN-4) virus reacted with the antigen in the nucleus as well as in the cytoplasm of DEN-4-infected mammalian and mosquito cells, as demonstrated by the peroxidase–antiperoxidase staining method. The intranuclear antigen appeared to accumulate at the nucleoli, forming spots, whereas the cytoplasmic antigen appeared to be localized mainly in large perinuclear foci in the infected cells. The MAb-reactive antigen was produced in the presence of actinomycin D, which caused the accumulation in the nucleus to be altered to a dispersed pattern. Radioimmunoprecipitation analysis of [35S]methionine-labelled purified virions and Western blot analysis of the antigens prepared from the infected mammalian and mosquito cells showed that the MAb was directed against the DEN-4 virus core protein (M r 15·5K). These results indicated that the DEN-4 virus core protein was partially transported, soon after its synthesis in the cytoplasm, into the nucleus and accumulated at the nucleoli.
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Detection of Dengue 4 Virus Core Protein in the Nucleus. II. Antibody against Dengue 4 Core Protein Produced by a Recombinant Baculovirus Reacts with the Antigen in the Nucleus
SUMMARYThe dengue 4 virus (DEN-4) core gene and part of the PreM genes were inserted into the baculovirus polyhedrin gene region. The recombinant baculovirus directed the synthesis of the DEN-4 core protein fused to a part of the polyhedrin protein (M r 25K), as determined by Western blot analysis using DEN-4 core monoclonal antibody. A mouse polyclonal antibody prepared against the DEN-4 core fusion protein showed antigenic reactivity with the authentic DEN-4 core protein (M r 15·5K) present in the nucleus as well as in the cytoplasm of DEN-4-infected Vero cells as demonstrated by the peroxidase-antiperoxidase staining method. This antibody did not react with cells infected with DEN-1, -2, -3 or Japanese encephalitis virus, or mock-infected cells.
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Polypeptides of Pneumonia Virus of Mice. I. Immunological Cross-reactions and Post-translational Modifications
R. Ling and C. R. PringleSUMMARYA murine polyclonal antiserum and monoclonal antibodies have been employed to identify pneumonia virus of mice (PVM) polypeptides in infected cells and to study post-translational modifications. Immunoprecipitation experiments using a murine polyclonal antiserum and a monoclonal antibody directed against a 39K protein have established an antigenic relationship between two PVM proteins and the N and P proteins of human respiratory syncytial virus. Although 20 virus-specific polypeptides have been identified in lysates of infected cells, evidence is presented that some of these are related and that the number of unique polypeptides probably does not exceed 11 or 12. The phosphoproteins of PVM have a pattern of mobilities more like that of the recently described pneumovirus causing rhinotracheitis in turkeys than that of human or bovine respiratory syncytial virus.
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Polypeptides of Pneumonia Virus of Mice. II. Characterization of the Glycoproteins
R. Ling and C. R. PringleSUMMARYThe kinetics of synthesis and the nature of the oligosaccharides of the glycoproteins of pneumonia virus of mice (PVM) were studied. Tryptic peptide mapping showed that the two major glycosylated polypeptides G1 and G2 were different forms of the same protein. G2 was derived from G1 which in turn appeared to be derived from an unidentified precursor. The G1/G2 protein of PVM is probably a haemagglutinin since a monoclonal antibody directed against it has a high haemagglutination inhibition titre. On the basis of experiments with inhibitors and glycosidases it was deduced that G1 and G2 have both N-linked and O-linked oligosaccharides. The putative fusion protein-equivalent of PVM was shown to possess N-linked oligosaccharides. In the presence of tunicamycin a high mobility form (F1t) appeared to be derived from a precursor (F0t) with the same mobility as the fully glycosylated protein. If by analogy with other paramyxoviruses this represents a cleavage event, the difference in mobility of the precursor and product suggests that the putative F2 product is smaller than the corresponding F2 protein of other paramyxoviruses. However, no F2 candidate protein was detected and evidence for an F1,2 dimer was inconclusive. The glycoproteins of PVM resemble those of respiratory syncytial virus in terms of their pattern of glycosylation, but differ in their processing.
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Synthesis of Immunogenic, but Non-infectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector
SUMMARYA baculovirus expression vector (AcLeon) derived from Autographa califomica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6·6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected insect cells made poliovirus proteins that included the structural proteins VP0, VP1 and VP3. Poliovirus particles were recovered from extracts of the infected cells and demonstrated to be free from detectable levels of RNA and to be non-infectious in tissue culture. After particle purification by CsC1 gradient centrifugation and immunization of outbred mice, antibodies to the structural proteins, including neutralizing antibodies, were obtained. Other recombinant baculoviruses, containing the majority of the capsid coding region of P3/Leon/37 (e.g. AcCAP21, nucleotide residues 742 to 3318), made an unprocessed precursor to the poliovirus structural proteins. These data suggested that processing of the poliovirus gene product by the AcLeon construct was catalysed by the poliovirus-encoded proteases. The results demonstrated that antigenic and immunogenic poliovirus proteins and empty particles can be made in insect cells by recombinant baculoviruses.
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Target and Effector Cell Fusion Accounts for B Lymphocyte-mediated Lysis of Mouse Hepatitis Virus-infected Cells
More LessSUMMARYIn confirmation of previous reports, we observed lysis of mouse hepatitis virus (MHV)-infected target cells in the presence of spleen and lymph node cells from non-immunized mice possessing a B cell surface phenotype (IgM+, IgG+, J11d+, Ia+, Fc+, Thy1–, MAC-1– and asialo-GM1–). Lysis was inhibited by MHV-specific antisera. The presence of immunoglobulin at the surface of B cells is not required for cytolysis since MHV-infected target cells are lysed in the presence of the B cell hybridoma Sp2/0, which fails to synthesize immunoglobulin. Using 51Cr-labelled Sp2/0 cells, both target and effector cells were shown to undergo cytolysis. Direct observation of target and effector cells co-incubated after labelling with different fluorescent dyes demonstrated that lysis correlates with the fusion of B cells and MHV-infected cells. These findings are consistent with the idea that the E2 protein of MHV, which is expressed on the infected cell surface and has receptor and membrane-fusion activities at neutral pH, selectively mediates fusion with cells of B lymphocyte lineage. This may represent a general mechanism by which enveloped viruses with fusion proteins that function at neutral pH can interfere with the function of subsets of immune cells.
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A Conformational Immunogen on VP-2 of Infectious Bursal Disease Virus that Induces Virus-neutralizing Antibodies that Passively Protect Chickens
More LessSUMMARYVP-2b, a major structural protein of infectious bursal disease virus (IBDV), and its precursor protein VP-2a were separated in a soluble form from the supernatant of ultracentrifuged viruses by using a monoclonal antibody specific for VP-3, the other major structural protein, to remove soluble VP-3 and remaining virus particles. The native VP-2a/2b inhibited the majority of virus-neutralizing (VN) activity in chicken anti-IBDV sera and chickens immunized with VP-2a/2b produced VN antibodies that passively protected susceptible chickens from infection. However, the separated VP2a/2b was not as immunogenic as intact virus particles. VP-2a/2b would appear to contain a conformational epitope which is destroyed by SDS and boiling and which may prove to be of critical importance in a subunit vaccine against type 1 IBDV,
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Neutralizing Epitopes of Type O Foot-and-Mouth Disease Virus. I. Identification and Characterization of Three Functionally Independent, Conformational Sites
More LessSUMMARYEleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.
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Neutralizing Epitopes of Type O Foot-and-Mouth Disease Virus. II. Mapping Three Conformational Sites with Synthetic Peptide Reagents
More LessSUMMARYFour neutralizing monoclonal antibodies (MAbs), recognizing three functionally independent, conformational sites on type O foot-and-mouth disease virus (FMDV) failed to react with immobilized structural proteins or synthetic peptides but bound to the isolated capsid protein VP1 and peptides in solution. Inhibition ELISA techniques were, therefore, applied using peptide antigens and anti-peptide sera to block MAb binding to virus particles, permitting the identification of those portions of the VP1 protein contributing to the epitopes. The binding site of one MAb, which neutralized a range of type O FMDV isolates, was shown to have components within regions 146 to 150 and 200 to 213 of VP1 with a critical involvement of the amino acids at positions 146 and 206 or 207. The determinants recognized by two other MAbs which were directed at similar, but not identical, epitopes from a second site included components from the 200 to 213 and 143 to 146 regions with amino acids 143 and 144, respectively, appearing critical for the inhibition of the virus binding of the two antibodies. These results demonstrate that the two previously identified immunogenic tracts of VP1 are brought into proximity in the quaternary structure of the virion to form an antigenic domain containing several conformational epitopes, some of which are functionally independent. A fourth, strain-specific MAb was effectively blocked from reacting with virus by peptides corresponding to residues 161 to 180 and 200 to 213.
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Specificity and Function of the Individual Amino Acids of an Important Determinant of Human Immunodeficiency Virus Type 1 that Induces Neutralizing Activity
More LessSUMMARYAn important antigenic determinant of human immunodeficiency virus type 1 that induces neutralizing activity in infected humans and chimpanzees was previously mapped with nonapeptides between amino acids 307 and 320 on the external envelope glycoprotein (gp120) of strain HTLV-IIIB (molecular clone BH 10) and amino acids 320 to 330 of strain HTLV-IIIRF. Using different sera we found different reactive nonapeptides that overlapped and shared a tetrapeptide, GPGR. This tetrapeptide, which is the same in HTLV-IIIB and HTLV-IIIRF, is flanked by amino acids that vary between virus strains. Because GPGR is predicted to form a β-turn and is flanked by two cysteine residues that may form a disulphide bridge, a hairpin-like structure is suggested for this part of gp120. The tetrapeptide GPGR and the reactive peptides are located on top of this structure, well exposed to antibodies. We determined the role of the individual amino acids in antibody binding using three sets of peptide analogues derived from three reactive nonapeptides (two of strain HTLV-IIIB which overlapped and one of strain HTLV-IIIRF). Each set contained peptide analogues in which each amino acid was replaced, one at a time, by all genetically encoded amino acids. At least five consecutive amino acids in each nonapeptide were essential for antibody binding. They include amino acids of GPGR and potentially provide the virus with ample opportunity to escape immune surveillance.
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Rabies Virus-specific Human T Cell Clones Provide Help for an in vitro Antibody Response against Neutralizing Antibody-inducing Determinants of the Viral Glycoprotein
SUMMARYHuman T cell clones were prepared from peripheral blood mononuclear cells from a vaccinated human donor and kept in culture in the presence of rabies virus antigen and growth factors. Phenotypic analysis of the T cell clones revealed expression of the CD3 and CD4 cell surface markers, but not of CD8, consistent with a phenotype of helper/inducer T cells. The rabies virus specificity of the T cell clones was established by virus-specific proliferation in response to the rabies virus Pitman-Moore strain (PM) produced in three different cell substrates. The clones also responded to the rabies virus strains Evelyn-Rokitnicki-Abelseth (ERA) and challenge virus standard (CVS), but not to the rabies virus-related Mokola and Duvenhage-6 virus strains. Proliferative responses of T cell clones required rabies virus antigen to be presented by autologous antigen-presenting cells in association with HLA class II molecules. When cultured with rabies virus antigen, but in the absence of growth factors, some of the T cell clones provided help for an antibody response of rabies virus immune B lymphocytes. Analysis of culture supernatant fluids showed that at least a part of this antibody response was directed against neutralizing antibody-inducing determinants of the viral glycoprotein.
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Mechanism of Immunity to Influenza: Maternal and Passive Neonatal Protection Following Immunization of Adult Ferrets with a Live Vaccinia–Influenza Virus Haemagglutinin Recombinant but Not with Recombinants Containing Other Influenza Virus Proteins
More LessSUMMARYNeonatal ferrets are protected against infection with influenza virus by milk-derived anti-influenza virus IgG after suckling on an immune mother. Live vaccines protect better than killed vaccines despite their stimulation of lower maternal haemagglutination-inhibiting antibody levels. This suggests that antibody to virus proteins other than the haemagglutinin may also be involved. To investigate this, adult ferrets were immunized intradermally with live vaccinia-influenza virus recombinants each expressing one of the 10 influenza virus polypeptides. Adult ferrets immunized with a recombinant expressing the H3 haemagglutinin were completely protected, and also passively protected their offspring, against a live challenge with clone 7a of the reassortant influenza virus A/Puerto Rico/8/34–A/England/939/69 (H3N2), immunity being mediated by IgG antibody. However, ferrets immunized similarly with recombinants expressing the H1 haemagglutinin, neuraminidase (N1 or N2), polymerases (PB1, PB2 or PAC), matrix protein (M1 or M2), nucleoprotein (NP) or non-structural proteins (NS1 or NS2) were completely susceptible to the influenza virus.
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Cross-protection against Microvariants of Influenza Virus Type B by Vaccinia Viruses Expressing Haemagglutinins from Egg- or MDCK Cell-derived Subpopulations of Influenza Virus Type B/England/222/82
More LessSUMMARYB/Singapore/222/79-like influenza viruses isolated from three patients during the winter of 1981 to 1982 and cultured in either embryonated hens' eggs or MDCK cells were studied. Sequence analysis indicated that the haemagglutinin (HA) genes of the six virus preparations contained at least four distinct HA1 sequences which differed by up to six amino acids. Only one pair of viruses had amino acid differences between the egg- and MDCK cell-derived viral subpopulations and this change did not affect a glycosylation site. Mice infected with previously described recombinant vaccinia viruses expressing either the egg- or MDCK cell-derived HA of B/England/222/82 developed neutralizing antibodies against all of the 1982 type B viruses and were protected against intranasal challenge with these viruses. Therefore, in this model system, the minor sequence variation between the HAs of egg- and MDCK cell-derived influenza B/England/222/82 virus had no detectable effect on the induction of cross-protection.
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The 1B (NS2), 1C (NS1) and N Proteins of Human Respiratory Syncytial Virus (RSV) of Antigenic Subgroups A and B: Sequence Conservation and Divergence within RSV Genomic RNA
More LessSUMMARYA 2330 nucleotide sequence spanning the 1B (NS2), IC (NS1) and N genes and intergenic regions of human respiratory syncytial virus strain 18537, representing antigenic subgroup B, was determined by sequencing cloned cDNAs of intracellular mRNAs. Comparison with the previously reported sequences for strain A2 of subgroup A showed that 1B, 1C and N were highly conserved at the nucleotide level (78, 78 and 86% identity, respectively) and at the amino acid level (92, 87 and 96% identity, respectively). The gene-start signals were exactly conserved between subgroups, and the gene-end signals contained only a single nucleotide substitution each in 1B and N. In most cases intergenic and non-coding gene sequences that were not part of presumed transcriptive signals were much less well conserved (generally 50 to 71%) than sequences that were part of translational open reading frames (82 to 86%). The nucleotide and deduced amino acid sequences of the N gene and protein of the Long strain of subgroup A were determined by sequencing cDNA clones of intracellular mRNA; the nucleotide sequence (representing all but the first 10 nucleotides of the gene) contained 15 differences from that of the A2 strain, but the deduced amino acid sequences were identical.
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Effect of Amino Acid Substitutions on Glycosylation of the Haemagglutinin–Neuraminidase Glycoprotein of Newcastle Disease Virus Strain Beaudette C
More LessSUMMARYThe nucleotide sequences of two monoclonal antibody-resistant mutant viruses predict changes from the wild-type in the number of potential glycosylation sites (Asn-X-Thr/Ser) in the mutant haemagglutinin–neuraminidase (HN) glycoproteins of the Beaudette C strain of Newcastle disease virus. The HN glycoproteins of these mutants, F5 and Z18, migrate either slower (F5) or faster (Z18) than that of the wild-type in SDS–PAGE. HN proteins synthesized in chick embryo fibroblasts following infection by either mutant or wild-type virus in the presence of tunicamycin (an inhibitor of glycosylation), comigrate on SDS–PAGE. These results confirm that the HN protein of the mutant virus, F5, has gained a glycosylation site at Asn(323)-Ser-Ser and that the conserved potential glycosylation site at Asn(481)-His-Thr is indeed glycosylated in the HN protein of the wild-type Beaudette C strain of Newcastle disease virus but is lost in that of the mutant virus, Z18.
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Morphology and Distribution of gp52 on Extracellular Human Cytomegalovirus (HCMV) Supports Biochemical Evidence that It Represents the HCMV Glycoprotein B
More LessSUMMARYGlycoprotein gp52 exists within the mature human cytomegalovirus (HCMV) envelope in heterodimeric, disulphide-linked complexes with glycoproteins gp95 and gp130. Biochemical studies involving immunoprecipitations and Western blots have demonstrated that gp52 is the glycoprotein B (gB) homologue of HCMV but that gp95 and gp130 are probably separate gene products. The distribution of this putative gB on extracellular HCMV particles was revealed by high resolution electron microscopy of preparations labelled with a monoclonal antibody, F5, directly coupled to colloidal gold. F5–gold probes, specific for HCMV gp52, bind to the distal end of 12 nm long, slender spikes projecting from virion and dense body envelopes. Labelled spikes were most often present in closely packed, homogeneous clusters and were frequently present on envelope protrusions. The degree of labelling on individual HCMV particles was highly variable. Both the morphology and distribution of HCMV gp52 show strong similarity with that previously reported for the gB of herpes simplex virus. Other morphologically distinct spikes occur in the HCMV envelope but these were not recognized by F5–gold probes.
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The Effect of Bovine Herpesvirus Type 1 Glycoproteins gI and gIII on Herpesvirus Infections
More LessSUMMARYWe expressed the bovine herpesvirus type 1 (BHV-1) glycoproteins, gI and gIII, in bovine cells using a bovine papillomavirus vector. The proteins expressed by these cells had the same M r as the native BHV-1 proteins and monoclonal antibodies detected no differences in their antigenic structure. Cells expressing gI were infected with either BHV-1 or herpes simplex virus type 1 (HSV-1). The number of plaques in gI-expressing cells was similar to that seen with normal fibroblasts infected with BHV-1 or HSV-1. However, BHV-1 or HSV-1 plaques produced in gI-expressing cells were smaller and darker than those seen in normal fibroblasts indicating an interference with cell-to-cell transmission or cellular lysis. Virus growth curves and [35S]methionine labelling of BHV-1-infected gI-expressing cells showed no difference in virus production, virus protein synthesis or cellular protein shutdown when compared to BHV-1-infected normal cells. This led us to conclude that the gI protein may interfere with a cellular protein(s) responsible for the cytopathic effects of BHV-1 infection. Cells expressing gIII were fully susceptible to BHV-1 infection.
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The Central Segment of Herpes Simplex Virus Type 1 Glycoprotein C (gC) Is Not Involved in C3b Binding: Demonstration by Using Monoclonal Antibodies and Recombinant gC Expressed in Escherichia coli
More LessSUMMARYThree monoclonal antibodies (MAbs) have been raised against cell membrane-derived herpes simplex virus type 1 glycoprotein C (gC-1). By using different DNA constructs of gC-1 expressed in Escherichia coli the sites recognized by these antibodies could be assigned to a peptide in the more hydrophobic and probably non-glycosylated middle third of the gC-1 molecule. This peptide segment corresponds to a 571 bp segment on the gC-1 gene located between the NcoI and the NruI restriction sites. None of the three MAbs interfered with the binding of the human serum complement component C3b to gC, which has been shown to be rather glycosylation-dependent. Since the data of other groups have suggested that antibodies directed against all regions of gC-1 inhibit C3b binding to gC-1, whereas our results suggest that the central part of gC-1 is not actually involved in C3b-binding activity, it can be inferred that the N- and C-terminal segments are involved in C3b binding by gC-1.
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Properties of the Herpes Simplex Virus Type 2 Trans-inducing Factor Vmw65 in Wild-type and Mutant Viruses
More LessSUMMARYA temperature-sensitive mutant (ts13) of herpes simplex virus type 2 (HSV-2) has a mutation which causes in vitro thermolability of the virion. This mutation lies within the gene encoding a virion structural protein of M r 65K which is known to stimulate immediate early transcription (the trans-inducing factor, 65K tif ). The results presented here show that the structural role of 65K tif is essential. The electrophoretic mobility of the 65K tif encoded by ts13 and a revertant of ts13 differed from that of the wild-type HSV-2 parent. Two monoclonal antibodies directed against 65K tif were shown to react with two different epitopes on this polypeptide, one of which was altered by the mutation in ts13. No differences were observed in the phosphorylation status of 65K tif from mutant- and wild-type-infected cells.
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Presence of Human Papillomavirus Type 18 DNA in Vulvar Carcinomas and Its Integration into the Cell Genome
More LessSUMMARYWe have screened 78 genital tract tumours from Italian female patients for the presence of human papillomavirus type 16 (HPV-16) and HPV-18 DNA. Dot and Southern blot hybridization experiments revealed that DNA sequences of HPV-16 and/or HPV-18 are present in 66% of tumour samples. HPV-18 was detected in 80% of vulvar carcinomas. In these tumours the integration of HPV-18 DNA seems to occur in the E1/E2 region of the virus genome with long deletions in almost all samples. The invariable retention of the non-coding region and the E6/E7 open reading frames suggests that the integration event and the possible expression of these sequences could play a role in some stages of cellular transformation.
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Characterization of Bovine Papillomavirus Type 1-transformed Clones which Show Distinct Transformed Phenotypes
More LessSUMMARYSeventeen independent cell clones were isolated from C127 cells transformed by bovine papillomavirus type 1 (BPV-1). Transformants showed differing degrees of expression of the transformed phenotype as monitored by saturation density, doubling time, growth in medium with a low serum concentration and colony-forming efficiency in soft agar. The degree of expression of the transformed phenotype did not correlate with either the BPV-1 copy number or levels of BPV-1-specific RNA in the transformed cell clones. A characteristic transformed cell clone, T1c, showed the lowest degree of expression of the transformed phenotype but contained the highest copy number of BPV-1 DNA and the highest level of BPV-1-specific mRNA. When we analysed different transformants by two-dimensional gel electrophoresis, we found that a set of six proteins changed quantitatively. Changes in the expression of these proteins were most consistent in clones expressing the greatest number of parameters of transformation, e.g. clone T4a. These data indicate that changes in the expression of cellular genes may correlate with the degree of expression of the transformed phenotype.
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Interaction between Rabies Infection and Oral Administration of Vaccinia–Rabies Recombinant Virus to Foxes (Vulpes vulpes)
More LessSUMMARYWe have investigated the influence of anti-rabies vaccination on the onset of the disease as well as the delay of death in foxes previously infected with rabies virus. A live vaccinia recombinant virus expressing the rabies virus glycoprotein (VVTGgRAB) was used as vaccine. Foxes were divided into six experimental groups of four animals. On day 0, each fox was experimentally infected with a rabies virus suspension. VVTGgRAB was administered by the oral route to each animal of three groups on day 0, 3 or 14. Foxes of other groups were used as unvaccinated controls or received, on day 0, the parental Copenhagen strain of vaccinia virus. The length of post-challenge survival and duration of clinical disease were recorded for each animal tested. All foxes, vaccinated or not, died from rabies as confirmed by fluorescent antibody tests. Animals vaccinated on days 0 and 3 died after a shorter period of incubation than unvaccinated controls. On the other hand, animals vaccinated on day 14 post-challenge died later than control animals. Foxes administered vaccinia virus died at the same time as unvaccinated controls. These results demonstrate that early and late death phenomena, as consequences of interactions between oral vaccination with VVTGgRAB and rabies infection, can occur in foxes.
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Antibody-dependent Enhancement of Yellow Fever and Japanese Encephalitis Virus Neurovirulence
More LessSUMMARYAntibody-dependent enhancement of yellow fever virus neurovirulence, as measured by a reduction in the average survival time of groups of mice, was demonstrated with wild-type or vaccine strains of yellow fever virus and with Japanese encephalitis virus using intraperitoneally administered monoclonal antibodies specific for the viral E glycoprotein of yellow fever virus. Enhancement of virulence could be induced by neutralizing, non-neutralizing or protective antibodies if the virus was allowed to establish a productive infection in the mouse brain before the antibody was administered. The implications of antibody-dependent enhancement in flaviviruses are discussed.
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- Plant
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Complementary DNA Cloning and Hybridization Analysis of Beet Western Yellows Luteovirus RNAs
More LessSUMMARYComplementary DNAs to the virion RNAs of the ST9 strain of beet western yellows luteovirus (BWYV) were cloned and used for hybridization analyses. These showed that the two major virion ssRNAs, the genomic RNA, approximately 6 kb, and the 3·1 kb ST9-associated RNA, do not show detectable sequence homology. Evidence was also obtained concerning an ssRNA molecule of approximate size 0·72 kb, which hybridized with selected cDNA clones to the BWYV 6 kb genomic RNA; small amounts of it are associated with virions of ST9 and ST9-M BWYV isolates. Extracts of plants infected by ST9 and other BWYV isolates contained ssRNAs of approximately 6 kb, 2·9 kb and 0·72 kb in size. Extracts of ST9-infected plants also contained RNAs of approximately 3·1 kb and 0·48 kb which hybridized with selected cDNA clones prepared from the ST9-associated 3·1 kb virion RNA. cDNA clones of the ST9 virion 6 kb RNA also hybridized with the genomic 6 kb RNA of other BWYV isolates. None of the clones hybridized with preparations of other luteoviruses tested. No RNA molecule with a sequence related to the 3·1 kb ST9-associated virion RNA was detected in virions or plant tissues infected by other isolates of BWYV or other luteoviruses.
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Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan
More LessSUMMARYViroids have been isolated from plum trees (Prunus salicina Lindley) affected with plum dapple fruit disease and from peach trees (Prunus persica Batsch) showing dapple fruit symptoms. The viroids were inoculated mechanically to cucurbitaceous plants, in which symptoms typical of hop stunt viroid (HSV) infection appeared. The complete nucleotide sequences of an isolate from plum and an isolate from peach (AF isolate) were shown to be identical, consisting of 297 nucleotides with a 93·6% sequence homology to HSV-hop. Another isolate from peach (A9 isolate) also consists of 297 nucleotides, but the sequence homology to HSV-hop is 99·7%, showing only one nucleotide replacement. These results indicate that these three viroids are strains of HSV, which we designate HSV-plum, HSV-peach (AF) and HSV-peach (A9), respectively. Comparative analysis of the nucleotide sequences of HSV strains from hop, grapevine, citrus, cucumber, plum and peach revealed variable and conserved regions in the HSV molecule. In Japan, these viroids are closely related not only to dapple fruit disease in plum cv. Taiyo, but also to dapple fruit symptoms on peach cv. Asama-Hakutou.
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Potato Spindle Tuber Viroid Does Not Complement Tobacco Mosaic Virus Temperature-sensitive Transport Function
SUMMARYIn mixed infections, the potato spindle tuber viroid (PSTV) does not complement the transport function of the tobacco mosaic virus (TMV) Lsl mutant, which has a temperature-sensitive transport function. This appears to be due to the fundamentally different mechanisms of virus and viroid transport. However, PSTV significantly enhances the accumulation of temperature-resistant TMV in mixed infections at a temperature non-permissive for Lsl.
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- Fungal
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Molecular Analysis of Agaricus bisporus Double-stranded RNA
More LessSUMMARYA detailed analysis was made of dsRNA molecules present in Agaricus bisporus (cultivated mushroom) affected and non-affected by La France disease occurring in The Netherlands. A specific pattern of 10 major dsRNA molecules was always associated with affected strains but non-affected strains also contained at least one of the dsRNAs. Cross-hybridization under stringent conditions showed that the dsRNAs were all unique.
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