- Volume 72, Issue 2, 1991
Volume 72, Issue 2, 1991
- Animal
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Cell-free translation and identification of the replicative form of Nudaurelia β virus RNA
More LessLarvae of the pine emperor moth, Nudaurelia cytherea capensis, infected with Nudaurelia β virus (NβV) consistently contained one species of dsRNA. This dsRNA was the correct size to be the replicative form of the NβV genome and, in Northern blots, it hybridized with 32P-end-labelled virion RNA. Other smaller dsRNAs were obtained in a non-reproducible manner but these had no sequences in common with the genomic probe; no dsRNAs were observed in extracts from virus-free larvae. Cell-free translation of NβV RNA resulted in the synthesis of only one major polypeptide, of M r about 71000, which could not be precipitated by an anti-NβV serum.
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Immunoelectron microscopic examination of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus-infected Lymantria dispar cells: time course and localization of major polyhedron-associated proteins
More LessImmunoelectron microscopy was employed to examine the temporal expression and localization of two proteins involved in baculovirus polyhedron assembly (polyhedrin and p10) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) in infected Lymantria dispar cells. In addition, the association of p10 with the polyhedron envelope (PE) protein was studied. The major capsid protein (p39) was also examined to investigate the association of virion structural proteins with polyhedron formation. In infected cells, p39 did not show a concentrated association with any infected-cell structures other than nucleocapsids and appeared to be randomly distributed over the nucleocapsid surface. Likewise, polyhedrin showed no major concentrations outside of developing or mature polyhedra. The p10 antibody cross-reacted with a protein associated with condensed chromosomes in uninfected cells. In infected cells, p10 is a component of the body of fibrillar structures. The PE protein has been shown to accumulate around the periphery of fibrillar structures. Cells infected with a polyhedrin-minus virus expressing the β-galactosidase gene under the control of the polyhedrin promoter were examined to determine whether the lack of polyhedra would influence the localization of major polyhedron-associated viral proteins. High concentrations of PE protein accumulating on the periphery of fibrillar structures appeared to be the major difference from wild-type virus-infected cells. The β-galactosidase protein appeared to be distributed throughout the nucleus and cytoplasm, in contrast with the specific localization of the viral proteins.
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Nucleotide sequence and transcript mapping of the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome
More LessThe organization of genes in the 2.95 kb EcoRI-SalI fragment (0.7 to 3.0 map units) located within the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome was studied by a combination of DNA sequencing, Northern blot analysis, S1 mapping and primer extension analysis. In addition to the two divergent overlapping transcripts [leftward early (ES1) and rightward late (ES2)] previously reported, a third transcript which is present at 18 h p.i. and which also runs leftward, overlapping ES1 by 1600 nucleotides (nt) at the 3′ end, was mapped to this region. The DNA sequence revealed the presence of three open reading frames (ORFs) of significant length. ORF-1 and ORF-2 correspond to the leftward transcripts, and code for potential polypeptides of 151 and 329 amino acids, respectively. ORF-3 which codes for a potential polypeptide of 167 amino acids is located on the opposite strand in a region for which no transcript mapping data are available. However, the conserved late gene promoter/cap site sequence (ATAAG) is present 23 nt upstream of the start of ORF-3.
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Variation in the characteristics of 10 mouse-passaged scrapie lines derived from five scrapie-positive sheep
More LessTen mouse-passaged scrapie lines were initiated from five sheep with clinical scrapie. Of the lines, five were initiated and passaged exclusively in mice with the s7s7 genotype and the remaining five lines were initiated in mice with the p7p7 genotype, with two of these lines subsequently being passaged exclusively in p7p7 mice and two being passaged mainly in p7p7 mice. Lines were passaged three or four times and two parameters were compared: incubation period and the induction of a weight increase during the preclinical period. Considerable variation in the incubation periods was found between the different passage lines at similar passage levels, with a range in s7s7 mice of 113 days to greater than 450 days and a range in p7p7 mice of 219 days to greater than 500 days. All of the lines passaged exclusively in s7s7 mice had shorter incubation periods in this mouse genotype than in p7p7 mice, whereas of the five lines initiated in p7p7 mice, two had shorter incubation periods in p7p7 mouse strains. C57BL mice were used as the indicator strain and most of the lines caused an increase in weight during the preclinical phase of disease compared to control mice injected with normal brain homogenates. For both parameters, incubation period and preclinical weight increase, differences were seen in lines that had identical passage histories, suggesting that an informational molecule separate from host genomic material must specify scrapie strain differences.
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Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase
More LessGlutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication.
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Integrated bovine leukosis proviral DNA in T helper and T cytotoxic/suppressor lymphocytes
More LessBovine leukosis virus (BLV) is associated with the disease complex enzootic bovine leukosis. The infection may remain clinically silent in the form of an aleukaemic state or emerge as a persistent lymphocytosis and more rarely as lymphosacroma. BLV has been considered classically to be a B lymphotropic virus, based upon the absolute increase in B lymphocytes in persistent lymphocytosis, the B lymphocyte phenotype of a majority of the cells making up lymphosarcomas and the identification of viral antigen expressed in B lymphocytes following in vitro culture of peripheral blood mononuclear leukocytes. This association of BLV with B lymphocytes is well established but the mechanism(s) of disease expression is not defined. To examine further the cellular tropism(s) of BLV, T lymphocyte subpopulations from 10 lymphocytotic cattle were established in vitro. Lymphocyte cultures were characterized by their subpopulation phenotype and DNA was extracted for identification of integrated provirus by Southern blot hybridization. Provirus was identified in T lymphocyte cultures derived from seven of 10 lymphocytotic cattle, with both T helper and T cytotoxic/suppressor subpopulations affected.
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Rhesus monkey macrophages infected with simian immunodeficiency virus cause rapid lysis of CD4-bearing lymphocytes
Inoculation of simian immunodeficiency virus into cultures of primary rhesus monkey macrophages or CD4-bearing transformed T lymphocytes resulted in persistent infection, with minimal virus replication in the macrophages and extensive replication in the lymphocytes. However, uninfected T cells added to infected macrophages underwent rapid fusion and lysis and were almost completely eliminated without the production of virus particles. Lysis required direct contact between the T cells and the infected macrophages, which enabled binding between CD4 on the former and viral gp120 on the latter to occur. This process was blocked by soluble CD4 and dextran sulphate. Neutralizing antibodies in the serum of an infected macaque prevented cell fusion by preventing infection of the macrophages. However, these antibodies did not prevent fusion when added to previously infected macrophages. Infected macrophages were incorporated into the syncytia of lymphocytes and continued incorporation of new lymphocytes into the syncytia required infected macrophages to be metabolically active. One inference from these studies is that infected macrophages in vivo could help mediate the well known depletion of T4 cells in patients with AIDS.
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Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3
More LessRotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5′ end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [α-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.
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The relationship between the flaviviruses Skalica and Langat as revealed by monoclonal antibodies, peptide mapping and RNA sequence analysis
More LessThe flavivirus Skalica was isolated from a bank vole in Czechoslovakia in 1976. It can be serologically distinguished from prototype strains of tick-borne encephalitis (TBE) virus and has a decreased virulence for adult mice. We have further defined the relationship of Skalica virus to other members of the TBE serocomplex (TBE European and Far Eastern subtypes, Langat and louping ill virus) by using a panel of 22 monoclonal antibodies, peptide mapping and RNA sequence analyses. By these criteria Skalica virus proved to be distinct from TBE virus and to be very closely related to Langat virus, differing by only two bases among a total of 416 nucleotides compared. The sequence of 22% of the Langat genome was determined and the encoded amino acid sequences were derived. Comparison of these with the corresponding amino acid sequences of TBE virus revealed a similarity of 85%, as opposed to 93% similarity between the European and Far Eastern subtypes of TBE virus.
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Restricted mumps virus infection of cells derived from normal human joint tissue
More LessMumps virus (MuV) is known to be associated with acute arthritis and may also have a role in chronic inflammatory joint disease. The mechanism of induction of joint inflammation is not known but may be associated with direct invasion of joint tissue. To investigate the possibility of persistent intra-articular infection, the interaction of MuV with primary cells from normal human joint tissue was examined. These mixed cultures of synoviral membrane cells and chondrocytes were found to be semi-permissive to the virus; only a small proportion of cells (5 to 20%) were infected and produced low titres of progeny virions. In addition, little viral antigen was detected on the cell surface relative to that found on Vero cells. This restricted infection of synovial membrane cells was related to a severely decreased synthesis of the viral glycoproteins, fusion and haemagglutinin—neuraminidase, and the membrane protein in comparison to the levels found in Vero cells. Persistent infections were readily established and could be maintained for 2 to 3 months. During the first month, the infection remained highly focal and supernatant viral titres were low. Thereafter both the percentage of infected cells and viral titres increased until finally the cultures were killed. No evidence was obtained for the generation of temperature-sensitive mutants or defective interfering particles during long-term infection, but the persistent virus derived from the cultures gave cloudy plaques and induced no fusion in Vero cells until passaged. This study has shown that human synoviral tissue cells have the intrinsic ability to support MuV replication and persistence which may be important in the pathogenesis of mumps arthritis.
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Respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (SH gene) and restriction mapping (N gene)
More LessThe genes encoding the small hydrophobic (SH) proteins of a series of respiratory syncytial (RS) virus strains were amplified using the polymerase chain reaction, cloned and sequenced. Analysis of the SH gene sequences from 12 RS virus strains isolated between 1956 and 1989 confirmed the homogeneity of the two subgroups, A and B, previously defined serologically. Although there is only 76% deduced amino acid sequence identity of SH proteins between subgroups, there was little variation in deduced amino acid sequences within the subgroups; nucleotide homologies within the subgroups ranged between 93% and 99%. Forty-two isolates of RS virus from a single epidemic season (autumn/winter 1989) were also examined to determine their relatedness. For these isolates regions of both the SH and nucleocapsid protein genes of each isolate were amplified and these regions were further analysed by direct nucleotide sequencing or restriction mapping. It was possible to discriminate at least six different lineages (or substrains) of RS virus circulating at the same time and in the same locality.
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Stable expression of rabies virus glycoprotein in Chinese hamster ovary cells
The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial N-linked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of N-linked glycosylation in trafficking and antigenicity of the rabies virus G protein.
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Induction of protective immunity with antibody to herpes simplex virus type 1 glycoprotein H (gH) and analysis of the immune response to gH expressed in recombinant vaccinia virus
Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) was found to protect mice from an HSV-1 strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV-1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus-infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses.
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The difference in sensitivity to cicloxolone sodium between herpes simplex virus types 1 and 2 maps to the locations of genes UL22 (gH) and UL44 (gC)
More LessCicloxolone sodium (CCX) is a broad spectrum anti-viral agent which has a largely non-specific and complex mode of antiviral action. However, the experimental finding that herpes simplex virus type 2 (HSV-2) (strain HG52) is consistently more sensitive to inhibition by CCX than HSV-1 (117 syn+) additionally implies the specific involvement of HSV genes. HSV-1/HSV-2 intertypic recombinants have been utilized to investigate this genetic difference by comparing their CCX ED50 concentrations. No short stretch of HSV-2 DNA was associated with its greater sensitivity to CCX, implying that two or more non-contiguously located HSV genes are involved. Correlating CCX sensitivity with recombinant virus genome structures allowed separate evaluation of the gene regions encoding glycoproteins gB, gC, gD, gE, gG, gH and gI and this suggests that the gene locations encoding gH and gC determine the CCX sensitivity difference. The selective inhibitor of Golgi apparatus glycosylation, monensin, gave results analogous to those obtained with CCX.
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Construction of herpes simplex viruses that are pseudodiploid for the glycoprotein B gene: a strategy for studying the function of an essential herpesvirus gene
More LessThe primary structure of glycoprotein B (gB) is conserved strongly among many members of the Herpesviridae, including some that differ vastly in their natural properties. To determine whether the structural similarity between the gBs of herpes simplex virus type 1 (HSV-1) and bovine herpesvirus type 1 (BHV-1) was reflected in functional homology, we constructed pseudodiploid HSV-1 virions which, in addition to their own gene encoding gB, also contained a gene for encoding BHV-1 gB. Two kinds of pseudodiploid viruses were constructed. In one, the coding sequences of the BHV-1 gB gene were linked to the 5′ flanking sequences of the HSV-1 thymidine kinase (TK) gene. In the other, the entire BHV-1 gB gene, including its own flanking sequences, was introduced into the TK gene. In cells infected with the viruses both HSV-1 and BHV-1 gB were made but they could be distinguished immunologically by monoclonal antibodies. Both glycoproteins were inserted into cellular and virion membranes but did not form oligomers with each other. A monoclonal antibody that binds to HSV-1 gB but not BHV-1 gB neutralized the parental HSV-1 and a revertant pseudodiploid virus from which the gene encoding BHV-1 gB had been excised, but was significantly less efficient at neutralizing the pseudodiploid viruses. This suggests that the BHV-1 homologue can complement the HSV-1 gB functions required for infectivity.
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The nucleotide sequence of the glycoprotein gB gene of infectious laryngotracheitis virus: analysis and evolutionary relationship to the homologous gene from other herpesviruses
More LessA 3698 bp region of the genome of infectious laryngotracheitis virus (ILTV) was sequenced and found to contain the entire glycoprotein gB gene and the C-terminal region of a gene homologous to the ICP18.5 protein gene of herpes simplex virus type 1. The ILTV gB gene encoded a protein with an M r of 100K possessing all the characteristics of a transmembrane glycoprotein. Alignment of the ILTV gB sequence with homologous sequences from six other herpesviruses revealed that 10 cysteine residues on the surface of the molecule were completely conserved and that the positions of several N-linked glycosylation sites were largely conserved. Evolutionary trees based on the gB amino acid sequences from a total of 13 herpesviruses were constructed and the relationships among these herpesviruses were examined.
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Immunological studies on the Epstein—Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells
More LessAntisera were raised against a purified recombinant form of the Epstein—Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli. These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji). Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV. Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an M r of approximately 55000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of post-translational modification during virus replication. The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum. A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase. Immunostaining of biopsies of oral ‘hairy’ leukoplakia with the antisera against EBV DNAse revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens.
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Cytotoxic T lymphocyte discrimination between type A Epstein—Barr virus transformants is mapped to an immunodominant epitope in EBNA 3
More LessAn immunodominant Epstein—Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68.
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Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins
More LessThree structural proteins (4a, 4b and 25K) located within the virion core of vaccinia virus are cleavage products of precursor polypeptides (P4a, P4b and P25K) synthesized late in viral infection. Pulse-chase labelling experiments revealed that cleavage of the core proteins lags considerably behind precursor synthesis and that processing requires continuous protein synthesis. The N-terminal sequences of 4b and 25K, but not 4a, were determined by microsequencing core proteins isolated from purified virions. Comparison of these data with the predicted amino acid sequence of P4b and P25K revealed a conserved Ala-Gly-Ala motif flanking the apparent N termini of both proteins, as well as several additional sequence similarities between the P4b and P25K precursors both upstream and downstream of the putative cleavage site. The Ala-Gly-Ala tripeptide signal was also found in the same region of the amino acid sequences of the homologous proteins of fowlpox virus.
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Natural and experimental infection of wild mallard ducks with duck hepatitis B virus
More LessWild duck populations were investigated over a 4 year period for duck hepatitis B virus (DHBV) infection and liver disease. It appeared that DHBV is endemic in wild migratory mallards from France and the U.S.A., although neither hepatocellular carcinoma nor viral DNA integration could be detected in liver samples examined. The follow up of natural infection indicated that wild mallards developed significantly higher serum titres to DHBV DNA than Pekin ducks. The results of experimental transmission demonstrated that such differences in viraemia were not related to the breed of ducks but to the virus isolate, since the wild mallard-isolated DHBV (DHBVwm) induced significantly higher viraemia in both mallard and Pekin ducklings compared to the domestic Pekin DHBV (DHBVp) isolate. The naturally infected mallard and Pekin ducks had only minor histological lesions of the liver compared with experimentally infected birds. There was no correlation between the intensity of viraemia and the severity of liver lesions, suggesting that as for mammalian hepadnaviruses the hepatic injury in DHBV-infected ducks is probably immunologically mediated.
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Production of polyclonal antibodies against the S and preS2 regions of woodchuck hepatitis virus: lack of detectable low glycosylated preS2 protein (GP33) in sera from infected animals
Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.
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Formation in vitro of the pTP-dCMP initiation complex of human adenovirus type 12
More LessWe report the covalent addition of [32P]dCMP to a protein from group A adenovirus 12 (Ad12)-infected human (KB) cells in vitro, using crude extracts. Synthesis of the 60K protein-dCMP complex required a DNA template containing a terminally located adenovirus replication origin; the protein-dCMP bond was alkali-labile but acid-stable. We therefore conclude that this product is the Ad12 terminal protein precursor (pTP)-dCMP initiation complex for DNA replication. Synthesis of Ad12 pTP-dCMP was specific for dCTP but was stimulated by dATP. In contrast to Ad2, the Ad12 initiation reaction required ATP. Antipeptide antiserum targeted to Ad DNA polymerase inhibited Ad12 pTP-dCMP synthesis in vitro, providing evidence that Ad DNA polymerase catalyses dCMP addition to pTP during initiation.
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Stabilization of human rhinovirus serotype 2 against pH-induced conformational change by antiviral compounds
More LessFour WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.
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Comparison of the nucleotide sequence of the SH gene and flanking regions of mumps vaccine virus (Urabe strain) grown on different substrates and isolated from vaccinees
More LessThe small hydrophobic (SH) protein gene and flanking regions of the Urabe Am9 vaccine strain of mumps virus were amplified by the polymerase chain reaction and sequenced directly by the dideoxynucleotide chain termination method. The 434 bp sequence was identical for the Urabe strain isolated from vaccines produced by three manufacturers and for virus isolated following post-vaccination parotitis. No changes were detected for coding, non-coding or intergenic regions between virus grown on different substrates. The Urabe virus SH coding region differed from the published sequence for strain SBL-1 by 14.4% at the nucleotide level and 24.6% at the amino acid level. The 5′ non-coding SH region was strongly conserved between the two strains (2% different), whereas the other non-coding regions were not.
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Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion
More LessVaccinia-measles recombinant viruses were used to examine the contribution of the individual measles virus glycoproteins in fusion. Although vaccinia virus recombinants expressing either the haemagglutinin or fusion proteins did not induce fusion in the cell lines examined, a double recombinant expressing both measles virus glycoproteins gave extensive syncytia in cells of human and simian origin. No fusion was observed in mouse, hamster or chicken cells. The fusion induced by the double recombinant could be specifically inhibited with either anti-fusion or anti-haemagglutinin monoclonal antibodies.
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The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus
More LessThe sequence of the H gene and flanking sequences in the F and L genes of canine distemper virus (CDV) have been determined. The H gene of CDV (1946 nucleotides) contains one large open reading frame starting at position 21 and terminating at position 1835, encoding a protein of 604 amino acid residues. This protein contains three potential glycosylation sites in the extracellular domain and, like all other paramyxoviruses, a N-terminal membrane-spanning hydrophobic anchor domain. The deduced H protein sequence shows an identity of 36% with rinderpest virus (RPV) and measles virus (MV). The identities at the nucleotide level are higher (RPV 52% and MV 53%). The amino acid sequence shows conservation of all the structural determinants with the H proteins of MV and RPV. The data also show that CDV is evolutionarily equidistant to RPV and MV with respect to the H gene.
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Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2
S. Oldfield, T. Hirasawa and P. RoyThe complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of bluetongue virus (BTV) United States serotype 13 was determined. The comparison of the predicted amino acid sequence of the encoded protein (VP5) with the sequences of VP5 from two BTV United States serotypes, 10 and 2, and two isolates of BTV serotype 1 (Australian and South African), revealed that the protein is highly conserved among the different serotypes despite their geographical separation.
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Antibodies to type D retrovirus in talapoin monkeys
More LessSera from 154 African non-human primates were screened for the presence of antibodies to type D retrovirus proteins. Four of five talapoin monkeys (Miopithecus sp.) captured in western Africa were positive for antibodies to type D retrovirus by ELISA and by immunoblot reactivity. Talapoins are the only African non-human primates that have so far shown evidence for type D retrovirus infection. Thus, talapoin monkeys appear to be a reservoir of type D retrovirus infection.
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Chemoprophylaxis of scrapie in mice
More LessThree applications of the polyanion pentosanpolysulphate about 2 months before infection of mice with scrapie completely protected animals infected with up to 100 LD50, and considerably prolonged the lifespan of those infected with 100 to 10000 LD50. The clinical diagnosis was confirmed by immunoblot analysis for the protein of scrapie-associated fibrils.
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Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia
More LessIsometric virus-like particles, 18 nm in diameter, have been isolated from banana (Musa spp.) affected by bunchy top disease in Australia. Banana bunchy top disease-associated virus-like particles (BBTV) banded as a single component with buoyant density of 1.28 to 1.29 g/ml in Cs2SO4 and sedimented at about 46S in isokinetic sucrose density gradients. The A 260/A 280 of purified preparations was about 1.33. A single coat protein of M r 20500 identified with antibodies to BBTV particles from Australia. Single-stranded DNA of about 1 kb as well as ssRNA smaller than 0.45 kb was also associated with the particles. A polyclonal antiserum to BBTV, suitable for use in ELISA, was prepared. Stability and antigenicity of purified BBTV was impaired by storage at pH ≥ 8.5 and freezing at -20 °C without protectants. BBTV was detected by double antibody sandwich-ELISA with monoclonal and polyclonal antibodies, in field-infected banana plants, single aphids from an infective colony, and in experimentally aphid-inoculated banana plants. After transmission of BBTV particles by aphids from a banana bunchy top disease-affected to an uninfected banana plant, the disease was induced and BBTV was detected by ELISA in symptomatic leaves only. BBTV isolates from Australia, Taiwan, People’s Republic of China, Tonga, Western Samoa and Hawaii were found to be serologically related, which suggests a common aetiology for the disease.
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Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA
More LessVirus-like particles were purified from banana plants with banana bunchy top disease. These particles were isometric with a diameter of 18 to 20 nm and a density of 1.28 to 1.30 g/ml in caesium sulphate. Associated with these particles were an ssDNA of about 1 kb and one major protein of M r 20100. DsDNA was synthesized from nucleic acid extracts from these particles and cloned. One clone, pBT338, hybridized specifically (i) with sap extracts from plants infected with banana bunchy top virus (BBTV) but not with sap extracts from healthy plants and (ii) with the small ssDNA in nucleic acid extracts from infected plants and virus-like particles. Banana bunchy top disease was transmitted from infected to healthy bananas by aphid inoculation and it was demonstrated that the small ssDNA was transmitted with the disease. It is probable that these particles represent the virions of BBTV containing small ssDNA and that the virus resembles subterranean clover stunt virus more than any other known virus.
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Resistance in Solanum brevidens to both potato virus Y and potato virus X may be associated with slow cell-to-cell spread
More LessA series of experiments was carried out to investigate the nature of the resistance of the wild potato species, Solanum brevidens, to potato virus X (PVX) and potato virus Y (PVY). In vitro inoculation of leaf protoplasts of S. brevidens and the virus-susceptible dihaploid S. tuberosum genotype PDH40 with PVX or PVY using polyethylene glycol showed that protoplasts of both species were similar in susceptibility. However, examination of protoplasts prepared from the leaves of S. tuberosum and S. brevidens inoculated 2 to 5 weeks earlier showed that the percentage of PVX- and PVY-infected leaf cells of S. tuberosum were, respectively, 45- to 100-fold and about 100-fold greater than the percentage of infected leaf cells of S. brevidens. These results suggest that resistance in S. brevidens to both PVX and PVY could be associated with slow cell-to-cell spread rather than with slow virus replication.
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Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus
The sequence of the 3′-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3′ terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3′ non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3′ non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3′ non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.
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Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter
More LessFull-length biologically active cDNAs of brome mosaic virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5′ ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower mosaic virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5′ end were infectious but RNAs transcribed from cDNAs with similar sequences were not.
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In vitro processing of the RNA-2-encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus
More LessIn vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, M r values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.
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Alfalfa mosaic virus RNA3 mutants do not replicate in transgenic plants expressing RNA3-specific genes
More LessThe RNA3 of alfalfa mosaic virus (AIMV) encodes the P3 protein and the virual coat protein (CP). RNA3 molecules transcribed in vitro replicated in protoplasts and plants when inoculated in mixtures with AIMV RNA1, RNA2 and CP. Transcripts with a deletion or inversion in the P3 gene replicated well in protoplasts but not in transgenic plants transformed with the P3 gene. Transgenic plants expressing the CP gene became infected after inoculation with a mixture of RNA1, RNA2 and wild-type RNA3 transcripts without addition of CP to the inoculum. Transcripts with a deletion in the CP gene replicated at a reduced level in protoplasts but not in CP-transformed plants. This suggests that P3 and CP are both required for cell-to-cell spread of AIMV and that mutations in the inoculum RNA could not be complemented in trans by the wild-type chimeric nuclear genes.
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Shortened forms of beet necrotic yellow vein virus RNA-3 and -4: internal deletions and a subgenomic RNA
More LessBeet necrotic yellow vein virus RNA-3 and RNA-4, produced as full-length biologically active transcripts in vitro, can undergo spontaneous internal deletions when inoculated onto Chenopodium quinoa leaves along with RNA-1 and -2. The deletion process is specific, giving rise to only a few major species, and can be rapid; deleted forms appear after only one or two passages in leaves. In one of the shortened forms of RNA-4, the deletion precisely eliminated one copy of a 15 nucleotide (nt) direct sequence repeat from the full-length prototype sequence, suggesting that ‘copy-choice’ switching of the replicase-template complex from one repeat to the other during RNA replication was responsible for the generation of this deletion. The deletion found in a major shortened form of RNA-3, on the other hand, did not occur near sequence repeats but began with GU and ended with AG like a nuclear intron sequence. Thus it is possible that the deleted sequence has been removed by splicing. However, two other deletions that were characterized were not associated with either of these types of sequence feature. An approximately 600 nt 5′-terminally truncated non-encapsidated form of RNA-3 was also detected in infected plant tissue. The evidence suggests that it is a subgenomic RNA derived from RNA-3.
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Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato spotted wilt virus
E. Maiss, L. Ivanova, E. Breyel and G. AdamLibraries of cloned cDNA were prepared from complete genomic RNA and isolated S RNA of the Bulgarian L3 isolate of tomato spotted wilt virus (TSWV-L3). Northern blotting of TSWV genomic RNA detected clones specific for the L, M and S RNAs in the library from complete RNA. S RNA-specific clones selected from both libraries covered approximately 2.8 kb (about 95%) of the S RNA. Sequencing of these clones showed TSWV-L3 S RNA to be ambisense. It contains two open reading frames (ORFs); one of 1401 nucleotides located on the viral RNA encodes an M r 52400 (52K) protein, and the other of 774 nucleotides on the complementary strand encodes an M r 28900 (29K) protein. Expression of the 29K ORF in bacteria and immunological analysis of the fusion protein synthesized confirmed that the 29K protein is the N protein of TSWV-L3. Comparison with the published sequence for the S RNA of a Brazilian TSWV isolate, CNPH1, revealed almost complete identity in the amino acid sequences for the 29K protein, but several clustered amino acid exchanges in the putative 52K protein. In addition, the separating non-translated intergenic region of the S RNA of the Bulgarian isolate is 81 nucleotides longer than that of CNPH1.
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Ambisense segment 3 of rice stripe virus: the first instance of a virus containing two ambisense segments
More LessThe complete nucleotide sequence of rice stripe virus (RSV) segment 3 shows that it has two open reading frames, one in the viral-complementary sequence, which codes for the nucleocapsid protein, and the other in the viral-sense sequence. The non-coding region between the ambisense genes in RSV segment 3 contains several U and A tracts, as do the ambisense S segments of phleboviruses and uukuviruses. As we have previously shown that RSV segment 4 has an ambisense nature, this is the first instance of a virus containing two ambisense segments.
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Nucleotide sequence of raspberry bushy dwarf virus RNA-3
More LessA nucleotide sequence is reported for RNA-3, the smallest of the three major RNA species found in particles of raspberry bushy dwarf virus (RBDV). The sequence of 946 nucleotides contains a single large open reading frame which encodes an M r 30509 polypeptide. In vitro translation of RNA-3 yielded an M r 30000 product that reacted specifically with antiserum to RBDV particles and we conclude that the amino acid sequence deduced from the sequence of RNA-3 is that of the RBDV coat protein, or an immediate precursor of it. No affinities were detected by comparing the nucleotide sequence of RNA-3 with the sequences of other plant viruses.
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