- Volume 70, Issue 12, 1989
Volume 70, Issue 12, 1989
- Review Article
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- Bacterial
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Analysis of the Complete Nucleotide Sequence of Chp1, a Phage which Infects Avian Chlamydia psittaci
More LessSUMMARYWe report the complete nucleotide sequence of bacteriophage Chp1. The genome was found to be 4877 bases long and it potentially codes for 11 proteins. Open reading frames (ORFs) 6 and 7 lie within ORFs 2 and 1 respectively but are in a second reading frame. No significant DNA homology was found when Chp1 was compared to the EMBL database. The N-terminal amino acid sequences of the three structural proteins VP1, VP2 and VP3 were determined and it was found that they were encoded by ORFs 1, 2 and 3 respectively. Amino acid homology studies revealed that VP1 has homology with the major structural protein of bacteriophages ϕX174 and S13, and that the protein inferred from ORF4 shows homology to the A proteins of ϕX174, S13 and G4. The genome of Chp1 has an organization similar to that of ϕX174 although it is 509 bases smaller. We propose that Chp1 is a member of the Microviridae but that it is sufficiently different to warrant its own subfamily which we have called the Chlamydiavirinae.
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- Animal
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Gene UL11 of Herpes Simplex Virus Type 1 Encodes a Virion Protein which Is Myristylated
More LessSUMMARYWe have investigated whether herpes simplex virus (HSV) contains structural polypeptides which are modified by myristic acid. We demonstrate that herpes simplex virions contain a family of myristylated proteins, M r approximately 13000 to 16000. These were mapped, using HSV-1/HSV-2 intertypic recombinants, to 0·130 to 0·204 map units on the virus genome. Using anti-peptide sera, raised against the carboxy-terminus of the predicted UL11 gene product, we have established that the myristylated virion polypeptides are products of the viral gene UL11.
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The Unique N-terminal Domain of the Large Subunit of Herpes Simplex Virus Ribonucleotide Reductase Is Preferentially Sensitive to Proteolysis
More LessSUMMARYUsing antisera made against peptides corresponding to different regions of the large subunit of herpes simplex virus type 1 ribonucleotide reductase we have probed proteolytic fragments of this protein and found that at least a part of its unique N-terminal domain is not necessary for enzyme activity. This non-essential region encompasses the domain previously predicted to be composed of β sheets with a well buried core of hydrophobic residues. Truncated forms of the large subunit are generated in vivo and are located almost exclusively in the nucleus.
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Presence of Antibody to Human Herpesvirus 6 in Monkeys
SUMMARYA serological survey of monkeys was conducted to determine the prevalence of antibody to human herpesvirus 6 (HHV-6). Two-hundred and fifteen sera from 10 species of monkeys were examined by an immunofluorescent antibody (IF) assay. The antibody was found in monkeys from eight of the 10 species examined, but was not detected in silvered lutongs or cotton-top tamarins. The prevalence of antibody was highest in squirrel monkeys. Sera with high antibody titres were examined further by Western blot analysis and the neutralizing antibody test and the antibody levels were compared with that from a patient with exanthem subitum. On Western blotting, monkey and human sera that were antibody-positive to HHV-6 antigen gave similar reactions with antigen components of almost the same M r. Furthermore, sera that were antibody-positive by the IF test were also positive by the neutralizing antibody test and their titres in the two tests were comparable. These results suggest the existence of HHV-6 or an HHV-6-related virus in monkeys.
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Preinfection Prophylaxis with Herpes Simplex Virus Glycoprotein Immunogens: Factors Influencing Efficacy
More LessSUMMARYUsing a guinea-pig model of genital herpes simplex virus (HSV) infection we explored the protection afforded by preinfection immunization with HSV glycoproteins. Glycoprotein immunogens prepared by recombinant DNA technology were found to be as effective as immunogens purified from HSV-infected cell cultures. Immunized animals developed less severe primary disease and also experienced less frequent recurrent infections. Protection was influenced by both adjuvant and route of administration. These studies suggest that recombinant HSV glycoproteins may be effective immunogens for human clinical trials, but that the development of an effective vaccine will require identification of new potent adjuvants that are safe for human use.
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Two Early Vaccinia Virus Genes Encode Polypeptides Related to Protein Kinases
More LessSUMMARYVaccinia virus particles contain a protein kinase with an M r of 62K calculated from sedimentation rate. We have sequenced the SalI G restriction fragment of the vaccinia virus genome near to the right inverted terminal repeat and have identified two genes which share 36% amino acid identity with each other and are related to the family of protein kinase genes. One gene, designated B1R, encodes a 34·2K protein which shares 27% identity with a protein kinase encoded by the herpes simplex virus type 1 US3 gene and contains conserved motifs characteristic of protein kinases of serine/ threonine specificity. The second gene, B12R, encodes a protein of 33·3K which is poorly related to known protein kinases and lacks specific amino acids at several highly conserved key positions. The deduced partial amino acid sequence of a gene in the corresponding region of the cowpox virus genome is identical to B12R except for one conservative amino acid substitution. Both of the vaccinia virus genes are transcribed towards the right-hand end of the genome early during infection. It is possible that the product of either or both of these genes associates to form a homo- or heterodimer that represents the 62K virion-associated protein kinase.
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The Adenovirus Type 40 Hexon: Sequence, Predicted Structure and Relationship to Other Adenovirus Hexons
More LessSUMMARYThe gene encoding the major capsid protein (hexon) of human adenovirus type 40 (Ad40) has been isolated and sequenced. Comparison of the predicted amino acid sequence of the Ad40 hexon with the corresponding polypeptide of the human enteric adenovirus, Ad41, reveals an overall identity of 88%. The majority of the changes in sequence are located in two areas, amino acids 131 to 287 and 390 to 425. Regions in the hexon protein that vary between Ad40 and Ad41 (subgroup F) were the same regions that varied between Ad2 and Ad5 (subgroup C) suggesting that these areas of the protein represent type-specific antigenic determinants. Other areas were conserved within members of a subgroup but varied between subgroups. Fitting of the Ad40 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that the variable regions are located in the l1, l2 and l4 loops that form the surface of the virion. Of major significance is the absence in Ad40 of the highly acidic region present in both Ad2 and Ad5. In Ad2 this region stretches down into the d-strand of the β-barrel forming the P1 domain. Molecular modelling indicates that the amino acids in Ad40 which correspond to the acidic region of Ad2 can also be accommodated in the eight-stranded β-barrel, thereby maintaining the integrity of the barrel. Since the acidic region is also absent from the hexon of Ad41, the sequence of amino acids that replaces the acidic residues may be responsible for some of the distinctive biological properties of the subgroup F adenoviruses.
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Characterization of the Adenovirus Proteinase: Development and Use of a Specific Peptide Assay
More LessSUMMARYAn assay system has been developed for the adenovirus endoproteinase which utilizes the synthetic peptide MSGGAFSW, derived from the cleavage site of the adenovirus type 2 (Ad2) protein pVI. MSGGAFSW was shown to be cleaved at the G–A bond when incubated with a source of Ad2 proteinase. Digestions were readily monitored by either fast protein liquid chromatography or thin layer electrophoresis, enabling the rapid production of quantitative data. Comparison of the peptide assay with a previously described [35S]methionine assay system showed it to be faster, cleaner and less prone to extreme conditions of pH and ionic strength. The effect on adenovirus proteinase activity of a number of inhibitors was assessed using both the [35S]methionine and peptide assays. Identical inhibitor profiles were obtained and these suggested that the adenovirus enzyme is a cysteine proteinase. The 23K gene product, thought to be the proteinase, contains cysteine and histidine residues at positions 122 and 54, respectively, that could constitute part of the active site of a cysteine proteinase. These amino acids and their surrounding residues are conserved in all serotypes examined and appear to bear some resemblance to those in the putative active sites of the 3C proteinases in picornaviruses.
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Characterization of the Adenovirus Proteinase: Substrate Specificity
More LessSUMMARYPeptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX↓G or M(L)XGG↓X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.
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Adenovirus Subviral Particles and Cores Can Support Limited DNA Replication
More LessSUMMARYAdenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA–complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn 2+ ions for Mg 2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6·4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for a least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.
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Phosphorylation of Adenovirus DNA-binding Protein
More LessSUMMARYEvidence is presented here which indicates that the adenovirus DNA-binding protein (DBP) is phosphorylated at a tyrosine residue early in infection. This was suggested by the discovery that a proportion of the label in 32P-labelled DBP was resistant to alkali, and was substantiated by acid hydrolysis of DBP immunoprecipitates and by immunoblotting with a monoclonal antibody against phosphotyrosine. Treatment of [35S] methionine-labelled DBPs with chymotrypsin produced fragments of apparent M r 45K and 39K whereas digestion of 32P-labelled DBP resulted in fragments of 45K and 26K. Consideration of the distribution of 32P label and its alkali stability in these fragments suggested that chymotrypsin cleaved populations of DBP at different sites depending on their phosphorylation states. The conservation, in all of the seven adenovirus serotypes sequenced, of a tyrosine residue (at amino acid 195 in adenovirus type 2) together with its surrounding residues, suggests that phosphorylation/dephosphorylation at this tyrosine residue may be important in various functions ascribed to the DBP.
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Polymerase Chain Reaction for Human Picornaviruses
More LessSUMMARYWe have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5′ non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes.
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The Nucleotide Sequence of Coxsackievirus A9; Implications for Receptor Binding and Enterovirus Classification
More LessSUMMARYThe complete nucleotide sequence of the genome of coxsackievirus A9 (CAV-9) has been determined from cDNA cloned in Escherichia coli. Excluding the 3′ poly(A) stretch, the RNA genome is 7452 nucleotides long and encodes a single polyprotein of 2201 amino acids. Comparison of the nucleotide and predicted amino acid sequences with those of the coxsackieviruses B1, B3 and B4 reveals a surprising degree of homology, with overall amino acid homologies of 86·9%, 86·2% and 87·0%, respectively. In contrast, there is much less homology to another coxsackie A virus, CAV-21, 60.4% overall amino acid homology. This demonstrates the high degree of diversity within the CAV group and indicates that the current classification does not directly correlate with molecular genetic properties. One major feature of CAV-9 is an insertion, relative to all other enteroviruses sequenced to date, which is located at the C terminus of VP1, and includes an arginine-glycine-aspartic acid tripeptide. Such sequences in a number of other proteins are known to have activity in promoting attachment to cell receptors and the implications for CAV-9 receptor binding are discussed.
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Selection of Antigenic Variants of Foot-and-Mouth Disease Virus in the Absence of Antibodies, as Revealed by an in situ Assay
More LessSUMMARYAntigenic variants of foot-and-mouth disease virus (FMDV) of serotype C (isolate C-S8c1) were selected upon serial passage of the virus in cell culture in the absence of anti-FMDV antibodies. The variants rose from frequencies of < 10–2 in the initial plaque-purified FMDV C-S8c1 preparation, to 0·1 to 1 in three passaged populations. The proportion of antigenic variants was quantified using a new in situ plaque immunotest. A nitrocellulose filter is applied to the agar overlay of a FMDV plaque assay, and allows recovery of infectious virus from individual plaques. A second filter is placed directly on the cell monolayer and binds enough virus to permit colorimetric visualization of plaques by an enzyme-linked assay using monoclonal antibodies (MAbs). Either all or a fraction of plaques from passaged FMDV failed to react with MAb 4G3, an antibody that recognizes an epitope located within residues 144 to 150 of capsid protein VP1. Some variants rapidly dominated the viral population, and others were maintained at low levels. RNA from unreactive viruses included mutations that resulted in amino acid substitutions at the epitope recognized by MAb 4G3. We discuss models for the selection of antigenic variants of FMDV in the absence of antibodies, and implications for the antigenic diversification of RNA viruses.
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Secretory Immunoglobulin A Antibody Response Is Conserved in Aged Mice following Oral Immunization with Influenza Virus Vaccine
More LessSUMMARYParenteral immunization of BALB/c mice at 3 months of age with inactivated influenza virus vaccine elicited a haemagglutinin (HA)-specific serum IgG antibody response. The magnitude of this response declined with advancing age at the time of vaccination. By contrast, HA-specific IgA and IgG antibody levels observed in lung lavage fluids of mice immunized at 1 and 2 years of age were comparable to those of 5 month old mice when inactivated influenza virus vaccine was administered intragastrically. The secretory immune response was not fully developed in the first 3 weeks of life. However, the HA-specific IgA and IgG responses to oral vaccination in sera were reduced in 1 or 2 year old mice when compared to 5 month old mice. These data demonstrated the preservation of the virus-specific secretory IgA response in the pulmonary fluids of aged mice after oral vaccination with inactivated influenza virus vaccine. An age-dependent difference of systemic and mucosal immunity was evident in orally immunized mice.
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Antigenic Conservation of H1N1 Swine Influenza Viruses
More LessSUMMARYInfluenza viruses of the H1N1 subtype have been continually circulating in pigs in the U.S.A. for at least 50 years. To examine the level of antigenic variation in these swine viruses, a panel of 60 monoclonal antibodies (MAbs) to the haemagglutinin (HA) of recent swine isolates was prepared. Evaluation of neutralization escape mutants selected with these MAbs defined four antigenic sites on the HA, two of which overlap. Swine viruses isolated over 24 years in an enzootic area in Wisconsin were examined by ELISA and haemagglutination inhibition (HI) with these MAbs and the results indicated that the antigenic sites defined by these MAbs were highly conserved in these viruses. In comparing recent H1N1 viruses from pigs, turkeys, ducks and humans, changes in the antigenic sites were detected on the basis of HI reactivity. However, results of ELISA with these viruses clearly showed that the antigenic sites were still present on almost all H1N1 viruses of swine origin; thus, altered reactivity of these viruses in HI tests with MAbs was not a reflection of changes in the antigenic sites defined by the MAbs. It seems likely that the variation detected in these viruses occurs by a mechanism other than immune selection.
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Mechanism of Recovery from Acute Virus Infection. IX. Clearance of Lymphocytic Choriomeningitis (LCM) Virus from the Feet of Mice Undergoing LCM Virus-specific Delayed-type Hypersensitivity Reaction
More LessSUMMARYAs shown previously, after inoculation into the footpad of a mouse the lymphocytic choriomeningitis (LMC) virus multiplies locally. Beginning on day 6 or 7 after infection, the foot undergoes a delayed-type hypersensitivity (DTH) reaction which consists of two distinct phases that are mediated by CD8+ cells and CD4+ cells, respectively, and at about the same time the virus is eliminated. In general, for terminating infection of the mouse with LCM virus the CD8+ cytotoxic/suppressive T lymphocyte (CTL) is essential; we have now determined the cells that mediate control of the virus in a tissue undergoing a specific DTH reaction. Depletion, in infected mice, of all T lymphocytes by treatment with anti-Thy-1 monoclonal antibody prevented virus elimination from the foot, and the same was true when the CD8+ CTLs were removed. Depletion of the CD4+ helper/suppressor subset only marginally impaired the ability of the mice to rid themselves of the virus. The conclusion that here too the principal antiviral element is the CD8+ CTL was confirmed by experiments in which footpad-infected mice were adoptively immunized with virus-immune splenocytes from syngeneic mice selected for subclasses of T lymphocytes, or from mice differing in defined regions of the major histocompatibility complex (MHC), and also by experiments in which monocytes were virtually absent. However, CD8+ CTL alone or cells from MHC recombinant mice with identity in class I loci were never as antivirally active as unseparated splenocytes from syngeneic donor mice. Since the CD8+ cells’ performance could be optimized by interleukin-2, we assume that the CD4+ T lymphocytes function as accessory cells; the same probably applies to monocytes.
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Cytotoxic T Lymphocyte Control of Acute Lymphocytic Choriomeningitis Virus Infection: Interferon γ, but Not Tumour Necrosis Factor α, Displays Antiviral Activity in vivo
More LessSUMMARYVirus-specific cytotoxic T lymphocytes (CTL) mediate their antiviral activity either by direct lysis of infected cells, or by the release of soluble lymphokines, or by a combination of the two. We have examined the role played by interferon-gamma (IFN-γ) and tumour necrosis factor (TNFα) in virus clearance. In vitro the amount of IFN-γ synthesized by some lymphocytic choriomeningitis virus-specific H-2-restricted CTL clones was quantitatively too small to correlate with a direct antiviral activity in vivo. However, treatment of mice with a neutralizing monoclonal antibody to IFN-γ significantly inhibited the clearance of virus from the spleens of acutely infected mice given adoptive transfers of immune spleen cells. Additionally, mice treated with exogenous recombinant murine IFN-γ 24 h before or at the same time as virus inoculation showed reduced virus titres in their spleens. Hence, IFN-γ displayed a direct antiviral effect in vivo. In contrast, treatment of mice with recombinant TNFα had no effect on virus clearance and thus TNFα is unlikely to play a significant role in this acute viral infection.
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Evaluation of Human and Simian Immunodeficiency Virus Plaque and Neutralization Assays
More LessSUMMARYA number of CD4+ T cell lines were compared for their ability to act as target cells for human immunodeficiency virus (HIV) infection in syncytium- and plaque-forming assays. MT-4 and C8166 cells were the most sensitive indicator cells for HIV- and simian immunodeficiency virus (SIV)-induced cytopathic effects, and gave rise to macroscopic (MT-4) and microscopic (C8166) plaques. The MT-4 plaque assay was evaluated for the measurement of HIV- and SIV-neutralizing antibodies.
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Mutation of Host Cell Determinants which Discriminate between Lytic and Persistent Mouse Hepatitis Virus Infection Results in a Fusion-resistant Phenotype
SUMMARYThe expression of mouse hepatitis virus (MHV) E2-specific mRNA, the E2 polypeptide and its associated cell fusing activity was monitored in various cell types inoculated with a recombinant vaccinia virus, designated vMS containing the E2 gene. The results suggest that host cell permissiveness to MHV infection correlates with cellular susceptibility to membrane fusion mediated by the MHV E2 glycoprotein. In addition, we utilized a genetic approach to the analysis of host cell functions involved in determining permissiveness to MHV. By using the chemical mutagen ethyl methanesulphonate, mouse fibroblast cell mutants were generated and selected for their resistance to cell killing by MHV. When challenged with MHV, all five mutants examined gave rise to persistent infections, in contrast to wild-type L-2 cells which were rapidly killed by the virus. The results provide genetic evidence in support of a previous correlation proposed between MHV permissiveness and two host determinants, namely susceptibility to MHV infection and to MHV-mediated cell fusion. Fusion resistance was specific to fusion mediated by the MHV E2 glycoprotein as shown in contact fusion assays between uninfected cells and cells infected either with MHV or with an E2-expressing recombinant vaccinia virus. In contrast, mutant cells were not resistant to fusion after treatment with polyethylene glycol. The observed high rate of generation of these mutants suggests that the conversion of a fully MHV-susceptible cell to a semi-resistant one is a fairly common event, possibly involving a single mutation. In this case, resistance to MHV infection and to E2-mediated membrane fusion may depend on a common host function. This result provides prospects for the precise genetic and biochemical characterization of the steps involved in host cell permissiveness to MHV infection.
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The Site of Bluetongue Virus Attachment to Glycophorins from a Number of Animal Erythrocytes
More LessSUMMARYBluetongue virus (BTV) was shown to agglutinate human, ovine and porcine erythrocytes. Removal of neuraminic acid (NA) from erythrocytes by Vibrio cholerae neuraminidase prevented their agglutination. Haemagglutination was also inhibited by N-acetyl neuraminic acid (NANA), N-glycolyl neuraminic acid (NGNA) and N-acetyl neuramin-lactose. The ability of BTV to agglutinate trypsin-treated human erythrocytes, which lack the amino-terminal domain and the single N-linked oligosaccharide of glycophorin A, suggests that the virus bound to human erythrocytes via NANA-containing, O-linked oligosaccharides. Glycoproteins with NA-containing oligosaccharides of known structure such as mucin, fetuin, alpha 1-acid glycoprotein, ovomucoid and ovine, porcine, human and equine glycophorin were examined for their ability to inhibit BTV-mediated agglutination of human, ovine and porcine erythrocytes. All glycoproteins containing NANA- or NGNAα2-6GalNAc were capable of inhibiting the agglutination of human and porcine erythrocytes. Treatment of human erythrocytes with Newcastle disease virus neuraminidase and of porcine erythrocytes with Clostridium perfringens neuraminidase to cleave preferentially the NANA- and NGNAα2-3Gal linkages respectively, were shown to have little effect on the ability of the erythrocytes to be agglutinated by BTV. The results suggested that BTV binds to NANA- and NGNAα2-6GalNAc residues in the O-linked oligosaccharides of human and porcine glycophorins respectively and indicated the presence of different binding sites on the virus for erythrocytes from other species.
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Asynchronous Mixed Infection of Culicoides variipennis with Bluetongue Virus Serotypes 10 and 17
More LessSUMMARYCulicoides variipennis (Diptera: Ceratopogonidae) the primary vector of bluetongue virus (BTV) in the U.S.A. were asynchronously mixedly infected with two BTV serotypes (BTV-10 and BTV-17); flies first ingested a blood meal that contained BTV-17 and 1, 3, 5, 7, and 9 days later selected flies ingested a second blood meal that contained BTV-10. Control flies ingested each parental virus separately, or both viruses simultaneously, in a single blood meal. Electrophoretic analysis of progeny virus clones indicated that superinfection with BTV-10 occurred when the flies ingested the second virus 1, 3 and 5 days post-initial infection. Parental BTV-17 and reassortant virus clones were isolated from these flies, but parental BTV-10 virus was not isolated from any flies. Reassortant clone frequencies were 67%, 71% and 17% when superinfection occurred on days 1, 3 and 5 after initial infection, respectively, as compared to 48% for simultaneously infected flies. Only parental BTV-17 clones were isolated from flies that ingested the second virus on days 7 and 9 after initial BTV-17 infection. The results indicated that interference to superinfection occurred in C. variipennis by 5 days and flies were refractory to superinfection by 7 days post-initial infection. Analysis of segregation of the parental origin of genome segments in the reassortant clones indicated selection against most segments of BTV-10 parental origin. This occurred both in individual flies and in individual groups. The fact that C. variipennis readily fed on a second blood meal and their ability to produce new viral genotypes suggested that these vectors are highly permissive hosts for evolution of BTV by genome reassortment.
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Identification and Primary Structure of the Gene Encoding the Berne Virus Nucleocapsid Protein
SUMMARYThe nucleotide sequence of the nucleocapsid (N) protein gene of Berne virus (BEV; proposed family Toroviridae) was determined from two independent clones of a cDNA library. From the deduced amino acid sequence a basic protein of 18.3K was predicted. In vitro transcription and translation, followed by immunoprecipitation, were used to identify the gene. The identification was confirmed by metabolic labelling, using the knowledge that cysteine residues are absent from the amino acid sequence of the N protein. Smaller N-related polypeptides encountered in BEV-infected cell lysates were shown to be probable products of aberrant translation, due to initiation on AUG codons further downstream in the N protein gene.
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Organ Distribution of Proteinase-resistant Prion Protein in Humans and Mice with Creutzfeldt-Jakob Disease
More LessSUMMARYWe attempted to clarify the organ distribution of human and murine proteinase-resistant prion protein (PrPCJD)in Creutzfeldt-Jakob disease (CJD), and to measure the concentration of PrPCJD, using a semi-quantitative Western blot analysis. Human PrPCJD was restricted to the central nervous system, whereas murine PrPCJD was present in the central nervous system and in the lymphoreticular system at the end stage of CJD. PrPCJD concentration in the central nervous system of mice was almost identical to that of humans. The minimum wet weight of an organ with a positive reaction was 0·3 mg for brain, 1 to 3 mg for spleen, 3 mg for spinal cord, 3 mg for lymph node, 10 mg for thymus and 10 to 30 mg for intestine of the CJD-infected mice. There were no immunoreactions in purified PrPCJD fractions from 300 mg of spleen, lymph node, liver or peripheral nervous systems of humans, nor in 300 mg of liver, lung or kidney of CJD-infected mice. Within the limits of our method, the distribution of murine PrPCJD differed from that of human PrPCJD. Antibodies on the Western blot membrane from murine spleen PrPCJD fractions stained the kuru plaques in the CJD-infected mouse brain. Therefore, PrPCJD in the murine spleen probably shares the epitopes of the antigen in the murine kuru plaques. Although the immunological detection of PrPCJD does have limits of sensitivity, PrPCJD concentrations did correlate with infectivity titres in scrapie-infected or CJD-infected mice.
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Detection of Defective Genomes in Hepatitis A Virus Particles Present in Clinical Specimens
More LessSUMMARYHepatitis A virus (HAV) particles harbouring a physically defective RNA genome have been reported to occur in all HAV-infected cell culture systems analysed so far. The most prominent defects consist of three distinct overlapping deletions in the region of the HAV genome encoding the structural proteins. By probing for the endpoints of these deletions in RNA samples using S1 nuclease and exonuclease VII mapping, we obtained suggestive evidence for the existence also of defective genomes in HAV particles present in faecal specimens, in viraemic blood collected in the course of hepatitis A virus infection in man, as well as in the liver of an experimentally infected marmoset monkey. The deletions identified extend from nucleotide (nt) 1200 to nt 3820 and from nt 1200 to nt 3240 of the HAV genome. They are compatible with two of the deletions detected in particles grown in vitro in cell cultures and shown to interfere with the replication of standard hepatitis A virions.
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Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization
More LessSUMMARYPersistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i. and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i. a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects.
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Immunological Response of Monkeys Infected Intranasally with Human Parainfluenza Virus Type 4
SUMMARYThis report describes our attempt to establish an experimental animal model for human parainfluenza virus type 4A (HPIV-4A) and 4B (HPIV-4B) infection, which was used to study the immune response to the viruses. Monkeys were inoculated intranasally with the viruses, and at 10 weeks post-infection they were re-infected with homologous subtype viruses. Virus-specific IgM and IgG serum antibodies were measured by ELISA. A small peak of IgM antibody was detected in the monkeys re-infected with HPIV-4B, whereas this response was not detected after re-infection with HPIV-4A. Virus-specific IgA and IgE antibodies were not detected in sera following infection and re-infection with HPIV-4. However virus-specific IgA and IgE antibodies were found in the saliva and nasal exudates of monkeys infected with either HPIV-4A or -4B. Re-infection of monkeys with HPIV-4B also stimulated an IgA and IgE response. To our knowledge this is the first description of a virus-specific IgE antibody response generated by a paramyxovirus infection of an experimental animal. The kinetics of haemagglutinin-inhibition and neutralization (NT) antibodies were similar to that of virus-specific IgG antibodies. The NT titres of sera from HPIV-4A-infected monkeys were enhanced by the addition of complement, whereas complement did not affect the NT activity of sera obtained from HPIV-4B-infected animals. Antigenic specificities of IgG antibody induced by HPIV-4 infection were analysed with radioimmunoprecipitation followed by SDS–PAGE. Anti-NP, -HN and -F antibodies appeared 2 weeks after infection, and the highest titres were found 2 weeks after re-infection. Anti-F antibody production followed a biphasic pattern previously observed in mumps virus infection.
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Na+ and K+ Concentration and Regulation of Protein Synthesis in L-A9 and Aedes albopictus Cells Infected with Marituba Virus (Bunyaviridae)
More LessSUMMARYInfection of L-A9 cells with Marituba virus produces a severe inhibition of protein synthesis. This inhibition is temporally correlated with an increase in the intracellular Na+ concentration and a decrease in the intracellular K+ concentration. However in Marituba virus-infected Aedes albopictus cells the intracellular level of Na+ and K+ ions and protein synthesis remained unaltered. Incubation of both cell types at high NaCl concentration facilitated the translation of viral RNA whereas the cellular protein synthesis was inhibited. Using a hypotonic medium, the opposite was found. Results are discussed in terms of a possible involvement of these ions in the viral translational process.
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Efficient Transfection of Insect Cells with Baculovirus DNA Using Electroporation
More LessSUMMARYEfficient transfection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus (AcNPV) DNA has been carried out using the technique of electroporation. The efficiency of transfection was monitored by assaying the extracellular virus in cell culture supernatants 3 days post-electroporation. Maximum titres of 2 × 109 p.f.u./ml AcNPV were obtained when using a pulse length of 7·7 ms and a field strength of 500 V/cm. This compared with a titre of 2 × 106 p.f.u./ml AcNPV using the standard calcium phosphate transfection method. Cotransfections of wild-type AcNPV DNA and the transfer plasmid pAcRP23-lacZ were also performed using electroporation and gave β-galactosidase recombinant virus titres of 5 × 104 p.f.u./ml; this compared with 5 × 102 p.f.u./ml using the calcium phosphate method. The maximum proportion of recombinant virus, 2·9%, was obtained by harvesting the transfection medium after 2 days, and using a pulse length of 2·8 ms and a field strength of 750 V/cm. We therefore conclude that electroporation provides a very efficient method for the transfection of insect cells with baculovirus DNA.
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Preliminary Studies on the Biology of Borna Disease Virus
More LessSUMMARYBorna disease virus (BDV) is an unclassified agent that causes neurological disease in a wide range of animal species and possibly in humans. The infectious nature of BDV has been long established but, despite extensive progress on the pathogenesis of the infection, the aetiological agent is still uncharacterized. Recent studies have shown that BDV replicates productively in cultures of foetal rabbit glial cells (FRG) which produce a virus-specific protein that is easily detected immunocytochemically. This provides a marker for BDV infectivity. This cell culture system was used to investigate the replication cycle of BDV. The agent required at least 1 h to bind to and penetrate the cells and the antigen was detected 24 h later. Cycloheximide and actinomycin D inhibited production of the antigen in inoculated cells, indicating that both protein synthesis and a DNA-dependent function were required for the production of viral antigen. Cocultivation of BDV-infected FRG cells with Vero cells resulted in a persistent productive infection in the latter. Use of these cells showed that the infectious agent matured exclusively in the cytoplasm and within the plasma membrane of the cell. Antigen-laden nuclei did not have infectivity. These studies showed that BDV has the physical and replicative properties typical of conventional viruses but its mechanism of replication and site of morphogenesis may be unique.
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Evidence that Beet Necrotic Yellow Vein Virus RNA-4 Is Essential for Efficient Transmission by the Fungus Polymyxa betae
More LessSUMMARYThe efficiency of transmission by Polymyxa betae of beet necrotic yellow vein virus (BNYVV) isolates containing different RNA components was compared using sugar-beet seedlings as test plants. Isolate S-4, containing RNA-1 +2 + 4, was transmitted by P. betae about 100 times more efficiently than isolate S-3 (RNA-1 +2 + 3) and about 1000 times more efficiently than isolate S-0 (RNA-1 + 2). Isolate S-34 (RNA-1 + 2 + 3 + 4) was transmitted even more efficiently than isolate S-4. Each isolate retained its characteristic RNA composition after transmission by P. betae. The virus content, measured by ELISA, of infected rootlets was S-34 > S-3 > S-4 > S-0. In inoculated leaves of Tetragonia expansa and Beta macrocarpa, isolates S-3 and S-34 multiplied more extensively than did S-4 and S-0. The inefficient transmission of isolate S-3 by P. betae, as compared with S-4, cannot be attributed to a poorer ability to spread in root tissue, but the difference in transmissibility of S-3 and S-0 may be explained in this way. These results show that RNA-4 of BNYVV is essential for efficient transmission by P. betae, and suggest that RNA-3 may influence the ability of the virus to spread in root tissue. RNA-4 and RNA-3 therefore seem to play important, but different, roles in virus survival and spread in nature.
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Production and Pathogenicity of Isolates of Beet Necrotic Yellow Vein Virus with Different Numbers of RNA Components
T. Tamada, Y. Shirako, H. Abe, M. Saito, T. Kiguchi and T. HaradaSUMMARYTen Japanese field isolates of beet necrotic yellow vein virus (BNYVV) were transmitted to Tetragonia expansa by inoculation with sap from rootlets of sugar-beet seedlings, to which the virus had been transmitted by the fungus Polymyxa betae. RNA extracted from BNYVV particles obtained from the T. expansa leaves was analysed by agarose gel electrophoresis. Some isolates contained RNA-1 (7·1 kb), RNA-2 (4·8 kb), RNA-3 (1·85 kb) and RNA-4 (1·5 kb) and the others contained, in addition, RNA-5 (1·4 kb). Further isolates, derived from single lesions produced by these isolates, had a variety of RNA compositions. Some contained only RNA-1 and RNA-2. Others contained, in addition, RNA-3, RNA-4, RNA-5 or RNA-6 (1·0 kb), or combinations of two or three of these components. Such isolates generally maintained their RNA composition on further subculture, and their particles had length distributions corresponding to their RNA components. Isolates containing RNA-1 + 2 + 3 caused yellow or strongly chlorotic local lesions in T. expansa, Beta vulgaris, B. macrocarpa and Chenopodium quinoa, and caused systemic stunting and yellow mosaic in B. macrocarpa and, occasionally, in B. vulgaris. In contrast, isolates containing RNA-1 + 2 + 4 or 1 + 2 + 5 induced chlorotic lesions, those containing RNA-1 + 2 + 6 or 1 + 2 induced faint chlorotic lesions, and none of these isolates easily infected B. macrocarpa systemically. Isolates containing different combinations of RNA-3,-4 and -5 induced more severe symptoms than those containing a single RNA. Such synergistic effects occurred between RNA-3 and RNA-4 or RNA-5, or between RNA-4 and RNA-5 or RNA-6, but not between RNA-3 and RNA-6, or between RNA-5 and RNA-6. These small RNA species therefore contain the genetic determinant(s) for lesion type and for ability to infect B. vulgaris and B. macrocarpa systemically. RNA-1 and RNA-2 are viral genome components. The other RNA components have some characteristics of viral satellite nucleic acids but they may not all be dispensable if the BNYVV isolates are to survive in nature.
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Two Related Viroids Cause Grapevine Yellow Speckle Disease Independently
More LessSUMMARYWe have confirmed that two closely related circular RNA molecules previously named grapevine yellow speckle viroid (GYSV) and grapevine viroid 1B (GV1B) are indeed viroids. Electron microscopy after spreading under non-denaturing conditions revealed that GYSV has a rod-like structure typical of viroids. Purified GYSV and GV1B replicated independently in inoculated grapevine seedlings and some of the infected plants developed yellow speckle symptoms indicating that both viroids can cause grapevine yellow speckle disease. Plus-sense RNA transcripts derived from a dimeric GYSV cDNA clone induced yellow speckle symptoms in a grapevine seedling confirming the role of GYSV in the yellow speckle disease. Two oligonucleotide probes were synthesized for the detection of the two related viroids. The probes which could detect each viroid individually were used to assess correlations between the occurrence of these viroids and the incidence of the disease.
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Host-dependent Suppression of Temperature-sensitive Mutations in Tobacco Mosaic Virus Transport Gene
More LessSUMMARYTobacco mosaic virus (TMV) mutants Lsl and Ni2519, temperature-sensitive (ts) in transport function in tobacco plants, were able to spread at high temperature (33 °C) in Amaranthus caudatus L. plants. On the other hand, TMV ts coat protein mutants retained the ts phenotype in both host plants. The ability of Lsl and Ni2519 to spread systemically in A. caudatus at high temperature is probably due to the functional stabilization of the transport proteins by a factor(s) provided by the host plant. In accordance with this, Lsl complemented the transport function of cucumber green mottle mosaic tobamovirus and red clover mottle comovirus; they acquired the ability to spread systemically at 33 °C in the A. caudatus plants preinfected with Lsl.
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Competition between Isolates and Variants of Cauliflower Mosaic Virus in Infected Turnip Plants
More LessSUMMARYThe Cabb S isolate of cauliflower mosaic virus (CaMV) is the only isolate that can be recovered from turnip plants mixedly infected with Cabb S and an infectious variant of Cabb S. Analysis of the progeny resulting from mixed infections of turnip plants was done for a better understanding of this dominance. Cabb S DNA was the dominant DNA recovered from plants after a mixed infection with Cabb S and D/H or W isolates of CaMV, whereas UM130 (derived from Cabb S by the insertion of a short oligonucleotide in open reading frame III) coexisted in turnip plants with D/H, NY8153, CM4-184 and W isolates. To determine whether there are additional sequences responsible for the ability of Cabb S to dominate over UM130, co-inoculation experiments were done using chimeric DNAs, obtained by in vivo recombination (IC141, IC143 and VR246) and by in vitro construction (W/CS and CS/W). Chimeras IC141, IC143 and VR246 coexisted in turnip plants with UM130. Chimera CS/W dominated over UM 130, whereas UM 130 dominated over W/CS. The location of Cabb S sequences in these chimeras suggests that more than one region is responsible for the competitive advantage of Cabb S DNA. A different modification of Cabb S DNA in open reading frame III also destroyed the ability of the DNA to dominate over the W isolate. In DNA from virions obtained after co-infection with UM130 and CS/W, more molecules were recovered with U M 130-specific sequences at position 1364 than with those at position 2040. This and similar results with progeny from W/CS and UM 130 co-infection suggest that genetic exchanges had occurred. However, exchanges were not detected in progeny from other mixed infections. Thus the dominance of the Cabb S isolate in mixed infections was due primarily to a better competitive ability of the Cabb S DNA. Plants inoculated first with UM130 and 2 to 8 days later with Cabb S contained only UM130 CaMV DNA, indicating that dominance was unable to overcome cross-protection.
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Further Evidence that Viroplasms Are the Site of Cauliflower Mosaic Virus Genome Replication by Reverse Transcription during Viral Infection
More LessSUMMARYCauliflower mosaic virus (CaMV) is a DNA plant virus that replicates its genome through an RNA intermediate. The cytoplasmic step of CaMV DNA replication was studied using a fraction consisting of purified viroplasms, which are virus-specific inclusion bodies accumulating in the infected plant cells. The isolated viroplasms retain a DNA polymerase activity able to synthesize CaMV DNA from endogenous templates. A further characterization of the viral DNA sequences produced in the isolated inclusion bodies indicates that newly synthesized DNA, mostly of polarity opposite to that of viral RNA, is single-stranded and partly associated with RNA by base-pairing. In addition to an RNA-dependent DNA polymerase activity, RNA molecules, which presumably originate from the viral RNA template used for reverse transcription, are found to accumulate in the purified inclusion bodies. Furthermore, a small DNA molecule strongly labelled in the purified fraction has been characterized and corresponds to the CaMV reverse transcription intermediate sa-DNA. These results provide further evidence that the reverse transcription of CaMV RNA occurs in the viroplasms. Additional data are presented which suggest that CaMV replication could occur in virion-related particles.
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Reaction of Coat Proteins of Two Comoviruses in Different Aggregation States with Monoclonal Antibodies
More LessSUMMARYMonoclonal antibodies were produced against two comoviruses, cowpea mosaic virus and cowpea severe mosaic virus. The antibodies were tested for their recognition of coat proteins of each virus in various conformational states by enzyme-linked immunosorbent assays. Three groups of epitopes were identified on the basis of antibody binding to native or denatured virions. Antibodies specific for epitopes exposed only after virus denaturation were cross-reactive, recognizing both viruses. Antibodies binding epitopes of the other two groups recognized intact virions and varied in their degree of cross-reactivity.
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Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses
More LessSUMMARYRabbit antisera produced against two comoviruses (cowpea mosaic virus and cowpea severe mosaic virus) were used in plate-trapped ELISA and liquid phase competition ELISA. In the latter, the top component competed against bound unfractionated virus more effectively than did nucleoprotein components. One murine monoclonal antibody elicited to cowpea mosaic virus and three monoclonal antibodies to cowpea severe mosaic virus exhibited differential binding to top component, relative to either middle or bottom components in plate-trapped or antibody-trapped ELISAs. Differential binding to centrifugal components of virus by two of these monoclonal antibodies was maintained in liquid phase competition assays. These data suggest that the encapsidation of RNA alters the configuration of the virion surface in the vicinity of the antibody epitopes. Ribonuclease digestion of intact or denatured virus did not affect the binding of monoclonal antibodies.
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Morphological Characteristics of Rice Stripe Virus
More LessSUMMARYThe morphological characteristics of particles of rice stripe virus (RSV) were examined. Each of four components (M1, M2, B and nB) of RSV, isolated by repeated cycles of sucrose density gradient centrifugation, contained circular filaments; the modal lengths were 510 nm for the M1 component, 610 nm for the M2 component, 840 nm for the B component and 2110 nm for the nB component. Each component was associated with a single-stranded species and a double-stranded species of RNA although a fifth component (the T component) did not form a distinct band after the density gradient centrifugation and was associated with circular filaments of 290 nm in length and a ssRNA species.
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Molecular Cloning and Terminal Sequence Determination of the S and M RNAs of Tomato Spotted Wilt Virus
More LessSUMMARYComplementary DNA to the genomic RNA of tomato spotted wilt virus (TSWV) was synthesized and cloned in either pUC19 or λgt10. Restriction endonuclease maps were constructed from cDNA clones specific for the S and the M RNA segments, extending 3·0 and 5·4 kbp respectively. The nucleotide sequences of the 3′ and 5′ termini of both RNA molecules were determined. The S and M RNAs contain inverted complementary repeats at their termini, probably involved in RNA replication and in the formation of circular nucleocapsids in virions. The terminal structures of the TSWV genome resemble, in these aspects, those of the Bunyaviridae.
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- Corrigendum
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