- Volume 68, Issue 3, 1987
Volume 68, Issue 3, 1987
- Review Article
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- Animal
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Synthesis in Escherichia Coli and Immunological Characterization of a Polypeptide Containing the Cleavage Sites Associated with Trypsin Enhancement of Rotavirus SA11 Infectivity
More LessSummaryAbout 45% of the rotavirus SA11 VP3 gene was inserted into a thermoinducible expression plasmid under the control of phage lambda PL promoter. The primary translation product predicted on the basis of the plasmid construction was a hybrid protein in which the 98 amino-terminal amino acids of phage MS2 polymerase were followed by amino acids 42 to 387 of the VP3 protein, which included the region containing the cleavage sites associated with trypsin enhancement of infectivity. On induction, a polypeptide that had the expected mol. wt. and contained VP3-related amino acid sequences as judged by immunological criteria, was synthesized to a level representing about 15% of the total bacterial protein. When a bacterial lysate enriched for the fusion polypeptide was injected into mice, it induced antibodies which inhibited haemagglutination and neutralized SA11 rotavirus infectivity.
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Serum Antibody Responses to Individual Viral Polypeptides in Human Rotavirus Infections
SummaryA radioimmunoprecipitation assay (RIPA) was used to study the serum antibody responses to individual polypeptides that developed after infection with viruses from human rotavirus subgroups I and II. Paired sera from eight children (1 to 8·5 years of age) were used in the study. Although all of the eight acute sera were negative by the complement fixation test, four of them were positive by RIPA, indicating a previous infection by rotavirus. A significant difference in the number of polypeptides immunoprecipitated was seen among the convalescent sera. The number of polypeptides immunoprecipitated was found to be related to previous infection experience. At most, ten different polypeptides were immunoprecipitated: seven structural polypeptides VP1 to VP7 and three non-structural polypeptides, NS1, NS2 and NS3. No sera immunoprecipitated VP8 or VP9. Acute sera positive by RIPA immunoprecipitated up to five polypeptides, VP1, VP2, VP3, VP4 and VP6. One of the non-structural proteins (NS2) was found to be particularly immunogenic, since antibodies to this polypeptide were detected in several convalescent sera. Among the structural proteins VP2 and VP6 were found to be the two immunodominant polypeptides which were recognized by all convalescent sera. Only three convalescent sera immunoprecipitated VP7, the major type-specific antigen responsible for inducing neutralizing antibodies. Three of four originally seronegative children with no reactivity in the convalescent sera to VP7 developed neutralizing antibodies to a single serotype. One child developed antibodies to two serotypes.
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Isolation and Characterization of Two Group A Rotaviruses with Unusual Genome Profiles
More LessSUMMARYAnalysis of the genomic dsRNA of rotaviruses isolated from calves with subclinical infections has revealed eight calves excreting group A viruses with unusual genome profiles. Two of these virus strains, C7–176 and C7–183, were grown and cloned by plaque purification in cell culture. Examination of their genome profiles after cloning showed that the unusual pattern had been retained. They were without RNA segment 11 but had an extra band (6a) migrating between segments 6 and 7. However, they contained the triplet of segments 7, 8 and 9, of similar size, which is characteristic of group A rotaviruses. The number and mol. wt. of the intracellular polypeptides induced by these viruses were similar to those of the bovine group A rotavirus UK strain. Analysis of the RNA transcripts produced by the transcription in vitro of purified C7–183 virus showed that segment 6a produced a large RNA transcript of corresponding size. After isolation, this transcript was translated in a rabbit reticulocyte lysate preparation and yielded a single polypeptide, vpll, equivalent to the product of segment 11 of rotaviruses with typical genome profiles.
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Genetic Relatedness of Corriparta Serogroup Viruses
More LessSUMMARYEight viruses of the Corriparta serogroup (Reoviridae: Orbivirus) that were known to be heterogeneous on the basis of serology and polyacrylamide gel electrophoresis were examined by reciprocal RNA-RNA blot hybridization of genomic RNA. Conserved and variant genes were identified by the degree of hybridization between cognate genes of different isolates. The eight viruses were divided into three subsets on the basis of the number of shared genes. Four of the viruses, isolated in Australia, formed one subset of related isolates and shared five conserved genes. Another isolate, Acado, was variant in all 10 genes and was considered to be a second subset. The remaining three isolates formed a third subset and shared four conserved genes. Genes 1, 3 and 10 were the most variable among the Corriparta serogroup isolates. Subsets of isolates within a serogroup which are highly related in the majority of the 10 genes and less related to serogroup viruses in another subset have not been reported previously. The phylogenetic relationship of Corriparta serogroup members suggested by the blot hybridization data is not apparent in the current taxonomic classification of these viruses which is based primarily upon serological data. The hybridization data on the Corriparta serogroup viruses are discussed and contrasted with other Orbivirus serogroups which have been examined similarly.
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Characterization and Physical Mapping of Molluscum Contagiosum Virus DNA and Location of a Sequence Capable of Encoding a Conserved Domain of Epidermal Growth Factor
More LessSummaryDNA from Molluscum contagiosum virus (MCV) isolates was analysed by restriction endonuclease cleavage, revealing two virus subtypes. Physical maps of cleavage sites for BamHI, ClaI and HindIII were constructed, and found to differ extensively between the two subtypes. MCV DNA was similar to Orthopoxvirus DNA with respect to size, terminal cross-linking and the presence of inverted terminal repetitions, but did not hybridize with vaccinia virus DNA. The genomes of the two MCV subtypes cross-hybridized and were colinear except for two small regions. There was sequence homology between DNA from corresponding map regions of the MCV subtypes but, in contrast to Orthopoxvirus DNA, no conservation of restriction sites. A synthetic oligonucleotide probe representing a conserved domain of epidermal growth factor, μ-transforming growth factor and the vaccinia growth factor identified equivalent regions of both MCV genomes as having the potential to encode this domain. This locus is similar to the position of the vaccinia growth factor gene in vaccinia virus DNA. Thus MCV may induce epidermal cell proliferation and tumourigenesis by expression of an epidermal growth factor-like polypeptide.
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Characterization of a Molecular Clone of RFM/Un Mouse Chromosomal DNA that Contains a Full-length Endogenous Murine Leukaemia Virus-related Proviral Genome
More LessSUMMARYA 12·4 kbp HindIII chromosomal DNA fragment harbouring an apparently intact 9·2 kbp endogenous murine leukaemia virus (MuLV)-related proviral genome was isolated from an RFM/Un strain mouse by molecular cloning and designated pRFM # 6. Nucleotide sequence analysis revealed the following characteristic features in the pRFM # 6 provirus: a distinct 200 bp sequence in the long terminal repeat (LTR) mid-U3 region, a primer binding site for glutamine tRNA, a 3′ pol region encoding an ‘endonuclease’ protein of 390 amino acids, and the mink cell focusforming virus type-specific sequence at the 5′ portion of the env gene. The 699 bp 5′ LTR and 700 bp 3′ LTR of pRFM # 6 provirus were identical except for three base changes in the U3 ‘enhancer’ region. At the cell-provirus DNA junction, 4 bp direct repeats were present. The proviral genome was found at the same chromosomal DNA site in BALB/c, AKR, C3H, CBA and RFM strain mice, but not in NFS/N or C57BL/6 strain mice.
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Incubation Periods and Survival Times for Mice Injected Stereotaxically with Three Scrapie Strains in Different Brain Regions
More LessSUMMARYIncubation period and survival time were determined in C57BL mice which had been injected stereotaxically with either the 139A, ME7 or 22L strain of scrapie in one of five different brain regions (cerebral cortex, caudate nucleus, thalamus, substantia nigra, cerebellum). The injection of 139A in the caudate nucleus, thalamus, substantia nigra or cerebellum resulted in significantly shorter incubation periods than following cerebral cortex injection. For ME7, mice injected in the thalamus and cerebellum had incubation periods approximately 2 weeks shorter than the cerebral cortex group. For 22L, the incubation periods after substantia nigra or cerebellum injection were significantly shorter than after cortex injection. The cerebellum injection group had a significantly shorter incubation period than the substantia nigra injection group. The survival times for mice injected with a particular scrapie strain were directly related to the incubation periods. The groups with the shortest incubation also had the shortest survival time (e.g. 22L in the cerebellum). On histological examination, 139A and ME7 produced brain lesions in all brain areas regardless of injection site. For the 22L strain, after cerebellum injection vacuolation was limited to the cerebellum, while injection into the cerebral cortex and other forebrain regions resulted in vacuolation in all brain regions examined. Despite the difference in the distribution of vacuolation seen in cerebellum- compared to cortex-injected (22L) mice, infectivity titres were similar in the cortex, cerebellum, cerebellar cortex and medulla plus pons.
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Induction of Demyelination by a Temperature-sensitive Mutant of the Coronavirus MHV-A59 is Associated with Restriction of Viral Replication in the Brain
More LessSUMMARYThe neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). The pathology of the demyelinating disease caused by one mutant, designated ts-342, was studied in detail. Pathological changes, starting 3 days post-inoculation (p.i.), were characterized by inflammation and demyelination in the CNS. Antibody responses directed against all virus-specific structural proteins were present at 7 days p.i. No virus particles were observed by electron microscopy at 14 days p.i. However, macrophages and lymphocytes were abundant in the areas of demyelination. The growth kinetics in vivo of wild-type virus, ts-342 and a revertant of ts-342 were compared. Wild-type virus and the revertant replicated rapidly in the brain and spread to the liver causing a lethal hepatitis. Ts-342, however, replicated to a much lesser extent within the brain and could not be detected in the blood or liver. The ts lesion in the genome of ts-342 seems, therefore, to determine the outcome of the infection.
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Herpes Simplex Virus Glycoproteins Associated with Different Morphological Entities Projecting from the Virion Envelope
More LessSummary: Spikes of different kinds, distinct in size and appearance were detected on the surfaces of herpes simplex virions by electron microscopy of negatively stained preparations. Use of monoclonal antibodies coupled to colloidal gold permitted identification of viral glycoproteins present in different structures projecting from the virion envelope. Antibodies specific for the glycoprotein designated gB bound to the most prominent spikes, which were about 14 nm long and, in side view, had a flattened T-shaped top. Antibodies specific for gC bound to structures that, in some instances, appeared to extend as much as 24 nm from the surface of the envelope and were too thin to resolve. Antibodies specific for gD bound to structures that extended as much as 8 to 10 nm from the surface of the envelope. The gB spikes were invariably clustered, usually in protrusions of the envelope varying from small bulbous distentions to long tail-like projections. The gC components were randomly distributed and widely spaced and the gD components were irregularly clustered in patterns distinct from those of the gB spikes. These three glycoproteins therefore form structures that are different in size, morphology and distribution in the envelope.
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DNA-binding Proteins of Herpes Simplex Virus Type 1-infected BHK Cell Nuclear Matrices
More LessSummaryThe nuclear matrix is involved in the replicative cycle of herpes simplex virus type 1 (HSV-1) and in at least some cases viral DNA has been shown to be closely associated with this structure. In this communication, we report the presence of five DNA- binding proteins in the nuclear matrix of HSV-1-infected BHK cells. These proteins (p114, p89, p77, p37 and p29) were detected by probing with 32P-labelled HSV DNA after Western blotting of nuclear matrix proteins. Three were identified as virion components: p89 as VP12, p77 as VP13 and p37 as the capsid protein VP22a. These polypeptides were detected in cells and nuclei and found to be associated with the nuclear matrix late during the lytic cycle, long after the onset of viral DNA replication.
The nuclear matrix-binding capacity of VP22a depended on viral DNA replication, since after DNA polymerase inhibition it was still synthesized and transported into the nucleus but was no longer associated with the nuclear matrix. After inhibition of viral DNA synthesis, VP13 was no longer found in cells, nuclei or nuclear matrices. These results suggest a possible involvement in anchoring viral progeny DNA to the nuclear matrix.
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Herpes Simplex Virus-induced Changes of the Keratin Type Intermediate Filament in Rat Epithelial Cells
More LessSUMMARYHerpes simplex virus type 1 (HSV-1) infection of human fibroblast cells grown in culture induces reorganization of the cytoskeleton fibrillar structures. Normal transport and insertion of HSV glycoproteins into the plasma membrane of the cells depend on the integrity of the microtubules. The natural host cells for HSV are epithelial cells, and an epithelial cell line established from rat palate was used in the present study. The effect of virus on the structure of the intermediate filaments and especially on the keratin proteins was studied. Two-dimensional gel electrophoresis of total cell extracts identified in uninfected cells two major acidic keratin proteins with apparent molecular weights of 44000 (44K) and 48K (pI 5·45 to 5·30, 5·50 to 5·35). A new keratin protein of 46K (pI 5·40 to 5·25) appeared in infected cells between 8 h and 12 h post-infection. Pulse-chase experiments identified the 46K protein as a processed form of the 48K keratin component, which was also cleaved in uninfected cells grown in the presence of cycloheximide. Partial proteolysis of the 46K and 48K keratins with Staphylococcus aureus V8 protease showed that the 48K and the 46K proteins differed in only one oligopeptide. The significance of the changed keratin composition of HSV- infected cells is discussed.
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Genomic Comparison of Herpes Simplex Virus Type 1 Isolates from Japan, Sweden and Kenya
SUMMARYOne-hundred and seventy-two epidemiologically unrelated herpes simplex virus type 1 (HSV-1) strains isolated in Japan (104 strains) and Sweden (68 strains) were compared by analysis of their genome structures using four restriction endonucleases, BamHI, KpnI, SalI and HindIII In addition, 32 Kenyan HSV-1 isolates previously compared to Japanese isolates were included for further comparison with the Swedish isolates. Remarkable and statistically significant differences were found between the HSV-1 isolates from the three countries. One-hundred and thirty cleavage sites were examined, and it was shown that isolates from two of the three countries were statistically distinguishable at 27 of these loci. Pairwise comparison between isolates from Japan and Sweden, Kenya and Sweden, and Japan and Kenya revealed variation in 18, 16 and 23 sites, respectively. By considering gains and losses of 19 sites, the total of 204 strains could be classified into 92 distinct cleavage patterns. Isolates from the three countries could be distinguished from each other by the pattern, except for one Swedish and two Kenyan isolates which shared a pattern. Twenty-one fragments that were present or absent only in individual isolates from one or other of the three countries could be detected. These results show that HSV-1 strains from geographically separate countries or anthropologically different races have distinct distributions of endonuclease recognition sites.
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A New Human Cytomegalovirus Isolate Has an Invertible Subsegment within Its L Component Producing Eight Genome Isomers
More LessSUMMARYA HindIII cleavage map of the genome DNA of a new isolate of human cytomegalovirus (HCMV), strain Tanaka, was constructed by cosmid cloning and Southern blot hybridization of virion DNA. The genome was found to be unique in that its long (L) component was composed of two subsegments, L1 and L2, and subsegment L2 underwent inversion relative to L1 at high frequency. In addition to the normal inversions of the L and short (S) components, this produced eight genome isomers. The novel invertible subsegment was flanked by an inverted sequence distinct from the inversion-specific a sequence present in the terminal and junction regions of the genome.
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Cytomegalovirus Strain AD169 Binds β 2 Microglobulin in Vitro after Release from Cells
More LessSummaryWe previously reported that a host protein, β 2 microglobulin (β 2 m) inhibited the detection of human cytomegalovirus (CMV) in urine specimens by enzyme immunoassay and postulated that β 2 m bound to the virus particle and masked the viral antigenic determinants. We report here that CMV strain AD169 grown in cell culture bound human β 2 m when this protein was added to cell culture fluids or when the virus was added to urine. Such binding was not seen with herpes simplex virus. CMV could also bind bovine β 2 m from foet al calf serum in cell culture fluids. The use of radiolabelled β 2 m in other experiments showed that CMV bound β 2 m after release from cells and that the bound β 2 m did not represent acquisition of class I HLA molecules during budding from host cell membranes. Immunoprecipitation studies showed that β 2 m was bound by two viral envelope proteins β 2 m BP1 (β 2 m-binding protein 1) and β 2 m BP2 of molecular masses 36000 and 65000 daltons respectively. β 2 m could not bind to separated viral proteins under reducing or non-reducing conditions. We propose that interaction of these two proteins on the viral surface is required to enable CMV to bind β 2 m.
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Cytomegalovirus in Urine Specimens Has Host β 2 Microglobulin Bound to the Viral Envelope: A Mechanism of Evading the Host Immune Response?
More LessSummaryWe have previously reported that human cytomegalovirus (CMV) from urine specimens cannot be captured onto a solid phase by CMV-specific monoclonal antibodies that can capture CMV grown in vitro. We report here that CMV exists in vivo in body fluids such as urine as β 2 microglobulin (β 2m)-coated particles. We have demonstrated the presence of β 2m on CMV purified directly from urine by Western blotting and have shown that the β 2m was associated with the viral envelope. Urinary CMV could be specifically bound by an affinity column comprising a monoclonal antibody specific for β 2m bound to Sepharose. The β 2m-coated urinary CMV could not be neutralized by hyperimmune globulin, human immune sera or murine monoclonal antibodies that could neutralize CMV grown in cell culture. We conclude that the binding of β 2m by CMV masks the important antigenic sites necessary for neutralization which are recognized by man’s immune response. We propose that CMV has evolved this mechanism of coating itself in a host protein as a mechanism of evading the host immune response and facilitating transmission between individuals.
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β2 Microglobulin Enhances the Infectivity of Cytomegalovirus and When Bound to the Virus Enables Class I HLA Molecules To Be Used as a Virus Receptor
More LessSUMMARYSUMMARY We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein β 2 microglobulin (β 2m) from body fluids or from cell culture media. In this report we have examined the effect of the β 2m on viral infectivity. We have shown that the addition of human purified β 2m, or a fraction of foetal calf serum corresponding to bovine β 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium. Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids. We concluded that the binding of β 2m by CMV increased its infectivity. We have shown that CMV and β 2m compete for binding sites on fibroblasts. As the main binding site on cells for /Cm is the class IHLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules. The binding of radiolabelled βm-coated CMV was significantly higher to Raji cells than to Daudi cells. Furthermore, CMV could compete with β 2m for binding to Raji cells, although the reverse was not true. These results demonstrate that CMV can use class I HLA molecules as a virus receptor. We propose that when coated with β 2Cm, CMV has the capacity to displace β 2m from the class I HLA heavy chain-βm dimer on the cell surface and bind to cells. The fact that β 2m enhances infectivity suggests that such binding leads to productive infection of cells.
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Human Epithelial Cell Expression of an Epstein-barr Virus Receptor
SUMMARYCultured human epithelium bound and internalized radiolabelled Epstein-Barr virus (EBV) within 1 h of exposure. A similar percentage of cultured cells also were reactive with monoclonal antibodies to the EBV/C3d receptor of B lymphocytes. In cross-sections of fresh frozen, stratified epithelium, receptor expression seemed limited to the less differentiated subpopulation of cells. These findings support the notion of direct infection of epithelial cells by EBV and suggest a viral life cycle in epithelium dependent on the stage of cell differentiation.
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Herpes Simplex Virus Type 1 Latency in Rabbit Corneal Cells in Vitro: Reactivation and Recombination Following Intratypic Superinfection of Long Term Cultures
More LessSUMMARYHerpes simplex virus type 1 (HSV-1) has been isolated from explanted human corneas after cultivation in vitro. To determine whether HSV-1 is persistent or latent in corneal cells, a system to study HSV-1 infection of rabbit corneal cells in vitro was developed. By elevation of the incubation temperature to 42 °C before and during HSV-1infection it was shown that both keratocytes and epithelial cells support a nonproductive rather than a productive infection. On subsequent temperature reduction to 37 °C, infectious virus was released from both cell types. Addition of the viral inhibitor acycloguanosine during the last 5 days of a 14 day incubation at 42 °C did not reduce the frequency of viral shedding following transfer to 37 °C, indicating that corneal cells support a latent as opposed to persistent infection. Cultures which failed to shed virus spontaneously up to 29 days post-inoculation were superinfected at 37 °C, with an Xba\ site deletion mutant of HSV-1. Restriction endonuclease analysis of progeny identified both the initial infecting virus and recombinants between the parental and superinfecting genomes.
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Different Roles for L3T4+ and Lyt 2+ T Cell Subsets in the Control of an Acute Herpes Simplex Virus Infection of the Skin and Nervous System
More LessSummaryRat monoclonal antibodies were used to deplete selectively Lyt 2 (cytotoxic) and L3T4 (helper) T cell populations in vivo. These antibodies produced >95 % depletion of the respective T cell subset as determined by fluorescent antibody and cytofluoro-graphic analyses. Antibody-treated mice were infected in the ear pinna with herpes simplex virus (HSV) and the induction of virus-specific T cell and antibody responses were monitored during the acute infection. Lyt 2-deficient mice produced delayed hypersensitivity and HSV-specific antibodies comparable to those in untreated animals. However, major histocompatibility complex class I-restricted T cell killing was abolished. In contrast, L3T4-deficient animals failed to produce either primary delayed hypersensitivity response or specific antibodies to the virus, but cytotoxic T cell responses were induced and even augmented in comparison with infected, normal animals. This observation clearly demonstrates that Lyt 2 cytotoxic T cells can be induced in a helper T cell-deficient environment. The ability of T cell subset-deficient mice to clear infectious virus was investigated in the skin of the ear and the part of the nervous system innervating the site of infection. L3T4-deficient animals showed a markedly delayed clearance of virus from the ear and also had a more florid infection of the nervous system. However, Lyt 2-deficient mice cleared the infection in the ear normally, but a severe infection of the nervous system was still observed. The implication of these observations to the pathogenesis of this virus is discussed.
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Role of Interferon in the Augmented Resistance of Trehalose-6,6′-dimycolate-treated Mice to Influenza Virus Infection
SUMMARYMice inoculated intravenously with 10 to 100 μg trehalose-6,6 ′-dimycolate in an oil- in-water emulsion (TDM emulsion) acquired high resistance to intranasal infection by influenza virus at 7 to 14 days, but not at 1 day, after treatment. Mice inoculated with an oil-in-water emulsion without TDM (control emulsion) did not resist infection. The activity of the reticuloendothelial system of mice inoculated with TDM emulsion or control emulsion was greatly stimulated 1 day and 14 days after treatment. Interferon production in response to influenza virus was augmented in lung and serum of TDM emulsion-treated mice. The augmented interferon production was greatly diminished in the TDM emulsion-treated mice by treatment with anti-Thy-1.2 monoclonal antibody. Production of haemagglutination-inhibiting antibody in the TDM emulsion- treated or control emulsion-treated mice was higher than that in untreated mice, although no difference was observed between the TDM emulsion-treated and control emulsion-treated mice. On the other hand, TDM emulsion treatment of mice did not influence the appearance of antibody-producing cells, nor the activity of natural killer cells in the mice. The enhanced resistance of mice was diminished by inoculating anti- interferon-α/β serum before influenza virus infection. No detectable interferon activity was observed in lung and blood of mice inoculated with anti-interferon-α/ β serum prior to influenza virus infection. These results suggest that the augmented early interferon production in T-lymphocytes of TDM emulsion-treated mice in response to influenza virus may play an important role in the enhanced resistance.
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Topological Mapping of Unique Epitopes on the Dengue-2 Virus NS1 Protein Using Monoclonal Antibodies
More LessSummaryMonoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42000 mol. wt.) from DEN-2- infected C6/36 (Aedes albopictus) cells. The virus specificity of NS 1 -reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.
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Protection of Mice Against Dengue 2 Virus Encephalitis by Immunization with the Dengue 2 Virus Non-structural Glycoprotein NS1
More LessSUMMARYImmunization of mice with the dengue 2 virus (DEN 2)-specified non-structural protein NS1 provided significant protection against intracerebral challenge with the virus in the absence of detectable neutralizing or other anti-virion antibody. NS1, purified from lysates of infected Vero cells by immunoaffinity chromatography, expressed an antigenic site(s) common to each of the four DEN serotypes, and hyperimmunization of rabbits with NS1 stimulated production of complement-fixing (CF) antibody with broad DEN serotype specificity. However, cross-protection was not observed: mice immunized with DEN 2 NS1 developed little or no heterologous CF antibody and were not protected against challenge with neurovirulent DEN 1. Induction of a protective immune response by NS1 suggests that it be considered for incorporation into possible synthetic or recombinant DNA DEN vaccines.
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Evidence for Antigenic Stability of Tick-borne Encephalitis Virus by the Analysis of Natural Isolates
More LessSUMMARYStrains of tick-borne encephalitis (TBE) virus isolated from ticks in natural foci in Austria were compared to strains isolated from the same foci 14 years previously. Comparative peptide mapping of the envelope (E) glycoproteins as well as analysis of the antigenic structure of the E proteins by the use of 14 monoclonal antibodies defining different epitopes did not provide evidence for antigenic variation. The same also holds true for isolates from a probably newly established natural focus in Western Austria. These results confirm previous data by showing that under natural ecological conditions TBE virus is quite stable and does not undergo major antigenic changes.
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Selection of Coxsackievirus B4 Variants with Monoclonal Antibodies Results in Attenuation
More LessSummaryInoculation of suckling mice with coxsackievirus B4 (CB4) results in the death of a majority of the animals. In this study we selected antigenic variants of CB4 in the presence of neutralizing monoclonal antibodies and tested them to see whether they were attenuated. Antigenic variants selected with a single antibody showed little or no attenuation by producing a high mortality (60 to 100 %). A double variant selected with two antibodies showed considerable attenuation by causing only 25% mortality. A triple variant selected with three antibodies was almost completely attenuated (killed only 5 % of the animals). Polypeptides from these variants were tested for their ability to interact with the monoclonal antibodies used for their selection. These studies showed that resistance of variant virus to neutralization in general was due to the inability of the antibody to bind to the virus. However, one of the antibodies could bind but not neutralize the virus, perhaps due to an alteration in the epitope. It is concluded that selection of CB4 variants using more than one neutralizing monoclonal antibody can lead to attenuation of the virus.
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Relationship between the Replication of Hepatitis B Virus and the Localization of Virus Nucleocapsid Antigen (HBcAg) in Hepatocytes
More LessSUMMARYAccording to the localization of hepatitis B virus (HBV) core antigen (HBcAg), detected by the avidin-biotin complex method, infected hepatocytes were classified into three types, i.e. those having nuclear (type I), nuclear and cytoplasmic (type II) or only cytoplasmic (type III) antigen. HBcAg-positive hepatocytes of all specimens (three) from non-specific reactive hepatitis and of most (five of seven) from chronic persistent hepatitis (CPH) patients were only type I; the other two CPH samples and all (seven) chronic active hepatitis samples were composed of a mixture of types I, II and III. Linear correlations between the frequency of type I, as well as that of all types (I, II and III) of the HBcAg-positive hepatocytes, and the amount of HBV DNA in serum were found. The relative HBV production of HBcAg-positive hepatocytes (serum HBV DNA amount/frequency of HBcAg-positive cells) was 0·11 in type I and 0·07 in all hepatocytes including types I, II and III. HBV core particles and complete HBV particles were found in type I hepatocytes. On the other hand, these particles were not found in a predominantly type III liver specimen. These results suggest that type I hepatocytes are more involved in the propagation of HBV than types II and III.
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Inhibition of BK Virus Haemagglutination by Gangliosides
More LessSUMMARYThe effect of gangliosides extracted from human group O Rh+ erythrocytes on haemagglutination by BK virus was investigated. Experiments were performed on both ganglioside mixtures and isolated fractions separated by column chromatography and characterized by thin-layer chromatography. These results were compared with those obtained with standard preparations of gangliosides, and the inhibiting activity was shown to be confined mainly to gangliosides with a R Flower than GM1. It was also observed that the insertion of gangliosides in liposomes increased the haemagglutination-inhibiting activity and that ganglioside coating restored the ability of glycosidase- treated human red blood cells to agglutinate.
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Structural and Functional Homology of Parvovirus and Papovavirus Polypeptides
More LessSUMMARYWe have compared the sequences of the putative polypeptides of the human pathogenic B19 parvovirus with protein sequences in the National Bethesda Research Foundation Library, and have discovered a significant homology between a B19 parvovirus non-structural (NS) protein and the T antigens of polyomaviruses and simian virus 40 (SV40) and the putative E1 proteins of papillomaviruses. The region of highest homology with the papovavirus proteins corresponds to the region that is most highly conserved in the NS1 proteins of several other parvoviruses. Studies with the T antigen of both polyomaviruses and SV40 have implicated this region as having an ATPase activity and nucleotide-binding function.
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Comparison of Porcine Parvovirus to Other Parvoviruses by Restriction Site Mapping and Hybridization Analysis of Southern Blots
More LessSummaryThe genomic relationship between porcine parvovirus (PPV) and several other autonomous parvoviruses was examined by restriction site and hybridization analysis. Restriction site maps of the PPV genome were prepared by digesting the doublestranded replicative form of the viral DNA with each of eight restriction enzymes. Subsequent comparison of such maps with those previously reported for PPV, canine parvovirus (CPV), feline panleukopenia virus (FPV), minute virus of mice (M VM), H-1 virus (H-1) and bovine parvovirus (BPV) revealed that while the maps of CPV, FPV, MVM and H-1 had a number of features in common, those of PPV and BPV were substantially different. For hybridization analysis radioactive probes prepared by nick translation of PPV, CPV and BPV genomes were tested under conditions of both low and high stringency for homologous hybridization and for heterologous hybridization with each of the other two viruses and with FPV. The results of these tests indicated homology among the genomes of PPV, CPV and FPV, but little or no homology between the genome of BPV and those of any of the other viruses tested. Additional tests with restriction fragments of PPV and a CPV probe indicated that heterologous hybridization was confined primarily to a segment of the genome between 1·85 and 2·7 kb from the 3′ end. Based on transcriptional maps previously determined for several of the rodent parvoviruses, this interval is likely to include part of the coding sequences for both non-structural and structural proteins and may be the genetic basis for the replicative as well as the antigenic similarities between PPV and both CPV and FPV.
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A Point Mutation in the Polyhedrin Gene of a Baculovirus, Autographa californica MNPV, Prevents Crystallization of Occlusion Bodies
More LessSummaryThe polyhedrin gene region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) morphology mutant M29 has been characterized by genetic and physical techniques. Recombination analysis of mutants M29 and AcM5polyl demonstrated that wild-type polyhedrin recombinants could be obtained and that the DNA restriction patterns of the recombinant viruses were identical to wild-type AcMNPV DNA. Marker rescue experiments using the wild-type AcMNPV EcoRI I fragment indicated that the morphology mutation responsible for the M29 phenotype was located in the 0·0 to 5·9% region of the genome. Direct DNA sequencing of the BamHI F fragment from M29 showed a single point mutation at position 253 of the polyhedrin gene. This mutation caused a substitution of phenylalanine for leucine at amino acid 84 of the M29 polyhedrin protein. These results indicated the necessity of amino acid conservation in the polyhedrin amino acid sequence for proper folding and assembly of the polypeptide into occlusion bodies.
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Restriction Endonuclease Analysis of Herpes Simplex Virus from Recrudescent Lesions, from Latent Infection and During Passage in the Skin and Nervous System of Mice
More LessSUMMARYThe stability of the restriction endonuclease profile of herpes simplex virus type 1 strain SC 16 in mice was studied. Virus isolated from skin during acute infection was compared with that from latently infected ganglia and with that from recrudescent lesions induced by trauma. In another experiment virus serially passaged only in skin was compared with virus that had also replicated in the nervous system. The loss or gain of specific restriction sites was not observed but in some cases the mobility of certain fragments decreased.
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Effects of the Epipodophyllotoxin VP-16-213 on Herpes Simplex Virus Type 2 Replication
More LessSummaryIt has been recently shown that VP-16–213, a semi-synthetic derivative of podophyllotoxin, inhibits the function of mammalian DNA topoisomerase II. In the present study, we examined the effects of VP-16–213 on the replication of herpes simplex virus type 2 (HSV-2). The compound did not inhibit the synthesis of early viral polypeptides at concentrations at which viral DNA synthesis was strongly suppressed, but induced double-strand breaks in newly synthesized HSV DNA. Electron microscopic examination of treated cells revealed the presence of a number of capsids with empty or partial cores. The level of topoisomerase II activity remained unaltered after infection, and all attempts to isolate VP-16–213-resistant mutants of HSV-2 have failed in spite of extensive efforts. It is suggested therefore that the mode of action of VP-16–213 may be inhibition of viral DNA synthesis by impairing the function of host cell topoisomerase II.
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The Morphology of Human Immunodeficiency Virus Particles by Negative Staining Electron Microscopy
More LessSUMMARYNegative staining electron microscopy was used to examine culture fluids from the H9/HTLV-III cell line after concentration by centrifugation. Characteristic retroviruslike particles bearing distinctive envelope projections were seen. The virion envelope was frequently extended in the form of a bleb or a tail. These particles were morphologically virtually indistinguishable from similar preparations of Friend murine leukaemia virus. H9/HTLV-III culture fluids contained, in addition, numerous comet-shaped particles with a dense head and flared tail. These particles were clumped by the addition of anti-HTLV-III-positive serum suggesting that they may represent intermediate forms of the virus.
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Growth of 293 Cells in Suspension Culture
More LessSummaryA subline of 293 cells able to grow in suspension culture has been developed by passage of 293 cells through nude mice. This new line, designated 293N3S, grows with a doubling time of approximately 30 h, continues to express adenovirus 5 early region 1 (E1) antigens, and remains permissive for adenovirus 5 host range mutants defective in E1 functions.
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Recombinant Human Interferon-γ Inhibits Adenovirus Multiplication in Vitro
More LessSummaryThe susceptibility of adenovirus to the inhibitory effect of human interferons in vitro was investigated. We tested recombinant human interferons-α 2, -β 1, and -γ against adenovirus serotypes 1 and 5 (group C), 3 and 7a (group B), and a wild strain isolated from an acutely ill child who later died. Pretreatment of WISH cells with interferon-γ for 24 h induced a dose-dependent inhibition of multiplication of all adenovirus strains tested, the TCID50 varying from 25 to 90 IU/ml. Human interferon-α 2 was unable to decrease adenovirus multiplication, while interferon-β 1, at 2000 IU/ml slightly lowered the yield of adenovirus. Similar results were obtained in HEp-2 and FS-4 cells, while A- 549 and peripheral blood mononuclear cells were insensitive to interferon-y. The difference between the effects of interferon-y and interferon-γ and β> on adenovirus multiplication in vitro suggests that its mechanism of antiviral action is different.
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A Recombinant Human Interferon-α B/D Hybrid with a Broad Host-range
More LessSummaryA recombinant interferon (IFN) hybrid has been found to have a broad host-range of activity in an antiviral assay (plaque reduction of vesicular stomatitis virus) and also high efficacy as an antiviral agent in at least 12 different animal cell species. The IFN hybrid consists of amino acids 1 to 60 from HuIFN-αB and amino acids 61 to 166 from HuIFN-αD. The profile of cross-species activity of the IFN-αB/D hybrid has been compared with that of HuIFN-αF, and of the parents HuIFN-αB and -αD. When both IFN-αB and -αD were active in a cell species, the hybrid IFN had comparable or better activity than the more active parental IFN. The hybrid shared a broad cross-species activity with IFN-αD. However, the IFN-αB/D hybrid was 10-fold more active on human cells, 30-fold more active on rabbit cells, and 50-fold more active on mouse cells than IFN-αD.
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Nucleotide Sequence of Beet Necrotic Yellow Vein Virus RNA-1
More LessSUMMARYThe complete nucleotide sequence of beet necrotic yellow vein virus RNA-1 is presented. The RNA molecule is 6746 nucleotides long excluding the poly (A) tail and has one long open reading frame encoding a polypeptide of M r 237 389. The 3′ terminal 60 residues of BNYVV RNA-1 display extensive sequence homology with the corresponding portions of BNYVV RNA-2, -3 and -4. Additional 3′ terminal homology exists between RNA-1 and -2. The sequence of the Afr 237389 RNA-l-encoded polypeptide shares domains of amino acid homology with polypeptides thought to be involved in replication of RNA from tobacco mosaic virus and several other viruses. Amino acid sequence homologies between two open reading frames of BNYVV RNA- 2 and two frames of RNA-β from barley stripe mosaic virus have also been detected.
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The Genome-linked Protein of Cowpea Mosaic Virus Is Bound to the 5′ Terminus of Virus RNA by a Phosphodiester Linkage to Serine
More LessSUMMARYThe linkage between the genome-linked protein VPg and the RNAs of cowpea mosaic virus has been analysed. The bond between VPg and the RNA chains was found to be resistant to mild acidic hydrolysis but sensitive to alkaline treatment. Hydrolysis of isolated VPg-phosphate with 5·6 m-HCl at 110 °C yielded phosphoserine as the sole phosphorylated amino acid. The data obtained show that VPg is linked to the 5′-terminal uridine of the genomic RNAs by the β-OH group of the serine located at the amino-terminal end of the protein.
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Ribonucleic Acid Polymerase Activity in Filamentous Nucleoproteins of Rice Grassy Stunt Virus
More LessSummaryFilamentous particles of rice grassy stunt virus, a member of the rice stripe virus group, were found to be associated with an RN A-dependent RNA polymerase activity. The general properties of this RNA polymerase were similar to those of that associated with particles of rice stripe virus. A minor polypeptide (mol. wt. 230000), which may be the polymerase, was detected in virus preparations.
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Physicochemical Characterization of Festuca Leaf Streak Virus
More LessSUMMARYParticles of festuca leaf streak virus (FLSV) contain three major proteins. One of these [mol. wt. 49000 (49K)] is the main constituent of the nucleocapsid, whereas the other two (mol. wt. 58K and 20K) were released from the nucleocapsid when particles were treated with non-ionic detergent. The 58K protein is glycosylated. The 58K, 49K and 20K proteins correspond to the G, N and M proteins of rhabdoviruses, respectively. Four minor proteins associated with the virus particles have mol. wt. of 189K, 117K, 101K and 41K. The 189K and 101K proteins are associated with the nucleocapsid, whereas the 117K protein was found in the soluble fraction after detergent treatment. Nucleic acid isolated from virus particles is probably RNA with an estimated mol. wt. of 4·3 × 106. The buoyant density of virus particles in sucrose was estimated to be 1·194 g/ml and the s 20 w, be 704S. The present results, together with previous information, make FLSV a definitive member of subgroup A of the plant rhabdovirus group of the family Rhabdoviridae.
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