- Volume 66, Issue 12, 1985
Volume 66, Issue 12, 1985
- Bacterial
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Characterization of Streptococcal Bacteriophage c6A
More LessSUMMARYBacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 ± 8 p.f.u. per infected cell after a latent period of 25 min at 30 °C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5′-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected.
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- Animal
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Agent Replication Dynamics in a Long Incubation Period Model of Mouse Scrapie
More LessSUMMARYThe dynamics of scrapie agent replication in brain and spleen following intracerebral or intraperitoneal infection were investigated in a model with particularly long incubation periods [IM mice (Sinc p7) infected with 87V scrapie]. In contrast to other mouse scrapie models previously investigated, there was no delay in onset of replication (‘zero phase’) in spleen using either route of infection, For both routes infectivity levels reached a plateau in spleen, which was maintained for at least 250 days in intraperitoneally infected mice. An infectivity plateau was also apparent in brain following intracerebral infection, suggesting that there may be constraints on agent replication in brain during the later part of the incubation period. In addition, the efficiency of the intraperitoneal route to produce disease was found to be particularly low for this model.
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Neutralization of Yellow Fever Virus Studied Using Monoclonal and Polyclonal Antibodies
More LessSUMMARYMonoclonal and polyclonal antibodies with known specificity for either the 54K envelope glycoprotein or the 48K non-structural glycoprotein of yellow fever (YF) virus-infected cells were studied in plaque reduction neutralization tests. Viruses employed in the tests comprised wild-type and vaccine strains of YF and a selection of other flaviviruses. Of 17 monoclonal antibodies examined, six of the 54K-specific antibodies neutralized at least one YF preparation. Both vaccine and wild-type YF viruses varied in their susceptibility to neutralization and there were also differences between individual 17D vaccine strains. The monoclonal antibodies produced a range of titres with the different viruses, the most potent, 864, leaving no non-neutralizable fraction. Addition of anti-globulin, complement or other antibodies did not affect the results. YF-neutralizing antibodies which bound to other flaviviruses did not necessarily neutralize them; hence, neutralization could be defined as either homotypic, heterotypic or both homotypic and heterotypic. A polyclonal antiserum and a broadly reacting monoclonal antibody produced almost identical neutralization results in tests with wild-type YF viruses. In contrast, the polyclonal antiserum produced higher titres with vaccine strains of YF. In mouse passive protection experiments on the other hand, the monoclonal antibody did not differentiate between these viruses.
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Cytotoxic Lymphocytes in the Lungs of Mice Infected with Respiratory Syncytial Virus
More LessSUMMARYMice infected with respiratory syncytial virus (RSV) developed cytotoxic lymphocytes in the lungs, which lysed RSV-infected, but not uninfected cells. Cytotoxic activity was greatest 7 to 9 days after infection, was virus-specific, MHC-restricted and abolished by treatment of lymphocytes with anti-Thy 1.2 or with anti-Lyt 2.2 sera and complement. There was a close temporal relationship between the appearance of these cytotoxic lymphocytes in the lung and clearance of virus. In contrast, RSV persisted in the lungs of athymic (nude) mice and such animals failed to develop RSV-specific cytotoxic lymphocytes. Thus, cytotoxic T-cells may have an important role in recovery from RSV infection.
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Delta-9-Tetrahydrocannabinol Decreases Host Resistance to Herpes Simplex Virus Type 2 Vaginal Infection in the B6C3F1 Mouse
More LessSUMMARYThe effect of delta-9-tetrahydrocannabinol (Delta-9-THC) on host resistance to herpes simplex virus type 2 (HSV-2) vaginal infection in the B6C3F1 mouse was determined. Animals were given Delta-9-THC or vehicle on days -1 to 2, or cyclophosphamide on day -1 or on days -1 to 2. HSV-2 was introduced intravaginally on day 0. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding and mortalities. Replicate groups of animals were bled on days 5, 8, 10, 14 and 21 post-viral inoculation to allow for screening for viraemia and for definition of the effect of Delta-9-THC on the humoral response. Animals were also employed for determination of delayed hypersensitivity responses (DHR). Virus-infected animals treated with 100 mg/kg Delta-9-THC exhibited greater severity of herpes genitalis, higher mortalities and higher mean titres of virus shed from the vagina. Suppression of the humoral response to HSV-2 occurred in animals treated with this dose of drug when compared to virus-infected vehicle controls. A delay in the onset of the DHR to HSV-2 was observed in animals receiving 100 mg/kg Delta-9-THC when compared with those receiving vehicle. These results indicate that Delta-9-THC decreases host resistance to HSV-2 vaginal infection in the B6C3F1 mouse. This decreased resistance is associated with suppression of the immune response to primary infection with HSV-2.
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The Cytotoxic Response to Murine Cytomegalovirus. III. Lymphokine Release and Cytotoxicity Are Dependent upon Phenotypically Similar Immune Cell Populations
More LessSUMMARYMurine cytomegalovirus (MCMV)-immune lymph node (LN) cells were generated following 4 days culture of draining popliteal LN cells removed after bilateral hind footpad inoculation of mice with 4 × 102 p.f.u. MCMV. This cell population expressed both cytotoxic activity against MCMV-infected mouse embryo fibroblasts (MEF) and the capacity to release lymphokine upon stimulation with MCMV-infected MEF. Cytotoxicity and lymphokine release were Class I, H-2-restricted, virus-specific and dependent upon Thy1.2+, Lyt2+ cells. Mixing of 5 × 105 MCMV-immune T cells with an excess of syngeneic MCMV-infected MEF stimulators produced detectable levels of the lymphokine interleukin-3 by 1 h, with a maximum rate of its release between 1 and 6 h and plateau levels by 14 h. The capacity to stimulate virus-specific MCMV-immune T cells was acquired by MEF at 1 h after MCMV infection with maximum stimulator ability attained by 8 h. In the presence of a fixed number of MCMV-immune T cells, and titration to excess of syngeneic 3 h or 18 h MCMV-infected MEF, lower interleukin-3 plateau levels were obtained with 3 h than with 18 h MCMV-infected stimulators. This suggested that fewer T cells responded to 3 h than to 18 h MCMV-infected MEF.
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Production of a Monoclonal Antibody against an Epitope on HeLa Cells that Is the Functional Poliovirus Binding Site
P. Nobis, R. Zibirre, G. Meyer, J. Kühne, G. Warnecke and G. KochSUMMARYCell lines of primate origin carry receptors on their plasma membrane which are responsible for the specific binding of poliovirus. This paper describes the isolation and characterization of a monoclonal antibody reacting with the plasma membrane of HeLa cells. The antibody (D171) was selected for its protection of HeLa cells against the cytopathic effect of poliovirus type 1. This protection was found to extend to all three viral serotypes, while the replication of five other viruses in HeLa cells was not affected. The 125I-labelled purified antibody did not react with cell lines derived from pig, dog or rodents but bound specifically to all lines of human or primate origin. Immunoglobulin or Fab fragments of D171 prevented the binding of 35S-labelled poliovirus to HeLa cells. Conversely, nearly all binding sites of 125I-labelled D171 immunoglobulins or Fab fragments could be blocked after preincubation of HeLa cells with poliovirus. These results indicate that D171 recognizes the poliovirus receptor site on different susceptible cells and that practically all D171 binding sites are involved in the specific attachment of poliovirus to the plasma membrane. To determine whether the epitope recognized by D171 could be separated from the receptor for poliovirus, human-mouse cell hybrids were prepared and analysed. In all 40 clones tested, the susceptibility to poliovirus correlated with the binding of D171.
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Two Initiation Sites for Foot-and-Mouth Disease Virus Polyprotein in vivo
More LessSUMMARYTypically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P16, are translated from the 5′ end of the coding region of the genome of foot-and-mouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (A10 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported.
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Oligonucleotide Fingerprint Analysis of Coxsackievirus A10 Isolated in Japan
More LessSUMMARYEight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co-electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes.
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On the Structure of the Acyl Linkage and the Function of Fatty Acyl Chains in the Influenza Virus Haemagglutinin and the Glycoproteins of Semliki Forest Virus
More LessSUMMARYThe acylation of the haemagglutinin (HA) of different influenza viruses and of the envelope glycoproteins of Semliki Forest virus (SFV) were analysed. The fatty acid linkage in these acylproteins was found to be resistant to a variety of organic solvents and combinations of these, even after pretreatment with various detergents. Fatty acids are released from influenza virus HA at a pH value between 11.8 and 12.1 at room temperature. Although this mild alkaline cleavage occurs rapidly, the release of fatty acids by treatment with hydroxylamine is time-, temperature- and concentration-dependent. By comparison with model esters the linkage in HA is suggested to be of the oxygenester type rather than a thioester linkage. To assay for possible functions of protein-bound fatty acids the biological activities of influenza virus (A/FPV/Rostock/34) and its solubilized spike glycoproteins were measured after deacylation. While viral haemagglutination activity was not hampered at all, its ability to haemolyse erythrocytes and infectivity were drastically reduced. Likewise, viral spike glycoproteins solubilized with detergents failed to induce haemolysis at low pH when fatty acids had been cleaved off. These results indicate the possible involvement of protein-bound fatty acids in fusion inducation through the acylated fusogenic spike glycoproteins.
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A Physical Map of the Oryctes Baculovirus Genome
More LessSUMMARYThe restriction endonucleases, BamHI, EcoRI, HindIII and PstI, cleave Oryctes baculovirus (strain PV505) DNA into 21, 43, 23 and seven fragments respectively. A large number of these fragments were cloned into the bacterial plasmids pUC8 and pBR328. These clones encompass 96% of the genome. The restriction sites for the four endonucleases were mapped using double and partial digestions of cloned fragments as well as hybridization of labelled fragments to Southern transfers of cleaved DNA. When HindIII fragment D and BamHI fragment D were hybridized to Southern transfers six regions containing reiterated sequences were found. The physical map for Oryctes baculovirus could not be orientated with respect to other published baculovirus maps because the BamHI fragment F of Autographa californica nuclear polyhedrosis virus which contains conserved polyhedrin gene sequences common to occluded baculoviruses did not bind to Oryctes baculovirus DNA.
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Intermolecular Recombination of the Herpes Simplex Virus Type 1 Genome Analysed Using Two Strains Differing in Restriction Enzyme Cleavage Sites
More LessSUMMARYIntermolecular recombination of herpes simplex virus type 1 (HSV-1) was studied by analysing the segregation of strain-specific restriction enzyme cleavage sites among progeny viruses produced after co-infection by two HSV-1 strains differing in eight restriction enzyme cleavage sites. Out of 93 progeny viruses examined, 51 clones were recombinant, and crossover sites of the recombinants were mapped on the HSV-1 genome. These sites were distributed evenly in the unique sequence of the L component (UL) and the recombination frequency in UL was estimated to be 1.12 per genome length, or 0.007 per kilobase pair. No evidence was obtained to support the existence of enhanced intermolecular recombination events in the regions containing inverted repeats and the L-S junction in comparison with the recombination frequency in UL. The finding of recombinants in an arrangement that minimized the number of crossover events suggested the participation of both of two arrangements of the L component of parental DNA (P or IS, and IL or ISL) in the generation of the recombinants. The possibility of a preference for P or IS over IL or ISL arrangements remains to be determined.
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The Structure of Adenovirus Chromatin in Infected Cells
More LessSUMMARYThe structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes. In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin. As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region. The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA. After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased. In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by [3H]thymidine incorporation after the cessation of cellular DNA synthesis. These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle. Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern. Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA. A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.
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A Proposal for Naming Adenovirus Genome Types, Exemplified by Adenovirus Type 6
Th. Adrian, B. Best and R. WigandSUMMARYA numerical code to denominate adenovirus (AV) genome types is proposed. Seven restriction endonuclease patterns are listed in alphabetical order (BamHI, BglII, BstEII, EcoRI, HindIII, KpnI, SmaI); patterns deviating from those of the prototype (1) are named 2, 3 etc. depending on the chronological order of the respective isolates. Thus AV6/3: 1231121 is a type 6 strain deviating from the prototype in three patterns, i.e. those of BglII, BstEII and KpnI. From 24 AV6 isolates, six were identical with the prototype, whereas the other strains represented 13 different genome types.
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Antibody to the 32K Structural Protein of Infectious Bursal Disease Virus Neutralizes Viral Infectivity in vitro and Confers Protection on Young Chickens
More LessSUMMARYChicken sera containing IgG antibodies specific for the 32000 (32K) mol. wt. structural polypeptide of infectious bursal disease (IBD) virus, as assessed by Western blotting, neutralized the in vitro infectivity of tissue culture-adapted IBD virus. When injected into young chickens, the serum passively protected them from challenge with pathogenic IBD virus. Chickens immunized with the 32K structural polypeptide of IBD virus, prepared by electroelution from SDS-PAGE gels, produced antibody detectable by ELISA and the virus neutralization assay, while chickens immunized with the 37K or 41.5K viral polypeptides synthesized antibody detectable by ELISA, but only very low levels of virus-neutralizing antibody. The immunoglobulin fraction of sera obtained from chickens immunized with the 32K polypeptide, but not the 41.5K polypeptide, passively protected chickens from infection with IBD virus. It is concluded that the 32K polypeptide is a major protective immunogen of IBD virus.
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Reassortment of Human Rotaviruses Carrying Rearranged Genomes with Bovine Rotavirus
More LessSUMMARYRotaviruses isolated from chronically infected immunodeficient children were previously shown to contain RNA yielding abnormal migration profiles on gels: normal RNA segments were lost or decreased in concentration, and additional bands of dsRNA were found which were derived (rearranged) from genome segments of lower molecular weight by concatemer formation. These viruses grew very slowly during passage in secondary rhesus monkey kidney cells. Upon superinfection with the tissue culture-adapted UK Compton strain of bovine rotavirus (BRV) extensive genome reassortment occurred. Clones with the following reassorted genome patterns were isolated: (i) RNA segments 5 or 6 of BRV were replaced by the corresponding RNA segments of human rotavirus; (ii) RNA segments 9 or 11 of BRV were replaced by different rearranged bands of RNA of human rotavirus; (iii) reassortants were observed containing more than one segment/rearranged band of human rotavirus RNA in different combinations. The reassortant viruses possessed functional proteins coded for by the genome segments and/or by rearranged bands of RNA of the human rotaviruses. Rearrangement of parts of the rotavirus genome may be a mechanism of evolution of these viruses.
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Release of Progeny Virus from Cells Infected with Simian Rotavirus SA11
More LessSUMMARYAnalysis of cells infected with simian rotavirus SA11 at late times of infection indicated that the particles were associated with membranes and the cytoskeleton. Although a large amount of cellular and non-structural viral proteins were released at these times, probably by cellular lysis, only virus with an outer layer was found outside the cells, while virus without an outer layer remained associated with the cells, probably with membranes and the cytoskeleton. Inhibition of glycosylation by tunicamycin did not abolish cell lysis but inhibited the liberation of particles and the non-glycosylated precursors of the structural and non-structural viral glycoproteins. These results indicate that immature virus was tightly associated with the structural matrix of the cell.
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Rapid Inactivation of Rotaviruses by Exposure to Acid Buffer or Acidic Gastric Juice
More LessSUMMARYInactivation rates of three bovine and several primate-origin rotaviruses were determined during exposure to acid buffers at pH 2.0, pH 3.0 or pH 4.0. Each rotavirus was inactivated at pH 2.0 (the acidity most resembling the normal fasting stomach) very rapidly, with half-lives for infectivity determined to be 1 min or less. Each rotavirus was inactivated at a much slower rate at pH 3.0; inactivation at pH 4.0 was minimal. No remarkable differences in acid resistance between different rotavirus strains were detected. Although these determinations were performed at room temperature (23 °C), experiments at diverse temperatures indicated an even more rapid rate of viral inactivation by acid at normal body temperature (37 °C). Studies of rotavirus exposed to natural human gastric juice at pH 1.8 or pH 2.1 revealed a rate of virus inactivation similar to that observed with glycine buffer of identical pH.
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Isolation of Feline Rotaviruses and Their Relationship to Human and Simian Isolates by Electropherotype and Serotype
More LessSUMMARYRotaviruses were detected by electron microscopy in the faecal specimens of six clinically well cats and virus was subsequently isolated from four of them. Analysis of the RNA of the isolates showed the existence of three electrophoretic types characteristic of the ‘long’ RNA electrophoretic pattern exhibited by rotaviruses. All feline isolates were neutralized only by antiserum to SA11 rotavirus, indicating that these isolates were serotype 3 rotaviruses. Antiserum prepared against a feline strain neutralized all the feline isolates as well as SA11 but showed no neutralizing activity against human isolates of serotype 1, 2 or 4.
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Survival Characteristics of Airborne Human Coronavirus 229E
More LessSUMMARYThe survival of airborne human coronavirus 229E (HCV/229E) was studied under different conditions of temperature (20 ± 1 °C and 6 ± 1 °C) and low (30 ± 5%), medium (50 ± 5%) or high (80 ± 5%) relative humidities (RH). At 20 ± 1 °C, aerosolized HCV/229E was found to survive best at 50% RH with a half-life of 67.33 ± 8.24 h while at 30% RH the virus half-life was 26.76 ± 6.21 h. At 50% RH nearly 20% infectious virus was still detectable at 6 days. High RH at 20 ± 1 °C, on the other hand, was found to be the least favourable to the survival of aerosolized virus and under these conditions the virus half-life was only about 3 h; no virus could be detected after 24 h in aerosol. At 6 ± 1 °C, in either 50% or 30% RH conditions, the survival of HCV/229E was significantly enhanced, with the decay pattern essentially similar to that seen at 20 ± 1 °C. At low temperature and high RH (80%), however, the survival pattern was completely reversed, with the HCV/229E half-life increasing to 86.01 ± 5.28 h, nearly 30 times that found at 20 ± 1 °C and high RH. Although optimal survival at 6 °C still occurred at 50% RH, the pronounced stabilizing effect of low temperature on the survival of HCV/229E at high RH indicates that the role of the environment on the survival of viruses in air may be more complex and significant than previously thought.
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LeDantec Virus: Identification as a Rhabdovirus Associated with Human Infection and Formation of a New Serogroup
More LessSUMMARYLeDantec virus, originally recovered in 1965 from a patient with a febrile illness in Senegal, was observed by thin section electron microscopy to be bullet-shaped, representative of members of the rhabdovirus group, with mean dimensions of 164 × 78 nm. Particles were observed budding from the plasma membrane and moderate numbers accumulated in the intercellular spaces. In three mammalian cell lines, LeDantec virus was rapidly cytopathic and replicated to moderately high titres. In complement fixation tests with other known rhabdoviruses, LeDantec was found to be related to Keuraliba virus, a previously ungrouped agent isolated from rodents in Senegal in 1968. We propose the formation of a new LeDantec serogroup comprising these two viruses.
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Detection and Localization of the v-myb(AMV) Gene Products of Avian Myeloblastosis Virus by a Synthetic Peptide Antiserum
More LessSUMMARYAn antiserum made against a synthetic peptide from an internal region of the predicted amino acid sequence of the avian myeloblastosis virus (AMV) transforming v-myb(AMV) gene identified two products, p46v-myb(AMV) and p32v-myb(AMV), which were localized in the nucleus of AMV-transformed myeloblasts. We propose that these proteins are the in vivo products of the v-myb(AMV) gene and thus the transforming protein(s) of AMV.
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Expression of Recombinant Vaccinia Virus-derived Alphavirus Proteins in Mosquito Cells
More LessSUMMARYA recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to (i) vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and (ii) using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.
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Identification of Dengue Type 2 Virus-specific High Molecular Weight Proteins in Virus-infected BHK Cells
More LessSUMMARYEvidence is presented for the production in dengue type 2 virus (DEN-2)-infected BHK cells of large virus-specific proteins with molecular weights up to 250000. These proteins were most prominent in lysates of cells which had been labelled with [35S]methionine for 7 to 15 min. During pulse-chase experiments, these high mol. wt. proteins appeared to be converted into smaller, more stable, proteins with mol. wt. between 10000 and 98000. Finally, inhibition of proteolysis prevented the chasing of label from the high mol. wt. proteins to the smaller viral proteins which normally accumulate in DEN-2-infected cells. These findings are consistent with the idea that processing of large polyprotein precursors plays an important role in the production of flavivirus proteins.
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Two Monoclonal Antibodies against La Crosse Virus Show Host-dependent Neutralizing Activity
More LessSUMMARYMammalian and arthropod cell cultures were used to assess the neutralizing activity of six monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus. Four antibodies, two neutralizing and two non-neutralizing, showed no host-dependent differences, giving similar results when post-treatment infectivity was determined using either Aedes albopictus cells or BHK-21 cells. For two other antibodies, however, dissimilar activities were observed between the vertebrate and invertebrate cell lines. One of these antibodies was positive when BHK-21 cells were employed as the post-treatment host and negative when mosquito cells were used; the other antibody was the converse. The epitope for this last antibody was present on all California serogroup viruses examined, which suggests that it may have a special significance in the natural life-cycle of the virus.
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Influence of Interferon on Persistent Infection Caused by Borna Disease Virus in vitro
More LessSUMMARYThe effect of interferon (IFN) on infection and maintenance of persistent infection of Borna disease (BD) virus in cell cultures was investigated. Acutely BD virus-infected primary rabbit brain and rat lung cells produced significant levels of interferon detectable 3 days post-infection in the culture supernatants. Rat brain and rat lung cells persistently infected with BD virus produced only moderate levels of IFN over a long period. In contrast, persistently infected Madin-Darby canine kidney (MDCK) cells did not produce detectable amounts of IFN. Exogenous homologous IFN completely inhibited the expression of BD virus antigen in acutely infected rabbit brain cells, when added during the first 24 h after infection. IFN added later (2 to 6 days post-infection) reduced virus titres to different degrees depending on the onset of treatment. However, IFN added to persistently infected rat lung cells did not appear to influence the degree or quality of BD virus antigen expression or the intracellular amount of infectious virus. Two facts indicate that IFN is not involved in the establishment or maintenance of persistent BD virus infection in vitro. Thus, MDCK cells, which could not be induced to produce IFN, can be readily persistently infected with BD virus in vitro, and exogenous IFN did not appear to influence persistent BD virus infection.
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Inhibition of Herpes Simplex Virus Type 1-induced Interferon Synthesis by Monoclonal Antibodies against Viral Glycoprotein D and by Lysosomotropic Drugs
More LessSUMMARYComponents of herpes simplex virus remained bound to the diploid cell membrane after nucleocapsid penetration into the cytosol. These components enabled the infected cells to induce interferon-alpha (IFN-α) in peripheral blood mononuclear cells even when the infected cells were fixed by glutaraldehyde. Monoclonal antibodies directed against the major viral glycoprotein D could neutralize their IFN-α-inducing capacity. Thus, the process of IFN induction does not require uptake and penetration of the inducer into the effector cells. The process was, however, sensitive to lysosomotropic drugs. These data suggest that a membrane receptor is involved in the IFN-α induction mechanism.
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Restriction Endonuclease Analysis of Bovine Herpesvirus 1 DNA and Nucleic Acid Homology between Isolates
More LessSUMMARYIsolates of bovine herpesvirus 1 (BHV-1) are associated with a variety of clinical manifestations. To determine if a single form of BHV-1 was responsible for the different virus-associated diseases or whether subpopulations of various isolates produced different clinical symptoms, studies were initiated to examine the DNA restriction enzyme patterns and nucleic acid homology between virus isolates from respiratory infections and other clinical syndromes. Differences between the genomes of several virus isolates were detected using DNA restriction enzyme analyses. However, nucleic acid hybridization studies of the virus DNAs using filter and liquid hybridization indicated at least a 95% genetic homology between the virus isolates from different types of infections. Additionally, these studies demonstrated that the DNA of BHV-1 had an average molecular weight of 84 × 106.
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- Plant
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Comparisons between the Primary Structure of the Coat Proteins of Turnip Yellow Mosaic Virus and Eggplant Mosaic Virus
More LessSUMMARYComparison of the primary structures of eggplant mosaic virus (EMV) and turnip yellow mosaic virus (TYMV) coat proteins shows that 32% of their amino acids are conserved. Alignment of the two sequences requires only one deletion near the N terminus and two insertions at the C terminus of TYMV coat protein. Although the coat protein of EMV is on average less hydrophobic than that of TYMV, structural predictions yield fairly similar conformations for the two proteins, apart from the N terminus. Neither coat protein possesses an accumulation of basic residues able to form the strong ionic RNA-protein interactions observed in serveral other isometric viruses. The nature of the amino acid exchanges seems to be different from that seen in families of homologous proteins. The highly conserved regions encompass a (probably weak) potential RNA-protein interaction site. Implications for the structure and stability of small isometric viruses are discussed.
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Particles of Rice Gall Dwarf Virus in Thin Sections of Diseased Rice Plants and Insect Vector Cells
More LessSUMMARYRice gall dwarf virus (RGDV) was studied in infected rice plants and in its principal insect vector, the green leafhopper Nephotettix nigropictus, by electron microscopy. In plants, virions were restricted to the cytoplasm and vacuoles of phloem-related cells and of gall cells generated from the phloem. In the insects, virions occurred in the cytoplasm of many different kinds of cells including those of the salivary gland, the gut, the fat body, muscles and nerves. Virions in both plant and insect cells had dense cores 40 to 50 nm in diameter which were surrounded by a less dense region; the total diameter was 60 to 70 nm. Viroplasms, crystals and tubules were observed in cells both of plants and insects. Although virions often occupied most of infected plant cells, they occurred only in restricted areas of insect cells. The restriction of virions to phloemrelated cells in infected plants probably explains why yields of purified RGDV are usually about 1/30 of those of rice dwarf virus, which invades many types of cell. About 3% of the virus particles seen in thin sections of infected plants appeared to be empty. Empty particles were purified from infected rice plants and were found to lack nucleic acid and much of the 56000 mol. wt. protein present in intact particles.
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Translation of Tobacco Ringspot Virus RNA in Reticulocyte Lysate: Proteolytic Processing of the Primary Translation Products
More LessSUMMARYTobacco ringspot virus (TobRV) RNA was translated efficiently in rabbit reticulocyte lysate and directed the synthesis of two principal polypeptides, M r 207 × 103 (207K) and 116K, corresponding to the translation products of RNA-1 and RNA-2 respectively. In addition, a 112K RNA-2-encoded polypeptide was sometimes detected. The 116K polypeptide was immunoprecipitated with anti-TobRV serum, suggesting that it was a precursor to coat protein. When translations were performed in the presence of dithiothreitol, the 207K polyprotein was apparently cleaved to yield 37K and 180K polypeptides, with additional processing into 65K and 128K polypeptides. Cleavage of the RNA-2-encoded polyprotein also occurred, although to a much lesser extent than that of the 207K polyprotein; polypeptides of 88K and 54K, both immunoprecipitated with antiviral serum, were identified as RNA-2-encoded cleavage products.
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Expression of High Molecular Weight Polypeptides by Carnation Mottle Virus RNA
More LessSUMMARYIn vitro translation of virion RNA from carnation mottle virus (CarMV) in a messenger RNA-dependent rabbit reticulocyte lysate (MDL) resulted in the synthesis of four virus-specific polypeptides of apparent M r 100000 (P100), 77000 (P77), 38000 (P38) and 30000 (P30). Partial peptide mapping experiments and in vitro translation in the presence of partially purified calf liver amber suppressor tRNA demonstrated that P30, P77 and P100 are a series of overlapping polypeptides generated by a double readthrough mechanism. In addition we report the infection of Chenopodium quinoa protoplasts with CarMV. The viral coat protein was detected in virus-infected protoplasts. Only one other infection-specific protein with an apparent M r of 100000 corresponded in size to any of the other in vitro translation products.
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- Fungal
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Naked dsRNA Associated with Hypovirulence of Endothia parasitica Is Packaged in Fungal Vesicles
More LessSUMMARYHypovirulence in Endothia parasitica is known to be associated with dsRNA. The characteristics of this cytoplasmically transmissible element suggest it might be viral in nature. Attempts to isolate viral particles indicate the presence of two particulate fractions in hypovirulent (HV) strains and a similar one in virulent (V) strains. Composition analyses of the fractions show these to be similar except that those from HV strains include dsRNA. Also present in these particles are carbohydrate, protein and chloroform-methanol-extractable substances. The protein composition is too low to indicate the presence of a capsid. The neutral sugars that are present (arabinose, mannose, galactose, glucose) in these particles are also present in the fungal cell wall. Silver- and ethidium bromide-stained polyacrylamide gels resolve several minor as well as the known major dsRNA components. These components are arbitrarily divided into four mol. wt. groups: 4.5 × 106 to 5.0 × 106, 1.2 × 106 to 1.5 × 106, 5.8 × 105 to 6.2 × 105 and < 4.6 × 105. Not all segments are present at all times, although the phenotype of the culture is not affected. Proteins are not detectable in vesicles of HV strains, but six proteins are detectable from V strains by gel electrophoresis. Assays for [32P]UMP incorporation indicate the presence of a RNA polymerase closely associated with the dsRNA. Our results suggest that the transmissible element responsible for hypovirulence is a naked dsRNA genome packaged within vesicles formed by the host.
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