- Volume 63, Issue 2, 1982
Volume 63, Issue 2, 1982
- Animal
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Molecular Cloning of Bovine Papillomavirus Genomes and Comparison of Their Sequence Homologies by Heteroduplex Mapping
More LessSummaryThe genomic DNAs of bovine papillomavirus (BPV) type 1, type 2 and type 4 were cloned in pAT153. BPV1 and BPV2 genomes were cloned using the single HindIII sites of the vector and virus DNAs, and BPV4 was cloned using the single BamHI sites. The orientation of the recombinant DNAs was established by restriction enzyme digestion, hybridization and heteroduplex analysis. The results showed that: (i) BPV1 and BPV2 DNAs are in register and are broadly homologous throughout most of their length when aligned at their single HindIII sites; (ii) depending on the degree of hybridization stringency used, the two DNAs show one major region and several minor regions of partial homology, mainly residing in the segment of the genomes believed to contain the structural genes; (iii) BPV4 DNA shares no homology with either BPV1 or BPV2 DNA.
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Murine Coronaviruses: Isolation and Characterization of Two Plaque Morphology Variants of the JHM Neurotropic Strain
More LessSummaryTwo plaque-size variants of the neurotropic JHM strain of mouse hepatitis virus have been isolated from the virus stock after eight serial passages in suckling mouse brain. One variant, JHM-DL, produces large plaques, while the other, JHM-DS, produces small plaques in tissue culture. DS replicates more slowly, has a lower virus yield in vitro, and is less virulent for mice than DL. They also differ in their pathogenicity for mice: JHM-DL infection results in acute encephalomyelitis while JHM-DS infection results in demyelination. Oligonucleotide fingerprint analysis of the RNA genomes of these two variants revealed that they had almost identical genetic sequences. Each variant, however, had a unique oligonucleotide spot not found in the other. The unique spot of the large plaque variant, JHM-DL, was localized at approximately 3 to 5 kb from the 3′ end, while the JHM-DS unique spot was mapped at 14 to 15 kb from the 3′ end of the genome. We have further shown that these oligonucleotide changes are not correlated with the plaque morphology. These two viruses may be useful for studying the molecular basis of virus-induced demyelination.
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Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1
More LessSummaryDeletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn +, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and BglII restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with BglII and nuclease BAL 31 followed by ligation and recleavage with BglII resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following BglII and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn + DNA in baby hamster kidney (BHK) cells to produce TK− deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively. 5-Bromo-2′-deoxyuridine, 5-bromo-2′-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK− recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK−, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK− mutant HSV-1 (17) dPyk−7. Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk−7 indicated that all TK− mutants except dPyK−7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.
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Monoclonal Antibodies to Three Non-glycosylated Antigens of Herpes Simplex Virus Type 2
More LessSummaryThe production and properties of three monoclonal antibodies, designated LP1, LP4 and AP2, directed against non-glycosylated polypeptides of herpes simplex virus type 2 are described. LP1 is specific for polypeptide VP16 and cross-reacts with HSV-1; LP4 reacts with the major DNA-binding protein and is type-specific. AP2 is directed against the major capsid antigen of HSV-1 and HSV-2.
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Low Infectivity of HSV-1 DNA Caused by Defective-interfering Genomes
More LessSummaryThe infectivity of herpes simplex virus, type 1, strain ANG progeny DNA from standard virus infections and of progeny DNA from infections involving defective-interfering virus particles (DI DNA) was compared in transfection assays. No difference in infectivity of virus DNA isolated either from infected cells or from progeny virus was found for a given type of infection. However, the values for the two types of infection differed markedly, with DI progeny DNA being less infectious by more than 2 log10. The low infectivity was mainly due to the presence of interfering DNA molecules in DI progeny DNA, regardless of whether intracellular DNA or DNA extracted from mature virions was analysed. The interfering capacity of DI progeny DNA did not depend on the integrity of the genomes. The physical proximity provided by simultaneous precipitation of infectious and of interfering DNA is an important factor influencing the degree to which DI DNA interferes. Interference by DI DNA in the transfection assay can be partly reversed by the addition of XbaI fragments of standard DNA; in control experiments this fragmented DNA was shown to lead to a reduction rather than to an enhancement of the infectivity of standard virus DNA.
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Experimental Infection of Inbred Mice with Herpes Simplex Virus. V. Investigations with a Virus Strain Non-lethal after Peripheral Infection
More LessSummaryThe herpes simplex virus type 1(HSV-1) strain ANG, unlike the majority of HSV-1 isolates, does not cause lethal encephalitis in various inbred mouse strains, when applied in doses up to 2 × 107 plaque-forming units intraperitoneally, intravenously, intravaginally, orally, or via the foot pads. We studied and compared the progress of infection by this non-lethal strain and by a lethal HSV-1 strain, and the components of the host defence mechanism involved. If injected intracerebrally, HSV-1 ANG replicated efficiently in mouse brain cells and led to encephalitis. Upon systemic or peripheral infection, it replicated in several mouse organs with a virulence similar to lethal HSV-1 isolates. It was clear that transport of HSV-1 ANG to the central nervous system (CNS) or replication in CNS tissue is efficiently restricted after peripheral infection. Conceivably, infection with lethal HSV-1 strains proceeds in two distinct steps, virus replication at the site of infection and in the spleen and, secondly, transport to CNS tissue and propagation in CNS cells. This second step is apparently blocked in infections of DBA/2 mice by HSV-1 ANG and can thus be studied separately. The blocking mechanism was not a function of interferon induction or sensitivity, nor was it due to an enhanced NK cell activation. Experiments with silica-treated mice, and with homozygous nude mice, which lacked T-lymphocytes, suggested that the observed restriction in virus transfer is independent of T-cell and macrophage functions. Yet, newborn mice were fully susceptible to intraperitoneal infection with HSV-1 ANG, suggesting that age-dependent defence mechanisms, the nature of which needs to be further examined, are of relevance in the restriction of peripheral infection by HSV-1 ANG in adult mice.
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An X-linked Locus Influences the Amount of Circulating Interferon Induced in the Mouse by Herpes Simplex Virus Type 1
More LessSummaryProduction of circulating interferon (IFN) was measured in inbred mouse strains following intravenous injection of herpes simplex virus type 1 (HSV-1). IFN titres reached maximal levels 2 to 3 h after injection of virus and a 10-fold difference was found between C57BL/6 mice and BALB/c, as high and low producers respectively. Mendelian analysis revealed that HSV-induced IFN production is governed by several loci, one of which is X-linked. The strain distribution pattern obtained from results in recombinant inbred lines and the results obtained in the congenic B6-C-H-28c-If-11 strain furthermore indicated an absence of close linkage to If-1. It is concluded that the levels of HSV-induced early IFN production are influenced by several autosomal loci and one X-linked locus.
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Characterization of Non-oncogenic Marek's Disease Virus-infected and Turkey Herpesvirus-infected Lymphocytes
More LessSummarySplenic leukocytes derived from N- and P-line chickens exposed to turkey herpesvirus (HVT-4), and from UCD-003 chickens exposed to the non-oncogenic SB-1 clone of Marek's disease virus were fractionated into subpopulations at various days post-exposure. The level of infection in these fractions was determined by enumeration of plaque-forming units after co-cultivation of leukocytes with virus-permissive chicken embryo fibroblasts. At 5 days post-exposure most HVT-4-infected lymphocytes were found to be of intermediate to low buoyant density (1.040 to 1.065 g/ml) and not to have detectable Fc receptors. They possessed surface Ia but were not IgM-bearing. At 15 days post-exposure HVT-4-infected lymphocytes were found to be of intermediate to high buoyant density (1.070 to 1.080 g/ml) and, at this time, virus isolation rates (p.f.u./106 cells) from fractions enriched in cells bearing Fc receptors were approximately equal to those leukocyte suspensions depleted of Fc receptor-bearing cells. Furthermore, the percentage reduction in infectivity caused by depletion of Ia-bearing cells at 15 days post-exposure was less than that at 5 days post-exposure. At both 5 and 15 days post-exposure carbonyl iron treatment failed to remove HVT-4-infected lymphocytes. These characteristics were similar whether HVT-4-infected lymphocytes were derived from N- or P-line chickens. SB-1-infected lymphocytes derived from UCD-003 chickens at 6, 7 and 14 days post-exposure were detected with equal frequency in fractions enriched or depleted of cells possessing Fc receptors. SB-1-infected lymphocytes for the most part lacked surface Ia and IgM, and were not depleted by carbonyl iron treatment. From these data it was concluded that most HVT-4- and SB-1-infected lymphocytes detectable by virus isolation were neither B cells nor macrophages.
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Adsorption and Penetration of Enveloped Herpes Simplex Virus Particles Modified by Tunicamycin or 2-Deoxy-d-glucose
More LessSummaryTritium-labelled, purified herpes simplex virus (HSV) enveloped particles produced in the presence of tunicamycin (TM) or 2-deoxy-d-glucose (DG) adsorbed to GMK cells equally as well as standard virus. However, in the presence of TM or DG, there was a reduced transport of virus DNA to cell nuclei and an increased sensitivity of attached particles to proteinase K. The reduced infectivity of HSV produced in the presence of glycosylation inhibitors is therefore probably due to an impairment in the fusion of virus envelope with plasma membranes.
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Measles Virus Persistence in Human Lymphocytes: A Role for Virus-induced Interferon
More LessSummaryBecause of the association of measles virus with persistent infections such as subacute sclerosing panencephalitis, and its possible involvement in multiple sclerosis, we have investigated the capacity of this virus to establish chronic infections in human peripheral blood lymphocytes (PBLs). We have demonstrated that a latent, persistent infection of human PBLs with measles virus results in low levels of infectious virus production in which large amounts of virus-induced interferon could be detected. Further, treatment of these silently infected cells with an anti-human leukocyte interferon serum results in a productive measles virus infection. The mechanism by which the anti-interferon serum shifts the virus-cell interaction from persistence to productive infection is discussed.
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Inhibition by Glucocorticosteroid Hormones of Interferon and Prostaglandin E Induction by Poly(rI).Poly(rC)
More LessSummaryWe have examined the relationship between induction of interferon (IFN) and prostaglandin E (PGE) production by poly(rI).poly(rC) in cultured human foreskin fibroblasts (FS11). Hydrocortisone and dexamethasone (2.5 × 10−7 m), which are known inhibitors of PGE synthesis, significantly decreased the induction of both IFN and PGE in IFN-pretreated (primed) cells. Desoxycorticosterone, progesterone and estradiol were devoid of this activity. Hydrocortisone also blocked the induction of IFN by double-stranded RNA (dsRNA), cycloheximide and actinomycin D in FS11 cells. Arachidonic acid overcame the inhibitory effect of hydrocortisone on PGE production, but failed to restore IFN production in the presence of the steroid. Moreover, the prostaglandin synthetase inhibitors, indomethacin, aspirin and flufenamic acid, did not change IFN production by dsRNA in primed FS11 cells, although prostaglandin synthesis was abolished. Although the induction of IFN and PGE by poly(rI).poly(rC) might be consequences of the same initial event in the cell, the accumulation of PGE does not seem to have a regulatory effect on the synthesis of IFN in this system.
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Enhancement of Plasminogen Activator Activity by the Culture Medium of Rous Sarcoma Virus-transformed Cells
More LessSummaryWe have tested the effect of the culture medium of chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) on plasminogen activator (PA) activity of normal and RSV-infected cells. The results obtained showed that fibrin digestion by cultures of normal uninfected and RSV-infected cells directly attached to a fibrin substrate was increased when the cells were exposed to the supernatant of an 18 h-culture of CEF transformed by RSV. Addition of cycloheximide to the supernatant completely abolished this effect. Similarly, exposure of both normal and RSV-infected cells to the culture medium of RSV-transformed CEF resulted in a marked increase of the soluble PA activity present in the supernatant. This effect, however, was not abolished by inhibition of protein synthesis. For both the cell-bound and soluble PA activities of normal and RSV-infected cells the stimulatory effect was transient, being maximal between 8 and 12 h of exposure to the transformed cell-conditioned medium and disappearing after 24 h. A possible correlation with transformation-enhancing factor(s), previously reported to be present in the culture medium of transformed cells, is discussed.
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Nature of the Antibody Response to the Foot-and-Mouth Disease Virus Particle, its 12S Protein Subunit and the Isolated Immunizing Polypeptide VP1
More LessSummaryInoculation of inactivated 146S foot-and-mouth disease virus particles into guineapigs elicited the formation of neutralizing antibody and the serum had a 10-fold higher titre in radioimmunoassay (RIA) with 146S particles than with the 12S virus subunit. In contrast, a single inoculation of the 12S subunit or the isolated polypeptide VP1 elicited the formation of antibody having a much lower titre in RIA with the 146S particle than with the 12S subunit and low or undetectable neutralizing activity. However, sera from guinea-pigs given two or more inoculations of the 12S subunit or VP1 had neutralizing activity. The level in the anti-VP1 serum was lower than that in the anti-12S serum and both were much lower than that in animals receiving two inoculations of the 146S particle. The neutralizing activity elicited by the three antigens was absorbed by the homologous antigen. In contrast, neither the 12S subunit nor VP1 absorbed the anti-146S neutralizing antibody and VP1 did not absorb the anti-12S subunit neutralizing antibody. However, the 12S subunit partly absorbed the neutralizing activity elicited by VP1. The results are compatible with a model in which the 146S particle elicits a spectrum of neutralizing antibodies which are completely absorbed by the homologous particle but only partially by the 12S subunit or VP1. The results are discussed in relation to the structural features required for the production of neutralizing antibody.
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African Swine Fever Virus DNA: Restriction Endonuclease Cleavage Patterns of Wild-type, Vero Cell-adapted and Plaque-purified Virus
More LessSummaryDNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 × 106 to 100 × 106. Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.
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Transformation of Human Embryonic Fibroblasts by BK Virus, BK Virus DNA and a Subgenomic BK Virus DNA Fragment
SummaryHuman embryonic fibroblasts (HEF) have been transformed by BK virus (BKV) DNA and by u.v.-inactivated or live BKV alone or in association with methyl-cholanthrene (MTC). The transformed cells produced BKV large T and small t antigens as well as the cellular 53 kdal protein, detected by immunofluorescence and immunoprecipitation. After an initial phase of lysis and virus shedding, virus or its coat protein antigen could not be detected in transformed cells. All human transformed cell lines could be superinfected by BKV or BKV DNA, but their susceptibility to superinfection was 20- to 500-fold lower than normal HEF. BKV could be rescued by fusion of transformed cells with normal HEF or Vero cells and by transfection of normal HEF with total DNA and DNA extracted from the Hirt supernatant of transformed cells. Blot hybridization analysis of DNA from transformed cells showed a considerable amount of free BKV DNA in monomeric and polymeric forms. Integrated BKV DNA was absent in most cell lines but present in only small amounts in BKV-transformed cells treated with MTC. Analysis of free BKV DNA with various restriction endonucleases and by blot hybridization showed that monomeric forms were complete BKV genomes, whereas polymers contained both complete and defective or rearranged BKV DNA. Transformation of HEF was also obtained with a 3.7 kilobase (kb) fragment of the BKV genome, produced by sequential digestion of BKV with the restriction endonucleases HhaI and EcoRI. This fragment extends clockwise on the virus genome from 0 to 72.2 map units and contains the entire early region. Blot hybridization analysis of cells transformed by the HhaI/EcoRI 3.7 kb fragment showed two separate integrations of BKV sequences without free virus DNA.
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Organization of Polyoma Virus DNA in Mammary Tumours of Athymic Mice
More LessSummaryRestriction mapping of polyoma virus DNA in mammary tumours of athymic mice gave patterns that varied with the tumour examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. All tumours contained tandemly integrated full-length and defective virus genomes. Some tumours also contained unintegrated virus DNA molecules, some full-length and others defective. The deletions were localized in the virus genomic sequences coding for the distal part of the large T antigen. After the first transplantation, the organization of polyoma virus genomes in tumours remained essentially unchanged through four successive transplantations. The tumour cells that initially contained free virus DNA molecules continued to possess such molecules during serial transplantations. The virus DNA molecules in transplanted tumours lacking unintegrated virus genomes were more methylated than in tumours containing unintegrated virus genomes.
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Characterization of the Oligosaccharides of Inkoo Virus Envelope Glycoproteins
More LessSummaryInkoo virus (a bunyavirus) was grown in BHK-21 cells and labelled with [35S]methionine or [3H]mannose. [35S]Methionine labelled the two envelope glycoproteins G1 (M r = 125000) and G2 (M r = 35000), as well as the nucleocapsid protein N (M r = 25000). Only G1 and G2 were labelled with the sugar precursor. The [3H]mannose-labelled virus was solubilized with detergent and digested with Pronase. The structure of the labelled glycopeptides originating from the mixture of G1 and G2 was studied by degrading the glycans stepwise with specific exo- and endoglycosidases, and by analysing the products by both gel and paper chromatography, as well as lectin-affinity chromatography. Three classes of N-glycosidic glycans were found: complex glycans with the monosaccharide sequence (NeuNAcαGalβGlcNacβ)≥2 (Man)3 (GlcNAc)2 (occurrence of fucose was not studied), high mannose-type chains with the average structure (Man)4-6 (GlcNAc)2, and endoglycosidase H-resistant small glycans which were partly susceptible to mannosidase. These latter types of oligosaccharide chains are a novel finding among virus glycoproteins. The relative ratio of the three types of oligosaccharide chains was roughly 4.6:1:1 respectively. The G1 glycoprotein carried most of the sugar chains, since it contained 85% of the [3H]mannose label. The results are discussed in relation to the site of virus maturation at smooth-surfaced vesicles in the Golgi region.
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Antiviral Effect of Prostaglandins of the A Series: Inhibition of Vaccinia Virus Replication in Cultured Cells
More LessSummaryProstaglandins of the A series potently inhibited the production of vaccinia virus in mouse L fibroblasts. With the highest non-toxic dose of PGA1, 4 µg/ml, the replication of the virus was inhibited by 95.3%. The antiviral activity was dose-dependent and specific for the A series. At the dose used, PGA1 was not toxic to uninfected cells and did not alter cell metabolism as measured by DNA, RNA and protein synthesis. PGA1 did not influence the adsorption of the virus by the host cells and the antiviral activity was not dependent on the presence of PGA1 during the early stages of infection. PGA treatment delayed and partially inhibited virus DNA synthesis and, while it did not produce any change in the pattern of protein synthesis in uninfected cells, it altered both the rate and the pattern of virus protein synthesis. We conclude that PGA1 selectively inhibits one or more steps involved in the replication of vaccinia virus in mouse L fibroblasts.
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Secretion of Immunoglobulins by Human Lymphocytes after Infection with Influenza Virus
More LessSummaryThe biosynthesis of IgM by the Epstein—Barr virus-negative RAMOS lymphoblastoid cell line infected with an influenza A virus, fowl plague virus Dobson strain (FPV-B), was investigated. The results show that FPV infection of RAMOS cells slightly inhibited overall cellular protein synthesis only at 24 h after infection, despite the synthesis of FPV-specific proteins. However, even at this time, the synthesis and secretion of IgM were not affected by virus infection. Secreted IgM contained a reduced amount of sialic acid. The quantity of the asialylated IgM increased proportionally to the amount of enzymically active neuraminidase, suggesting that the asialylation of IgM is due to the action of virus neuraminidase. No such asialylated IgM was observed in RAMOS cells infected with measles virus, which does not possess neuraminidase. These results, together with a previous observation of ours that asialylated immunoglobulins acquire an altered antigenicity, suggest that the modulation of enzyme activities in B lymphocytes in response to an exogenous aggression may lead to disturbances in the structure and in the antigenic properties of immunoglobulins.
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Localization of Rotavirus Antigens in Infected Cells by Ultrastructural Immunocytochemistry
More LessSummaryVirus structural antigens were localized within a line of monkey kidney (MA104) cells infected with the simian rotavirus SA11 using electron microscopic immunoperoxidase techniques. When hyperimmune guinea-pig anti-SA11 serum was used, virus particles, membranes of virus-associated endoplasmic reticulum, and viroplasmic inclusions were most heavily labelled. A general cytoplasmic reaction (ribosomes, intracytoplasmic membranes, etc.) with anti-SA11 serum was also observed, but nuclei were unstained. In addition, several other virus-induced structures were found to contain rotavirus proteins, including convoluted smooth membrane within the endoplasmic reticulum, aberrant virus-like particles, and 15 to 20 nm diam. cytoplasmic tubules. Monospecific antiserum to VP7 (outer capsid glycoprotein, mol. wt. 38000) reacted strongly with virus particles and the virus-associated endoplasmic reticulum, but reacted poorly with viroplasmic inclusions. The nucleus and general cytoplasm were unstained with anti-VP7. In contrast, monospecific antisera to VP2 and VP6 (inner capsid proteins, mol. wt. 94000 and 41000 respectively) reacted very strongly with viroplasmic inclusions. Virus particles, endoplasmic reticulum and cytoplasmic ribosomes were also labelled with these sera. These results indicate that rotavirus inner capsid proteins are synthesized throughout the cytoplasm and become concentrated in viroplasmic inclusions, while the outer capsid glycoprotein is synthesized primarily on ribosomes of the rough endoplasmic reticulum. Thus, the outer capsid layer appears to be acquired during virus budding into cisternae of the endoplasmic reticulum.
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Proteolytic Activation of Influenza WSN Virus in Cultured Cells is Performed by Homologous Plasma Enzymes
More LessSummaryThe effect of chick embryo allantoic fluid, porcine plasma or canine plasma on virus progeny was studied in cultured chicken, porcine and canine cells infected with influenza WSN virus. Cells incubated either without plasma or with heterologous plasma produced virions which had uncleaved haemagglutinin and low infectivity. Cells incubated with homologous plasma produced highly infectious virions with cleaved haemagglutinin. Little increase of progeny virus infectivity was observed in canine cell-porcine plasma and porcine cell-canine plasma host systems. The addition of protease inhibitors to culture containing homologous plasma, in particular ε-amino-n-caproic acid (an inhibitor of plasminogen activation), suppressed cleavage of haemagglutinin, and virions which had uncleaved haemagglutinin and low infectivity were produced by the cells. It therefore follows that haemagglutinin cleavage and activation of influenza WSN virus infectivity in cultured cells is most efficiently performed by homologous plasma proteolytic enzyme(s). The mechanism of selective plasma-mediated influenza virus proteolytic activation in homologous cells is discussed.
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Dissemination of Herpes Simplex Virus in Nude Mice after Intracutaneous Inoculation and Effect of Antibody on the Course of Infection
More LessSummaryDissemination of herpes simplex virus (HSV) in nude mice after intracutaneous inoculation in the midflank, and the effect of passively administered antibody on the course of infection were investigated. In untreated infected mice the skin lesions developed rapidly and HSV could first be recovered from the homogenate of the dorsal root ganglia on day 3 after infection, from the spinal cord on day 7 and from the brain on day 11. HSV could not be recovered from the blood, spleen or liver. In mice passively immunized with human gamma globulin, development of the skin lesions was rather slow and HSV could not be recovered from the homogenate of the dorsal root ganglia until day 16. From the results of explant culture of the ganglia, HSV was found to have reached the ganglia as early as 48 h after infection, even in mice administered human gamma globulin. The protective action of antibody seems to originate from the inhibition of virus growth not only at the inoculation site but also in the dorsal root ganglia.
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Identification of the Envelope Surface Glycoproteins of Equine Herpesvirus Type 1
More LessSummaryThe structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1–2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.
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Immunochemical Studies of Polioviruses: Identification of Immunoreactive Virus Capsid Polypeptides
More LessSummaryInvestigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and nonneutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The noncorrelation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.
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Vesicular Stomatitis Virus Can Establish Persistent Infections in Syrian Hamsters
More LessSummaryPersistent infections by vesicular stomatitis virus (VSV) of the Indiana serotype were readily established in adult Syrian hamsters following intraperitoneal injection of the virus. Plaque-forming virus, identified as VSV by serological and physical criteria, was isolated from brain homogenates of five hamsters that were tested 3 to 8 months after infection. Four of these animals had exhibited either transient or permanent paralysis, whereas the fifth appeared healthy, during the period of observation. At the time of sacrifice all hamsters had high titres of anti-VSV-neutralizing antibodies in their sera.
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Mumps Virus-persistently Infected Cell Cultures Release Defective Interfering Virus Particles
More LessSummaryTwo human cell cultures, HEp-2 and L-41, persistently infected with mumps virus, produced small amounts of slowly replicating small-plaque infectious virus and detectable amounts of defective interfering virus particles which were similar to standard mumps virus in polypeptide composition, contained subgenomic size RNAs and interfered with standard mumps virus replication in susceptible cells.
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Indomethacin and Aspirin Do Not Inhibit the Antiviral or Anti-proliferative Actions of Interferon
SummaryNeither indomethacin nor aspirin, at concentrations which inhibited the formation of prostaglandins and prevented the interferon-induced increase in the intracellular concentration of cyclic GMP, had any significant effect on the development of the interferon-induced antiviral state either in mouse L1210 cells challenged with vesicular stomatitis virus or in mice infected with encephalomyocarditis virus. Furthermore, neither drug had any significant effect on the interferon-induced inhibition of cell multiplication in cultures of mouse leukaemia L1210 cells. The differences in the effects of these cyclo-oxygenase inhibitors on different interferon effects may provide some insight into the different pathways of interferon action.
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A Human Amniotic Cell Line Yielding High Titres of Human Fibroblast Interferon
More LessSummaryA line of human amniotic cells (UAC) was found to yield large amounts of fibroblast interferon (IFN-β; 100 to 150 IU/103 cells) upon induction with Newcastle disease virus (NDV). UAC cells have a doubling time of about 24 h, and do not require foetal calf serum for growth or optimal IFN yield. The IFN produced was shown to be HuIFN-β by assaying it on homologous and heterologous cells, and by neutralization tests with specific antisera. It could be purified to a specific activity of 0.6 × 107 IU/mg protein by chromatography on Blue Sepharose. Addition of 8 µg poly(A+) RNA from NDV-induced UAC in 100 µl of reticulocyte lysate resulted in the production of 510 ± 340 IU/ml of an IFN that was neutralized only by anti-HuIFN-β serum.
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A Viable Simian Virus 40 Variant with a Deletion in the Overlapping Genes for Virion Proteins VP1, VP2 and VP3
More LessSummaryNucleotide sequence analysis was used to determine the exact location of a deletion in the late region of the SP2 mutant of simian virus 40 (SV40), a viable small-plaque variant isolated from a persistent infection of rhesus monkey kidney cells. The results indicate that six base pairs are deleted from that part of the SV40 genome in which the coding regions for the three virion proteins, VP1, VP2 and VP3, overlap. This implies that all three virion proteins are affected by the deletion. This finding is discussed with respect to the viability of SP2.
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Recombinational Joints in a Simian Virus 40 Variant Generated in a Persistent Infection
More LessSummarySP1, a viable simian virus 40 (SV40) variant isolated from a persistent infection of rhesus monkey kidney cells, contains sequence rearrangements in the untranslated region of the SV40 genome which are transcribed into late mRNA leader sequences and in the region which encodes the large T antigen. Nucleotide sequences about the recombinational junctions in SP1 were determined. The sequence data show that in most instances there was not extensive homology between recombining sequences. The recombinant sequences are discussed with respect to the mechanisms by which they might have been generated.
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Satellite RNA in Particles of Strawberry Latent Ringspot Virus
More LessSummaryRNA from particles of isolate H of strawberry latent ringspot virus (SLRV) comprises species of mol. wt. 2.9 × 106 (RNA-1), 1.4 × 106 (RNA-2) and 0.4 × 106 (RNA-3). Isolates obtained by inoculating plants with RNA-1 plus RNA-2 lacked RNA-3, even after repeated subculturing. RNA-3 was produced when it was inoculated in mixtures with the RNA of such isolates, but not when it was inoculated alone. Most of the nucleotide sequence of RNA-3 was absent from either RNA-1 or RNA-2. RNA-3 is therefore a satellite RNA. SLRV isolates containing or lacking satellite RNA induced indistinguishable symptoms in test plants. Like the genome RNA of SLRV, satellite RNA was found to be polyadenylated and to be bound covalently to protein. Satellite RNA induced the synthesis of a 38000 mol. wt. polypeptide in rabbit reticulocyte lysates. Several properties of SLRV satellite RNA therefore resemble those of tomato black ring virus satellite RNA.
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Infection of Protoplasts from Tobacco Suspension Cultures by Tobacco Mosaic Virus
More LessSummaryProtoplasts isolated from suspension-cultured tobacco cells were inoculated with tobacco mosaic virus (TMV) using a procedure similar to that for tobacco mesophyll protoplasts. Fifty to 70% of protoplasts were infected, as determined by staining with fluorescent TMV antibody. The formation of progeny virus particles was substantiated by electron microscopy of sectioned protoplasts, and the virus yield was assessed by assaying the infectivity of protoplast extracts. The course of TMV multiplication was studied by following the incorporation of [3H]uridine into virus RNA. The results indicated that infection in this system is synchronous and that the rate of virus multiplication is comparable to that in mesophyll protoplasts. A system of undifferentiated, growing plant cells was thus established for one-step growth of TMV.
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