- Volume 47, Issue 2, 1980
Volume 47, Issue 2, 1980
- Articles
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- Animal
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Synthesis of Virus DNA and Polypeptides by Temperature-sensitive Mutants of Rabbitpox Virus
More LessSUMMARYEighteen temperature-sensitive (ts) mutants of rabbitpox virus were examined for defects in synthesis of DNA and protein. Two mutants (ts-3 and ts-16) were defective in DNA synthesis (DNA−), since both incorporated significantly less than wild-type amounts of labelled thymidine into acid-precipitable material when infected cells were incubated at the restrictive temperature. Both these mutants gave only the ‘early’ class of virus polypeptides when infected cell extracts were examined by SDS-polyacrylamide slab gel electrophoresis following incubation at 40 °C. Nine of the remaining sixteen DNA+ ts mutants (ts-1, ts-2, ts-6, ts-12, ts-15, ts-17, ts-31, ts-32 and ts-33) synthesized wild-type levels of most virus polypeptides at 40 °C; six DNA + ts mutants (ts-7, ts-8, ts-9, ts-11, ts-23 and ts-24) were defective in the post-translational cleavage of the polypeptides involved in membrane stabilization and particle assembly; one DNA+ ts mutant (ts-14) synthesized only the ‘early’ class of virus polypeptides, implying that either replicated DNA was not fully functional or that a specific early function was required for late transcription.
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A Comparison of New World Alphaviruses in the Western Equine Encephalomyelitis Complex by Immunochemical and Oligonucleotide Fingerprint Techniques
More LessSUMMARYWe have examined the molecular basis for the observed antigenic differences between isolates of western equine encephalomyelitis (WEE) virus and those of a serologically related alphavirus from the eastern United States designated Highlands J (HJ). The structural proteins of WEE virus isolates have mol. wt. of 55 × 103 (E1), 47 × 103 (E2) and 33 × 103 for the nucleocapsid. The E1 glycoprotein had an isoelectric point (pI) of 6.4 and induced haemagglutination-inhibiting (HI) antibody which was specific for WEE virus. The E2 glycoprotein of WEE virus had a pI of 8.4 and induced antibody which was virus specific by neutralization (PRNT) but cross-reacted with HJ virus in the radioimmune precipitation (RIP) test. Envelope glycoproteins of HJ virus isolates had mol. wt. of 58 × 103 (E1) and 49 × 103 (E2) respectively. The E1 glycoprotein from HJ virus had a pI of 6.8 and induced antibody which reacted specifically in the HI, PRNT and RIP tests. Isolated E2 protein of HJ virus had a pI of 9.1 and induced antibodies which were reactive at equal titre with both WEE and HJ viruses by RIP.
Two-dimensional gel electrophoresis of RNase T1 oligonucleotides of WEE virus and HJ virus genome RNase T1 oligonucleotides revealed that the primary structures of the RNAs of these two serologically related alphaviruses were very distant. The fingerprints of the oligonucleotides from 16 WEE viruses from western and central North America, Mexico and South America were similar to each other and easily distinguished from those of the eight HJ viruses isolated in the eastern United States from Massachusetts to Louisiana.
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Detection of Avian Oncovirus Group-specific Antigens by the Enzyme-linked Immunosorbent Assay
More LessSUMMARYA three-step sandwich enzyme-linked immunosorbent assay was developed for the detection of avian oncovirus group-specific (gs) antigens. The assay procedure was to coat the wells of microtitre plates with hamster anti-gs IgG, react with crude or purified antigen and finally with hamster anti-gs IgG linked to horseradish peroxidase. The sensitivity was 8 picograms (pg) of input avian myeloblastosis virus (AMV) protein, with negligible background. As the ELISA takes less than 2 h to perform, large-scale screening for infected birds is feasible. A blocking assay was also developed for detecting anti-gs antibodies by adding unlabelled antiserum after the antigen step.
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Identification of a Virus-specified Protein in the Nucleus of Vaccinia Virus-infected Cells
More LessSUMMARYA new protein has been detected in the nuclei of vaccinia virus-infected cells. This protein has an apparent mol. wt. of 28000 (VP28) on SDS-polyacrylamide gels and has been detected in Triton X-100-treated nuclei of infected BSC-40, L-929 and CVC cells. Within the infected cells, VP28 was synthesized maximally at 1 to 2 h p.i. in the cytoplasm and accumulated in the nuclei at 4 to 5 h p.i. The appearance of VP28 was not affected by cytosine arabinoside (25 µg/ml), an inhibitor of virus DNA synthesis, or rifampicin (100 µg/ml), an inhibitor of vaccinia assembly, but was inhibited by irradiation of the infecting virions; thus classifying it as an early vaccinia virus gene product. Nuclear-cytoplasmic mixing experiments suggested that the nuclear location of VP28 was not an artefact of the cell fractionation techniques employed. VP28 did not appear to be phosphorylated.
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Isolation and Partial Characterization of Two Forms of Cytoplasmic Nucleocapsids from Measles Virus-infected Cells
More LessSUMMARYTwo species of measles virus nucleocapsids with distinct buoyant densities were isolated from infected AV3 cell homogenates by isopycnic CsCl gradient centrifugation. The more buoyant or ‘light’ form of the nucleocapsid had a density of 1.26 to 1.28 g/ml, whereas the less buoyant or ‘heavy’ nucleocapsid species had a density of 1.30 g/ml. Analysis of the two nucleocapsid species by SDS-polyacrylamide gel electrophoresis showed that both forms possessed two phosphorylated polypeptides with mol. wt. of 69000 and 60000. The heavy form of nucleocapsid consisted solely of these two polypeptide species, while the light form of nucleocapsid had two additional associated polypeptides (VP4, mol. wt. 52000, and 45K, mol. wt. 45000). Ultrastructural and immunofluorescent studies suggest that the two isolated capsid forms represent the two morphologically distinct capsid species observed in vivo. This paper discusses the possible relationship between the two capsid forms and assembly of the virus.
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Detection of a Precursor-like Protein of Bovine Leukaemia Virus Structural Polypeptides in Purified Virions
More LessSUMMARYGel filtration chromatography of disrupted bovine leukaemia virus (BLV) resulted in the isolation of the 25000 mol. wt. major internal protein (p25), two previously uncharacterized proteins of mol. wt. 65000 (p65) and 12000 (p12), and a mixture of p12 and a protein of mol. wt. 15000 (p15). The p65 protein does not bind to concanavalin A and its antigenicity is ether resistant. Therefore, this polypeptide is different from the previously described glycoprotein associated with BLV. Radioimmunoprecipitation and competitive radioimmunoassays indicated that the p65 protein shares antigenic determinants with the p25, p15 and p12 proteins, respectively. Furthermore, tryptic peptide mapping demonstrated that p65 contains p25, p15, p12 and a BLV protein of mol. wt. 10000 (p10). These results are consistent with the view that p65 is the precursor of gag gene-derived core proteins of BLV.
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Some Characteristics of an Early Protein (ICP 22) Synthesized in Cells Infected with Herpes Simplex Virus
More LessSUMMARYIn Vero cells incubated at 40°C or treated with azetidine at 37°C, synthesis of a polypeptide (‘C’) of apparent mol. wt. 66000 was stimulated. It was not phosphorylated and was found in the cytoplasmic fraction of cell lysates. In cells infected with herpes simplex virus type 1 (HSV-1) in the presence of azetidine, synthesis of cellular proteins, including polypeptide C, was suppressed and infected cell polypeptides ICP 4, 0, 22 and 27 (apparent mol. wt. 170000, 120000, 75000 and 60000, respectively) were made. All were phosphorylated and accumulated in the nucleus. Messenger RNA for the same four polypeptides was made in cells infected in the presence of cycloheximide. Thus, ICP 22 is distinct from cellular polypeptide C and is probably a virus-specific α polypeptide, although it differs from α ICP 4, 0 and 27 in that its rate of synthesis does not decline rapidly when later polypeptides are produced. It is modified after synthesis in at least two steps, the second of which may require a later virus-specific polypeptide. In cells infected with HSV-2 the synthesis of a polypeptide analogous to ICP 22 could not be detected.
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Alterations in Virus Protein Synthesis and Capsid Production in Infection with DI Particles of Herpesvirus
More LessSUMMARYHigh multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (ρ = 1.724 g/ml) than the DNA (ρ = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al., 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.
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A Sensitive, Single-Radial Diffusion Autoradiographic Zone Size Enhancement Technique for the Assay of Influenza Haemagglutinin
More LessSUMMARYA novel radial-diffusion technique in agarose gels is described which is applicable to the assay of small concentrations of influenza haemagglutinin (HA) antigen. The method is based on single-radial-diffusion zone size enhancement (ZE), using autoradiography to demonstrate antigen-antibody reactions. The ZE technique is capable of reliably assaying concentrations of haemagglutinin as low as 0.1 µg/ml, which represents a 20- to 40-fold increase in sensitivity over conventional single-radial-diffusion techniques. The ZE response is dependent upon the antigenic identity of the 125I-labelled ‘marker’ HA and the HA antigen being assayed. Haemagglutinins showing variation within a subtype (e.g. H3) show reduced ZE responses and thus the method has potential value for distinguishing between closely related HA antigens.
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Two Particle Types of Avian Infectious Bronchitis Virus
More LessSUMMARYTwo distinct types of avian infectious bronchitis virus (IBV) particles were isolated on sucrose density gradients. The higher density particles banded at 1.18 g/ml, had typical coronavirus morphology and contained all the structural polypeptides and a complete genome. The less dense particles of density 1.13 g/ml appeared to have typical coronavirus morphology, although they were much more flattened than the more dense particles. Furthermore, these particles lacked the ribonucleoprotein polypeptide and the genome, although all the other polypeptides were present in the same amounts as in the denser particles.
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Inhibition of RNA and Protein Synthesis in Interferon-treated HeLa Cells Infected with Vesicular Stomatitis Virus
More LessSUMMARYSynthesis of vesicular stomatitis virus RNA and protein is almost completely inhibited in HeLa cells treated with relatively high doses of human fibroblast interferon. With lower concentrations of interferon virus replication is inhibited, but near normal amounts of virus RNA are found in cells infected at a m.o.i. of 10. All the virus RNA species are found in these cells with the exception of genomic size RNA. In contrast, synthesis of all the virus proteins is equally inhibited in proportion to the interferon concentration used to treat the cells. This inhibition is due to a decline in the rate of protein synthesis, which occurs in interferon-treated cells sooner after infection than in untreated cells. The decreased rate of protein synthesis is accompanied by a change in the polysome pattern of infected cells, characterized by polysome run-off and increase in 80S ribosomes. At the same time, a larger proportion of the virus poly(A)-containing RNA is not associated with polysomes in interferon-treated cells than in control cells. The non-polysomal virus RNA has a sedimentation rate identical with that of polysomal virus RNA. Possible causes for the decline in the rate of protein synthesis observed in interferon-treated cells are discussed.
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Symmetrical Arrangement of the Heterologous Regions of Rabbit Poxvirus and Vaccinia Virus DNA
More LessSUMMARYCleavage sites for the restriction endonucleases EcoRI, KpnI and XhoI were mapped on rabbit poxvirus and vaccinia virus DNA. These physical maps were used to analyse the structural variations between the two DNAs. Two specific heterologous regions, symmetrically arranged at each end of the genomes, have been identified. Region 1, representing the exterior part of the terminal repetition, appears to contain unrelated sequences in each DNA and accounts for the difference in length of the two genomes. Region 2, separated from region 1 by a conserved part of the terminal repetition, is located at the transition from repeated to unique DNA sequences. Its overall length of about 4 megadaltons is well conserved and it contains individual DNA-specific as well as conserved restriction sites. The major central part of the genomes (over 100 megadaltons) contains very few, widely dispersed restriction site variations.
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The Prevalence of Naturally Occurring Antibodies to Human Syncytial Virus in East African Populations
More LessSUMMARYA seroepidemiological study of naturally occurring antibodies to the human syncytial virus has been carried out by means of an indirect immunofluorescence test on 639 East Africans, consisting of 493 normal Ugandans, 66 Kenyan patients with nasopharyngeal carcinoma (NPC), and 80 Kenyan and Tanzanian patients with various other tumours or non-cancerous conditions. It was found that 3.4% of the normal individuals had antibodies to the virus and serial serum samples were available from 14 of these, permitting the study of antibody class in seroconversion and antibody persistence. As in an earlier survey, a significantly higher incidence of antibodies was found amongst NPC patients. Blocking and indirect immunofluorescence tests with simian foamy viruses (SFV) showed some cross-reactivity between SFV 6 and the human syncytial virus, but not identity. The results are discussed in relation to the very real occurrence of natural infection by human syncytial virus in certain geographical regions.
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Biological and Molecular Biological Characterization of the Virus Progeny from Transformed Clones MuSV-124 and MuSV-349: Evidence for MuLV-specific Nucleotide Sequences in the MoMuSV Size Class of RNA from MoMuSV-124
More LessSUMMARYThe genetic information of MoMuSV-349 and MoMuSV-124, two clones of productively transformed TB cells, was distributed between two size classes of RNA (mol. wt. 2.9 × 106 and 1.9 × 106) in the proportions of 5:1. Some preparations of MoMuSV-124 lacked the large RNA. The virions produced by both clones also contained all the nucleotide sequences of Moloney leukaemia virus and the ratio of MuSV:MuLV produced by the two clones differed markedly. The distribution of the sequences specific for Moloney murine leukaemia virus (MoMuLV) between the two size classes of RNA was studied using molecular hybridization to DNA probes complementary to and representative of: (i) the Moloney murine sarcoma virus (MoMuSV) RNA genome (mol. wt. 1.9 × 106); (ii) those nucleotide sequences shared by MoMuSV and MoMuLV; (iii) nucleotide sequences specific for MoMuSV; (iv) nucleotide sequences specific for MoMuLV. The only detectable Moloney leukaemia virus-specific nucleotide sequences present in MoMuSV-124 virions were in the RNA of mol. wt. 1.9 × 106, whereas these sequences were detected in the RNA of mol. wt. 2.9 × 106 isolated from MoMuSV-349 virions. The biological properties of the replicating information in MoMuSV-124 suggest that, consistent with the small size of RNA, it is defective, whereas MoMuSV-349 produces virions containing an intact MoMuLV genome, competent for replication.
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Preparative Isoelectric Focusing of Poliovirus Polypeptides in Urea-Sucrose Gradients
More LessSUMMARYIsoelectric focusing in urea using small density gradient columns for dissociated poliovirus resulted in a complete separation of the four virus polypeptides. Identification and purity of VP1, 2, 3 and 4 was shown by SDS-polyacrylamide gel electrophoresis. The isoelectric points were determined and compared with the values obtained in gels. The recovery of the individual polypeptides was about 80%. Up to 500 µg of poliovirus per tube could thus be separated in a one-step procedure giving pure and SDS-free poliovirus polypeptides.
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The Use of an Indirect Enzyme-linked Immunosorbent Assay to Detect Baculovirus in Larvae and Adults of Oryctes rhinoceros from Tonga
More LessSUMMARYAn indirect sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the baculovirus of Oryctes rhinoceros in purified virus preparations and in extracts of O. rhinoceros larvae and adults collected in the field in Tonga. A 20 ng amount of purified virus was detected unequivocally but precise quantitative determinations of virus in insect extracts proved difficult because virus in solutions containing host protein gave lower absorbance values than a comparable purified virus standard. The implications for use of ELISA in ecological studies are discussed together with the choice of appropriate standards.
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Differential Host Ranges for in vitro Infectivity of Mouse Mammary Tumour Viruses
More LessSUMMARYHost-range variants of mouse mammary tumour viruses (MMTVs) have previously been shown to productively infect cells of several species in vitro (Howard & Schlom, 1978). We report here that cell lines have been identified which exhibit differential restriction for replication of different MMTV variants. In addition, a cell line has been identified that changes as a function of passage in culture from being permissive to being restrictive to infection with MMTVs. MMTVs propagated in both murine and non-murine cells retained their antigenic reactivities in a group-specific radioimmunoassay for MMTVs and demonstrated no evidence for the presence of type-C viruses as determined by a variety of techniques. These studies thus establish in vitro cell substrate tropisms that can be used to differentiate between MMTVs.
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Quantification of Influenza Virus Messenger RNAs
More LessSUMMARYThe amounts of specific influenza virus cRNAs present on polyribosomes of cells treated with cycloheximide at various times after infection were measured by quantitative hybridization with 125I-labelled virus RNA segments. All species of cRNA were found associated with polyribosomes at every time point analysed. The relative abundance of the specific RNAs was in the same order as the reported relative synthesis of the influenza virus proteins. However, the concentrations of the cRNAs spanned a considerably smaller range than published protein synthetic rates, suggesting that both transcriptional and translational controls operate to regulate the final levels of influenza virus polypeptide synthesis.
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Multiplication of Human Rotavirus in Cultured Cells: An Electron Microscopic Study
J. Esparza, M. Gorziglia, F. Gil and H. RömerSUMMARYHuman rotaviruses were capable of efficient multiplication in LLC-MK2 cells when the inoculum was pre-treated with trypsin, centrifuged on to the cell monolayer and the infected cells maintained in a medium containing trypsin. However, not all of the human rotavirus isolates used to infect cells resulted in efficient virus production. The ability of human isolates to multiply in cultured cells was studied by direct observation of virus in the electron microscope, by radioactive labelling with 3H-uridine of the newly synthesized virus and by electron microscopic examination of thin sectioned infected cells. With one of the specimens used (F-617) only 5 to 10% of the cells showed evidence of virus multiplication, with the great majority of the infected cells showing numerous complete (double-capsid) virus particles scattered in the cytoplasm. When cells were inoculated with another human specimen (SIB-I), infected cells were more abundant, reaching a maximum of 60%; however, a variety of particle types, probably representing different subviral structures or different steps of rotavirus morphogenesis, were commonly observed. The presence of these aberrant or incomplete virus structures may represent a manifestation of the defectiveness of this virus and may explain the difficulties encountered in its serial passage.
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Volume 105 (2024)
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