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Volume 68,
Issue 6,
1987
Volume 68, Issue 6, 1987
- Review Article
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Some Highlights of Animal Virus Research in 1986
More LessIn 1986 we published a review article entitled ‘Some highlights of animal virus research in 1985’, in which we attempted to summarize important and interesting research on animal viruses published in 1985 (McGeoch et al., 1986b). The limitations of the exercise were recognized: coverage was selective rather than systematic and, outside clearly pre-eminent subjects, was overtly influenced by personal scientific interests. In this article we now present a similar account of animal virology in 1986. Much the same ground rules have been retained. Selectivity remains of the first importance, and our treatment concerns work published in 1986 as far as possible, but with a little flexibility where necessary.
As with 1985, animal virology in 1986 was dominated by work on acquired immune deficiency syndrome (AIDS) retroviruses. Outside this area, however, we were not able to identify any small set of truly outstanding advances, although work in many virus systems clearly made excellent progress. This evaluation posed problems for a review of the type undertaken, in particular for achieving a balanced selection of topics. With this background we also found it more necessary than before to address virus groups in separate sections (representing a step back from the previous, integrative stance). We have chosen to present aspects of research on, first, the AIDS viruses, and then in succession on hepatitis viruses, herpesviruses, poxviruses, papovaviruses, picomaviruses and negative strand viruses, and to finish with a section on virion structure.
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- Bacterial
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Transmembrane Cation Movements during Infection of Lactobacillus lactis by Bacteriophage LL-H
More LessSummaryPhage LL-H-induced cation (K+, Na+, Mg2+, Ca2+, Cd2+) movements in Lactobacillus lactis bacteria have been studied. The effects of the m.o.i. and external cation concentration have been quantified. LL-H-induced effluxes showed cation specificity: K+ but practically no Mg2+ was lost during LL-H infection at low and moderate m.o.i. (up to about 100). Simultaneously to K+ efflux, divalent cation influxes were observed. These were dependent on the m.o.i. and on concentrations of external divalent cations and were concomitant with phage DNA transport, as concluded from the timing of the first phage-promoted biochemical changes in host cell metabolism and from electron microscopical observations. Host energy was not mobilized with phage-induced divalent cation influx. Several features of divalent cation influxes support the view that divalent cations have to be cotransported into the cell as counterions of LL-H DNA. Phage DNA associated with divalent cations may be the basic feature of the divalent cation dependence of LL-H infection.
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- Animal
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Characterization of Monoclonal Antibodies Against the Human Immunodeficiency Virus (HIV) gag Products and Their Use in Monitoring HIV Isolate Variation
More LessSummaryMonoclonal antibodies were raised against the gag proteins of a British isolate of the human immunodeficiency virus (HIV), CBL-1. Seven of the monoclonal antibodies recognized HIV gag-coded proteins of 55000 mol. wt. (p55) and 24000 mol. wt. (p24), three recognized p55/pl8 and three pi8 alone. Cross-competition assays suggested that at least two independent epitopes exist on the p24 cleaved from the p55 precursor, whereas a cluster of closely related epitopes was recognized by the p55/p18 and p18 antibodies. The panel of monoclonal antibodies was then used to compare by immunofluoresence the expression of gag-encoded antigenic determinants on HIV isolates from different geographical locations. Minor changes in epitope expression were observed in isolates from the U.S.A. and Haiti with the most notable changes being detected in isolates from Zaire, Tanzania and Uganda.
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Fine Structure of Cells Infected with Human Cytomegalovirus After Treatment with 9-(1,3-Dihydroxy-2-propoxymethyl)guanine
More LessSummaryInfection with human cytomegalovirus (CMV) is characterized by cytological changes which are readily visualized by electron microscopy using ultrathin sections of infected cells. Treatment of such cells with 9-(l ,3-dihydroxy-2-propoxymethyl)guanine (DHPG), a potent inhibitor of CMV, is effective when initiated at early or late times after infection and the response to such treatment has been studied by fine structural analysis. Inhibition of viral DNA synthesis by DHPG treatment (50 μm) late in virus infection resulted in a cessation of virus growth accompanied by a lack of development and possible regression in skein-like intranuclear inclusions together with a depletion in cytoplasmic dense bodies. Such changes were accompanied by the appearance of nuclear dense bodies. These were also present when virus growth was reduced (5 μm-DHPG) rather than completely inhibited (50 μm-DHPG) by treatment initiated from the time of infection. The nuclear bodies were predominantly of a reticular type structure after the early treatment but mainly of a homogeneous form when virus growth was interrupted at late times. Their presence appeared to be connected with the ability of infected cells to initiate the synthesis of late proteins and their morphology may relate to the extent of such protein synthesis. Unlike cytoplasmic dense bodies, provisional findings on the characterization of the nuclear bodies suggested that the 69K matrix protein was not present in abundance.
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Synthesis of Cytomegalovirus DNA Is an Antiviral Target Late in Virus Growth
More LessSummaryThe mechanism of action of 9-(l,3-dihydroxypropoxymethyl)guanine (DHPG) and phosphonoformic acid (PFA) but not 5-fluorouridinedeoxyribose (FUdR), provides selective action against cytomegalovirus (CMV)-coded events and this was used to demonstrate that the synthesis of viral DNA was continuous during the extended phase of virus growth. The synthesis de novo of viral DNA was measured by restriction enzyme analysis after exposure to [32P]orthophosphate and its interruption by DHPG or PFA resulted in a cessation in the extrusion of infective virus from treated cells. The rate of decline in infectivity appeared to correspond to the failure of cells to maintain the synthesis of late proteins once DNA synthesis was blocked. Thus, regulation of late protein synthesis appeared to be linked to synthesis de novo of viral DNA even at late stages in CMV growth. The synthesis of the polyamines spermidine and spermine, considered obligatory for CMV growth, was unaffected by early or late inhibition of viral DNA and this showed that some virus-induced events were unaffected by the restriction on virus growth by DHPG. This provided evidence that polyamine biosynthesis was a target independent of viral DNA synthesis per se, which may be important in future considerations of combined drug therapies.
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Monoclonal Antibodies to the Latent Membrane Protein of Epstein-Barr Virus Reveal Heterogeneity of the Protein and Inducible Expression in Virus-transformed Cells
More LessSummaryMonoclonal antibodies specific for the ‘latent membrane protein’ (LMP) of Epstein-Barr virus (EBV), one of the effector proteins of EBV-induced B cell transformation, have been generated from mice immunized with a β-galactosidase fusion protein containing the carboxyl half of the B95. 8 strain LMP sequence. Four monoclonal IgG1 antibodies, designated CS. 1, CS. 2, CS. 3 and CS. 4, which together recognized at least three different epitopes on the molecule, were used to examine various aspects of LMP expression in B cell lines transformed in vitro. The pooled CS. 1 to 4 reagent detected the LMPs encoded by each of 20 geographically distinct EBV isolates, despite a degree of inter-isolate heterogeneity in the size and antigenicity of the protein. In cell lines carrying the prototype B95. 8 virus strain, particularly if these were virus producers, an additional lower molecular weight LMP was also detected; this appeared to correspond to the truncated form of the protein already predicted to exist from the analysis of B95. 8 lytic cycle mRNAs. Attempts were made to identify an analogous truncated form of LMP in cell lines carrying other virus isolates after treatment with phorbol ester and/or sodium butyrate to induce virus production. Surprisingly these experiments showed that expression of the full length LMP molecule was itself strongly inducible by these agents; when monitored at the single cell level, this was a generalized response and was not restricted to cells entering a lytic cycle. Expression of LMP in EBV-transformed B cells therefore appears to be subject to a distinct type of regulation.
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Nuclear DNA-binding Proteins Determined by the Epstein-Barr Virus-related Simian Lymphotropic Herpesviruses H. gorilla, H. pan, H. pongo and H. papio
More LessSummaryNuclear DNA-binding proteins were extracted from lymphoblastoid cell lines transformed with Epstein-Barr virus (EBV) or with the related lymphotropic herpesviruses of gorilla (Herpesvirus gorilla), chimpanzee (H. pan), baboon (H. papio) or orang-utan (H. pongo). They were immunoblotted with the sera of all four simian species in comparison with EBV antibody-positive human sera. Eight nuclear proteins were identified, and were designated GONA-1 and GONA-2 for H.gorilla-determined nuclear antigens, PANA-1A, PANA-1B and PANA-2 for H. pan, PONA-2 for H. pongo and HUPNA-1 and HUPNA-2 for H.papio-determined nuclear antigens. One of two tested HUPNA-2-positive baboon sera and one PONA-2-positive orang-utan serum also reacted with EBNA-2 in EBV-transformed cells. A human serum that contained antibodies to all five EBNA proteins cross-reacted only with PANA-2 and PONA-2. Monospecific anti-peptide antibodies against EBNA-2, type A, also reacted with PONA-2, but not with the other simian nuclear antigens. The data provide evidence that EBV-like simian lymphotropic herpesviruses induce EBNA-like nuclear antigens and that EBNA-2 and some simian EBNA-related proteins contain an epitope that has been conserved during the evolution of the EBV family of viruses.
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Characterization of Bovine Viral Diarrhoea-Mucosal Disease Virus-specific Proteins in Bovine Cells
More LessSummaryThe presence of virus-specific polypeptides in bovine viral diarrhoea-mucosal disease (BVD) virus-infected bovine cells was studied by radiolabelling in the presence of a hypertonic initiation block (HIB) and by analysis by SDS-PAGE. These experiments were complemented by radioimmunoprecipitations with anti-BVD hyperimmune serum of infected cells labelled under isotonic conditions. A total of 12 polypeptides (M r 165, 135, 118, 80, 75, 62, 56 to 58, 48, 37, 32, 35 and 19, all × 10−3) were identified in infected cells. Time course analysis of the induction of the viral polypeptides indicated that they could be detected as early as 4 h post-infection and their synthesis reached a plateau between 12 and 20 h post-infection. The most abundant polypeptides were the ones that could be detected earliest. HIB was found to be an excellent adjunct to existing techniques in the identification of viral polypeptides. Seven of these polypeptides had not been reported previously. This is the first report of the direct detection of BVD virus-induced polypeptides in infected cells without the aid of immunoprecipitation. The sum of the molecular masses of these polypeptides is greater than the coding capacity of the genome; therefore precursor-product relationships must exist between these polypeptides.
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Glycoproteins of Bovine Viral Diarrhoea-Mucosal Disease Virus in Infected Bovine Cells
More LessSummaryBovine cell cultures infected with bovine viral diarrhoea-mucosal disease (BVD) virus were radiolabelled with l-[35S]methionine or d-[2-3H]mannose followed by analysis of the labelled polypeptides by radioimmunoprecipitation and polyacrylamide gel electrophoresis in one and two dimensions. Six glycoproteins were detected in infected cells. Two abundant species had M r of 48K and 56K to 58K while the less abundant species had M r of 118K, 75K, 65K and 25K. When cells were radiolabelled with l-[35S]methionine in the presence of tunicamycin 56K to 58K migrated with apparent masses of 54K (a minor species) and 48K to 50K (the major molecular species) in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4K to 6K reduction in the apparent molecular mass of 56K to 58K yielding a single 52K digested product, indicating that theheterogeneity of 56K to 58K was due to differences in the oligosaccharide moieties. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a critical role in the infectious cycle of BVD virus.
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Replication of Heliothis zea Nuclear Polyhedrosis Virus in Insect Cell Culture: Virus-induced and Structural Proteins
More LessSummaryThe replication of Heliothis zea nuclear polyhedrosis virus was studied in the H. zea (IPLB-HZ-1075A) insect cell line. Infectious extracellular virus was first detected at 24 h post-infection (p.i.) and reached a maximum titre of 1 × 107 p.f.u./ml at 90 h p.i. Viral DNA synthesis was detected by 14 h p.i. and increased exponentially until 32 h p.i. Using [35S]methionine pulse-labelling, 38 virus-induced proteins were identified, ranging in mol. wt. from 13K to 128K. Virus-induced protein synthesis was temporally regulated, with new proteins detected between 8 and 28 h p.i. Host cell protein synthesis continued throughout the virus replication cycle (48 h), although at gradually decreasing rates. Fifteen extracellular virus structural proteins were identified, and all but one comigrated with virus-induced proteins. Thirty-two structural proteins of tissue culture-derived, occluded virus were identified using silver staining procedures.
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Expression of Bluetongue Virus Group-specific Antigen VP3 in Insect Cells by a Baculovirus Vector: Its Use for the Detection of Bluetongue Virus Antibodies
More LessSummaryDNA representing RNA segment 3 of bluetongue virus (BTV) serotype 17, corresponding to the gene that codes for a group-specific antigen VP3, has been inserted into a baculovirus transfer vector in lieu of the 5′ coding region of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). After cotransfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived recombinant transfer vector DNA, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with one of these recombinant viruses, a protein that was similar in size and antigenic properties to the BTV VP3 protein was synthesized. Antibodies raised in mice or rabbits to the baculovirus-expressed VP3 protein immunoprecipitated the VP3 protein of BTV-17 as well as that of BTV-10. The expressed antigen reacted with antisera representing four U.S.A. BTV serotypes in an indirect ELISA test.
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Neutralization of Foot-and-Mouth Disease Virus Can Be Mediated Through Any of at least Three Separate Antigenic Sites
More LessSummarySeven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformation- dependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP1. The sequence of most or all of the VP1 coding region of 10 escape mutants of that site plus three parental isolates was determined by primer extension sequencing. At least five amino acids were found to be involved but in only one case (residue 148 of VP1) did a change at that residue produce complete resistance to neutralization. Partial resistance was produced by changes at residues 144,154 or 208 of VP1 or another residue(s), as yet undefined, that is probably in one of the other capsid polypeptides. Thus the site defined by these mutants was made up of at least three regions, the region involving residues 144 to 154 of VP1, the region encompassing residue 208 from the COOH terminus of VP 1, plus a region, probably of VP2 or VP3, encompassing the undefined residue(s).
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Surface Structure and RNA-Protein Interactions of Foot-and-Mouth Disease Virus
More LessSummaryThe surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54 % of the residues of VP 1, 15 % of the residues of VP2 and 37 % of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VP1, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.
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The Effect of L3T4 T Cell Depletion on the Pathogenesis of Theiler’s Murine Encephalomyelitis Virus Infection in CBA Mice
More LessSummaryTheiler’s murine encephalomyelitis virus (TMEV) gives rise to a biphasic disease of the central nervous system (CNS) following intracranial inoculation of susceptible strains of mice. The early phase, during the first month, resembles poliomyelitis and in the late phase the mice suffer from inflammatory demyelination reminiscent of multiple sclerosis. In order to investigate the role of helper T cells in the acute and chronic phases of the disease we depleted mice of their L3T4 T cells in vivo with rat monoclonal antibodies, prior to infection and prior to the onset of clinical signs of demyelination. Mice depleted of their helper cells failed to produce antibodies to TMEV and consequently were unable to clear virus from the CNS and died within the first month of infection. Depletion of T cells before the demyelinating phase of the disease resulted in a marked decrease in the incidence of disease from 77% of the immunocompetent animals with clinical signs of paralysis to 36%. Immunocompetent TMEV-infected mice also developed antibodies against myelin suggesting that autoimmune mechanisms may play a role in TMEV-induced demyelination.
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Antigenic Structure of Human Interferon ω1 (Interferon α II1): Comparison with Other Human Interferons
More LessSummaryRecombinant human interferon (IFN) ω1 (IFN-α II1) was compared with other human IFNs with regard to antigenic structure, using polyclonal and monoclonal antibodies. Antisera to recombinant IFN-α1 or IFN-α2c cross-neutralized the antiviral activity of other species of IFN-α, but not that of IFN-ω1. Antisera to IFN-β or IFN-γ likewise had no effect on IFN-ω1. Conversely, antiserum to IFN-ω1 did not neutralize any of the other IFNs examined (IFN-α1, -α2c, -α10, -αB, -αF, -β, -γ). None of four monoclonal antibodies to IFN-α, including a broad-spectrum antibody that neutralized all five recombinant species of IFN-α tested, had any effect on the biological activity of IFN-ω1. Various immunoassays for IFN-α did not recognize IFN-ω1. In immunopre-cipitation experiments, radiolabelled IFN-α2c was not precipitated by antiserum to IFN-ω1. In contrast, antisera to human leukocyte IFN or ‘lymphoblastoid’ IFN did neutralize IFN-ω1, although with titres significantly lower than those towards the immunogen or recombinant species of IFN-α. In conclusion, human IFN-ω1 is antigenically different from human IFN-α, IFN-β or IFN-γ, but is a component of ‘natural’ mixtures of IFN species produced by virus-induced leukocytes or Burkitt’s lymphoma cells.
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Rat Glial C6 Cells are Defective in Murine Coronavirus Internalization
More LessSummaryRat C6 glial cells were resistant to infection by several strains of murine coronaviruses. The restriction was not at the adsorption stage, since virus adsorbed to the C6 cells in a similar manner to mouse L cells which supported a lytic infection. The virus could not be internalized by the C6 cells. However, if the virus was introduced into the C6 cells by polyethylene glycol fusion, viral replication occurred and progeny virions were released from the infected cells. These studies indicated that the C6 cells were restrictive to coronavirus replication by preventing the early penetration stage of the viral replicative cycle.
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Sequence and N-terminal Processing of the Transmembrane Protein E1 of the Coronavirus Transmissible Gastroenteritis Virus
More LessSummarySequencing of part of a clone from a transmissible gastroenteritis virus genome cDNA library led to the identification of the gene encoding the E1 matrix protein. The amino acid sequence of the primary translation product predicts a polypeptide of 262 residues which shares many features with the previously characterized murine hepatitis virus and infectious bronchitis virus E1 proteins. However, N-terminal amino acid sequencing revealed that a putative signal peptide of 17 residues was absent in the virion-associated polypeptide. The predicted mol. wt. of the mature unglycosylated product, 27 800, is in agreement with the experimental M r value.
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Fusion Glycoprotein of Measles Virus: Nucleotide Sequence of the Gene and Comparison with Other Paramyxoviruses
More LessSummaryThe sequence of the fusion (F) glycoprotein mRNA of the Hallé strain of measles virus was determined from a cDN A clone representing the entire length of the mRNA. It contained 2384 nucleotides, excluding poly(A), with a 5′ consensus sequence typical of paramyxoviruses and a 3′ terminus found in measles virus mRNAs. The coding sequence was preceded by an unusually long (580 nucleotide) 5′ non-translated region, which contained 44% cytosine. The longest open reading frame coded for a polypeptide of 553 amino acids with a predicted molecular weight of 59·84K. Comparison of the sequence with that of the Edmonston strain of measles virus showed that the gene is highly conserved. No amino acid differences were observed between the two strains. The F polypeptide had three regions of high hydrophobicity: an N-terminal signal peptide, the N-terminus of FI and a C-terminal membrane-spanning region. The four potential asparagine-linked glycosylation sites (one in the signal peptide) were all in the F2 subunit. Comparison of the measles virus F amino acid sequence with other paramyxoviruses revealed homologies with these viruses. Certain regions such as the N terminus of F1 and ten cysteine residues which probably impose structural restraints were highly conserved.
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Appearance of a Soluble Form of the G Protein of Respiratory Syncytial Virus in Fluids of Infected Cells
More LessSummaryWe examined the antigenic reactivities and virion associations of glycoproteins that were released into the culture fluids of cells infected with respiratory syncytial (RS) virus. Culture fluids and cell extracts were obtained from cells 24 to 30 h after they were infected with the Long strain of RS virus. Radioimmune precipitation of [3H]glucosa- mine-labelled glycoproteins by large glycoprotein (G)-specific or fusion protein (F)- specific monoclonal antibodies (MAbs) revealed that the G, F1 and F2 proteins were present in cell extracts but only the G protein was clearly evident in culture fluids. A glycoprotein (M r 43K) which may be a precursor or a breakdown product of the G protein was also precipitated by the G-specific MAb from cell extracts and culture fluids. The G protein in culture fluids was slightly smaller (M r 82K) than the G protein in cell extracts (Afr 88K). An abundant or heavily labelled, 18K glycoprotein in the fluids of virus-infected but not of mock-infected cells was weakly precipitated by the F- specific MAb; this suggested that the 18K protein shares epitopes with the fusion protein of RS virus. The absence of F1 and F2 polypeptides from culture fluids is evidence that the cells, which contained an abundance of these proteins, were intact. To determine whether any of the viral glycoproteins released by infected cells were soluble (non-virion-associated), culture fluid was subjected to rate zonal centrifugation in a 10 to 50% sucrose gradient. An assay of fractions using a MAb-capture ELISA for the nucleocapsid (N) and F proteins revealed a peak of activity, due to virions, in the centre of the gradient, and a strong signal for the N protein at the top of the gradient suggesting that N protein was released from intact cells. Radioimmune precipitation of glycoproteins from the fractions at the top of the gradient using a hyperimmune guinea- pig serum revealed the G protein and a heterogeneous band which had the electrophoretic mobility of the 43K protein. Neither the F1 nor the F2 protein was present in these fractions thus suggesting that virions had remained intact. These results showed that a soluble form of the G protein of RS virus is released into the culture fluids of intact, infected cells. Several theories concerning viral and non-viral origins for the 18K protein are discussed.
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Virological Aspects of Measles Virus-induced Encephalomyelitis in Lewis and BN Rats
More LessSummaryLewis and Brown Norway (BN) rats which are susceptible or resistant to autoimmune reactions against brain antigen, respectively, were inoculated intracerebrally with a neurotropic measles virus. Suckling rats died from a rapidly fatal acute encephalopathy (AE). With increasing age Lewis rats developed a subacute measles encephalomyelitis (SAME) whereas BN rats showed a clinically silent encephalitis (CSE). Infectious virus could occasionally be recovered from SAME animals using cocultivation techniques but not from BN rats with CSE. With monoclonal antibodies against measles virus, viral proteins were localized in brain tissue. Nucleocapsid and phosphoprotein were detected in infected brain cells of all animals with AE, SAME and CSE, whereas measles virus haemagglutinin, fusion and matrix proteins were either reduced or absent, suggesting a restricted synthesis of measles virus envelope proteins. These data suggest that the different diseases of the two rat strains are related to the immunogenetic background rather than to the replication of measles virus in the central nervous system. This animal model provides the opportunity to investigate further the events occurring during establishment of measles virus persistence in the brain, and the genetic control of associated immunological and immunopathological reactions.
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ICTV Virus Taxonomy Profile: Rhabdoviridae 2022
Peter J. Walker, Juliana Freitas-Astúa, Nicolas Bejerman, Kim R. Blasdell, Rachel Breyta, Ralf G. Dietzgen, Anthony R. Fooks, Hideki Kondo, Gael Kurath, Ivan V. Kuzmin, Pedro Luis Ramos-González, Mang Shi, David M. Stone, Robert B. Tesh, Noël Tordo, Nikos Vasilakis, Anna E. Whitfield and ICTV Report Consortium
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