- Volume 69, Issue 8, 1988
Volume 69, Issue 8, 1988
- Review Article
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Biochemistry and Immunology of Infectious Bursal Disease Virus
More LessIntroduction. The aetiological agent of infectious bursal disease (IBD), IBD virus (IBDV), belongs to a new group of viruses referred to as ‘birnaviruses’ (Dobos et al., 1979), which has been characterized only recently (Brown, 1986). There are excellent reviews dealing with the clinical, pathological, serological and epidemiological aspects of IBDV infection (Faragher, 1972; Becht, 1980; Okoye, 1984; Cummings et al., 1986). The molecular biology of birnaviruses has also been reviewed (Dobos & Roberts, 1983) but with an emphasis on infectious pancreatic necrosis virus (IPNV), the birnavirus genus prototype. The purpose of the present review is to compile information on structural and immunological aspects of IBDV. These are subjects of much recent interest, and have great relevance to the control of IBD in chickens.
IBD is a highly contagious viral disease of young chickens which is characterized by destruction of the lymphoid cells in the bursa of Fabricius; other lymphoid organs are also affected but to a lesser degree (Cheville, 1967).
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Appearance of Influenza A Virus Antigenic Variants after Treatment of Infected MDCK Cells with Human Leukocytes
More LessSummaryExudation of polymorphonuclear leukocytes (PMN) from the infected mucosa is a characteristic feature of influenza virus infection. Since reactive oxygen species generated by PMN can be strong mutagens, the possibility of production of antigenic variants of the virus by virus-PMN interaction was investigated. Cloned influenza A NWS (H1N1) virus multiplying in Madin-Darby canine kidney cells was treated with human peripheral PMN. Assays in the presence and absence of monoclonal antibody to the cloned virus showed a seven- to ten-fold increase in the frequency of variants in the presence of PMN. The mutagenic effect was abolished by addition of superoxide dismutase to the culture.
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Membrane Fusion by Peptide Analogues of Influenza Virus Haemagglutinin
More LessSummaryWe have studied the interactions of synthetic peptides corresponding to the sequence of the amino terminus of the HA2 subunit of influenza virus haemagglutinin with artificial lipid membranes. The peptides could fuse cholesterol-free liposomes at neutral as well as acid pH; however, liposomes containing cholesterol could only be fused below pH 6. The fusion process caused leakage of aqueous liposomal contents. Peptides with amino acid substitutions had fusion properties similar to whole haemagglutinin molecules with the corresponding sequence changes. Non-fusogenic peptides still interacted with the membrane but did not cause leakage of liposomal contents. A correlation between the α-helical content of peptide and its fusogenicity was noted, but this was not absolute. The results reported here support suggestions for a role of the amino terminus of HA2 in virus-endosome fusion.
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The Intracellular Distribution of Influenza Virus Matrix Protein and Nucleoprotein in Infected Cells and Their Relationship to Haemagglutinin in the Plasma Membrane
More LessSummaryPre- and post-embedding immune electron microscopy techniques employing ferritin and large and small gold markers to detect cell surface and intracellular antigens respectively, have been combined in a study of influenza virus-infected cells. This has permitted, for the first time, the simultaneous detection of intracellular virus matrix protein (M), nucleoprotein (NP) and membrane haemagglutinin (HA). The technique facilitated an investigation of the possible physical interrelationship between these three proteins both in the infected cell, and on the infected cell membrane. Electron-dense bodies uniformly labelled by antibody to M protein were observed in the nucleus and cytoplasm. Similarly, NP was detected in both the nucleus and cytoplasm. Approximately 50% of the nuclear NP was located in close proximity to the M protein-containing dense bodies but mainly on the perimeter of the structures. A similar relationship of NP to the M-containing dense bodies was observed in the cytoplasm. M protein and NP were readily detected in sections of budding virions. Labelling of these proteins was also observed on the cytoplasmic face of the plasma membrane but the density of labelling only occasionally approached that of newly formed virions. These findings suggest that budding occurs very quickly after the internal proteins arrive at the plasma membrane. Double labelling experiments on the cell surface indicate that NP and HA behave as independent molecules and do not form tight complexes with each other.
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The IgG Subclass Responses Induced by Wild-type, Cold-adapted and Purified Haemagglutinin from Influenza Virus A/Queensland/6/72 in CBA/CaH Mice
More LessSummaryThe IgG subclass and IgA responses were investigated in CBA/CaH mice after inoculation with wild-type (wt) and cold-adapted (ca) derivatives of influenza A/Queensland/6/72 virus, and with purified haemagglutinin (H3) derived from the wt strain of the same virus. Intranasal inoculation of the wt and ca viruses resulted in responses dominated by IgG2a in serum, saliva and lung secretions, whereas an intramuscular injection of purified H3 elicited the production of all four IgG subclasses in serum and IgG2b and IgG3 in saliva and lung secretions. The source of IgG on mucosal surfaces was from local production and was not a transudate from serum, as demonstrated by the lack of albumin in saliva and lung secretions, and by the appearance in saliva and lung samples of IgG subclasses not present in serum at the time of sampling. The level of IgA on mucosal surfaces was influenced by the growth restrictions of intranasally inoculated ca virus, resulting in higher levels of IgA in saliva, whereas wt virus, able to replicate at higher temperatures, induced higher levels of IgA in lung secretions. The purified H3 inoculated by the intramuscular route elicited lower levels of IgA in serum, saliva and lung secretions than either the wt or ca viruses.
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Mechanism of Recovery from Acute Virus Infection. VI. Replication of Lymphocytic Choriomeningitis Virus in and Clearance from the Foot of the Mouse
More LessSummaryThe hind foot was chosen for study of the mechanism by which adult mice clear lymphocytic choriomeningitis (LCM) virus. The T cell-mediated swelling that follows the local inoculation of virus allows parallel investigation of the infectious process and delayed-type hypersensitivity in one organ. A dose of 105 mouse infectious units (IU) was optimal, and in all mouse strains tested foot swelling commenced 6 days after injection, with maximal response on days 7 and 8. When mice were sensitized by intravenous (i.v.) infection and challenged locally with infectious virus, the extent of swelling depended on both doses of virus and was most extensive when the interval between primary inoculation and local elicitation was 10 days. The rates of replication of the virus and its clearance were similar in the feet of mice of four strains tested, varying with regard to LCM virus-specific cell-mediated immunity. In CBA/J mice, virus elimination from the foot was followed for a longer time period and was incomplete up to 100 days after infection. A protocol for determining adoptive immunization was established; local inoculation of 105 IU was followed 22 h later by i.v. infusion of 1 × 108 unselected or 4 × 107 T cell-enriched cells from the spleens of syngeneic donors that had been infected i.v. 7 or 8 days previously with 103 IU. The concentrations of virus in the recipients’ feet began to decline 2 to 3 days thereafter. Adoptive immunization by local inoculation of immune spleen cells was less successful, apparently because the virus multiplied in transferred cells.
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Deleted Viral RNAs and Lymphocytic Choriomeningitis Virus Persistence in vitro
More LessSummaryLymphocytic choriomeningitis virus (LCMV) infection of most tissue culture cell lines results in a non-cytopathic persistent infection. Persistent infections in vitro share many characteristics with persistent LCMV infection of mice; both are associated with decreased titres of infectious virus, restricted accumulation of viral glycoproteins at the surface of infected cells and the generation of interfering particles. We have used gel electrophoresis and hybridization techniques to analyse LCMV gene expression during persistent infection of a number of tissue culture cell lines. Our study has demonstrated that, although deleted viral RNAs can be detected during persistent LCMV infection in vitro, there may not be an obligatory association between deleted RNAs and persistence. In addition, we have found that LCMV interfering activity can be produced in the apparent absence of deleted intracellular viral RNAs.
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Genetic Variation of Murray Valley Encephalitis Virus
More LessSummaryThe genomes of 21 isolates of Murray Valley encephalitis virus (MVE) from Australia and Papua New Guinea were characterized and compared using RNase T1 oligonucleotide fingerprinting. Most Australian isolates grouped in clusters that were linked with a similarity coefficient of greater than 75%, indicating substantial homogeneity. Two isolates grouped as a cluster that linked with other isolates at a level of 67%. These two isolates, one from the north and one from the south-east of Australia were very similar and could demonstrate the movement of MVE between these areas. This notion is substantiated by genetic homogeneity of isolates from the Kimberley region and from south-eastern Australia. One Australian isolate (OR 156) and the Papua New Guinea isolate (MK 6684) were substantially different from each other as well as from the other isolates. No evidence was found for a poly(A) tract in the genome of MVE.
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Detection of Virus-specific Antigen in the Nuclei or Nucleoli of Cells Infected with Zika or Langat Virus
More LessSummaryTwo monoclonal antibodies (MAbs) with molecular specificities for either the viral envelope glycoprotein (MAb 541) or the non-structural NS1 glycoprotein (MAb 109) were derived using West Nile and yellow fever (YF) viruses respectively. Their antigenic reactivity with a large number of flaviviruses was tested by indirect immunofluorescence microscopy. Both produced cytoplasmic fluorescent staining patterns with the homologous virus against which they were raised. Additionally, MAb 541 reacted with two substrains of YF virus whereas MAb 109 reacted with Bussuquara, YF and Ntaya viruses. These reactions were exclusively cytoplasmic. Two unexpected patterns of fluorescent labelling were observed when the antibodies were tested with Zika and Langat viruses. MAb 541 produced fluorescent staining of the nuclei, but not the cytoplasm, of cells infected with Zika virus and MAb 109 labelled only the nucleoli of cells infected with Langat virus. Double-labelling experiments showed that the nuclear fluorescent label was confined to virus-infected cells, and antibody absorption experiments with virus-infected cell packs confirmed the virus specificity of the nuclear antigen. The unexpected presence of virus-specific antigen in the nuclei or nucleoli of Zika or Langat virus-infected cells brings into question the role of the nucleus in flavivirus replication.
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Dengue 2 Virus Envelope Protein Expressed by a Recombinant Vaccinia Virus Fails to Protect Monkeys against Dengue
SummaryA cDNA copy of the dengue (DEN) 2 virus genome region encoding the virion capsid, membrane and envelope structural proteins has been inserted into vaccinia virus (VV) DNA under the control of its 11K late promoter. The DEN-2 envelope protein was expressed and processed in cells infected with the VV recombinant (VV/D2S). No DEN-2 virus antibody response was detected in mice, hamsters or monkeys vaccinated with VV/D2S. Furthermore, a viraemia was observed in recombinant-vaccinated monkeys after challenge with infectious DEN-2 virus.
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Stable Expression of the Hepatitis B Virus Surface Antigen Containing Pre-S2 Protein in Mouse Cells Using a Bovine Papillomavirus Vector
More LessSummaryThe large Bg/II fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.
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Antigenic Variation among 173 Strains of Type 3 Poliovirus Isolated in Finland during the 1984 to 1985 Outbreak
More LessSummaryAntigenic properties of 128 clinical type 3 poliovirus isolates of the 1984 to 1985 Finland outbreak from 95 persons and 45 strains from sewage water specimens were evaluated using five neutralizing monoclonal antibodies (MAbs) directed against an antigenic site (designated site 1) on VP1 at amino acids 89 to 100. All five MAbs neutralized the type 3 poliovirus strains used in the vaccines, P3/Saukett and P3/Sabin, but none of them neutralized the prototype strain of the outbreak (P3/Finland/23127/84). Forty-six percent of the clinical isolates resembled the prototype strain (class A) while the rest of the isolates were neutralized by one or more of the MAbs (classes B to D). Although an antigenic drift from A to one of the other classes was observed in sequential specimens from several individuals, no clear-cut overall change in the class distribution was found within the 3 months time span of the outbreak. Homogeneous virus populations were isolated from the sewage specimens using a microtitre endpoint dilution method. The last positive sewage specimens which were obtained in January to February 1985 still had a majority of the class A strain. Some of the clinical isolates were also tested using MAbs directed against distinct antigenic sites. These studies showed that strains that gave the same pattern of reactivity with site 1 MAbs could be differentiated using antibodies directed against other sites. Fifteen strains belonging to different antigenic subclasses were subjected to partial RNA sequencing of the genome region coding for antigenic site 1. The antigenic variation was usually, but not always associated with corresponding amino acid substitutions in antigenic site 1. These results indicate that the antigenic sites of type 3 poliovirus vary extensively within a given outbreak and even during replication in a given host. This variation may have both pathogenetic and epidemiological significance.
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Conservation of Antigenic Properties and Sequences Encoding the Envelope Proteins of Prototype Hantaan Virus and Two Virus Isolates from Korean Haemorrhagic Fever Patients
SummaryViruses isolated from the blood of two Korean haemorrhagic fever patients were propagated in cell culture and compared to prototype Hantaan virus which was isolated from Apodemus mice. The antigenic properties of the human isolates were found to be closely related to Hantaan virus by plaque reduction neutralization, haemagglutination inhibition and fluorescent antibody staining with both polyclonal and monoclonal antibodies. The medium genome segment of each human isolate was sequenced and compared to that of Hantaan virus. Nucleotides comprising the Hantaan virus G1 and G2 envelope protein-coding regions differed from those of the other viruses by only 5.4% and 5.7%. The human isolates differed from one another by 1.6%. The nucleotide differences resulted in predicted amino acid variations of 1.3% to 2.3% among the three viruses, with the majority occurring as conservative substitutions in G1.
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Physicochemical Properties of Marburg Virus: Evidence for Three Distinct Virus Strains and Their Relationship to Ebola Virus
SummaryThe physicochemical and antigenic properties of three groups of Marburg (MBG) virus isolates, separated temporally and geographically, were compared to each other and to another member of the same family, Ebola (EBO) virus. Each MBG isolate contained seven virion proteins, one of which was a glycosylated surface protein. Peptide mapping of glycoproteins, nucleoproteins (NP) and viral structural protein (VP40) demonstrated extensive sequence conservation in the proteins of viruses isolated over a 13-year period, but homology was not evident in VP24. Some homology between the NPs of MBG and EBO was observed. A close antigenic relationship between MBG strains was found by radioimmunoassay but no evidence was found of antigenic cross-reactivity with EBO viruses. MBG virion proteins are produced from virus-specific monocistronic mRNA species. Five of the seven viral proteins were produced by in vitro translation of these RNAs. MBG virions contained one RNA species with an M r of 4.2 × 106 and virions had a density of 1.14 g/ml in potassium tartrate. Virus isolates from different outbreaks had distinct T1 oligonucleotide maps, but had approximately 95% homology in base sequence. No two geographically distinct virus pairs were more closely related to each other than to a third virus isolate. MBG viruses are thus similar to EBO viruses in morphology and other physicochemical properties and are very similar to each other in RNA and protein composition. Each of the three geographically and temporally distinct MBG virus outbreaks appears to have been due to a genetically distinguishable, but antigenically closely related virus strain. In addition, these studies confirm the belief that MBG and EBO viruses are members of the new virus family, the Filoviridae.
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Reconstitution with T Lymphocytes Protects Nude Mice from a Central Nervous System Disorder Induced by a Temperature-sensitive Vesicular Stomatitis Virus
More LessSummaryA temperature-sensitive mutant of vesicular stomatitis virus (VSV), tsG31-KS5 VSV, intracerebrally inoculated into BALB/c (+/+) or Swiss outbred mice yielded a clinically asymptomatic persistent infection of the central nervous system (CNS). BALB/c nude (nu/nu) mice infected with tsG31-KS5 VSV, however, all perished within 26 days of infection. All the nude mice were afflicted with a slowly progressing CNS disorder, with symptoms including lethargy, curvature of the spine, hind-limb paralysis and other neurological disorders, before they succumbed to the infection. Wild-type (wt) VSV infection of either normal or nude mice, on the other hand, invoked a rapidly lethal disease with all animals dying within 4 days of infection. When nude mice were reconstituted with 5 × 106 syngeneic T lymphocyte-enriched splenocytes, over 70% of them not only survived the tsG31-KS5 VSV infection but appeared to be free of any neurological disorders. Only 20% of these reconstituted mice infected for 20 days with tsG31-KS5 VSV endured a wt VSV challenge. In contrast, BALB/c (+/+) mice infected for 20 days with tsG31-KS5 VSV all survived a wt VSV challenge. Reconstitution of nude mice with 5 × 106 T lymphocytes did not elicit a vigorous secondary humoral antibody response against VSV. All the animals reconstituted with 5 × 107 T lymphocytes and infected with tsG31-KS5 VSV, however, had both late and early humoral responses that equalled antibody responses of BALB/c (+/+) mice. Reconstitution with either 5 × 106 or 5 × 107 T lymphocytes afforded the nude mice equivalent protection from the CNS disorder triggered by tsG31-KS5 VSV. Reconstitution with 5 × 106 T lymphocytes, therefore, protected nude mice from the neurological disease induced by the persistent virus without eliciting a robust humoral antibody response. Infectious, temperature-sensitive VSV was retrieved from the CNS of the nude mice that had been reconstituted with 5 × 106 T lymphocytes and infected for up to 30 days with tsG31-KS5 VSV. The CNS-isolated VSV was less temperature-sensitive than tsG31-KS5 VSV. When the CNS-isolated VSV was intracerebrally inoculated into Swiss outbred mice, an aggressive disease ensued with most of the mice developing a CNS disorder. In comparison, Swiss outbred mice were asymptomatically infected with tsG31-KS5 VSV. The VSV isolated from the CNS was more lethal to the mice than tsG31-KS5 VSV possibly because it was less temperature-sensitive.
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Antiviral Activity of Recombinant Rat Interferon Gamma in Immunologically Impaired and Immunosuppressed Rats
More LessSummaryTreatment of Wag/Rij rats with recombinant rat interferon gamma (rRIF-γ) resulted in complete protection against a lethal pseudorabies virus (PRV) infection. To investigate whether the protection resulted from direct inhibition of virus replication or from a stimulation of immune mechanisms, we tested rRIF-γ activity in naturally immunocompromised and artificially immunosuppressed rats. The antiviral effect of rRIF-γ was not abolished in silica- and carrageenan-treated, phagocyte-depleted rats. Immunologically immature newborn and T cell-deficient nude rats were also protected under a regime of rRIF-γ treatment as well as whole body gamma-irradiated rats. Sera of the protected rats were devoid of PRV-neutralizing antibodies. Our results indicate that the protective activity of rRIF-γ is based on direct inhibition of virus replication; stimulation of the immune system is not required but may be responsible for protection upon challenge several weeks after infection.
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Antibody Responses to Murine Cytomegalovirus in Genetically Resistant and Susceptible Strains of Mice
More LessSummaryThe role of antibodies as mediators of genetically determined resistance to murine cytomegalovirus (MCMV) in mice has not been elucidated. The ability of mice with different MCMV resistance phenotypes to produce an antibody response to MCMV was investigated in order to assess whether the host genotypes that control resistance also influence antibody production. Antibodies to MCMV in the sera of resistant (BALB.K, CBA/CaH, B10.BR) and susceptible [BALB/c, BALB.B, C57BL/10ScSn (B10), B10.D2, B10.A, A/J] mice were determined by ELISA and/or a complement-requiring neutralization assay. IgM antibodies were produced by all strains of mice as early as 3 to 5 days post-infection (p.i.) with maximum titres observed after 10 days p.i. for some strains, whilst IgG antibodies were produced by 5 to 7 days p.i. with maximum titres at 20 days p.i. IgA antibodies were not detected in the sera of MCMV-infected mice. Virulent MCMV induced higher antibody titres than either attenuated or u.v.-inactivated forms of the virus. Although high doses of virulent virus delayed the early production of IgM antibody they did not adversely affect the kinetics of IgG antibody production. High titres of neutralizing antibodies were detected as early as day 3 post-inoculation of virulent virus; when attenuated virus was used in the neutralization assay, this was found to be more easily neutralized than salivary gland-derived virus. Interestingly, although guinea-pig complement greatly enhanced antibody-mediated neutralization of MCMV, mouse complement was also effective at enhancing neutralization. Although genetically determined resistance to MCMV is an early event with the resistant phenotype being demonstrable during the first few days of the infection, there was no evidence that antibodies were responsible for this resistance since neither antibody titres nor the time of first appearance of antibody correlated with resistance status. However, these results do not exclude a more general role for antibody in limiting MCMV infection, especially in immunity to re-infection since passively transferred antibodies from resistant or susceptible mouse strains lowered virus titres in MCMV-infected animals.
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Effect of Macrophage Activation on Resistance of Mouse Peritoneal Macrophages to Infection with Herpes Simplex Virus Types 1 and 2
More LessSummaryTo define the effect of heterogeneity of murine peritoneal macrophages (Mø) on intrinsic resistance to herpes simplex virus (HSV) infection, several Mø populations were characterized for their response to infection with HSV type 1 (HSV-1) and HSV-2. Steady-state resident Mø (Res Mø) were compared in parallel with Mø activated with Corynebacterium parvum (now designated Propionibacterium acnes) (CP Mø) and thioglycollate-elicited inflammatory Mø (TG Mø). Res Mø were completely non-permissive for productive virus infection and showed no c.p.e. The intrinsic resistance of CP Mø to HSV infection was similar to that of Res Mø, in that the infection was non-productive for infectious virus, but CP Mø showed marked c.p.e. TG Mø showed semi-permissiveness, with virus yields at least 10-fold higher than those in Res Mø and CP Mø, and marked c.p.e. The three distinct intrinsic response patterns were maintained regardless of whether Mø were derived from CD-1 or B6C3F1 mice, or whether the infecting virus was HSV-1 or HSV-2. To define the level at which Mø restrict HSV replication, immunofluorescence assays for viral antigens and hybridization analyses for viral DNA were performed. All Mø populations showed immediate early and early virus polypeptides. Res Mø and CP Mø showed no viral DNA replication, but TG Mø showed moderate levels of viral DNA synthesis that paralleled the infectious virus titres produced. Investigation of the mechanism for the heterogeneous intrinsic antiviral response among the Mø revealed that interferon was not involved, because antiserum to mouse α/β interferon did not alter the intrinsic resistance patterns. Induction of c.p.e. in Mø required live, replication-competent HSV. The involvement of tumour necrosis factor (TNF) in c.p.e. was found to be unlikely; no significant amounts of TNF were detected in the culture medium of the Mø, and inclusion of anti-TNF antibody did not inhibit c.p.e.
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Specific Induction of Cellular Gene Transcription in Herpes Simplex Virus Type 2-Transformed Cells
More LessSummaryIn spite of much work, the mechanism of oncogenic transformation by herpes simplex virus (HSV) is as yet unknown. It has been proposed that HSV type 2 (HSV-2) can transform cells by a ‘hit and run’ mechanism. In the past we have demonstrated that several polypeptides can be immunoprecipitated from HSV-2-transformed cells, but not from control cells or adenovirus-transformed cells, by rabbit hyperimmune sera to HSV-2. It is possible that the expression of these proteins might be the result of activation of cellular genes during transformation. We have now isolated cDNAs representing transcripts of genes that are expressed at higher levels in HSV-2-transformed hamster embryo fibroblasts than in the parental cells. Cytoplasmic transcripts and genomic sequences homologous to three clones (pAA8, pHD1 and pLC7) were analysed. Northern blot analyses showed that 0.75 kb transcripts which hybridize to the three cDNAs were present in HSV-2-transformed cells and were completely absent or present at low levels in control hamster fibroblasts. These transcripts were not present in mouse cells transformed by other DNA viruses or by a chemical carcinogen. The expression of these transcripts seemed to be confined to certain HSV-2-transformed cell lines. Southern blot analysis suggested that the 0.75 kb transcripts corresponding to these cDNAs may have arisen from a single gene. Nuclear run-off experiments indicated that activation occurred at the level of transcription. The activation of the gene or genes corresponding to these cDNAs may be an integral part of the mechanism of transformation by HSV-2.
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Immunological Conservation between Epstein-Barr Virus and Herpes Simplex Virus
More LessSummaryWe have analysed Epstein-Barr virus (EBV)- and herpes simplex virus (HSV)-infected cells for evidence of antigenic conservation of virus-coded proteins. Immunofluorescence and Western blot analyses of EBV-transformed cell lines demonstrated the presence of proteins that are antigenically related to the HSV alkaline DNase, infected cell-specific protein 34/35, glycoprotein B, thymidine kinase and the major DNA-binding protein. These proteins were characterized on the basis of M r and possible kinetic class.
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Gene Sequence and Mapping Data from Marek’s Disease Virus and Herpesvirus of Turkeys: Implications for Herpesvirus Classification
SummaryPurified DNAs from Marek’s disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M13 bacteriophage. Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences. The predicted amino acid sequences from these gammaherpesviruses were compared to known sequences from other herpesviruses using computer analysis. Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus. However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences. A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA. The results were consistent with the MDV and HVT genomes being collinear with VZV.
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Molecular Cloning and Sequence Analysis of the Mumps Virus Gene Encoding the P Protein: Mumps Virus P Gene Is Monocistronic
More LessSummaryThe nucleotide sequence of the P (phosphoprotein) gene of two strains of mumps virus has been determined from overlapping cDNA clones. The P gene contained a single open reading frame coding for a protein of 391 amino acids with a calculated M r of 41587, in good agreement with the value (40K to 45K) estimated from electrophoretic mobility on SDS-polyacrylamide gels. No open reading frame analogous to the C gene of other paramyxoviruses existed in the mumps virus P gene region. Comparison of the amino acid sequence of the mumps virus P protein with that of Newcastle disease virus showed a limited sequence homology.
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Comparison between Parainfluenza Virus Type 2 and Simian Virus 5: Monoclonal Antibodies Reveal Major Antigenic Differences
More LessSummaryMarked differences in the apparent M rs of the HN, NP and F proteins of simian virus 5 (SV5) and parainfluenza virus type 2 (PF-2) were revealed by SDS-PAGE. To examine the antigenic relationships between SV5, PF-2 and other paramyxoviruses, monoclonal antibodies (MAbs) specific to PF-2 were isolated. These antibodies had specificities for the HN, NP and P proteins and together with 54 MAbs to SV5 were tested for their ability to react with SV5, PF-2, PF-3, mumps and measles virus proteins. Most of these MAbs (55 out of 60) reacted with homologous virus only. However, five reacted with both SV5 and PF-2. These antibodies had reactivities to the NP, M and P proteins. Furthermore, one of the antibodies with reactivity to the P protein also reacted with mumps virus. Although none of the 21 MAbs with specificities for the HN protein of either SV5 or PF-2 cross-reacted with heterologous virus, some antigenic similarities between the HN protein of SV5 and PF-2 could be detected. This was demonstrated by raising a series of polyclonal antisera to purified preparations of SV5 or PF-2 HN proteins in BALB/c mice, and testing for their ability to neutralize both SV5 and PF-2 and also to immunoprecipitate the HN proteins of these viruses. Surprisingly, while low levels of cross-neutralizing antibody could be detected in some sera (e.g. neutralization of SV5 1:1600 and of PF-2 1:80), other sera with similar neutralization titres against homologous virus failed to neutralize heterologous virus. Furthermore, only a minority of the anti-HN antisera showed any immune-precipitating activity against the heterologous HN protein.
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Haemagglutinin of Measles Virus: Purification and Storage with Preservation of Biological and Immunological Properties
More LessSummaryMeasles virus envelope haemagglutinin (H) was purified rapidly with Triton X-100-solubilized virions by a two-step anion-exchange chromatography using fast protein liquid chromatography. The purity of the glycoprotein in its dimeric form was demonstrated by SDS-PAGE followed by silver staining or autoradiography. The purified H glycoprotein was further freed from contaminating detergent by dialysis of octylglucoside detergent. This purification procedure, together with subsequent lyophilization and storage at -70°C of the H glycoprotein which was incorporated into phospholipid vesicles allowed the full preservation of its haemagglutinating activity, its reactivity with a monoclonal anti-H antibody that recognized a conformational epitope and its capacity to elicit anti-H antibodies with haemagglutination-inhibiting and neutralizing activities.
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Canine Distemper Virus (CDV) Immune-stimulating Complexes (Iscoms), but Not Measles Virus Iscoms, Protect Dogs against CDV Infection
More LessSummaryThe potential of immune-stimulating complexes (iscoms), a novel form of antigenic presentation, for the induction of protective immunity against morbillivirus infection was shown by immunizing dogs with canine distemper virus (CDV) iscoms, which contained the fusion (F) protein and a minor amount of the haemagglutinin of the virus. The immunized dogs developed CDV-neutralizing antibodies but, in contrast to non-immunized dogs, did not develop viraemia or clinical signs of infection upon intranasal challenge with the virulent Snyder Hill strain of CDV. Immunization of dogs with measles virus (MV) iscoms, prepared either from affinity-purified MV F protein or from purified whole virus, resulted in partial protection against challenge with CDV. The data presented clearly show that the iscom form of antigenic presentation may be considered a serious candidate for subunit vaccines against morbillivirus infection.
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Oligo-2′,5′-adenylate Synthetase Activity in K562 Cell Lines Persistently Infected with Measles or Mumps Virus
SummaryFluctuation of oligo-2′,5′-adenylate synthetase (2-5AS) activity was examined in K562 cells infected with vaccine strains of measles virus (strains AIK-C and CAM-70) and mumps virus (strains Torii and Miyahara). Persistent infection was easily established in the mumps virus-infected cells without significant cytolysis or cell killing. In contrast, most of the cells infected with measles virus were killed by extensive cytolysis within 3 to 4 days. The small number of cells that did survive became persistently infected. That these persistently infected cells carried a virus antigen was confirmed by fluorescein isothiocyanate-labelled anti-measles virus rabbit antiserum and anti-mumps virus rabbit antiserum. The cells produced infectious progeny virus as well as interferon (IFN). Little induction of 2-5AS activity by IFN was demonstrated during the early stages of infection by these viruses. Similar results were observed in some of the persistently infected cells but not, however, K-CMP cells (K562 cells persistently infected with CAM-70) or K-MMP cells (K562 cells persistently infected with Miyahara). Failure to induce 2-5AS activity was unchanged in cells cultured for more than 6 months. The decrease of 2-5AS activity observed in K-MTP cells (K562 cells persistently infected with Torii) was the result of suppression of transcription of 2-5AS mRNA. On the other hand, a normal level of mRNA was found in K-AKP cells (K562 cells persistently infected with AIK-C). Therefore, it is suggested that the decrease of 2-5AS activity in K-AKP cells may be due to a failure to translate 2-5AS mRNA.
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Prevention of Epstein-Barr (EB) Virus-induced Lymphoma in Cottontop Tamarins by Vaccination with the EB Virus Envelope Glycoprotein gp340 Incorporated into Immune-stimulating Complexes
More LessSummaryExperimental induction of malignant lymphomas can be achieved in the cottontop tamarin by inoculation with Epstein-Barr (EB) virus. This system provides an animal model for assessing the efficacy of vaccine protection against the virus which is intended to reduce the incidence of human tumours associated with EB virus infection, namely endemic Burkitt’s lymphoma and undifferentiated nasopharyngeal carcinoma. Cottontop tamarins have been vaccinated with the major envelope glycoprotein of EB virus, gp340, incorporated into immune-stimulating complexes (iscoms) and were thereby protected against a 100% lymphomagenic dose of virus. The gp340 iscoms are highly immunogenic, requiring only a few micrograms of immunogen to induce protective immunity and thus would be a strong candidate for further development as an EB virus vaccine for use in man.
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Neutralization and Sensitization of Lactate Dehydrogenase-elevating Virus with Monoclonal Antibodies
More LessSummaryMonoclonal antibodies directed against VP3, the envelope glycoprotein of lactate dehydrogenase-elevating virus (LDV), were found to neutralize a large proportion of the virus population. This effect of monoclonal anti-VP3 antibodies was significantly increased by a murine monoclonal rheumatoid factor, indicating that the same antiviral antibodies can either neutralize or sensitize different fractions of the virus. This observation could be explained by heterogeneity in LDV particles, resulting in diverse responses to antibodies and therefore to the persistence of the virus in vivo.
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Synergistic Interactions of Anti-NS1 Monoclonal Antibodies Protect Passively Immunized Mice from Lethal Challenge with Dengue 2 Virus
More LessSummaryNon-neutralizing, serotype-specific anti-NS1 monoclonal antibodies partially protected passively immunized mice from lethal dengue 2 virus intracerebral challenge. There was no apparent correlation between complement-fixing activity and protective capacity among individual anti-NS1 monoclonal antibodies. Immunization with specific combinations of non-protective or partially protective antibodies resulted in prolonged survival or reduced mortality. Solid protection, equal to that achieved after immunization with neutralizing polyclonal antibody, was achieved only with an antibody pair which individually fixed complement to high titre with homologous virus. Some groups of mice had increased morbidity after immunization with combinations of protective monoclonal antibodies that bind to overlapping epitopes. These results may affect the design of recombinant dengue vaccines which may require the inclusion of serotype-specific antigenic domains.
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Monoclonal Antibodies Directed against Human Immunodeficiency Virus (HIV) gag Proteins with Specificity for Conserved Epitopes in HIV-1, HIV-2 and Simian Immunodeficiency Virus
More LessSummaryMonoclonal antibodies (MAbs) were raised against gag proteins of human immunodeficiency virus type 1 (HIV-1), strain HTLV-IIIB. One of 29 antibodies was specific for p17 of HIV-1. Twenty of 28 MAbs reactive with the major core protein p24 of HIV-1 showed cross-reactivity with HIV-2, and five of these also detected the corresponding antigens of simian immunodeficiency virus (SIVmac). The MAbs were reactive in several tests, i.e. ELISA, immunostaining of Western blots, immunofluorescence, alkaline phosphatase-anti-alkaline phosphatase immunocytochemistry and immunoelectron microscopy. The submembrane protein p17 was clearly localized within the virion.
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Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus
More LessSummaryThe binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the α-peptide of β-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.
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Differentiation of Junin Virus and Antigenic Variants Isolated in vivo by Kinetic Neutralization Assays
More LessSummaryThe major natural reservoir of Junin virus, the aetiological agent of Argentine haemorrhagic fever, is the cricetid Calomys musculinus. Neonatal animals experimentally infected with Junin virus (XJCl3 strain) developed typical disease and approximately 80% of them died. Most survivors become persistently infected. Antigenically variant viruses were isolated from the blood and brain of infected cricetids during the acute and chronic stages of the disease. These variants could be distinguished from the parental strain by kinetic neutralization assays using polyclonal antibodies. Some biological properties were shared with the parental virus strain including its virulence for newborn C. musculinus. These variant viruses may play a major role in chronic disease since we have shown that a viral isolate from an infected brain was poorly neutralized by serum obtained from the same animal.
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Large Scale Production of Hepatitis A Virus in Cell Culture: Effect of Type of Infection on Virus Yield and Cell Integrity
More LessSummaryApproaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells. The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period. In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later. Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus.
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Nucleotide Sequence and Evolutionary Relationships of Cucumber Mosaic Virus (CMV) Strains: CMV RNA 2
More LessSummaryThe nucleotide sequence of RNA 2 of the Fny strain (Subgroup I) of cucumber mosaic virus (CMV) was determined and compared at both the nucleic acid and protein level with the previously determined corresponding sequence of RNA 2 of the Q strain (Subgroup 2) of CMV. Fny-CMV RNA II 2 consisted of 3050 nucleotides and contained a single open reading frame (ORF) of 2571 nucleotides, whereas Q-CMV RNA 2 consists of 3035 nucleotides and contains a single ORF of 2517 nucleotides. At the nucleotide level, there was 71% sequence homology between the two RNAs, while at the protein level sequence homology was 73%. Protein homology was greater (89%) in the central third than in either the N-terminal (64%) or the C-terminal (56%) thirds. The secondary structures of the 3′ end of the RNAs were very similar, even though the nucleotide sequence homology between the 3′-terminal 180 nucleotides was only 62%. By contrast, there was 80% sequence homology between the 5′-terminal 86 residue, non-translated regions of the two RNAs. The evolutionary relationships and the divergence and retention of specific sequences among the two CMV strains and other plant viruses are discussed.
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The Complete Nucleotide Sequence of Potato Virus X and Its Homologies at the Amino Acid Level with Various Plus-stranded RNA Viruses
More LessSummaryDouble-stranded cDNA of potato virus X (PVX) genomic RNA has been cloned and sequenced. The sequence [6435 nucleotides excluding the poly(A) tract] revealed five open reading frames (ORFs) which were numbered one to five starting at the 5′ terminus of the RNA. They encoded proteins of M r 165588 (166K), 24622 (25K), 12324 (12K), 7595 (8K) and 25080 (coat protein), respectively. ORFs 1 and 2 were inphase coding regions. The ORF 1 product contained domains of homology with the tobacco mosaic virus 126K and 183K products. The ORF 2 and 3 products showed homologies with the barley stripe mosaic virus 58K and 14K proteins, the beet necrotic yellow vein virus 42K and 13K products and the white clover mosaic virus 26K and 13K products, respectively. The significance of these homologies with respect to putative functions of the PVX-encoded proteins are discussed.
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Antigenic Characterization of Potato Virus X with Monoclonal Antibodies
More LessSummaryA panel of mouse monoclonal antibodies (MAbs) against potato virus X (PVX) was obtained and three of these which had high affinity to the antigen were characterized in detail. These three antibodies defined two epitopes on PVX and recognized native virus, viral coat protein and denatured viral coat protein in various immunological assays. Two of the MAbs and rabbit anti-PVX polyclonal antibodies bound to the 68 amino acid N-terminal peptide of the PVX coat protein. This implies that the N terminus of the PVX coat protein is exposed at the virus surface and forms a highly immunogenic antigenic determinant. In double antibody sandwich (DAS) ELISA, MAbs and their horseradish peroxidase conjugates reacted with PVX at 10 to 20 ng/ml. Monoclonal antibodies to PVX reacted with virus in potato leaves and tubers and detected the virus in DAS ELISA in various combinations, including in combination with polyclonal antibodies.
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The Subcellular Location of the Gene 1 Product of Cauliflower Mosaic Virus Is Consistent with a Function Associated with Virus Spread
More LessSummaryUsing immunogold cytochemistry, plasmodesmata have been identified as subcellular locations of the protein product (P1) of cauliflower mosaic virus gene 1 in infected turnip tissue. Thin sections, from tissue adjacent to chlorotic local lesions on systemically infected fully expanded leaves, were probed with antiserum to a protein product derived from a lacZ-gene 1 fusion, and with control sera. Modified plasmodesmata between infected mesophyll cells, and plasmodesmata in the end walls of phloem parenchyma cells were specifically labelled with anti-P1 serum. In the former case, the position of the label suggested that P1 was extracellular and had formed a structural component of the modified plasmodesmata. Cell walls at the corners of cells in both healthy and infected tissue were also labelled. Anti-P1 serum also reacted with nuclei and the leaf cuticle, in both healthy and infected tissue. These observations provide strong circumstantial evidence that P1 is involved in the cell-to-cell movement of cauliflower mosaic virus.
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In vitro Biological Activity Associated with the Aphid Transmission Factor of Cauliflower Mosaic Virus
More LessSummaryAn assay system was developed to enable cauliflower mosaic virus (CaMV) isolate Cabb B-JI to be acquired by Myzus persicae feeding through membranes on crude extracts and subcellular fractions of infected turnip plants. Optimum transmission of the virus was from solutions containing 200 mm-Tris-HCl pH 7.6, 100 mm-EGTA and 50 mm-MgCl2. The rates of transmission by single aphids from such extracts were similar to or higher than those of single aphids from injected plants to healthy plants. Similar transmission efficiencies following feeds lasting 1 min, 15 min or 3 h show that the aphid-virus association is not diminished by long acquisition feeds. Subcellular fractionation showed that transmission was related to the presence of virus particles and aphid transmission factor (ATF) but not to the inclusion body protein. It is suggested that the transmitted agents were virus particles linked to ATF. Although figwort mosaic virus (FMV) was not transmitted by aphids from extracts of infected plants it became aphid-transmissible when the extracts were mixed with similar extracts of plants infected with CaMV-Cabb B-JI, perhaps because CaMV ATF assists in the transmission of FMV.
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The Structure of Particles of Tobacco Ringspot Nepovirus: Evidence from Electron Microscopy
More LessSummaryParticles of tobacco ringspot nepovirus from purified preparations were trapped on grids by immunosorbent electron microscopy and then either negatively stained, or freeze-dried and shadowed with uranium, for structural studies. Particle dimensions differed considerably with different stains and methods of preparation, but no obvious substructure was apparent. In contrast, particles which were freeze-dried and then shadowed exhibited either fivefold or threefold symmetry and had a structure resembling that of models made up of 60 units in clusters of five arranged in a T = 1 lattice. Both chemical and morphological evidence are compatible with this structure.
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