- Volume 71, Issue 5, 1990
Volume 71, Issue 5, 1990
- Animal
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Characterization of the DA26 Gene in a Hypervariable Region of the Autographa Californica Nuclear Polyhedrosis Virus Genome
More LessA region of the baculovirus Autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. Sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated DA26. The DA26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. Northern blot analysis of DA26 showed that it was expressed very early after infection. DA26-specific transcripts could be detected after the 1 h viral adsorption period upon infection of cultured Tricho- plusia ni cells. These transcripts were mapped by nuclease protection assays. A recombinant virus was constructed in which DA26 was disrupted by insertion of the Escherichia coli lacZ gene. This virus was viable in both T. ni and Spodoptera frugiperda cells and analysis of the kinetics of protein synthesis revealed no differences between wild-type and recombinant viruses. The disruption of DA26 also did not interfere with the ability of the virus to infect T. ni or S. frugiperda larvae.
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The Effect of Host Resistance to Tick Infestation on the Transmission of Thogoto Virus by Ticks
More LessTick-borne virus transmission was examined using guinea-pigs and hamsters previously infested with ticks. Guinea-pigs developed immunity to Rhipicephalus appendiculatus after a single exposure to the ticks. Nymphal and adult stages that fed on resistant guinea-pigs had increased mortality during feeding, and reduced engorged weights. Egg production from female ticks fed on resistant hosts fell by at least 50%. Guinea-pigs maintained high levels of immunity to tick infestation for at least 210 days after the initial exposure. In contrast, hamsters did not develop resistance to ticks even after three or four infestations. R. appendiculatus adults infected with Thogoto (THO) virus (donors) were allowed to co-feed with uninfected nymphs (recipients) on either resistant or naive guinea-pigs. The number of recipient ticks that acquired virus was significantly reduced on resistant guinea-pigs. In contrast, feeding on pre-infested hamsters did not affect tick-borne transmission of THO virus. Host resistance to tick infestation, if prevalent in nature, may severely limit the spread of tick-borne viruses. Such an effect could result directly from a reduction in the number of ticks that acquire virus, or indirectly from poor egg production (in the case of viruses maintained in ticks by vertical transmission) and reduced survival of ticks fed on resistant hosts.
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Heterologous Reassortment of Bunyaviruses in Aedes Triseriatus Mosquitoes and Transovarial and Oral Transmission of Newly Evolved Genotypes
More LessAedes triseriatus mosquitoes were orally infected with two different California serogroup bunyaviruses (La Crosse and snowshoe hare viruses) and high frequency reassortment occurred in these mosquitoes. Increased viral replication and subsequent gene segment reassortment was noted in the ovaries of mosquitoes that had ingested multiple blood-meals. To determine whether newly generated reassortant viruses could be transmitted transovarially to progeny mosquitoes, adult female mosquitoes were inoculated with the two temperature-sensitive (ts) parental viruses, and allowed to blood-feed and oviposit. Of 58 infected progeny mosquitoes assayed, six (10 %) contained non-ts viruses, and three of these transmitted non-ts viruses to a susceptible host. Selected viruses of the non-ts phenotype, which were isolated from mosquitoes and from mice fed upon by the mosquitoes, were demonstrated to be reassortant viruses by oligonucleotide fingerprinting.
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Evidence for Chromosomal Transmission of Polydnavirus DNA
More LessHeterogeneity in polydnavirus DNA was exploited as a means of following the transmission of viral genomes in parasitoid populations. Parasitoid lines isogenic for viral DNA markers were established from both a braconid (Cotesia melanoscela) and an ichneumonid (Hyposoter fugitivus) species. In crossing experiments these markers routinely segregated in Mendelian (chromosomal) fashion, suggesting that the structure of polydnavirus genomes is probably determined by the integrated form of viral DNA, rather than by extrachromosomal molecules.
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Production, Purification and Biological Properties of an Escherichia Coli-derived Recombinant Porcine Alpha Interferon
More LessRecombinant plasmids for intracellular synthesis of mature porcine interferon alpha 1 (IFN-α1) in Escherichia coli were constructed. High amounts of antiviral activity were obtained [up to 4 × 105 international units (IU) per ml of bacterial culture]. Recombinant porcine IFN-αl (rIFN-αl) was purified to homogeneity by monoclonal antibody immunoaffinity and was found to have the expected M r (17·5K) and N-terminal sequence (except for the apparent lack of an N-terminal methionine). Its specific antiviral activity was 5 × 107 to 10×l07 lU/mg MDBK cells. In vitro biological properties of this purified rIFN-α1 were compared to those of virus-induced porcine leukocyte interferon: the two interferons shared similar antigenic determinants and had the same ability to induce a cytocidal effect on primary cultures of pig kidney epithelial cells. However, rIFN-α1 was at least six times more active in inducing an antiviral state on homologous porcine cells. These properties are discussed in the light of a possible in vivo use of the purified recombinant molecule.
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Nucleotide Sequence of the Gene Encoding the Spike Glycoprotein of Human Coronavirus HCV 229E
More LessThe gene encoding the spike glycoprotein of the human coronavirus HCV 229E has been cloned and sequenced. This analysis predicts an S polypeptide of 1173 amino acids with an Mr of 128600. The polypeptide has 30 potential N -glycosylation sites. A number of structural features typical of coronavirus S proteins can be recognized, including a signal sequence, a membrane anchor, heptad repeat structures and a carboxy-terminal cysteine cluster. A detailed, computer-aided comparison with the S proteins of infectious bronchitis virus, feline infectious peritonitis virus, transmissible gastroenteritis virus and murine hepatitis virus, strain JHM is presented. We have also done a Northern blot analysis of viral RNAs in HCV 229E- infected cells using synthetic oligonucleotides. On the basis of this analysis, and by analogy to the replication strategy of other coronaviruses, we are able to propose a model for the organization and expression of the HCV 229E genome.
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Enzyme-linked Immunosorbent Assay of Antibodies to Rabbit Haemorrhagic Disease Virus and Determination of its Major Structural Proteins
More LessAn ELISA was developed for the determination of antibodies to rabbit haemorrhagic disease virus (RHDV) in whole blood and blood serum of rabbits. Naturally acquired antibodies were detected in 19·4% of blood samples collected from 1461 rabbits in 43 farms apparently free of the disease, 19·7% samples were doubtful and 60·9% of the rabbits were free of antibodies to RHDV. Their presence has a considerable effect on the resistance of rabbits to infection with RHDV. Antibodies were also found in rabbit blood serum samples collected up to 12 years before the first outbreaks of RHD were reported. Up to 14 viral protein antigens were determined by PAGE and Western blot analysis, of which three with M r values of 6IK, 38K and 52K were major proteins, the 61K being dominant. Our hyperimmune sera, a Chinese reference serum and sera with positive antibody titres, including those collected several years before the first outbreaks of RHD, reacted identically with these antigens in the Western blot analysis. The data obtained suggest that naturally acquired antibodies are a product of a specific response to prior infection with an avirulent strain of the virus.
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Disoxaril Stabilization and Immunogenicity of Poliovirus Procapsids
More LessDisoxaril (5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]- heptyl]-3-methylisoxazole) protects poliovirus procapsids against alkaline dissociation and thermal denatur- ation up to 42 °C. When added during the purification of procapsids, it enhances their yield and antigenic quality. Disoxaril increases the immunogenicity of both purified virions and procapsids in mice. Whereas untreated procapsids mainly elicit H-specific antibodies, disoxaril-treated procapsids yield high titres of neutralizing antibodies. The prospect of using disoxaril-treated procapsids in vaccines is discussed.
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Rotavirus RNA Replication: Single-stranded RNA Extends from the Replicase Particle
More LessThe rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used nondenaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SA11-infected MA104 cells and resolved on 0·6% agarose gels containing 50 mm-Tris-glycine pH 8·8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.
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Establishment and Characterization of Canine Parvovirus-specific Murine CD4+ T Cell Clones and Their Use for the Delineation of T Cell Epitopes
Canine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV- immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thyl+, CD4+, CD8− and proliferated specifically in response to CPV antigen. After stimulation with CPV antigen in culture the T cell clones produced IL-2 and proliferated in the absence of exogenous IL-2. Naive mice to which CPV-specific T cell clones had been adoptively transferred developed a CPV-specific delayed type hypersensitivity reaction upon simultaneous intracutaneous injection of CPV in their ears. The ability of recombinant viral fusion proteins, representing the VP2 capsid protein of the antigenically closely related feline panleukopenia virus and of synthetic peptides derived from the amino acid sequence of the VP2 of CPV, to stimulate these T cell clones enabled the identification of T cell epitopes.
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Failure to Demonstrate Human T Cell Lymphotropic Virus Type I in multiple Sclerosis Patients
More LessThe polymerase chain reaction (PCR) technique was employed in searching for human T cell lymphotropic virus type I (HTLV-I) gag, env and pol sequences in samples of DNA prepared from two HTLV-I seropositive patients with tropical spastic paraparesis (TSP), the Swedish multiple sclerosis (MS) patients who recently have been reported to be PCR-positive for HTLV-I gag and env sequences, and eight healthy individuals. Precautions were taken in order to reduce the risk of cross-contamination in the PCR. In the two TSP patients strong signals were obtained with gag, env and pol amplification primers and detection probes. In MS patients and healthy individuals, no signals were obtained with gag and env. In occasional experiments, weak signals were seen for the pol segment for a single MS patient and/or healthy individuals, but these signals were not reproducible in subsequent experiments. Thus, the present data do not confirm the presence of HTLV-I sequences in MS patients.
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Presentation and Immunogenicity of Viral Epitopes on the Surface of Hybrid Hepatitis B Virus Core Particles Produced in Bacteria
More LessWe recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.
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Transcript Analysis of The Equine Herpesvirus 1 Glycoprotein B Gene Homologue and Its Expression by a Recombinant Vaccinia Virus
More LessTranscript mapping of the equine herpesvirus 1 (EHV- 1) glycoprotein B (gB) gene homologue by Northern blot, SI nuclease and primer extension analyses indicated that two overlapping transcripts of 3·4 and 4·6 kb originated from the same strand and were transcribed from left to right between coordinates 0·40 and 0·43 of the EHV-1 genome. The 3·4 kb transcript encoded EHV-1 gB and the 5′ RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inserted into vaccinia virus by homologous recombination. Cells infectedwiththerecombinantvirussynthesizedEH V -1 gB antigen, which was detectable in the cytoplasm and on the cell surface by immunofluorescence using an EHV-1 neutralizing horse serum and EHV-1 monoclonal antibodies. On Western blots, bands of 138K to 143K, 80K to 90K and 55K to 57K were identified in recombinant virus-infected cells, by both EHV-1 monoclonal antibodies and the polyclonal horse serum. These were similar in M r to bands identified by these sera in EHV-1-infected cells. Mice vaccinated with the recombinant virus produced antibodies which recognized proteins of the same M r as EHV-1 gB, on Western blots, but did not have in vitro neutralizing activity.
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The Pathogenesis of Equine Herpesvirus Type 1 in the Mouse: A New Model for Studying Host Responses to the Infection
More LessAn infection was established in adult BALB/c mice by means of intranasal inoculation of the AB4 strain of equine herpesvirus type 1 (EHV-1). The acute infection was confined to the respiratory tract and blood. Virus was shown to replicate in the nasal mucosa, trachea and lung for several days producing clinical signs of disease. Viraemia was also detected and a small proportion of peripheral blood cells contained virus at the peak of the infection. Histological and electron microscopic evidence were obtained which proved that productive virus replication occurred in the ciliated epithelial cells lining the bronchi and in pneumocytes in the lung, resulting in the destruction of these cells. Both humoral and cell-mediated responses to the infection were detected and monitored. By means of immunoprophylaxis or chemotherapy it was possible to modify the course of the infection. This infection model has many striking features in common with that observed in the natural host and the observations suggest that the mouse is a convenient and relevant model in which to study both host responses to EHV-1 infection and modification of the pathogenesis by means of immunoprophylaxis or therapy.
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Pseudorabies Virus Glycoprotein gI: in Vitro and in Vivo Analysis of Immunorelevant Epitopes
Overlapping fragments of the gene encoding glycoprotein gl of pseudorabies virus (PRV; herpesvirus suis 1) were expressed in bacteria. Using the fusion proteins and a panel of monoclonal antibodies (MAbs) against gI as well as swine sera we found that the N-terminal part of gI (residues 33 to approximately 100) contains a highly antigenic and immunogenic domain. Transfer of antibodies binding to this region as well as vaccination with fusion proteins containing the N terminus of gI are able to confer protection to mice against a lethal challenge of virus. The results show that gI, which is non-essential for virus replication in tissue culture, can induce neutralizing and protective antibodies. The potential suitability of fusion proteins encompassing N-terminal parts of gI as diagnostic tools is demonstrated.
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Sequence Comparison of Five Polymerases (L proteins) of Unsegmented Negative-strand RNA Viruses: Theoretical Assignment of Functional Domains
More LessThe large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.
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Sequence Analysis of the HN Gene of Parainfluenza Virus Type 2
More LessA cDNA library was constructed in λgt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43 % amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.
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Characterization and Immunological Properties of Influenza A Virus Nucleoprotein (NP): Cell-associated NP Isolated from Infected Cells or Viral NP Expressed by Vaccinia Recombinant Virus do not Confer Protection
More LessA nucleoprotein (NP) preparation purified from the chorioallantoic membrane of chicken eggs infected with fowl plague virus (A/FPV/Rostock/34, H7N1) yielded, in addition to the commonly known 56K protein, a 42K component that could not be detected in virus particles. After testing with a series of NP-specific monoclonal antibodies it was found that some reacted with both proteins and others were bound only by the 56K protein. Among both types of NP-specific monoclonal antibodies only a limited number were bound to infected murine cells. Some antibodies bound to cells infected with a given subtype failed to react with the surface of cells infected with a different subtype. Binding was demonstrated by cellular ELISA, radioimmunoassay and immunofluorescence. The results indicate that only restricted antigenic domains of the native NP and perhaps NP fragments are exposed at the surface of infected murine cells. Additionally, the purified NP preparation was used to immunize mice in order to determine the protective capacity of cell- associated NP. In parallel, and as a relevant control, mice were immunized with a vaccinia virus recombinant containing the gene for NP prior to challenge with infectious virus. High levels of monospecific antibodies and a cytotoxic T cell activity was found in mice immunized with purified NP or infected with the vaccinia recombinant after secondary restimulation in vitro. After treatment with specific antibodies the cytotoxic cells were shown to be classical CD8+ cytotoxic T lymphocytes. Despite the elicitation of a humoral and a cellular immune response by the forms of NP employed mice were not protected from influenza virus infection.
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Fusion Characteristics of Influenza C Viruses
More LessA number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.
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Monoclonal Antibodies to Three Structural Proteins of Newcastle Disease Virus: Biological Characterization with Particular Reference to the Conformational Change of Envelope Glycoproteins Associated with Proteolytic Cleavage
More LessMonoclonal antibodies (MAbs) to the haemagglutinin—neuraminidase (HN), fusion (F) and matrix (M) proteins of Newcastle disease virus were prepared and characterized. At least three non-overlapping or partially overlapping antigenic sites were delineated on the HN, three on the F and three on the M proteins by competitive binding assays. Antigenic sites on the HN and F proteins roughly represented functional domains defined by serological tests. Two antigenic sites on the F protein were involved in virus neutralizing and haemolysis-inhibiting activity. These antigenic determinants were readily affected by treatment with certain surfactants and acetone. Proteolytic cleavage of the HN and F proteins was associated with conformational change, revealed by altered reactivity with MAbs and by altered topological arrangements of some epitopes. None of the anti-M MAbs inhibited any biological activities of the virus.
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Protective Effect of MonoclonalAntibodies to Newcastle Disease Virus in Passive Immunization
More LessA series of monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins and the matrix (M) protein of Newcastle disease virus (NDV) were tested for protective effects in passive immunization of newborn chickens against challenge with a virulent heterologous strain of NDV (Italien). MAbs with high virus-neutralizing activity directed to one antigenic site of the HN protein delayed virus growth and significantly prolonged survival time, but all chickens eventually succumbed to infection. MAbs directed to two antigenic sites of the F protein completely suppressed virus growth and prevented death of chickens, although the neutralizing activities of these anti-F MAbs were lower than those of the above anti-HN MAbs. Combined administration of the anti-HN and anti-F MAbs had a synergistic protective effect, but no protective effects were shown by MAbs against the M protein.
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Degradation of Herpes Simplex Virions by Human Polymorphonuclear Leukocytes and Monocytes
The degradation of herpes simplex virus particles after uptake by phagocytes was studied, but, since lysis of the phagocyte also resulted in damage to the viral envelope, measurement of viral infectivity as a criterion of viral degradation after phagocytosis was not possible. Therefore we focused on later events in viral destruction, namely the degradation of macromolecules. We have demonstrated that polymorphonuclear leukocytes (PMN) and monocytes (MN) can rapidly degrade the membrane proteins of the phagocytosed herpesvirus virions. PMN and MN from a patient with chronic granulomatous disease showed a similar rate of degradation compared to PMN and MN from healthy donors, which excludes an important role for toxic oxygen species in viral protein degradation. Experiments using toxic oxygen species-generating systems supported this observation. In contrast to PMN, MN are also effective in the digestion of viral DNA. We conclude that PMN and MN are able to neutralize large amounts of phagocytosed HSV, so their role in antiviral defence has again been demonstrated.
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Identification of the Genes Encoding Two Capsid Proteins of Herpes Simplex Virus Type 1 by Direct Amino Acid Sequencing
More LessAmino-terminal amino acid sequencing was carried out on proteins purified from herpes simplex virus type 1 capsids. The sequences of two capsid proteins (VP19C and VP23) showed them to be encoded by genes UL38 and UL18, respectively. The product of UL38 has been shown to be essential for capsid assembly, but no role has previously been assigned to the product of UL18.
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Expression of Bovine Herpesvirus Type 1 Glycoprotein gI in Transfected Bovine Cells Induces Spontaneous Cell Fusion
More LessBovine MDBK cells were transfected with Rous sarcoma virus-based vectors for constitutive expression of the bovine herpesvirus type 1 (BHV-1) glycoprotein, gI. Cell lines stably expressing recombinant gI were cloned and characterized. Recombinant gI was localized intracellularly, predominantly in a perinuclear region, and on the cell surface. Cells expressing gI exhibited spontaneous polykaryon formation, thus confirming the fusogenic activity described previously in gI-expressing transfected murine LMTK- cells. The recombinant form of gI synthesized in transfected MDBK cells was similar in Mr to the form expressed in BHV-1-infected MDBK cells, unlike the recombinant form of gI expressed by LMTK− cells which is deficient in N -linked glycosylation. It was concluded that cell fusion associated with the expression of BHV-1 gI in transfected mammalian cells is a reproducible phenomenon in a number of cell types and is not due to species-specific factors or expression of abnormally glycosylated gI. Cell fusion is a useful in vitro marker for gI function and may contribute to the spread of BHV-1 infections in vivo.
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Comparison of Heparin-sensitive Attachment of Pseudorabies Virus (PRV) and Herpes Simplex Virus Type 1 and Identification of Heparin-binding PRV Glycoproteins
More LessTo determine whether heparan sulphate residues on the cellular surface could serve as an attachment receptor for pseudorabies virus (PRV), the effect of heparin on PRV in plaque reduction and adsorption tests was investigated. PRV was significantly less sensitive to heparin than was herpes simplex virus type 1 (HSV-1). At concentrations of 500 µg/ml heparin the number of plaques formed by PRV was reduced to 7 % of the untreated control whereas the number of plaques formed by HSV-1 was reduced to below 0·1%. Adsorption of PRV to host cells was also less sensitive to heparin treatment than was adsorption of HSV-1. Experiments concerning the binding sites of PRV showed that heparin binds to the disulphide-linked glycoprotein complex gII (PRV gB), gIII (PRV gC) and probably gV.
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Cytopathic and Non-cytopathic Biotypes of Border Disease Virus Induce Polypeptides of Different Molecular Weight with Common Antigenic Determinants
More LessTen monoclonal antibodies have been raised against lysates of cells infected with cytopathic border disease virus (BDV). These antibodies all recognize non- cytopathic BDV and react with a number of different strains of bovine viral diarrhoea virus (BVDV). Studies with radiolabelled cell lysates show that all the antibodies precipitate two polypeptides of apparent M r 80000 and 130000 from cells infected with cytopathic virus and a single polypeptide of apparent M r 120000 from cells infected with non-cytopathic virus. Two of the monoclonal antibodies react on immunoblots and show the same pattern of reactivity indicating that these three polypeptides are antigenically related.
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Lactate Dehydrogenase-elevating Virus Induces Antibodies Reactive with a Surface Antigen of Aetiologically Unrelated Murine Cell Transformants
More LessMice infected with lactate dehydrogenase-elevating virus (LDV) developed antibodies reactive with a tumour cell surface antigen (TSA) of Moloney sarcoma virus (Mo-MSV)-transformed mouse cells (Sac). We demonstrate that STU mice infected with LDV were protected against growth of syngeneic Sac tumour cells as early as 23 days post-infection (p.i.) and up to 5 months p.i. Nine LDV strains, including the neurovirulent LDV-C, elicited production of anti-TSA antibodies, which were restricted to the IgM isotype. Monoclonal anti-TSA antibodies were raised 4 days after infection of STU mice with LDV. When tested against several transformants of STU and BALB/c mouse origin they were found to react with Mo-MSV transformants (PV-TC-77, STU mouse origin; MSV85 C1 3, BALB/c mouse origin), methylcholanthrene-transformed MethA cells (BALB/c origin) and L929 cells. We suggest that the well known tumour growth inhibition by LDV is due to LDV-induced anti-TSA antibodies.
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Identification of the Major Immunogenic Structural Proteins of Human Foamy Virus
More LessWe have identified the major immunogenic structural proteins of the human foamy virus (HFV), a distinct member of the foamy virus subfamily of Retroviridae. Radiolabelled viral proteins were immunoprecipitated from HFV-infected cells by foamy virus antisera of human and non-human primate origin. Precipitated viral proteins were in the range of 3IK to 170K. Labelling of proteins with [I4C]glucosamine or with [35S]methionine in the presence of tunicamycin, as well as endo- β - N -acetylglycosaminidase H and F treatment of [35S]methionine-labelled proteins, revealed three viral glycoproteins of approximately 170K, 130K and 47K, most likely representing the env gene-encoded precursor, the surface glycoprotein and the transmembrane protein of HFV, respectively.
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Uniformity of the Splicing Pattern of the E6/E7 Transcripts in Human Papillomavirus Type 16-transformed Human Fibroblasts, Human Cervical Premalignant Lesions and Carcinomas
We utilized the RNA polymerase chain reaction (PCR) to analyse the transcripts of the E6/E7 open reading frames of human papillomavirus type 16 (HPV-16). Total RNA was isolated from 14 cervical squamous carcinomas, nine cervical intraepithelial neoplasias and from human fibroblasts transformed with different HPV-16 constructs. In all specimens two spliced transcripts were detected. Sequence analysis of the cloned PCR products showed that both transcripts were generated by splicing out an intron in E6, from nucleotides (nt) 226 to 409 in one transcript and from nt 226 to 526 in the other. The major transcript present in all RNA specimens had the smallest intron in E6. The RNA PCR described here is the method of choice for analysing splice and donor sites in tissue specimens where a limited amount of RNA is available. Results obtained with transformed cells revealed no difference in splicing whether HPV-16 was controlled by its homologous promoter or by a heterologous promoter, the Rous sarcoma virus long terminal repeat.
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The S RNA Segment of Tomato Spotted Wilt Virus has an Ambisense Character
More LessThe complete nucleotide sequence of the S RNA of tomato spotted wilt virus (TSWV) was determined. The RNA is 2916 nucleotides long and has an ambisense coding strategy. The sequence contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted Mr of 52·4K and one in the viral complementary sense which encodes the viral nucleocapsid protein of Mr 28·8K. Both proteins are expressed by translation of two subgenomic RNA species that possibly terminate at a long stable hairpin structure, located at the intergenic region. The structure of this RNA segment resembles that of the arthropod-borne phleboviruses (family Bunyaviridae). The absence of significant sequence homology between TSWV and bunyaviruses infecting animals suggests that TSWV should be considered as a representative of a new genus within the Bunyaviridae.
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Similarities Between Putative Transport Proteins of Plant Viruses
More LessThe nucleic acids of many plant viruses encode proteins with one or more of the following properties: an M r of approximately 30000, localization in the cell wall of the infected plant and a demonstrated role in cell-to- cell transport of infection. A progressive alignment strategy, aligning first those sequences known to be similar, and then aligning the resulting groups of sequences, was used to examine further the relatedness of the amino acid sequences of putative transport proteins of caulimoviruses, of proteins similar to the putative transport protein of alfalfa mosaic virus (A1MV) and of those similar to the tobacco mosaic virus (TMV) 30K protein. The strategy first identified regions in which multiple dipeptides of one group were similar to those of another group. The regions of similarity were brought into alignment by the conservative introduction of gaps. The positions of the introduction of gaps were adjusted to optimize similarity.
Statistical significances of the resulting alignments, determined both by comparison with shuffled amino acid sequences and with the sequence alignment off-set by 1 to 15 residues in each direction, suggest that the amino acid sequences of the three groups of viruses are distantly related. Nevertheless, significant relationships between members of the caulimoviral group of sequences and members of each of the AIMV-like and TMV-like groups were found. These relationships and the analysis of the number of insertions/deletions between present sequences and a hypothetical common ancestor suggest that the sequences of the caulimoviral proteins are less diverged from the ancestor than either the AIMV-like or TMV-like proteins. The alignment identified common regions of predicted secondary structure and regions of similar hydropathy, regions possibly crucial for proper functioning of the proteins.
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Nicotiana Velutina Mosaic Virus: Evidence for a Bipartite Genome Comprising 3 kb and 8 kb RNAs
More LessDNA complementary to Nicotiana velutina mosaic virus (NVMV) RNA was qloned and five segments larger than 0·9 kb were used in Northern blot hybridization analysis to identify two virus-specific RNAs, approximately 8 kb (RNA 1) and 3 kb (RNA 2) in size. The clones selected as probes did not hybridize with RNA from various tobamoviruses, or from beet necrotic yellow vein (BNYVV) and peanut clump furoviruses. In an attempt to determine the taxonomic position of the virus, about 75 % of the NVMV RNA 2 was sequenced and four open reading frames (ORFs) were identified. ORFs 1,2 and 3 encode proteins of M r 20K, 39K and 13K, whereas ORF 4 was incomplete. ORFs 2, 3 and 4 overlapped in an arrangement closely resembling the triple gene block identified in BNYVV RNA 2, barley stripe mosaic virus (BSMV) RNA 2, potato virus × and potato virus M RNA. The presumed coat protein gene of NVMV RNA 2 (ORF 1) is situated to the 5′ side of the triple gene block as for BNYVV and BSMV RNA 2. Amino acid homologies were detected among the 13K and 14K proteins of NVMV RNA 2, BNYVV RNA 2 and BSMV RNA 2. Significant homology was also detected between the 39K protein of NVMV RNA 2 and the 42K protein of BNYVV RNA 2, with a motif specific for ATP- and GTP-binding (NTP-binding motif), and a conserved viral DNA polymerase domain. The presence of a triple gene block in NVMV RNA 2 indicates that NVMV has affinities with members of the hordei-, furo-, potex- and carlavirus groups but not with the tobamovirus group. The divided RNA genome of NVMV, and the sizes of the two RNAs suggest that NVMV is most closely allied to the furoviruses, but the unique nature of its different biological properties and lack of any serological relationships with furoviruses lead us to conclude that NVMV has no clear relatedness to any taxonomic group of plant viruses.
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Evidence for the Role of Subgenomic RNAs in the Production of Potato Virus S coat Protein During in Vitro Translation
More LessPurified virus particles of potato virus S (PVS) when disrupted yielded only one readily resolved band of 7·5 kb in ethidium bromide-stained agarose gels. However Northern hybridization of viral RNA, probed with a clone specific to the viral coat protein gene, revealed a region of subgenomic RNAs of approximately 1·3 kb. Sucrose gradient fractionation of subgenomic RNA revealed that it coded for viral coat protein when translated in vitro in rabbit reticulocyte lysate. Virus particle lengths suggest that this subgenomic RNA may be encapsidated in 100 to 220 nm particles.
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