- Volume 70, Issue 3, 1989
Volume 70, Issue 3, 1989
- Animal
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Most Alphaviruses Share a Conserved Epitopic Region on Their Nucleocapsid Protein
More LessSUMMARYFourteen hybridoma cell lines secreting antibodies against the Semliki Forest virus nucleocapsid protein were established and employed for identification of conserved epitopes among 27 alphavirus types and subtypes. Using an antibody capture test, the antibodies were found to cross-react to variable degrees with alphaviruses belonging to the Semliki Forest, western encephalitis and eastern encephalitis complexes, as well as Middelburg and Ndumu. None of the antibodies reacted with either Venezuelan equine encephalitis or Barmah Forest virus. Due to their reactivity with Fort Morgan, Y62-33, Whataroa and chikungunya, the monoclonal antibodies were divided into six reactive types. Competition assays showed that the epitopes for all the types were either identical or clustered on a single domain of the nucleocapsid protein.
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Loss of Functional Voltage-gated Sodium Channels in Persistent Mumps Virus-infected PC12 Cells
More LessSUMMARYRat pheochromocytoma (PC12) cells, persistently infected with mumps virus (MV), failed to generate full-sized stimulus-evoked action potentials (SEAPs) when examined by intracellular electrophysiological recording techniques. Application of tetrodotoxin (TTX) had little or no effect on MV-reduced SEAPs, indicating that the number of functional voltage-gated Na+ channels was decreased or their operation was blocked by the virus. In contrast, MV-infected cells generated normal Ca2+ spikes when bathed in a solution containing TTX, tetraethylammonium ions and a high concentration (20 mM) of Ca2+. In addition, when infected cells bathed in TTX were superfused with Co2+ the SEAP profile reverted to that typical of PC12 cells with functional voltage-gated K+ channels only. These observations indicate that MV affects voltage-gated Na+ channels, but spares voltage-gated Ca2+ and K+ channels of persistently infected cells.
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Synthesis of Respiratory Syncytial Virus RNA in Cell-free Extracts
More LessSUMMARYA cell-free system has been used to synthesize respiratory syncytial virus (RSV) RNA in vitro. Polyadenylated species representing all size classes of RSV mRNAs were labelled. Some of the labelled RNA was seen in a CsCl gradient at the density characteristic of negative-strand viral nucleocapsids. Experiments using a thiotriphos-phorylated nucleotide indicate that RNA chains are initiated de novo in the cell-free system.
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A Model for Persistent Murine Coronavirus Infection Involving Maintenance via Cytopathically Infected Cell Centres
More LessSUMMARYThe relatively cell impermeable hygromycin B was found to inhibit viral but not cellular protein synthesis when added to cultures of murine hepatitis virus (MHV)-infected or mock-infected mouse L-2 fibroblasts. Membrane permeability, as judged by influx of sodium ions, has previously been demonstrated to be an MHV E2 glycoprotein-mediated, cytopathic effect of MHV infection in L-2 cells. It is therefore likely that the selective effect of hygromycin B on viral protein synthesis is a reflection of an increased drug penetration into virus-infected cells. Using hygromycin B as a marker for MHV-induced cell membrane cytopathology, the effects of drug treatment on a persistent MHV infection in mouse LM-K fibroblasts were investigated. MHV persistence in LM-K cells, which normally involves a steady state infection of 0·1 to 1 % of the cells in culture, was found to be cured by hygromycin B treatment, as measured by the elimination of infectious virus from the supernatant medium. Hygromycin B also resulted in the eradication of MHV-specific RNA from LM-K cells, arguing against the presence of a non-cytopathically or latently infected subpopulation of cells.
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Analysis of Transcription Initiation in the Panolis flammea Nuclear Polyhedrosis Virus Polyhedrin Gene
More LessSUMMARYThe nucleotide sequence of the polyhedrin gene of Panolis flammea multiple nucleocapsid polyhedrosis virus (PfMNPV) has been determined. The coding sequences of this gene shared 82% similarity at the DNA level and 88% similarity at the protein level with the polyhedrin gene from Autographa califomica (Ac) MNPV. A single nucleotide deviation from the consensus transcription initiation sequence for baculovirus very late genes was identified in the PfMNPV polyhedrin gene. RNA was prepared from Mamestra brassicae larvae infected with PfMNPV and compared with RNA harvested at 24 h post-infection from AcMNPV-infected Spodoptera frugiperda cells using Northern blotting with an AcMNPV polyhedrin gene-specific probe. The PfMNPV mRNA was estimated to be 1·0 kb compared with a larger size of 1·15 kb for the AcMNPV polyhedrin mRNA. A cDNA copy of the 5′ end of the PfMNPV polyhedrin mRNA was made using the technique of primer extension and sequenced to demonstrate that the point of transcription initiation was similar to that of AcMNPV polyhedrin mRNA.
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- Plant
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Characterization of Single- and Double-stranded RNAs in Particles of Rice Stripe Virus
More LessSUMMARYA rice stripe virus isolate (RSV isolate T), which was first isolated from an RSV-resistant cultivar of rice, induced atypically severe symptoms in wheat plants. The isolate also produced larger amounts of the fastest sedimenting nucleoprotein component (nB) in wheat plants than in rice plants. Four species of dsRNA of M r 5 × 106,2·5 × 106,1·8 × 106 and 1·5 × 106, as well as four species of ssRNA were detected in RNA extracts of purified filamentous virus particles of isolate T. The M r values of the ssRNAs were estimated by agarose gel electrophoresis to be approximately 3 × 106,1·6 × 106, 1·1 × 106 and 0·9 × 106. These values are significant revisions of those published earlier. Northern blot hybridization showed that each ssRNA of RSV isolate T had a unique sequence. Shorter viral RNAs were neither collinear with longer RNAs nor subgenomic RNAs. Each dsRNA of RSV isolate T contained the sequence of the ssRNA with the same strand length.
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The Complete Nucleotide Sequence of Plum Pox Virus RNA
SUMMARYThe complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5′ terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3′ terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted M r of 353·8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5′ non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3′ non-coding region of PPV-NAT and several other members of the potyvirus group.
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Competitive Multiplication of RNA3 Species of Different Strains of Alfalfa Mosaic Virus
More LessSUMMARYCompetition between RNA3 from alfalfa mosaic virus (A1MV) strain S (RNA3-S), strain B (RNA3-B) and strain 425L (RNA3-L) was studied. The identification of the RNA3 species multiplying in infected leaves was possible since the RNA3 5′ non-coding leader sequences in strains S, B and 425L differ in length. RNA3 present in total RNA from infected tobacco leaves was detected, and strains were identified from the length of the cDNA reverse-transcribed from RNA primed with a specific oligonucleotide. In competition experiments the inoculum, containing known amounts of RNA1, 2, 3 and 4 of one strain, was complemented with various amounts of heterologous RNA3 and inoculated to a systemic host. It is shown that RNA3-S was better replicated in vivo by the A1MV replicase of strain B than was RNA3-B itself, and to a lesser extent better replicated by the A1MV replicase of strain L than was RNA3-L. Comparison of genetic information carried by the RNA3 species present in the inoculum suggests that the more efficient multiplication of RNA3-S is related to the structure of the leader sequence of RNA3-S.
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Identification and Characterization of the Potato Leafroll Virus Putative Coat Protein Gene
More LessSUMMARYComplementary DNA clones representing approximately 6100 nucleotides of potato leafroll virus (PLRV) were generated, restriction-mapped, and partially sequenced. Within one of the cDNA clones an open reading frame (ORF) encoding a 23K protein was identified and further characterized. Amino acid sequence comparison of this protein showed significant homology (47·1%) with the barley yellow dwarf virus (BYDV-PAV) coat protein. This and other observations suggested that this gene encodes the PLRV coat protein. Other similarities were observed between PLRV and BYDV sequences in this region of their genomes, including an ORF of 17K within the ORF encoding the 23K putative coat protein.
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