- Volume 70, Issue 10, 1989
Volume 70, Issue 10, 1989
- Animal
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Porcine Parvovirus: DNA Sequence and Genome Organization
More LessSUMMARYWe have determined the nucleotide sequence of an almost full-length clone of porcine parvovirus (PPV). The sequence is 4973 nucleotides (nt) long. The 3′ end of virion DNA shows a Y-shaped configuration homologous to rodent parvoviruses. The 5′ end of virion DNA shows a repetition of 127 nt at the carboxy terminus of the capsid proteins. The overall organization of the PPV genome is similar to those of other autonomous parvoviruses. There are two large open reading frames (ORFs) that almost entirely cover the genome, both located in the same frame of the complementary strand. The left ORF encodes the non-structural protein NS1 and the right ORF encodes the capsid proteins (VP1, VP2 and VP3). Promoter analysis, location of splicing sites and putative amino acid sequences for the viral proteins show a high homology of PPV with feline panleukopenia virus and canine parvoviruses (FPV and CPV) and rodent parvovirus. Therefore we conclude that PPV is related to the Kilham rat virus (KRV) group of autonomous parvoviruses formed by KRV, minute virus of mice, Lu III, H-1, FPV and CPV.
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Localization of Human Papillomavirus Type 16 DNA Using the Polymerase Chain Reaction in the Cervix Uteri of Women with Cervical Intraepithelial Neoplasia
SUMMARYThe localization of human papillomavirus type 16 (HPV-16) DNA throughout the cervix uteri of women with cervical intraepithelial neoplasia (CIN) was studied by utilizing the polymerase chain reaction technique directly on histologically defined sections of paraffin-embedded cervical tissue obtained by conizations. HPV-16 DNA was detected only in the sections that contained CIN lesions and/or koilocytes. No HPV-16 DNA was detected in sections that contained only normal epithelium. This is in accordance with HPV-16 playing a role in the development of CIN lesions.
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Identification of Virus-specific Polypeptides by Monoclonal Antibodies against Serotype 2 Marek’s Disease Virus
More LessSUMMARYA total of 41 antibody-secreting hybridoma cells against the HPRS24 strain of Marek’s disease virus (MDV) type 2 (MDV2) have been isolated. Of these monoclonal antibodies (MAbs), 24 were found by immunofiuorescence tests to react specifically with MDV2-infected cells, but not MDV type 1 (MDV1)- or herpesvirus of turkeys (HVT)-infected cells, while eight reacted with MDV1- or MDV2-infected cells and nine with MDV1-, MDV2- or HVT-infected cells. By using these MAbs, seven classes of MDV type-specific or cross-reactive polypeptides were characterized by immuno-precipitation followed by SDS‒PAGE. Among them, a 28K/32K glycoprotein differed from the previously identified gA and gB. The 28K/32K glycoprotein was found on the surface of MDV2-infected cells and in the cytoplasm by an immunofluorescence test with MAbs. In addition, a cross-reactive polypeptide of 25K/29K was also detected in MDV1-infected cells with MAbs reactive with the 28K/32K glycoprotein of MDV2.
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Immunoblot Analysis of the Antibody Response to Murine Cytomegalovirus in Genetically Resistant and Susceptible Mice
More LessSUMMARYThe murine model of human cytomegalovirus infection was employed to analyse the kinetics of antibody production to murine cytomegalovirus (MCMV) structural and immediate early (IE) polypeptides following MCMV infection of genetically resistant and susceptible strains of mice. A total of 22 structural and six non-structural, IE proteins were identified. Analysis of immunoblots by densitometry identified four patterns of antibody reactivity to MCMV structural polypeptides during primary and secondary antibody responses over a period of 5 weeks post-infection (p.i.). Firstly, antibodies were strongly reactive with an 83K protein soon after infection, with levels which decreased with time; antibodies to a second group of viral proteins were also recognized soon after infection, but consistent levels of reactivity were maintained. Viral proteins that were recognized beyond day 14 p.i. or following a second MCMV infection formed the third group; the fourth group consisted of viral proteins that were detected at variable times p.i. by antisera from different mouse strains. The kinetics and intensity of the antibody response to individual viral proteins were influenced by virus dose, time p.i. and by a second MCMV infection. In addition, the genetic constitution of the host influenced the antibody response to MCMV proteins both quantitatively and qualitatively. In particular, sera from mice possessing C57BL but not BALB/c genes detected a 56K M r viral protein during seroconversion. Antibody reactivity to this protein was shown to segregate among sera from CXB mice, with a strain distribution pattern which indicated a linkage with the b locus on chromosome 4. Finally, expression of the 56K protein was detected in B10.BR but not BALB/c embryo fibroblasts in vitro, with expression being a dominant trait in (BALB/c × B10.BR) F1 embryo fibroblasts. Thus, host genes may influence the expression of this structural viral protein.
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Recombinant Vaccinia Virus Expressing the Herpes Simplex Virus Type 1 Glycoprotein C Protects Mice against Herpes Simplex Virus Challenge
SummaryThe gene encoding the herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) was isolated and cloned into a vaccinia virus insertion vector, and the resulting vaccinia-gC vector was used to construct a recombinant vaccinia virus that expressed gC (VVgC5). Infection of cells with VVgC5 resulted in cell surface expression of authentic HSV-1 gC. HSV-1 gC-specific neutralizing antibodies were produced in VVgC5-immunized mice, and lymphocytes exhibited an HSV-1-specific proliferation response in vitro following infection. More importantly, VVgC5-immunized mice were resistant to subsequent lethal HSV-1 challenge.
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Analysis of Reassortment and Superinfection during Mixed Infection of Vero Cells with Bluetongue Virus Serotypes 10 and 17
More LessSUMMARYThe reassortment of genome segments during mixed infection of Vero cells with bluetongue virus (BTV) serotypes 10 and 17 was investigated, using non-selective conditions for analysis of the progeny of mixed infections. Reassortment was found to be an early event in the BTV replication cycle, indicating that progeny BTV genomes undergo a single round of reassortment. Non-random segregation of individual genome segments was observed in crosses at equal multiplicity of infection, and was confirmed in crosses performed at unequal multiplicity. Asynchronous infections showed that superinfection exclusion resulted in the failure of the superinfecting virus to contribute genome segments to reassortants if the second virus followed the first by more than 4 h. The significance of these results for the evolution and epidemiology of BTV is discussed.
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Characterization of Monoclonal Antibodies against Four Structural Proteins of Rinderpest Virus
SUMMARYTwenty-four monoclonal antibodies (MAbs) against rinderpest virus (RPV) were established and characterized by several serological tests. Of the 24 MAbs, 10 recognized the nucleoprotein (NP), six the phosphoprotein (P), four the haemagglutinin (H), and two the fusion (F) protein as determined by radioimmunoprecipitation assay. The specificities of the remaining two MAbs could not be determined. From a competitive binding assay using MAbs against each structural protein, at least five, four and two separate antigenic sites were identified on the NP, P and H proteins, respectively. MAbs against the H protein neutralized the infectivity of the virus, but those against the F protein were only neutralizing in the presence of guinea-pig complement. The reactivities of each of the MAbs for other strains of morbillivirus were tested using an indirect immunofluorescent antibody assay. The MAbs against four out of five antigenic sites on the NP showed cross-reactivity amongst all the strains of morbillivirus tested whereas the fifth antibody reacted only with RPV. Of the antibodies specific for the P protein, the antibody against one site was cross-reactive with all the strains of RPV, measles virus (MV) and canine distemper virus (CDV), the antibody against another site was reactive with RPV and MV but not with CDV, and the antibodies against the other two sites were specific for RPV.
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Protection of Mice from Lethal Influenza by Adoptive Transfer of Non-neutralizing Haemagglutination-inhibiting IgG Obtained from the Lungs of Infected Animals Treated with Defective Interfering Virus
More LessSUMMARYThe lungs of C3H/HeMg mice infected with a lethal dose of the influenza virus A/WSN (H1N1) contained antibody to the viral neuraminidase glycoprotein but not to the haemagglutinin (HA). When coinoculated with a life-saving amount of defective interfering (DI) WSN, lungs were found to contain both anti-neuraminidase and haemagglutination-inhibiting (HI) antibodies which peaked at 3 and 5 days post-infection (p.i.), respectively. Mice coinoculated with WSN and β-propiolactone-inactivated DI WSN had only anti-neuraminidase antibody in the lungs. The HI activity was unusual in that there was no detectable neutralizing activity. The HI activity has been affinity-purified by adsorption to and elution from WSN HA and consisted entirely of IgG, comprising isotypes IgG1, IgG2a and IgG2b. The antibody was specific for WSN HA, of low avidity and had disappeared from the lungs by 20 days p.i. Transfer of affinity-purified HA-specific, non-neutralizing antibody to mice 24 h before infection with WSN alone protected 60% from death. We conclude that the antibody contributes significantly to the life-saving activity of DI WSN virus and that the ability to stimulate such antibody represents a novel property of this DI virus.
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Characterization of a Novel Human Respiratory Syncytial Virus Chimeric FG Glycoprotein Expressed Using a Baculovirus Vector
More LessSUMMARYHuman respiratory syncytial virus (RSV) codes for two glycoproteins (F and G) which have been shown to be the major targets for the host antibody response. We have expressed a novel chimeric glycoprotein (FG) in insect cells using a baculovirus vector. The chimeric glycoprotein contains the signal and extracellular regions of the RSV F glycoprotein linked to the extracellular region of the RSV G glycoprotein. Beginning at the amino terminus, the chimeric glycoprotein consists of amino acids 1 to 489 from RSV F followed by amino acids 97 to 279 from RSV G. The chimeric FG glycoprotein did not contain an anchor region and was efficiently secreted into the medium of recombinant baculovirus-infected insect cells. The FG glycoprotein ranged in size from 69K to 91K and was heterogeneous with respect to isoelectric point. The cleavage site present on the F glycoprotein was recognized on the chimeric FG, and the glycoprotein appeared to be antigenically similar to the native RSV F and G glycoproteins.
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Protection of Cotton Rats against Human Respiratory Syncytial Virus by Vaccination with a Novel Chimeric FG Glycoprotein
More LessSUMMARYThe cotton rat model of experimental human respiratory syncytial virus (RSV) infection was used to study the efficacy of FG, a novel chimeric glycoprotein which was expressed in insect cells using a baculovirus vector. FG contained the extracellular regions of the F (fusion) and G (attachment) glycoproteins of RSV. Vaccination with FG resulted in induction of neutralizing antibody and was correlated with protection of lung tissue from RSV challenge against both serogroup A and B virus strains. Both crude FG taken from supernatants of insect cells and affinity-purified FG were immunogenic and active against RSV. FG vaccination was effective by three routes of administration, following a single dose, and when administered with different adjuvants.
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Antibody-forming Cell Assays of Avian Paramyxoviruses: Immunization of Chickens Demonstrates Two Serotype Groups
More LessSUMMARYChickens were immunized with eight different serotypes of avian paramyxovirus (PMV). Ninety percent of splenic IgG antibody-forming cells (AFC) were serotype-specific with respect to the glycoprotein and nucleoprotein-polymerase antigens in an indirect immunoperoxidase binding system. The IgG AFCs which cross-reacted between serotypes divided the serotypes into two mutually exclusive super serogroups composed of PMV-1, -3, -4, -7 and -9, and PMV-2, -6 and -8. PMV-5 was not tested.
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Infection of Brain Cells by Diverse Human Immunodeficiency Virus Isolates: Role of CD4 as Receptor
More LessSUMMARYCell lines originally derived from malignant tumours of the brain were infected by diverse human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) isolates. By surface immunofluorescence it was shown that susceptible cells did not bear the CD4 antigen. They were also non-permissive for the formation of plaques by vesicular stomatitis virus pseudotypes and did not form syncytia with HIV-producing cells. Virus production was of low titre, and reverse transcriptase and the p24 antigen were consistently undetectable in the culture supernatants. Output virus could be detected by cocultivation with a sensitive T cell line, C8166, by the culture of supernatant medium with T cells and by detection of proviral HIV DNA after amplification. A higher multiplicity of input virus was required to establish a brain cell infection than was required for T lymphocytes or monocytes. Some HIV-susceptible brain cells contained mRNA for CD4 but infection was not blocked by anti-CD4 antibodies. Apparently HIV infection of these cells does not involve CD4 as the cellular receptor.
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Variations in CD4 Expression by Human Monocytes and Macrophages and Their Relationship to Infection with the Human Immunodeficiency Virus
More LessSUMMARYThe expression of CD4 antigen on the surface of LeuM3-positive human blood monocytes was found to be variable with 65 to 90% of cells from 46 normal human volunteers being positive by dual staining flow cytometry. When monocytes adhered to plastic (but not when cultured on Teflon), a marked decrease in CD4 expression was observed between 1 and 24 h post-adherence. CD4 expression could not be detected in macrophages adhered to plastic for 5 days by using four anti-CD4 monoclonal antibodies in flow cytometry or direct immunofluorescence. Conversely an increasing proportion of adherent cells expressed LeuM3 and OKM5 surface antigens over the 5 days. CD4 mRNA levels were measured by slot-blot and Northern hybridization, and total cellular CD4 protein levels by immunoprecipitation. Both cellular mRNA and CD4 levels remained constant throughout the 5 day period but membrane CD4 protein levels were greatly reduced indicating that the down-regulation of CD4 was posttranslational. Infection with two of six fresh human immunodeficiency virus (HIV) isolates showed different kinetic patterns when tested on purified monocytes recently adhered to plastic and macrophages adherent for 5 days. HIV antigen and reverse transcriptase levels in infected monocyte cultures remained high for 3 to 4 weeks before detachment and necrosis of the cells occurred. Infection of macrophages generated much lower levels of antigen and reverse transcriptase which declined to very low or undetectable levels over 2 weeks, leaving persisting viable macrophages. One week after infection HIV nucleic acid was detected in 69 ± 7% of monocytes and 6 ± 3% of macrophages by in situ hybridization. Blocking experiments with anti-Leu3a monoclonal antibody suggested that HIV infection of 5 day adherent macrophages occurred mainly by a mechanism other than binding to CD4.
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Inhibition of Human Immunodeficiency Virus Type 1-mediated Cytopathic Effects by Poly(l-lysine)-conjugated Synthetic Antisense Oligodeoxyribonucleotides
More LessSUMMARYThe ability of poly(l-lysine)-conjugated and methylphosphonate-modified synthetic human immunodeficiency virus type 1 (HIV-1) antisense oligodeoxyribonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection was studied. The abundance of viral antigens in oligomer-treated cultures indicated that the oligomers did not significantly affect viral infectivity. Similarly, no significant effects on relative viral RNA accumulation were apparent. The presence of poly(l-lysine)-modified oligomer complementary to the HIV-1 splice donor site resulted in a significant reduction in the production of viral structural proteins and virus titre in infected cultures. In addition, these cells were protected from HIV-1-mediated cytopathic effects while the other cultures rapidly succumbed to the cytotoxic effects of HIV-1 infection. The presence of poly(l-lysine)-conjugated oligomer resulted in the establishment of a persistent HIV-1 infection characterized by a highly productive virus infection in the absence of cell death while treatment of persistently infected cells with phorbol ester resulted in renewed cytopathicity. These results demonstrate the ability of synthetic antisense oligonucleotides to protect susceptible host cells from the cytopathic effects of HIV-1 infection.
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Vesicular Stomatitis Virus RNA Replication: a Role for the NS Protein
More LessSUMMARYSynthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.
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Altered ATP Function of a Vesicular Stomatitis Virus Mutant Detected by Kinetic Analysis of the Transcriptase Using Phosphorylated Ribavirin
More LessSUMMARYWe have studied the effect of phosphorylated ribavirin on the vesicular stomatitis virus (VSV) in vitro polymerase reaction by analysis of kinetic data obtained by varying the concentration of nucleoside triphosphates. The wild-type VSV had previously shown a competitive inhibition with the four natural nucleoside triphosphates with the use of ribavirin diphosphate (RDP) or ribavirin triphosphate (RTP). In contrast, when RDP (or RTP) was added to a transcription assay system using the polR1 mutant of VSV, a non-competitive or mixed type of inhibition was observed when the concentration of ATP was varied. Our results indicate that polR1 has an altered ATP function in addition to the previously described phenotypic characteristics of this mutant, which include synthesis of readthrough products of the leader/nucleocapsid (N) gene junction and a decreased ATP requirement for transcription. We have also studied CsCl-purified in vitro transcription products by primer-extending leader or N mRNA transcripts and found that the ratio of leader/N mRNA for VSV polR1 (1·3:1) was lower than values obtained previously for wild-type (3·7:1).
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Sequences in the 5′ Non-coding Region of Human Rhinovirus 14 RNA that Affect in vitro Translation
More LessSUMMARYA subgenomic cDNA clone from human rhinovirus 14 (HRV-14), comprising the 5′ non-coding region and the first 1182 nucleotides of the coding sequence, has been inserted into a vector under the control of the T7 promoter, and RNA was transcribed. Deletions in the 5′ non-coding sequence modulated viral polyprotein synthesis significantly in a reticulocyte lysate system. Removal of the first 491 nucleotides had little effect, but deletion of a further 55 nucleotides (491 to 546) significantly increased the efficiency of the translation process. Further deletion to nucleotide 621 almost abolished translation, suggesting an essential role for the 546 to 621 nucleotide sequence. The efficiency of the translation process can also be influenced by the addition of ribosomal salt wash prepared from uninfected HeLa cells.
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Nucleotide Sequence of High-passage Hepatitis A Virus Strain HM175: Comparison with Wild-type and Cell Culture-adapted Strains
More LessSUMMARYThe nucleotide sequence of cDNA from a high-passage, cell culture-adapted variant of hepatitis A virus strain HM175 was compared with the previously determined sequences of wild-type virus and two other cell culture-adapted variants. A total of 42 nucleotide changes were detected when the sequence was compared with wild-type virus. Five of these changes were common to all cell culture-adapted strains and a further two changes were shared by the strains that had experienced the greatest number of cell culture passages. The mutations were distributed throughout the genome coding for amino acid substitutions in regions 2B, 2C and 3D with silent changes in 1C and the 5′ non-coding region. The possible relevance of these mutations to cell culture adaptation and attenuation is discussed.
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Sequence Analyses of Thogoto Viral RNA Segment 3: Evidence for a Distant Relationship between an Arbovirus and Members of the Orthomyxoviridae
More LessSUMMARYThe genome of Thogoto (THO) virus, an unclassified tick-borne virus, comprises six segments of single-stranded RNA. The complete sequence of the third largest RNA segment has been determined from overlapping cDNA clones and by primer extension studies. Segment 3 RNA consists of 1865 nucleotides (approx. 6·2 × 105 M r). It has a large open reading frame (ORF1 ; 597 amino acids, 68·6K) in its virus-complementary sequence, confirming that the RNA has a negative-sense coding strategy. A transcription termination (polyadenylation) site located after the end of ORF1 has been identified. A second ORF (ORF2; 98 amino acids in length), overlapping ORF1, is also present in the virus-complementary sequence although whether it is translated is not known. The 3′ and 5′sequences of the segment 3 RNA are complementary and similar to those of the tick-borne Dhori (DHO) and the mammalian and avian influenza viruses. Protein database searches have identified regions of homology between the sequence of the THO ORF 1 gene product and regions of the PA protein of influenza virus strain A/NT/60/68 (approx. 20% aligned homology) and the corresponding protein of influenza B/Sing/222/79 virus (approx. 15% aligned homology). Although the THO protein sequence is not as closely related to those of the influenza viruses as they are to each other (40% aligned homology), the indicated sequence data provide further evidence of relationships between the tick-borne THO and DHO viruses and the vertebrate orthomyxoviruses.
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Kunjin Virus Isolates of Australia Are Genetically Homogeneous
More LessSUMMARYThe genomes of 22 isolates of Kunjin virus (KUN) from Australia were characterized and compared using RNase T1 oligonucleotide fingerprinting. The results show that all isolates belonged to one topotype, the distribution of which covered the entire Australian continent. This finding is similar to that of Murray Valley encephalitis virus, but in contrast to the results reported for some other flaviviruses such as Saint Louis encephalitis virus.
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- Plant
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Synthesis of RNAs Specific to Citrus Exocortis Viroid by a Fraction Rich in Nuclei from Infected Gynura aurantiaca: Examination of the Nature of the Products and Solubilization of the Polymerase-Template Complex
More LessSUMMARYViroid-specific polymerase activity was detected in preparations rich in nuclei from Gynura aurantiaca infected with citrus exocortis viroid (CEV). The polymerase catalysed the synthesis of several RNAs, shown to be viroid-specific since they could not be observed in control experiments with healthy plants, and they contained CEV-specific sequences most of which were of the same polarity as the viroid RNA. The synthesis of the CEV-specific RNA species was greatly reduced in the presence of 1 μm-α-amanitin, suggesting the involvement of RNA polymerase II in this process. The structure of the viroid-specific RNA species was studied by chromatography on non-ionic cellulose, digestion with RNase under low and high ionic strength conditions, and analysis by polyacrylamide gel electrophoresis in non-denaturing and denaturing systems. The results showed that these RNAs synthesized in vitro contain unit and longer than unit length linear viroid strands forming multistranded complexes with single- and double-stranded regions. The RNAs therefore have the same structural properties as deduced for RNAs isolated from viroid-infected tissues which are the presumed replicative intermediates of the rolling circle mechanism proposed for viroid synthesis. A soluble fraction containing the polymerase-template complex responsible for the synthesis of the CEV-specific RNAs was isolated by treatment of the nuclei-rich preparation with heparin and DNase. This soluble fraction could be of interest in further studies to characterize the components of the polymerase-template complex involved in CEV replication.
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Properties of a Viroid-replicating Complex Solubilized from Nuclei
More LessSUMMARYAn organelle-free fraction with the ability to synthesize citrus exocortis viroid (CEV) RNA was prepared from nuclei-rich samples taken from CEV-infected Gynura aurantiaca D.C. leaf tissue. This extraction was accomplished in the presence of the detergent sarkosyl. Characterization of the viroid synthetic reaction demonstrated that the solubilized complex retained the properties displayed by intact nuclei. These include optima of 1 mm-MnCl2, 10 mm-MgCl2 and 25 mm-(NH4)2SO4, as well as inhibition by α-amanitin (90% at 4 μg/ml) and by the chelators o-phenanthroline (78% at 5 mm) and hydroxyquinoline (45% at 5 mm). Nucleic acid-binding agents such as ethidium bromide and actinomycin D showed a low, non-specific inhibition at relatively high concentrations. Sucrose gradient sedimentation analysis of the sarkosyl supernatant (SSN) components before and after the RNA synthesis showed that the bulk of the progeny remained associated with the viroid complex which sedimented faster than phenol-treated viroid RNA. A high percentage of the CEV RNA molecules migrated in electrophoresis as the circular form. This suggests that all elements necessary for the synthesis of viroid RNA and processing to circular structures are present in the SSN. This endorses the potential of these subnuclear preparations for the study of the processes involved in viroid replication.
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Phylogeny of Geminiviruses
More LessSUMMARYAmino acid sequences of 16 geminivirus replication-associated proteins and 15 coat proteins were aligned and a new computer program was used to calculate the minimum mutation distances for all possible pairwise comparisons. These data were used to construct phylogenetic trees. Trees based on coat proteins had two main branches which were positively correlated with vector specificities of the viruses. Trees based on replication-associated proteins also had two main branches which were positively correlated with viral host specificities for either monocotyledonous or dicotyledonous plants. Therefore, evolutionary pressures on coat proteins and replication-associated proteins are probably highly influenced by vectors and hosts, respectively. Geminiviruses that infect dicotyledonous plants may be divided further by geographical origins into Old World and New World viruses. These results suggest the possible geographical origins of some geminiviruses, that new taxa should be erected, and have implications for distinguishing viruses and strains.
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Indian Cassava Mosaic Virus: Ultrastructure of Infected Cells
More LessSUMMARYUltrastructural examination of leaf tissue of Nicotiana benthamiana infected with Indian cassava mosaic virus (ICMV) revealed abnormalities in phloem and, occasionally, xylem cells. Nuclei contained granular inclusion bodies which seemed to be largely composed of virus-like particles and were shown by immunogold labelling (IGL) to be rich in ICMV coat protein. Later in infection, hollow spheres made up of fibrillar material were produced. Virus coat protein was not a necessary component of these structures but was sometimes found in their hollow centres. Cytoplasmic abnormalities were uncommon but a few vascular parenchyma cells contained paracrystalline aggregates which seemed to be made up of hollow tubes 30 to 40 nm in diameter. Some of these same cells also had large areas of cytoplasm which contained numerous randomly orientated tubules about 20 nm in diameter. No ICMV coat protein could be detected by IGL in either the paracrystalline or the tubule-containing inclusions. The ultrastructural effects of ICMV resemble those of other whitefly-transmitted geminiviruses, but with some differences of detail, and they also include types of abnormality not previously recorded.
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Immunogold Localization of Parsnip Yellow Fleck Virus Particle Antigen in Thin Sections of Plant Cells
More LessSUMMARYThe distribution of parsnip yellow fleck virus particle antigen in infected cells of Nicotiana clevelandii or Spinacia oleracea was examined by immunogold labelling of ultrathin sections. Best results were obtained by pretreating sections with Decon 75 followed by long incubation times on antiserum (16 h) and gold probe (6 h). The cytoplasmic inclusions induced by infection have three main components: accumulations of 20 to 30 nm diameter tubules, granular bodies and amorphous matrix material. Much gold label was located over the areas of amorphous matrix material whereas the other components of the inclusions were not labelled. Specific but less dense labelling was observed over virus-induced cell wall outgrowths, and over other areas of cell wall and some nuclei in infected cells. Virus-like particles found in 45 nm diameter tubules within the cell wall outgrowths were not labelled, perhaps because they were inaccessible to the antibody. The results indicate that large amounts of virus particle antigen are present in cells. However, the number of recognizable virus particles was considerably less than expected from the amount of virus extracted from leaves.
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Plant Virus Transport Function: Complementation by Helper Viruses Is Non-specific
More LessSUMMARYThe possibility of complementation of the cell-to-cell spread (within the inoculated leaf) between different related and unrelated plant viruses has been studied. Various tobamoviruses (tobacco mosaic, sunn-hemp mosaic, cucumber green mottle mosaic viruses and ‘tobamovirus from orchids’) can facilitate each other’s replication in non-permissive hosts or at a temperature non-permissive for transport of one of the virus partners, probably by complementation of transport functions. Complementation of movement also occurred between some, but not all unrelated viruses tested. The complementation in transport function seems to be non-specific: it can occur between viruses even if their putative transport proteins significantly differ in structure. Consequently these viruses were classified tentatively into different ‘transport groups’.
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The Outer Capsid Protein of Rice Dwarf Virus Is Encoded by Genome Segment S8
SUMMARYThe nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an M r of 46422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.
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Symptom Production on Tobacco and Tomato Is Determined by Two Distinct Domains of the Satellite RNA of Cucumber Mosaic Virus (Strain Y)
More LessSUMMARYComplementary DNAs of two different satellite RNA isolates from cucumber mosaic virus (CMV), Y from Japan and Ra from France, have been cloned in a transcription vector containing the Pr promoter. When inoculated on plants with CMV RNA (strain KIN), the transcripts of the cloned Y satellite cDNA elicit a bright yellow mosaic on tobacco and a lethal necrosis on tomato. Addition of the transcripts of the Ra satellite cDNA to an inoculum of CMV RNA resulted in symptom attenuation on both tobacco and tomato, in agreement with the characterized symptoms of the natural satellite. Recombinant molecules involving these two satellites have been constructed in order to determine which parts of the Y satellite RNA are involved in symptom induction. The determinant for symptom production on tobacco lies in the region between nucleotides 1 and 219. The domain for necrotic symptoms on tomato resides on the 3′ half of the molecule beyond nucleotide 219.
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The Use of 3′ Non-coding Nucleotide Sequences in the Taxonomy of Potyviruses: Application to Watermelon Mosaic Virus 2 and Soybean Mosaic Virus-N
More LessSUMMARYThe sequence of the 3′ 1106 nucleotides of the watermelon mosaic virus 2 (WMV 2) genome has been determined. The sequence contains the complete coding region of the viral coat protein followed by a 3′ untranslated sequence of 251 nucleotides. When these sequences were compared with the equivalent regions of the N strain of soybean mosaic virus (SMV-N), the coat protein coding regions were 82% homologous, whereas the 3′ untranslated sequences were 78% homologous. Optimal alignment of the 3′ untranslated regions of RNA from 13 strains of seven other distinct potyviruses revealed that the degree of homology between strains was in the range 83 to 99%. In contrast, the sequences from distinct viruses had identities in the range 39 to 53%, comparable to the level of identity found between the 3′ non-coding regions of viruses from unrelated plant virus groups. On the basis of these results, WMV 2 and SMV-N could be regarded as strains of one virus. These results also suggest that the sequence of the 3′ untranslated region of the potyvirus genome may be an accurate marker of genetic relatedness and could serve as an aid to identification and classification of potyviruses.
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The Genome-linked Protein and 5′ End RNA Sequence of Plum Pox Potyvirus
More LessSUMMARYThe infectivity of plum pox potyvirus (PPV) RNA was decreased by treatment with proteases. Ribonuclease digestion of iodinated PPV RNA yielded material which had an electrophoretic mobility corresponding to M r 22000. This protein presumably corresponds to the protease-sensitive structure needed for infectivity. A protein-linked RNase T1-resistant oligonucleotide, 38 nucleotides long, was sequenced and shown to correspond to the 5′ terminus of the RNA by sequence comparison to the RNAs of two other potyviruses, tobacco etch virus and tobacco vein mottling virus. A 12 nucleotide block was found to be completely conserved in the RNAs of the three viruses.
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Volume 73 (1992)
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Volume 72 (1991)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)