- Volume 71, Issue 12, 1990
Volume 71, Issue 12, 1990
- Animal
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Inhibition of Primary Transcription of Vesicular Stomatitis Virus by Prostaglandin A1
More LessProstaglandin A (PGA) exhibits antiviral activity against RNA and DNA viruses. The effect of PGA, on vesicular stomatitis virus (VSV) was investigated. When VSV-infected L-1210 cells were kept in the presence of PGA 2 the amount of all five viral proteins and their respective mRNAs was dose-dependently decreased. To determine whether the effect was on viral transcription or translation, the temperature-sensitive VSV mutant tsG 41 was employed. This is a good model system for the investigation of primary transcription; at the restrictive temperature of 39 °C, tsG 41 is unable to replicate but can transcribe viral mRNA. Mutant mRNA synthesis was strongly inhibited by PGA, at this temperature, indicating that the major effect is on primary transcription. This conclusion is supported by data demonstrating that in vitro transcription of viral genomic RNA was also inhibited by PGA1
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Effect of Protein Kinase C Inhibitors on Interferon-β Production by Viral and Non-viral Inducers
More LessInduction of interferon-β (IFN-β) in human (BG-9), simian (CV-1) and mouse (L-929) cell lines by Sendai virus and by poly(rI). poly(rC) has been studied for its possible dependence on protein kinase C (PKC) through the use of pharmacological inhibitors (K252a and H-7) of PKC. Exposure of BG-9, CV-1 or L-929 cells to K252a (⩾0·025 μm), a staurosporine derivative, 24 h before or after induction of IFN with poly(rI).poly(rC), inhibited by >95% the production of IFN-β. In contrast, virus-induced IFN production was enhanced threefold or more by K252a in BG-9 and L-929 but not in CV-1 cells. A naphthalene sulphona-mide inhibitor of PKC, H-7, at ⩾5 μm, decreased poly(rI). poly(rC)-induced IFN production in BG-9 and CV-1 cells by 75 to 94%, but had no effect on IFN production in L-929 cells. Viral induction of IFN was not affected significantly by H-7 in BG-9, CV-1 and L-929 cells. In contrast to these results, the calmodulin inhibitor, trifluoperazine (5 to 15 μm) did not affect IFN-β production by poly(rI). poly(rC) but significantly enhanced IFN production by Sendai virus in both human and murine cell lines. Thus, in human and simian fibroblasts the induction of IFN-β by poly(rI).poly(rC) appears to be PKC-dependent, whereas viral induction of IFN-β is not. Results with K252a implicate PKC in non-viral induction of IFN in mouse fibroblasts, as well. Direct measurements of PKC activity in BG-9 cells exposed to several concentrations of K252a showed that the membrane PKC activity is significantly more sensitive to inhibition by K252a than is cytosolic PKC activity. In L-929 cells, K252a inhibited membrane PKC activity similarly, but was less effective as an inhibitor of cytosolic enzyme activity than in BG-9. These studies support an intregral role for PKC activity, particularly membrane-associated activity, in non-viral [poly(rI). poly(rC)] induction of IFN-β in human, simian and mouse fibroblasts.
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Characterization of the Infection Cycle of the Orgyia Pseudotsugata Multicapsid Nuclear Polyhedrosis Virus in Lymantria Dispar Cells
More LessTo characterize the infection cycle of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus in Lymantria dispar cells, the time course of DNA synthesis and polyhedron production, and the onset and rate of budded virus production were investigated at three different m.o.i. (5,10 and 100). In addition, the time course of expression of three proteins (gp64, p39 and polyhedrin) representative of three temporal classes of baculovirus genes was also analysed using Western blot analysis. DNA synthesis began at 12 to 18 h post-infection (p.i.). The rate of budded virus (BV) production reached maximal levels at 24 to 36 h p.i. and continued at high levels indicating that BV production was not turned off late in infection. Polyhedra were first observed at 48 h p.i. The m.o.i. appeared to influence the magnitude but not timing of early events in the viral infection cycle (gp64 expression and DNA synthesis) and also influenced the initial levels of BV production and the percentage of cells containing occlusion bodies. The m.o.i. had little influence on the final rates of BV production and the time of detection of p39 and polyhedrin on Western blots.
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Localization of Adenovirus-encoded DNA Replication Proteins in the Nucleus by Immunogold Electron Microscopy
More LessThe distribution of three adenovirus-encoded DNA replication proteins in the nucleus of human 293 cells was studied by immunogold electron microscopy. The infected nuclei contained four morphologically distinct inclusions. They were highly electron-dense granules (type I), compact fibrogranular masses of medium electron density (type II), filamentous masses of low electron density (type III) and large polygonal crystals (type IV). In immunogold labelling studies, antibodies to the adenovirus single-stranded DNA-binding protein (DBP) and antibodies to single-stranded DNA showed extensive binding to the type III inclusions. The antibodies to the adenovirus DNA polymerase (AdPol) and terminal protein (TP) predominantly labelled type II inclusions. Double immunogold labelling studies detected low levels of AdPol and TP in type III inclusions and DBP in type II inclusions. The selective distribution of DNA replication proteins suggests that the type II and III inclusions represent two functionally different entities that may be involved in two different aspects of adenovirus DNA replication, i.e. chain initiation and elongation.
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Characterization of Antibody and Cytotoxic T Lymphocyte Responses to Human Influenza Virus H3 Haemagglutinin Expressed from the Haemagglutinin Locus of Vaccinia Virus
More LessAntibody and cytotoxic T lymphocyte (CTL) responses to the haemagglutinin (HA) of human H3N2 influenza virus were analysed, using recombinant vaccinia viruses containing the influenza HA gene inserted into the HA gene locus of vaccinia virus. The recombinant vaccinia viruses elicited a high haemagglutination inhibiting (HI) antibody response to the homologous influenza virus in mice. In addition, HI antibody generated by the recombinant vaccinia virus reacted with antigenic variants of human H3N2 influenza virus in a manner similar to that elicited by the HA vaccine. Mice with a high response to influenza virus HA vaccine were highly responsive to the HA expressed from the recombinant vaccinia virus, as measured by HI antibody production. The immunogenicity of the influenza virus HA expressed by the recombinant seems to be attributable to the intrinsic immunogenicity of the HA molecule. The recombinants primed mice for an influenza virus H3-specific CTL response and primed CTLs recognized the target cells in a subtype-specific manner. The results indicate that a recombinant vaccinia virus derived by the insertion of a foreign gene into its HA gene locus is a potent live vaccine not only for eliciting a high antibody response but also for priming a specific CTL response.
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Theiler’s Murine Encephalomyelitis Virus-binding Activity on Neural and Non-neural Cell Lines and Tissues
More LessThree categories of cell lines are described with respect to their activity in binding Theiler ’s murine encephalomyelitis virus (TMEV). High, medium and low densities of viral receptors can be detected on cell lines from different species and origins by using an immunological binding assay. Nevertheless, TMEV acts as a fastidious virus that only infects a few cell types productively. No correlation between virion binding and degree of permissiveness to infection could be detected. The presence of viral receptors in both susceptible and resistant strains of mice seemed to have a widespread tissue distribution, the thymus being an exception. When primary cerebral cultures, enriched in neurons, astrocytes or oligodendrocytes, were checked in the immunological assay, a higher density of viral receptors was detected in the neuronal population. The number of virus-binding sites in the BHK-21 cell line is reported here to be 5 × 103 per cell; approximately 15 × 103 and 2·5 × 103 are the estimates of binding sites per cultured neuron and macroglial cell, respectively.
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Analysis of the Fowlpox Virus Genome Region Corresponding to the Vaccinia Virus D6 to A1 Region: Location of, and Variation in, Non-essential Genes in Poxviruses
More LessThe DNA sequence of the fowlpox virus genome corresponding to the vaccinia virus D6 to A1 region has been determined. Translation of this sequence reveals fowlpoxvirus gene homologues corresponding to the D6, D7, D9, DIO, Dll, D12, D13 and A1 genes of vaccinia virus. In contrast, no gene homologue for the non-essential vaccinia virus D8 gene was present in fowlpox virus. Instead, a gene transcribed from the opposite strand to the vaccinia virus D8 gene showing no homology to any previously sequenced poxvirus gene was present. The amino terminus of the fowlpox virus D9 homologue had undergone substantial changes, including frameshifts which would be predicted to inactivate the gene. Insertion of a gene cartridge composed of the vaccinia virus p7 · 5 promoter and the lacZgene into the fowlpox virus D8, D9 and DIO genes in vitro, followed by recombination into fowlpox virus, was carried out. Stable insertion mutants with the correct genotype were obtained for D8 and D9 which, when tested in chickens did not appear to have been attenuated. No stable insertion mutants were obtained for DIO, indicating that this gene probably encodes a function which is essential for virus replication. The D8 and D9 genes of fowlpox virus represent useful insertion sites for the construction of recombinant fowlpox virus vaccines.
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A 39000 M r Immunodominant Protein of Fowlpox Virus Contains Multiple Copies of a 12 Amino Acid Repeat Sequence
More LessThe nucleotide sequence of an unusual fowlpox virus gene which maps immediately upstream from the fowlpox virus 4b gene has been determined. The 34000 M r protein predicted to be encoded by the gene contains 11 copies of a 12 amino acid serine-rich repeat sequence. The seven amino-terminal copies of the repeat sequence are perfectly conserved but variation exists in the four carboxy-terminal copies. Three peptides were synthesized which contained either one copy of the repeat sequence, two copies of the repeat sequence or a hydrophilic amino-terminal region of the protein. All three peptides when injected with adjuvant into rabbits gave rise to antibodies which reacted strongly on Western blots of purified fowlpox virus proteins with a 39000 M r protein. When directly compared in Western blots the antipeptide sera were shown to recognize a protein comigrating with one of the two immunodominant proteins recognized by chicken anti-fowlpox virus sera taken 2 weeks postinfection. The virion protein is removed by treatment with sodium deoxycholate suggesting that it is located at or near the surface of the virus.
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Glycosylation Governs the Binding of Antipeptide Antibodies to Regions of Hypervariable Amino Acid Sequence within Recombinant gp120 of Human Immunodeficiency Virus Type 1
More LessAntibodies raised to an overlapping series of peptides following the amino acid sequence of the external envelope glycoprotein (gpl20) of human immunodeficiency virus type 1 (HIV-1) recognize eight regions in recombinant gpl20 molecules. If the recombinant molecules are glycosylated, three of these regions show a reduced capacity to bind antibody. Of the other five regions, two are strain-specific and carbohydrate restricts antibody binding to their N-terminal flanks, and three can be recognized by antibodies in recombinant gpl20 from an unrelated strain of HIV-1. Antibodies in sera from HIV-l-infected patients bind at high levels to peptides from five regions of gpl20. Of these regions, two coincide with those recognized by antibodies raised to peptides. Four of the five epitopes recognized by the rat antipeptide sera whose ability to bind antibody is influenced most by glycosylation, and three of the five regions which induce high levels of antibodies in patients’ sera, contain putative glycosylation sites which are variable between strains of HIV-1. Such sites flank the putative neutralization and CD4-binding regions of gpl20. It is suggested that changes in the number and position of carbohydrate moieties following mutation can alternately mask and reveal epitopes. Masking an epitope can render a virus resistant to neutralization, whereas virus which binds antibody without being neutralized is able to gain entry to cells bearing antibody and complement receptors. Changes in the glycosylation pattern of gpl20 may therefore contribute to the control of HIV-1 spread within its host.
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Liposomes Modulate Human Immunodeficiency Virus Infectivity
We have investigated the effects of the fusion of liposomes with human immunodeficiency virus type 1 (HIV-1Lva) on the ability of the virus to infect CD4+ and CD4− cells. Fluorescence dequenching measurements indicated that HIV-1 fuses with liposomes composed of either cardiolipin (CL) or N-[2,3-(dioley- loxy) propyl]- N,N,N -trimethyl ammonium chloride (DOTMA) but not appreciably with dioleoylphospha- tidylcholine (DOPC) liposomes. Pre-incubation of HIV-1 with DOTMA liposomes enhanced virus production (measured by p24 gag antigen production in the culture medium and in situ) in CD4+ A3.01 and H9 cells in a concentration-dependent manner, but did not mediate the infection of the CD4− cell line, K562. Pre-incubation of HIV-1 with between 10 and 30 | μ m- DOTMA liposomes, and subsequent incubation with A3.01 cells, resulted in the production of about 30-fold greater levels of virus than controls. The presence of DOTMA liposomes during the incubation of A3.01 cells with HIV-1 enhanced the infectivity of the virus up to 90-fold compared to controls. Conversely, preincubation of HIV-1 with CL liposomes inhibited infection of A3.01 cells, dependent on the concentration of liposomes; DOPC liposomes did not alter the infectivity of the virus under any of the incubation conditions. Our results thus indicate that fusion of HIV-1 with liposomes alters the ability of the virus to infect its target cells.
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Measurement of Antibody-dependent Infection Enhancement of Four Dengue Virus Serotypes by Monoclonal and Polyclonal Antibodies
More LessAlthough its underlying mechanisms are poorly understood, data comparing each of the four dengue virus serotypes suggest that in vitro antibody-dependent infection enhancement is a reproducible and measurable phenomenon related to other serological measures of antibody-virus binding. Information characterizing infection enhancement may provide clues to disease pathogenesis for dengue and other viruses that exhibit antibody-enhanced infection. We propose criteria for the detection and quantification of in vitro antibody- dependent enhancement of flavivirus infection based on observations using all four dengue virus serotypes, macrophage-like cell lines and human peripheral blood monocytes, and various immune sera and monoclonal antibodies. It is proposed that antibody-dependent infection enhancement is defined by the following findings: (i) significantly increased virus production is measured in quantitative assays at different points on the growth curve; (ii) assays of the virus output of cells infected with mixtures of constant amounts of virus and serial dilutions of the pre-existing antibody source produce characteristic ‘enhancement profiles’ of rising and falling virus output over at least a 10−3-fold dilution range; (iii) for each enhancing antibody source the dilution producing maximal infection enhancement is related to other serological measures of binding to the envelope, or another virus component; (iv) infection enhancement is detected with different antibody sources and virus strains (when available) tested over a range of m.o.i.; (v) other causes of enhanced virus production are ruled out.
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Genetic Variation of Japanese Encephalitis Virus in Nature
More LessForty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
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Epitope Analysis of the Envelope and Non-structural Glycoproteins of Murray Valley Encephalitis Virus
More LessPrevious studies have shown that antibodies produced against strategic flavivirus epitopes play an important role in recovery and immunity. Definition of the conformation and location of these epitopes and the degree of their conservation among flaviviruses is important to understanding the humoral response to flavivirus infection. In this study we have examined epitopes recognized by 14 monoclonal antibodies (MAbs) produced to the envelope (E) and nonstructural (NS1) proteins of Murray Valley encephalitis virus (MVE). These antibodies were analysed for specificity, neutralization, haemagglutination inhibition (HI) and competitive binding. We have identified six distinct epitopes on the E protein which are located in four non-overlapping domains. MAbs to epitopes in one domain neutralized virus, were specific for MVE and Japanese encephalitis virus, and reacted with epitopes resistant to reduction. Two other E domains, one specific to MVE and the other shared by all flaviviruses, also contained neutralization sites and were stabilized by disulphide bonds. The fourth domain on E was conserved among the flaviviruses, sensitive to SDS denaturation and did not induce neutralizing antibody. Studies with MVE NS1 MAbs revealed that they were mostly type-specific, unreactive with conserved epitopes, and unreactive in HI and neutralization tests. The six epitopes identified on NS1 did not overlap and represent antigenic domains either resistant or sensitive to reduction. Immunoblotting of viral proteins in MVE-infected C6/36 cells revealed two distinct forms of NS1 and high M r proteins of 97K and 108K that represented disulphide-linked heterodimers of E and NS1.
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Insertion of DNA Sequences at a Unique Restriction Enzyme Site Engineered for Vector Purposes into the Genome of Herpes Simplex Virus Type 1
More LessWe describe the construction of a novel herpes simplex virus (HSV) vector containing a unique XbaI restriction enzyme cloning site in an intergenic position in the short unique genome region. Sequences can be inserted at this site with high efficiency by ligation with XbaI-digested vector DNA. A series of plasmids has been designed for use with this vector. These allow protein coding sequences to be placed under the control of various transcriptional regulation signals and then isolated as XbaI fragments ready for insertion into the vector. The XbaI fragments also contain the β- galactosidase gene thereby facilitating selection of recombinant virus by screening for blue plaques. A variant of the vector has been made based on the temperature-sensitive (ts) mutant tsK, which expresses only immediate early (IE) genes at non-permissive temperatures. Chloramphenicol acetyltransferase was used as a reporter gene to assess the fidelity of expression of sequences cloned into this position. Under these circumstances IE and early HSV promoters were shown to behave as expected in both wild-type and ts vectors.
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The Herpes Simplex Virus Type 1 Alkaline Nuclease is Not Essential for Viral DNA Synthesis: Isolation and Characterization of a lacZ Insertion Mutant
More LessHerpes simplex virus type 1 (HSV-1) encodes a novel enzyme activity, the alkaline nuclease, whose precise role in the viral replication cycle remains obscure. The alkaline nuclease gene corresponds to the UL12 open reading frame, which is predicted to encode a protein of 626 amino acid residues. We describe the isolation and characterization of a null mutant of the gene for the viral alkaline nuclease in which 917 bp from the N- terminal half of the gene (corresponding to residues 70 to 375) were deleted and replaced by the insertional mutagen ICP6::lacZ. The resulting mutant virus, AN- 1, was propagated in helper cells (S22) which express the wild-type version of the alkaline nuclease gene. Mutant AN-1 growth in Vero cells is severely restricted, although small amounts of infectious virus are produced. On the other hand, wild-type levels of viral DNA and late viral proteins are expressed in virus AN- 1-infected Vero cells. These results indicate that the HSV-1 alkaline nuclease gene product is not essential for viral DNA synthesis but may play a role in the processing or packaging of viral DNA into infectious virions. Possible roles in the viral infectious cycle will be discussed.
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Identification and Characterization of the Virion Protein Products of Herpes Simplex Virus Type 1 Gene UL47
More LessWe have identified two encoded proteins with an antiserum raised against a synthetic oligopeptide corresponding to amino acids 671 to 684 of the predicted protein product of gene UL47 of herpes simplex virus type 1 (HSV-1). They have apparent Mrs of 82000 and 81000 and are both major virion components located in the tegument. The 82/8IK proteins were first detected in infected cells in minor amounts 6 h after infection at 37 °C but were later (from 10 h until 24 h after infection) present in large amounts. UL47 regulation was investigated using phosphonoacetic acid (PAA), an inhibitor of DNA synthesis: the amounts of the 82/8IK protein synthesized were compared with those of 65KDBP, an early gene product, and 21K/22K, a true late gene product. The data showed that UL47 is regulated as a true late gene.
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Inactivation of the Shutoff Gene (UL41) of Herpes Simplex Virus Types 1 and 2
More LessGene UL41 of herpes simplex virus type 1 (HSV-1) and the corresponding gene of HSV-2, which control the virion-mediated early suppression of cellular protein synthesis, have been inactivated by inserting a β- galactosidase expression cassette into their coding regions. The resulting recombinants grew well in tissue culture, although with the type 2 recombinant viral protein synthesis was slightly delayed. As a result of inactivation of UL41 host protein synthesis was not suppressed in the presence of actinomycin or early in normal infection, although it declined at a late stage. Polyribosomes were not broken down early in infection, cellular DNA synthesis was not inhibited and in the presence of cycloheximide stable alpha (immediate early) mRNA accumulated, in marked contrast to that of the parent HSV-2 strain. Comparison of the proteins of purified virions of HSV-1 and shutoff-defective recombinant virus revealed discrepancies consistent with the presence of the UL41 gene product in the enveloped virion.
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Equine Herpesvirus Type 1 Unique Short Fragment Encodes Glycoproteins with Homology to Herpes Simplex Virus Type 1 gD, gI and gE
More LessThe nucleotide sequence of a 6·4 kbp portion of the 10·6 kbp BamHI fragment D contained in the unique short region of the equine herpesvirus type 1 (EHV-1) genome has been determined. Analysis of this sequence revealed five open reading frames (ORFs), four complete and one incomplete, which were encoded by the same sense strand. Comparison of the EHV-1 DNA sequence with that encoding glycoproteins of other alphaherpesviruses has revealed no significant homologies. Comparison at the amino acid level, however, has demonstrated regions of significant sequence similarity between the three complete EHV-1 ORFs 2, 3 and 4, and the herpes simplex virus type 1 (HSV-1) glycoprotein gD encoded by the US6 gene, the HSV-1 glycoprotein gI encoded by the US7 gene and the HSV-1 glycoprotein gE encoded by the US8 gene, respectively. The interrupted ORF 5 was found to display partial homology with the HSV-1 US9- encoded protein, but no homology was found between the protein encoded by ORF 1 and other proteins. The three collinear EHV-1 ORFs encoding putative glycoproteins with homology to the HSV-1 glycoproteins were therefore designated EHV-1 gD, gI and gE, respectively. Moreover, further similarities were found between EHV-1 gD and pseudorabies virus (PRV) gp50, between EHV-1 gI and PRV gp63 and varicella- zoster virus (VZV) gpIV, and between EHV-1 gE and PRV gI and VZV gpI. It is concluded that EHV-1, PRV, HSV-1 and VZV encode homologous glycoprotein genes in the small unique components of their genomes and that the genetic organization of these regions is conserved.
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Herpesviral Deoxythymidine Kinases Contain a Site Analogous to the Phosphoryl-binding Arginine-rich Region of Porcine Adenylate Kinase; Comparison of Secondary Structure Predictions and Conservation
More LessTwelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple alignment. Six highly conserved sites were observed. Site 1 corresponded to a glycine-rich loop that forms part of the ATP-binding pocket in porcine adenylate kinase (PAK), and site 5 corresponded to a region in PAK, located on one lobe of the cleft, that contains arginine residues that bind substrate phosphoryl groups. Site 3, consisting of the motif -DRH-, is thought to be involved in thymine/deoxythymidine recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2 and 6 have not been identified. Secondary structure predictions were made by the Gamier method and averaged for each position in the multiple alignment. The structure predicted for all six sites was typically a short flexible region (turn or coil) at or adjacent to the site, flanked by rigid structures (helix or sheet) on either side.
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The Mechanism of the Antiherpetic Activity of Zinc Sulphate
More LessThe molecular mechanism of the effects of zinc ions against herpes simples virus (HSV) infection was investigated. Zinc sulphate (100 µm) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1 %0 of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mm-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein- dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.
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The Product of Varicella-zoster Virus Gene 62 Autoregulates Its Own Promoter
More LessVaricella-zoster virus (VZV) gene 62 encodes a protein with a predicted M r of 140000 (140K) which has considerable amino acid identity with the major immediate early (IE) protein Vmwl75 (ICP4) of herpes simplex virus type 1 (HSV-1). Vmwl75 is an essential virus polypeptide with a pivotal role in the activation of early and late viral gene expression and also in the repression of IE gene expression. The VZV 140K protein has been shown to function as a strong transcriptional activator in transfection assays and largely complements for the loss of Vmwl75 function in HSV-1. We report the results of cotransfection experiments which demonstrate that the 140K protein strongly represses expression from its own promoter, that of gene 62, thus establishing further functional similarity between it and Vmwl75. However, whereas Vmwl75 can substitute for the 140K protein in repression of the gene 62 promoter, the 140K protein does not repress the HSV-1 IE3 promoter in the reciprocal experiment. The integrity of a domain of Vmwl75 (designated region 2), previously shown to be crucial for repression of the HSV-1 IE3 promoter, is also required for repression of the gene 62 promoter. Moreover, a similar requirement for the highly similar region 2 of the 140K protein for repression is demonstrated, suggesting that VZV 140K protein and HSV-1 Vmwl75 autoregulate IE gene expression by a related mechanism.
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Differential Expression of Two Immediate Early Genes of Herpesvirus Saimiri as Detected by in Situ Hybridization
More LessTranscripts from two immediate early (IE) genes have been identified in cells infected with the gammaherpes- virus, herpesvirus saimiri. One is a 1·3 kb RNA transcribed from the HindIII-G fragment of virus DNA (IE-G), the other is a 1·6 kb RNA from the gene for the IE 52K phosphoprotein. Labelled oligonucleotide probes specific for each of these RNAs have been used in in situ hybridization experiments to compare their expression in individual cells in infected populations. In the presence of cycloheximide, the IE-G RNA accumulates synchronously throughout the population of infected cells and prior to the asynchronous accumulation of RNA from the gene for the IE 52K protein in the same population of cells. This heterogeneity in the timing of expression of RNA from the IE 52K gene is paralleled by the asynchronous accumulation of the protein product. We conclude that transcription of the IE-G RNA is independent of expression of the IE 52K gene and that expression of the 52K gene requires (or is prevented by) factors which do not affect accumulation of the RNA from the IE-G gene.
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Sequence Comparison Between the Fusion Protein of Human and Bovine Espiratory Syncytial Viruses
More LessThe nucleotide sequence was determined for the fusion (F) protein-coding mRNA of the bovine respiratory syncytial virus (strain RB 94) and the amino acid sequence of the F protein was deduced for comparison with the sequence of human respiratory syncytial virus subtypes A and B (RSS-2 and 18537 strains). The human and bovine RS virus F proteins (excluding the cleaved signal peptide) share 83 to 84% homology. The greatest divergence occurred within the F2 subunit in the region preceding the cleavage activation site.
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The Two Open Reading Frames of the 22K mRNA of Human Respiratory Syncytial Virus: Sequence Comparison of Antigenic Subgroups A and B and Expression in Vitro
More LessThe sequence of the 22K mRNA of strain 18537 of antigenic subgroup B of human respiratory syncytial virus (RSV) was determined by sequencing cloned cDNAs of intracellular mRNA. Comparison with the corresponding sequence of the A2 strain of subgroup A showed that there is 78% nucleotide sequence identity overall, that the amino acid sequence of the 22K protein is 92% identical between subgroups and that the 22K mRNA of both subgroups contains a second, internal, overlapping open reading frame (ORF) whose length, nucleotide sequence and potential translational start and stop sites were highly conserved and whose predicted product has 62% amino acid identity between subgroups. Sequence analysis of 36 cDNAs of intracellular 22K mRNA of strain A2 did not detect nucleotide insertions or deletions in the region of overlap between the two ORFs, indicating that the majority of intracellular 22K mRNA is a faithful copy containing the two distinct ORFs. Translation in vitro of mRNAs transcribed from engineered cDNAs showed that an mRNA which contained only the second, internal ORF directed the synthesis of a previously unidentified polypeptide of the predicted size, whereas mRNA representing the complete gene directed the synthesis of both the 22K protein and the product of the internal ORF. This latter species was synthesized in vitro as a discrete, separate protein rather than as a fusion protein. It is not yet known whether this protein is synthesized in RSV-infected cells.
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Antisense Oligodeoxyribonucleotides Inhibit the Expression of the Gene for Hepatitis B Virus Surface Antigen
More LessThe effect of a series of antisense oligodeoxyribonucleotides [oligo(dN)] on the expression of the surface antigen (HBsAg) gene of human hepatitis B virus (HBV) was examined using hepatocellular carcinoma cells that contain integrated HBV genomes. Of a number of antisense oligo(dN)s tested, synthetic 15-mers directed at the cap site of mRNA and regions of the translational initiation site of the HBsAg gene were found to be highly effective and inhibited viral gene expression by as much as 96 %. The inhibition was specific to the HBsAg gene and appeared to be at the level of translation. These results suggest a therapeutic potential for antisense oligo(dN) in the treatment of patients who are chronically infected with HBV.
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The Putative Nucleocapsid and Envelope Protein Genes of Hepatitis C Virus Determined by Comparison of the Nucleotide Sequences of Two Isolates Derived from an Experimentally Infected Chimpanzee and Healthy Human Carriers
cDNA fragments of a 5′-terminal region of the hepatitis C virus (HCV) genome were isolated by the reverse polymerase chain reaction from RNA extracted from plasma samples of healthy Japanese carriers. Their nucleotide sequence was compared with that of the original isolate which had been passaged twice in chimpanzees. No deletions or insertions were observed between the two sequences in the regions examined. Both the 5′ untranslated and putative nucleocapsid (core) protein regions were highly conserved (99 % and 91% nucleotide identities, respectively). In contrast, the region immediately downstream which encodes a putative envelope glycoprotein(s) showed only 74% nucleotide identity between the two isolates. At the polypeptide level, the core and envelope domains showed 97% and 75% amino acid identities, respectively. This envelope variation may reflect the adaptation of HCV to the different hosts and/or the result of immunological selection. The highly conserved nucleotide sequence of the 5′ untranslated and core regions may play an important regulatory role in the life cycle of HCV.
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Nucleotide Sequence and Transcriptional Analysis of the HindIII P Region of a Temperature-sensitive Mutant of Autographa Californica Nuclear Polyhedrosis Virus
More LessDNA sequence analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus confirmed the specific amplification of 1·4 kb of viral DNA from this region of the genome. The sequenced region included an open reading frame, translated in a counterclockwise direction, which would potentially encode a 74K protein. The amplified DNA was contained within this open reading frame, resulting in in-frame amplifications of a domain within the protein. Transcription studies revealed the presence of a ladder of viral RNA species corresponding to a 2·5 kb transcript carrying tandem repeats of about 1·4 kb. This indicated that the duplicated DNA was transcribed in the same orientation as the p10 gene. We predict that transcripts synthesized from the opposite DNA strand also consist of a ladder of related mRNAs which would be translated to produce a family of p74 proteins with multiple internal domains.
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Cooperation Between Bovine Papillomavirus Type 4 and ras in the Morphological Transformation of Primary Bovine Fibroblasts
More LessPrimary bovine fibroblasts derived from foetal palate can be transformed by bovine papillomavirus type 4 DNA only in the presence of an activated ras gene, indicating that the virus does not encode all the information required for morphological transformation of non-established cells. A subgenomic fragment containing the complete E8 and E7 open reading frames (ORFs) induces transformation in cooperation with activated ras but transformation is abolished when the E7 ORF is deleted at the 3’ end, showing that this ORF encodes a necessary transforming function. Transformation is more aggressive when the E8 and E7 ORFs are placed under the transcriptional control of the long terminal repeat of the mouse Moloney leukaemia virus, suggesting that the degree of transformation is dependent on the level of expression of these genes.
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Translocation and Cleavage of Rubella Virus Envelope Glycoproteins: Identification and Role of the E2 Signal Sequence
More LessThe structural proteins of rubella virus (RV) are translated as a large polyprotein precursor, p110, which is processed to produce the mature virion components, the 33K capsid protein (C) and the two envelope glycoproteins, E1 (58K) and E2 (42K to 47K). The precise processing mechanism has not been elucidated; however it must include at least two proteolytic cleavages to release the individual virion components from the polyprotein, and it must provide for their dichotomous intracellular distribution. The C protein remains in the cytoplasm where it participates in the formation of nucleocapsids, while the envelope glycoproteins enter the cellular secretory pathway and are N-glycosylated and cleaved. Sequence analysis of the 24S mRNA encoding the polyprotein precursor suggests that both E1 and E2 are preceded by signal peptides for translocation across the membrane of the rough endoplasmic reticulum. A recent study has provided direct evidence that the putative signal peptide preceding E1 can in fact mediate translocation of E1. In this study, we have used in vitro translation- translocation assays to examine further the processing of RV glycoproteins. We have shown that the putative signal sequence preceding E2 can mediate translocation of the E2 protein in the absence of an intact E1 signal peptide. The experiments also revealed that cleavage of the E2-E1 polyprotein requires (i) the E2 signal peptide, (ii) microsomal membranes and (iii) sequences beyond the proximal half of the E1 signal peptide. Together these results suggest that separation of the E2 signal sequence as well as the proteolytic cleavage of El from E2 is performed by the cellular enzyme, signal peptidase.
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Neutralizing Antibodies to Human Rhinovirus Produced in Laboratory Animals and Humans that Recognize a Linear Sequence from VP2
More LessSynthetic peptides representing amino acids 156 to 170 of virus capsid protein VP2 from human rhinovirus (HRV) type 2 have previously been used to elicit a neutralizing antibody response; polyclonal antisera against this peptide have been compared with a virusneutralizing monoclonal antibody (8F5) which recognizes the same linear sequence. The neutralizing activity of both antibodies against virus is abolished by an amino acid substitution in VP2 at position 163 but only the peptide antiserum neutralizing activity is affected by a change in VP2 at position 158. The minimal binding site of 8F5 has been mapped, using overlapping peptides, to a sequence TRLNPD covering VP2 residues 160 to 165. Using affinity purification it has been shown that 8 to 10% of total neutralizing activity of polyclonal rabbit or guinea-pig antivirus antiserum is due to recognition of this linear VP2 156 to 170 determinant. Furthermore, a similar population of neutralizing antibodies appears to be associated with an immune response to recent HRV infection in humans. These results confirm the existence of linear determinants on the surface of HRV which may be mimicked by suitable synthetic peptides.
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Purification and Initial Characterization of Human Placental Trophoblast Interferon Induced by Polyriboinosinic · Polyribocytidylic Acid
Human placental trophoblast interferon (tro-IFN), induced in trophoblast cultures by a superinduction procedure, was purified to a homogeneous product with retention of biological activity. The problems associated with isolation from serum-containing medium were overcome by a combination of Blue Sepharose affinity chromatography and reversed-phase HPLC (RP-HPLC) on Separon SGX C-18. This two-step purification procedure yielded tro-IFN with a specific activity of 3·4 × 107 international units/mg of protein. The overall recovery of interferon activity was 66·7%. The purified tro-IFN was shown to be a glycoprotein with an Mr of 24K on native and SDS-PAGE. Its antiviral activity was stable at pH 2·0 at 37 °C but was sensitive to heat at 56 °C for 1 h and was neutralized by antibodies to human IFN-β.
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Interferon Production by Cultured Human Trophoblasts and Choriocarcinoma Cell Lines Induced by Sendai Virus
More LessHuman term-placental trophoblasts in primary culture were studied for an interferon (IFN) response when challenged with Sendai virus and compared to three choriocarcinoma cell lines, placental fibroblasts and placental macrophages. Normal trophoblasts were high producers and released both IFN-α and IFN-β. In contrast, one choriocarcinoma cell line was a low producer and all malignant lines produced only IFN-β. Circulating monocytes produce IFN-α but placental macrophages secreted IFN-β and some IFN-α, suggesting that IFN production may be dependent on the stage of differentiation. A role for trophoblast IFNs in protection of the foetus against virus infections is proposed.
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Suppression of Interferon-induced Oligo-2′,5′-adenylate Synthetase Induction in Persistent Infection
Persistent infections with several strains of mumps virus (strains Torii and Miyahara), measles virus (strains Edmonston, CAM-70, AIK-C and Schwarz) and subacute sclerosing panencephalitis (SSPE) virus (Hälle and Mantooth) were established in various cell lines (FL, KB, A549, SK-AS, 293, K562, Ramos and NC-37). Oligo-2′,5′-adenylate synthetase activity was demonstrated to be only slightly induced by interferon in cytoplasmic and nuclear fractions of cell lines persistently infected with mumps virus. In these cells, resistance to vesicular stomatitis virus infection was not induced by interferon treatment. Treatment of the persistently infected cells with interferon for 10 and 24 h did not stimulate an increase in the amount of synthetase mRNA. In cells persistently infected with measles and SSPE viruses, reduced induction of the enzyme varied with host cell types. Induction of the enzyme was not found in K562, SK-AS and KB cells, but was recognized in NC-37 and FL cells.
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Heptad Repeat Sequences are Located Adjacent to Hydrophobic Regions in Several Types of Virus Fusion Glycoproteins
More LessExtensive regions of heptad repeat units consistent with an α-helical coiled coil conformation are located adjacent to hydrophobic, potentially fusion-related regions in the amino acid sequences of paramyxovirus fusion and retrovirus envelope glycoproteins. Similar arrangements of hydrophobic peptides and heptad repeat units exist in coronavirus peplomer proteins and influenza virus haemagglutinins. This suggests that there may be similarities in the structures of these proteins and in the functions of the hydrophobic fusion-related regions during virus entry.
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Mutational Analysis of Plum Pox Potyvirus Polyprotein Processing By the NIa Protease in Escherichia Coli
More LessA binary Escherichia coli expression system has been used to study the pathway for proteolytic processing of the plum pox potyvirus (PPV) polyprotein. Trans cleavage at the carboxyl end of the cylindrical inclusion protein occurred, although with lower efficiency than that at the large nuclear inclusion protein-capsid protein junction. No trans cleavage at the carboxyl end of the small nuclear inclusion protein (NIa) was detected. The proteolytic activities at different cleavage sites of several deletion and point mutations of NIa protein have been analysed. The large ΔSX deletion and two different point mutations at His 239 abolished proteolytic activity at all sites. The effect of other mutations, particularly a Glu substitution for Asp 274, depended on the particular cleavage site analysed. The results obtained with the PPV NIa protein mutants were similar to those reported for comparable mutations in the tobacco etch virus 49K protease, despite differences in the sequences recognized for processing. No evident competitive inhibition of the proteolytic activity of PPV NIa protease by the presence of an excess of the different protease mutants could be demonstrated.
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Nucleotide Sequence of Barley Yellow Mosaic Virus RNA 1: A Close Evolutionary Relationship with Potyviruses
More LessThe complete nucleotide sequence of barley yellow mosaic virus (BaYMV) RNA 1 was obtained by analysis of overlapping cDNA clones and by direct RNA sequencing. The sequence is 7632 nucleotides in length, excluding a 3′ poly(A) tail. The first AUG codon at nucleotide 172 appeared to be the initiator for a single long open reading frame encoding a protein of 2410 amino acids with an Mr of 270755. Amino acid sequence comparisons revealed that the BaYMV 270K protein contains three regions upstream of the C- terminal capsid protein which share significant homologies with the cytoplasmic inclusion and two nuclear inclusion proteins of potyviruses thus indicating their similarities in genetic organization. However, the apparent low levels of homology in the corresponding proteins of BaYMV and potyviruses are in contrast with the high conservation among potyviruses. Moreover, our data indicate that BaYMV RNA 1 has no counterpart to the two cistrons located in the 5′- terminal region of the potyvirus genome. Although the data suggest a close evolutionary relationship between BaYMV and potyviruses, the striking differences set BaYMV apart from potyviruses.
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Nucleotide Sequences of Coat Protein Genes for Three Isolates of Barley Yellow Dwarf Virus and their Relationships to Other Luteovirus Coat Protein Sequences
More LessBarley yellow dwarf virus (BYDV) can be separated into two groups based on, among other criteria, serological relationships that are presumably governed by the viral capsid structure. Nucleotide sequences for the coding regions of coat proteins of approximately 22 K were identified for the MAV-PS1, P-PAV (group 1) and NY-RPV (group 2) isolates of BYDV. The MAV-PS1 and P-PAV coat protein sequences shared 71 % deduced amino acid similarity whereas that of the NY-RPV isolate shared no more than 51 % similarity with either the MAV-PS1 or the P-PAV sequence. Other comparisons showed that these and other BYDV coat protein sequences examined to date share a high degree of identity with those identified from other luteoviruses. Among luteovirus coat protein sequences in general, several highly conserved domains were identified whereas other domains differentiate MAV- PS1 and PAV isolates from NY-RPV and other luteoviruses. Sequence similarities and differences among BYDV coat proteins (approx. 22K) are consistent with the serological relationships exhibited by these viruses. Amino acid sequence comparisons between BYDV isolates that share common aphid vectors indicate that it is unlikely that these coat proteins are involved in aphid specificity.
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Serological Differentiation of 20 Isolates of Tomato Spotted Wilt Virus
More LessTwenty tomato spotted wilt virus (TSWV) isolates were serologically compared in ELISA employing five different procedures using a rabbit polyclonal antiserum against nucleocapsid proteins (NuAbR) and mouse monoclonal antibodies (MAbs), two directed to nucleocapsid proteins (N1 and N2) and four directed to glycoproteins G1 to G4. All the antisera were raised against TSWV-CNPH1. The 20 isolates were differentiated into two distinct serogroups. Serogroup I consisting of 16 isolates strongly reacted with NuAbR. The other four isolates were poorly recognized by NuAbR and were placed in another serogroup, designated II. The panel of MAbs differentiated the TSWV isolates into three serotypes. The 16 isolates forming serogroup I reacted strongly with the MAbs generated and were identified as serotype I isolates. The four isolates which made up serogroup II were split into serotypes II and III. The serotype II isolates did not respond or responded poorly with MAbs N1, N2 and G3. The two other isolates placed in serotype III were recognized by N1 but not by N2 and G3. Two isolates became defective after several mechanical passages and failed to respond or responded very poorly with MAbs directed to glycoproteins. Our results show that ELISA employing polyclonal and monoclonal antisera is a useful tool to differentiate TSWV isolates and to detect defective forms. The results also strongly suggest that TSWV nucleocapsid proteins are less conserved than the glycoproteins.
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Differentiation and Antigenic Characterization of Closely Related Alfalfa Mosaic Virus Strains with Monoclonal Antibodies
More LessA panel of 15 mouse monoclonal antibodies (MAbs) was raised against five strains of alfalfa mosaic virus (AMV) which were closely related antigenically but biologically distinct. A wide diversity of MAb specificity was revealed by screening them in three formats of indirect ELISA, using native and glutaraldehyde- fixed AMV particles as well as isolated coat protein preparations. Of these MAbs, seven reacted specifically with only one AMV strain in at least one ELISA format and at least one MAb was capable of identifying each of the strains. One of the MAbs reacted with a cryptotope, whereas the other recognized different subtypes of either metatopes or neotopes, indicating that the AMV particle has a complex antigenic structure. Only two of the MAbs precipitated AMV in agarose gels. Another two, which recognized epitopes on coat protein subunits, also reacted well in immunoblots. One of the precipitating MAbs recognized an epitope which appears to be common to AMV and cucumber mosaic virus.
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Complementarity Between the 5′- and 3′-terminal Sequences of Rice Stripe Virus RNAs
More LessThe 5′ and 3′ termini of four ssRNA species of rice stripe virus (RSV) isolate T were sequenced. The 3′ termini of the three smallest ssRNAs, i.e. RNAs 2, 3 and 4, had the sequence 5′ GACUUUGUGU 3′; that of ssRNA 1 had the sequence 5′ GACUAUGUGU 3′. The 5′-terminal sequences of all four ssRNAs were
5′ ACACAAAGUCC 3′. The 5′- and 3′-terminal sequences of about 20 bases of each ssRNA were almost complementary to each other. It is possible that RSV RNAs form panhandle structures characteristic of the RNA of negative-strand viruses.
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Segment 5 of the Rice Dwarf Virus Genome Encodes a Protein Highly Conserved within the Phytoreoviruses
More LessThe complete nucleotide sequence of segment 5 (S5) of the rice dwarf virus (RDV) genome was determined. RD V S5 is 2571 bp in length and has a single long open reading frame which encodes a polypeptide of 801 amino acids (Mr 90495). When compared to the wound tumor virus genome S5, there was 56·9% and 52·8% similarity in the nucleotide and amino acid sequences, respectively. This high similarity suggests that the S5 proteins of these viruses are functionally similar.
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The Primary Structure of the Virion Protein Gene and Encoded Protein of Erysimum Latent Tymovirus
More LessThe nucleotide sequence of the virion protein (VP) gene of erysimum latent tymovirus (ELV) has been determined and the amino acid sequence of the VP deduced and confirmed by peptide analysis. The ELV VP is larger than the VPs of other tymoviruses because it has, unexpectedly, 11 more amino acid residues at its N terminus. The amino acid sequences of the VPs of ELV and four other tymoviruses align unequivocally and their relationships, as assessed from the percentage of identical residues, correlate well with previously reported serological tests which have shown ELV to be distant from other tymoviruses.
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