- Volume 72, Issue 9, 1991
Volume 72, Issue 9, 1991
- Animal
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The herpes simplex virus ribonucleotide reductase is required for ocular virulence
More LessWe used a herpes simplex virus (HSV) type 1 ribonucleotide reductase (RR) null mutant (ICP6Δ) to study the role of HSV-1 RR in ocular HSV infections. We found that ICP6Δ was unable to induce vascularization of the cornea or stromal keratitis following inoculation into the cornea of BALB/c mice, but was able to induce a transient mild blepharitis. The parental strain (HSV-1 KOS) and a revertant of ICP6Δ, ICP6Δ+ 3.1, both caused severe ocular disease, indicating that HSV-1 RR is required for ocular virulence in mice. ICP6Δ grew poorly in vitro (Vero and BALB/c 3T3 fibroblasts) and in vivo (eye, trigeminal ganglia and brain) compared to ICP6Δ+3.1 and HSV-1 KOS, suggesting that the avirulence of ICP6Δ is due to poor growth in the host. ICP6Δ also grew less well in primary human corneal fibroblasts, suggesting that RR may be required for virulence in humans. These results indicate that drugs inhibiting the function of RR might be effective in treating ocular HSV infections.
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Variation in resistance to herpes simplex virus type 1 of oligodendrocytes derived from inbred strains of mice
More LessPrimary oligodendrocyte (OL) cultures from three inbred strains of mice with known differences in resistance to herpes simplex virus type 1 (HSV-1) infection in vivo (A/J, susceptible; BALB/cByJ, moderately resistant; C57BL/6J, resistant), also display a similar pattern of resistance in vitro. The nature of the in vitro resistance at the cellular level was investigated. Virus production at different m.o.i.s indicated that the differences in HSV-1 replication are m.o.i.-dependent. Overall, virus yield from the OL cultures infected at a multiplicity of 1 increased 48 h post-infection (p.i.); no additional enhancement occurred 72 h p.i. However, the difference in the replication capacity of the three OL cultures observed at 24 h p.i. persisted at 48 and 72 h p.i. Serial electron microscopy studies on infected OL cultures derived from the different murine strains suggested that the resistance to HSV-1 infection occurs at different stages during the replicative cycle. Virus was detected at the nuclear membrane 5 min p.i. in A/J cells, but was not observed until 120 min p.i. in BALB/cByJ cells, whereas virus could not be detected at the nuclear membrane of C57BL/6J cells, even at 24 h p.i. Virus adsorption, determined by assay of residual non-adsorbed virus infectivity and cell-associated, radiolabelled HSV-1, did not differ in the OL cultures. The cumulative data suggest that A/J cells display the same replication pattern as permissive CV-1 cells, whereas the major replicative blocks in the other two murine strains occur at the level of the cytoplasmic membrane in C57BL/6J OLs, and at the level of the nuclear membrane in BALB/cByJ cells.
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Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells
More LessWe have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD14+ cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.
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A human monoclonal antibody against varicella-zoster virus glycoprotein III
Hybridomas producing human monoclonal antibodies (HMAbs) against varicella-zoster virus (VZV) were generated by fusing murine myeloma cells with human lymphocytes immunized in vitro. An assay system was developed to select anti-glycoprotein (gp)III HMAbs from the pool of anti-VZV HMAbs. A murine anti-gpIII MAb, 4B7, did not react with a VZV-infected cell homogenate, but did react with a VZV-infected cell monolayer, whereas anti-gpI and anti-gpII MAbs reacted with both antigens. Hybridomas were screened to obtain HMAbs having a reaction profile similar to that of 4B7 and one such clone, V3, stably produces human IgG1 (κ). HMAb V3 immunoprecipitated a VZV antigen of 115K to 120K, which was not immunoabsorbed by an anti-gpII HMAb, implying that V3 recognizes gpIII. V3 neutralized VZV independently of complement, unlike anti-gpI and anti-gpII HMAbs. All five strains of VZV tested were completely neutralized by V3, and the dose of V3 required to reduce the number of virus plaques by 50% ranged from 0.027 to 0.15 µg/ml. V3 was also able to inhibit the spread of virus infection from infected to uninfected cells, whereas anti-gpI and anti-gpII HMAbs could not. In addition, V3 mediated antibody-dependent cellular cytotoxicity but not complement-dependent cytotoxicity of VZV-infected cells. The results suggest that an anti-gpIII HMAb may provide a new means of passive immunoprophylaxis and also help to identify an antigenic epitope appropriate for a subunit vaccine.
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Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies
More LessA panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar M r to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced M r (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in M r. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.
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Cleavage of the bovine herpesvirus glycoprotein B is not essential for its function
More LessHerpes simplex virus glycoprotein B (HSVgB) and its bovine herpesvirus homologue (BHVgB) share similar primary structures. These glycoproteins are present in the envelope of the virion and are believed to initiate infection by fusing the virus envelope with a host cell membrane. BHVgB, like the membrane-fusing glycoproteins of most enveloped viruses, is normally cleaved and is present as a disulphide-linked complex in the virus envelope and host cell membranes. HSVgB, however, remains uncleaved, presumably because it lacks a similar protease recognition sequence. To determine whether the cleavage of BHVgB is essential for its role in initiating infection, we altered the coding sequence of this glycoprotein by removing the protease cleavage site and making this region similar to that of HSVgB. The mutant BHVgB gene was expressed by an HSV recombinant virus in mouse L cells and produced an uncleaved BHVgB. The uncleaved BHVgB could complement the function of HSVgB which had been neutralized by monoclonal antibody H233. When expressed in mouse L cells, the uncleaved mutant BHVgB retained its ability to fuse membranes.
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Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses
More LessWe have previously classified isolates from a respiratory syncytial (RS) virus epidemic into distinct lineages by restriction mapping and nucleotide sequencing of parts of the nucleocapsid protein and small hydrophobic protein genes, which are areas of the genome not considered to be under immunological pressure. This study has now been extended by the determination of the nucleotide sequences of the attachment (G) protein genes of isolates from each subgroup A lineage. Deduced amino acid identities of the G proteins ranged between 80% and 99%, corresponding closely to the previously determined relatedness of the lineages. The amino acid variability was not evenly distributed; in the extracellular part of the protein there was a sharply defined hypervariable domain which was separated from a more extended variable domain by a highly conserved region. Most nucleotide changes in the variable domains were in the first and second positions of the codon triplets. These results suggest that there may be considerable immunological pressure for change in certain areas of the G protein and this may account for the ability of this virus to reinfect individuals repeatedly. The results presented here reflect the pattern of published data comparing prototype strains of the A and B subgroups.
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Antigenic variation of European haemorrhagic fever with renal syndrome virus strains characterized using bank vole monoclonal antibodies
More LessMonocloral antibodies (MAbs) against Puumala (PUU) virus, the aetiological agent of nephropathia epidemica, were produced by fusing activated spleen cells from a bank vole (Clethrionomys glareolus) with the mouse myeloma cell line SP2/0. This novel approach, utilizing the natural vector of PUU virus for hybridoma production, proved to be highly efficient, and eight stable PUU virus-specific heterohybridomas were isolated and characterized. The bank vole MAbs were all specific for the nucleocapsid protein (N) of PUU virus, as determined by immunoprecipitation. When evaluated by additivity immunoassays, the MAbs were found to recognize several different, distinct or overlapping, epitopes on N. The MAbs were used in immunofluorescence assays to compare eight PUU-related virus isolates, and the prototype Hantaan, Urban rat and Prospect Hill viruses. The reactivity varied among the different MAbs and could be classified into five groups. One MAb reacted exclusively with PUU-related viruses; two MAbs reacted with all PUU-related virus strains tested, as well as Prospect Hill virus, but did not react with Urban rat virus and Hantaan virus; one MAb reacted with all PUU-related virus strains tested and weakly with Hantaan virus, but not with Urban rat and Prospect Hill viruses; two MAbs reacted with all the virus strains tested. Two virus strains, K-27 and CG-1820, isolated in the western U.S.S.R., were distinguished from the other PUU-related virus strains by two MAbs, suggesting that the large group of independently isolated PUU-related viruses may be more heterogeneous than previously believed.
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Typing of hepatitis C virus genomes by restriction fragment length polymorphism
More LessRecently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the K1 type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5′-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1- and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively.
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The trans-activating C-type retroviruses share a distinct epitope(s) that induces antibodies in certain infected hosts
More LessUsing sera from hosts infected with bovine leukaemia virus (BLV), human T cell lymphoma virus types I and II (HTLV-I and -II), or simian T cell lymphoma virus type I (STLV-I), we found that the major gag proteins of these viruses cross-react immunologically. The specificity of this cross-reactivity was demonstrated by absorption using purified viral proteins, virus lysates and extracts of infected cells. The data strongly suggested that the cross-reacting epitope(s), referred to as CE, differs from those responsible for cross-reactions between the major gag proteins of HTLV-I, HTLV-II and STLV-I, and between those of BLV and HTLV-I reported previously. The prevalence of antibodies to CE was low, even amongst infected hosts with high titres to other epitopes present in the major gag proteins of the homologous viruses. CE was not detected in any of the other C- or D-type retroviruses, or lentiviruses examined. Therefore, it is likely that CE can be used to define serologically a subgroup of C-type retroviruses, the genomes of which display unique features and functional activities.
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A mutant of canine adenovirus type 2 with a duplication of the E1a region exhibits altered expression of early region 4
More LessThe genomic DNA of a vaccine strain of canine adenovirus type 2 (Vaxitas; ICI Tasman) has been shown to contain two copies of the E1a region, the second being at the far right end of the genome. DNA sequence analysis of the right terminal 2.8 kbp of this vaccine strain showed that numerous point mutations have occurred in the second copy, which would preclude the synthesis of any functional products. However, expression vectors in which the E1a promoter from the right terminus were linked to the chloramphenicol acetyltransferase gene showed that the promoter was fully functional. Furthermore, the activity of the reiterated E1a promoter was considerably greater than that of the normal E4 promoter. This dramatic change in the regulation of E4 expression may be an important factor in determining the altered host cell specificity displayed by this vaccine strain virus.
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Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures
More LessIn this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5′ end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3′-terminal sequences and may form a very stable panhandle structure (ΔG -242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (ΔG -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.
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Primary amines enhance the antiviral activity of interferon against a membrane virus: role of intracellular pH
More LessInhibition of vesicular stomatitis virus (VSV) replication in LB cells by interferon (IFN) resembles the action of IFN on some retroviruses, in that the incorporation of glycoprotein into virions is defective. Primary amines added between 1 and 2 h post-infection significantly enhanced (five- to 1000-fold) the antiviral activity of IFN against VSV, but no enhancement of the antiviral activity of IFN against encephalomyocarditis virus, a virus with no membrane component, by primary amines was seen. SDS-PAGE and immunofluorescence analysis of viral proteins, and Nycodenz gradient fractionation, suggested that both IFN and primary amines inhibited the transport of VSV glycoprotein (G) to the plasma membrane; instead, G accumulated in the trans-Golgi network (TGN). Using sensitive intracellular pH (pHi) indicators, we found that IFN treatment significantly raised the pHi. A further increase in pHi was seen with a combination of IFN and primary amines; the increase in pHi correlated with an enhancement of the antiviral activity of IFN by primary amines. Amiloride inhibited the IFN-induced increase in pHi and a concomitant increase in the concentration of Na+ ions; this observation suggested that IFN induced cytoplasmic alkalinization by activating an Na+/H+ antiporter system. These results indicated that the IFN-induced increase in pHi may be responsible for the accumulation of G in the TGN, thereby producing G-deficient virus particles with reduced infectivity.
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A comparison of WIN 51711 and R 78206 as stabilizers of poliovirus virions and procapsids
More LessThe thermal denaturation of poliovirus virions and procapsids in the 42 to 48 °C range was studied using N- and H-specific monoclonal antibodies. The half-life of the N antigen of Mahoney and Sabin 1 virions was extended 50- to 250-fold by either 10 µm WIN 51711 or R 78206. The minimum concentrations required for full stabilization at 46 °C (1.0 µm for WIN 51711, 0.2 µm for R 78206) were independent of the strain or serotype of the virus; 30 to 60 molecules of stabilizer per virion were required for full protection. R 78206 was the most efficient stabilizer of Mahoney procapsids; the half-life of the N-specific epitopes of these particles at 44 °C was extended from less than 1 min to 1 day.
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Identification and characterization of incomplete hepatitis A virus particles
More LessThe range of hepatitis A virus (HAV) particles generated during persistent infection of different cell lines was studied. Buoyant density and sedimentation analyses of cell extracts revealed a uniform profile of particles in all cell lines analysed except for BS-C-1 cells. The virion itself usually represented less than 50% of the total mass of virus antigen. A major portion of the antigen was associated with non-infectious, empty particles, which banded at 1.305 g/ml and 1.20 g/ml CsCl, and sedimented in sucrose gradients at 76S and 59S. Empty HAV particles were similar to those of poliovirus with respect to their physical stability and had the characteristic capsid protein content (VP0, VP1 and VP3). An additional RNA-containing particle, probably the provirion, represented only a minor species characterized by a buoyant density of 1.32 g/ml in CsCl and sedimenting at 130S.
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A recombinant vaccinia virus expressing hepatitis A virus structural polypeptides: characterization and demonstration of protective immunogenicity
A recombinant vaccinia virus containing most of the P1 region of hepatitis A virus (HAV) was constructed. Cell lysates of cultures infected with the virus contained HAV proteins detectable by radioimmunoassay. Western blot analysis revealed the presence of a single protein of M r 60K to 62K, bearing epitopes from structural polypeptides VP4, -3 and -2, and the N terminus of VP1. The size of the protein suggests that at least some of the vaccinia virus thymidine kinase is also expressed. Inoculation of tamarin monkeys with the recombinant virus resulted in the development of a specific anti-HAV immune response which was protective against challenge with a virulent strain of HAV. Recombinant viruses expressing the above region of HAV or the proteins expressed by such viruses may be useful in the development of a vaccine suitable for use in man.
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Analysis of Borna disease virus-specific RNAs in infected cells and tissues
More LessBorna disease virus (BDV) is an infectious agent that causes profound disturbances in motor function and behaviour in a wide range of animal species and possibly humans. The infectious nature of BDV has long been established, but the aetiological agent has not been isolated or classified. Recently, we have reported the isolation of BDV-specific cDNA clones using subtractive libraries constructed from mRNA from infected material. Here we describe studies on one of these cDNA clones, B8, and confirm its specificity by in situ hybridization on sections of BDV-infected brain. The complete nucleotide sequence of BDV-specific clone B8 was determined. Oligonucleotides of positive and negative polarity synthesized from sequences from the 5′ and 3′ ends, as well as the central part, of clone B8 identified both positive- and negative-strand BDV-specific RNAs in infected rat brain. All B8 sequences used as oligonucleotide probes were found to be contained in the larger positive- and negative-strand RNAs. Thus, the structure of the BDV-specific RNAs appears to be a nested set of multiple, overlapping subgenomic positive- and negative-strand RNA transcripts.
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Actin-independent maturation of rabies virus in neuronal cultures
More LessThis study outlines the effects of a modification of the actin-based cytoskeleton on the maturation of rabies virus in human neuroblastoma cell and primary rat cortical neuron cultures. In a Ca2+-depleted or an EGTA-containing medium, disruption of microfilaments did not affect intracellular viral nucleoprotein synthesis, as demonstrated by dual-immunofluorescence microscopy, and caused no change in the extracellular titre of rabies virus. Furthermore, the continuous presence of the anti-calmodulin drugs trifluoperazine (1 to 20 µm) and chlorpromazine (1 to 30 µm), or the l-type Ca2+ channel antagonist nifedepine (1 to 10 µm) or the Ca2+-specific ionophore A23187 (0.05 to 1.0 µm), did not modify the extracellular titre of rabies virus significantly over a 48 h period. The inference from these studies is that the maturation of rabies virus is independent of the integrity of the microfilament structures and calmodulin-dependent processes of neuronal cells.
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Localization of the non-structural protein NS3 in bluetongue virus-infected cells
More LessThe localization of the blue tongue virus (BTV) non-structural proteins NS3 and NS3a has been identified using immunoelectron microscopical techniques. NS3 and NS3a have been observed in the plasma membrane of BTV- and recombinant vaccinia virus (expressing NS3)-infected cells. The NS3 protein was associated with areas of membrane perturbation. There was a good correlation between the presence of NS3 and NS3a and BTV release. The NS3 protein was associated with membrane fragments and the inability to detect it on the extracellular aspect of intact cells suggested that the protein was not exposed extracellularly. Electron microscopical and biochemical evidence suggested that fragments of plasma membrane containing NS3 and NS3a were released from infected cells. Collectively, the data indicate that NS3 and NS3a may be involved in the final stages of BTV morphogenesis, i.e. the release of BTV from infected cells.
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Immunofluorescent detection of bovine papillomavirus E4 antigen in the cytoplasm of cells permissive in vitro for viral DNA amplification
More LessThe E4 gene of several human papillomavirus types is expressed in association with vegetative viral DNA synthesis in differentiated epidermal cells. To develop reagents to study expression of the bovine papillomavirus type 1 (BPV-1) E4 gene in warts and in virus-transformed cell lines, rabbit polyclonal antiserum was raised to the BPV-1 E4 antigen produced as a fusion polypeptide in Escherichia coli. By immunoblotting analysis of productively infected bovine fibropapilloma tissue, E4-related proteins of 16K, 21K, 30K and 42K were detected. In some but not all C127 cell lines transformed by BPV-1 or by a replication-competent BPV-1 deletion mutant, cytoplasmic E4 antigen with a predominantly perinuclear localization was detected by immunofluorescence analysis in a subpopulation of cells in stationary-phase cultures. The E4-expressing cells were identified by their grossly enlarged size to represent the same cell subpopulation shown earlier to support BPV-1 DNA amplification. The observation of synthesis of the E4 protein in association with viral DNA amplification in this system provides further evidence that there is a switch in viral early region gene expression in a subpopulation of division-arrested cells, which may accurately reflect events occurring during the vegetative phase of BPV-1 replication in terminally differentiated cells in vivo.
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The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes
More LessTwo independent non-overlapping epitopes could be demonstrated on the structural protein VP3 of infectious bursal disease virus by non-neutralizing monoclonal antibodies produced against serotypes I and II. Both serotypes have one epitope in common, whereas the second epitope is distinct for serotype I and serotype II.
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Nucleotide sequence of the genes encoding the matrix protein of two wild-type measles virus strains
More LessThe nucleotide sequences of the matrix protein (M) genes of two wild-type measles virus (MV) isolates (JM and CM) have been determined and shown to differ in 56 positions; 31 of these differences are located in the non-coding region and 25 in the coding region of the gene. Most (80%) of the mutations in the coding region are changes to the third base of a codon. A maximum parsimony analysis of the available M gene nucleotide sequences allowed the construction of a tree with at least three lineages or subtypes. One wild-type strain (JM) was very similar to a subacute sclerosing panencephalitis virus strain (case B); the second wild-type strain, CM, showed nucleotide sequence similarity with MV from a case of measles inclusion body encephalitis. Both wild-type virus sequences are distinct from those so far determined for vaccine strains.
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Sequence characterization of the matrix protein genes of parainfluenza virus types 4A and 4B
The complete nucleotide sequences of the matrix protein (M) genes of parainfluenza virus types 4A and 4B (PIV-4A and -4B) were determined from cDNA of the mRNA, and found to be 1548 bases in length, exclusive of poly(A) sequences. The sequences contained a large open reading frame of 1146 nucleotides encoding 362 amino acids. A high degree of identity (96.1%) was observed between the amino acid sequences of PIV-4A and PIV-4B M. These M sequences were compared with those of 10 other paramyxoviruses and a phylogenetic tree was constructed.
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Molecular relationships between human parainfluenza virus type 2, and simian viruses 41 and 5: determination of nucleoprotein gene sequences of simian viruses 41 and 5
The nucleotide sequences of cDNAs of the simian virus 5 (SV5) nucleoprotein (NP) gene, and the 3′ end of the genome and NP gene of SV41 were determined. The open reading frames of the SV5 and SV41 NP genes encode polypeptides with M rs of 56582 and 60575, respectively, values which are consistent with those estimated by SDS-PAGE. The NP of human parainfluenza virus type 2 (hPIV-2) was more closely related to that of SV41 (amino acid sequence identity 70.5%) than that of SV5 (57.0%); the amino acid sequence identity between the NPs of SV41 and SV5 was 63.3%. The sequence of the 3′ end of the genome of SV41 showed a high level of similarity to that of hPIV-2, the terminal 18 nucleotides being identical. It is concluded from these findings that SV41 is related most closely to hPIV-2, even though SV5 had been thought to be an animal type of hPIV-2.
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The unique region of the human herpesvirus 6 genome is essentially collinear with the U l segment of human cytomegalovirus
More LessThe entire genome of human herpesvirus 6 (HHV-6) strain U1102 was cloned as overlapping fragments in cosmid and plasmid vectors. Cleavage maps were constructed for the restriction endonucleases BamHI, EcoRI, NotI and SmaI. The genome of HHV-6 U1102 is a linear dsDNA of 163 kbp, consisting of a long unique 142 kbp region flanked by direct terminal repeats of 10.5 kbp. Short stretches (290 to 470 nucleotides) of DNA, four from the terminal repeats and 55 from the U l region, were sequenced and compared by computer with the known herpesvirus amino acid sequences. Homologies were found for 10 open reading frames that are scattered over the U l region of HHV-6. Their relative positions and orientations indicate that the unique region of HHV-6 is essentially collinear with the U l region of human cytomegalovirus (HCMV), but is not collinear with the other human herpesviruses. It confirms and extends earlier observations that HHV-6 is more closely related to the β-herpesvirus HCMV, suggesting that HHV-6 may be considered as the prototype of a new β2-herpesvirus subgroup.
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Conserved domains of glycoprotein B (gB) of the monkey virus, simian agent 8, identified by comparison with herpesvirus gBs
More LessThe herpesvirus simian agent 8 (SA8) gene which corresponds to the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) was localized, cloned and sequenced. Comparison of its deduced amino acid sequence with those of its counterparts in 12 other distinct herpesviruses was used to evaluate their homology and phylogenetic relationship. The results emphasized that SA8 gB is more closely related to those of HSV-1 and -2, and bovine herpesvirus 2 than to the homologous proteins of other herpesviruses. Furthermore, the alignment showed several regions of domains conserved in the closely related sequences, including four conserved in all the herpesvirus gB sequences examined. The conservation of 10 cysteine residues and most of the proline residues, as well as several potential N-glycosylation sites, suggested that the secondary and tertiary structures of these gBs were similar.
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The RL neurovirulence locus in herpes simplex virus type 2 strain HG52 plays no role in latency
More LessWe have demonstrated that the variant JH2604 of the herpes simplex virus type 2 (HSV-2) strain HG52 is completely avirulent in BALB/c mice following intracranial inoculation, with an LD50 of > 107 p.f.u./mouse compared to the wild-type LD50 of < 102 p.f.u./mouse. In JH2604, a 1.5 kbp deletion extends from the DR1/Ub junction of the ‘a’ sequence to 511 bp upstream of the 5′ end of IE1 in both long repeats. We have since constructed a second variant (2701) in which only 850 bp are removed from the RL. This deletion lies entirely within the sequences deleted in JH2604 and leaves intact most of a short 189 bp open reading frame (ORF) highly conserved between HSV-1 and HSV-2. Like JH2604, 2701 shows wild-type growth characteristics and is neither host range-nor temperature-restricted. This was most noteworthy in the case of mouse 3T6 cells. 2701 has an LD50 of 5×105 p.f.u./mouse on intracranial inoculation, a value intermediate between those of HG52 and JH2604. In assays for intracranial replication, JH2604 exhibits no detectable growth with a rapid decline in virus titre, 2701 shows limited growth over the first 24 to 36 h post-inoculation before the titre again declines and HG52 grows rapidly, reaching a high titre until the mice die. Taken together these results suggest that a region of the genome upstream of IE1 encodes a gene product essential for HSV replication in neurons of the central nervous system. It is highly likely that the conserved ORF is in an important region of a polypeptide essential for neurovirulence, although the upstream sequences present in 2701 but absent from JH2604 must also play a role. Although JH2604 and 2701 are avirulent, they both establish latent infection in the dorsal root ganglia of BALB/c mice and reactivate in vitro in a manner indistinguishable from HG52. This suggests a distinct separation of the factors involved in neurovirulence and the establishment of/reactivation from latency.
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Sequence analysis of the herpes simplex virus type 1 strain 17 variants 1704, 1705 and 1706 with respect to their origin and effect on the latency-associated transcript sequence
More LessThe precise endpoints of the deletions/insertions in three variants (1704, 1705 and 1706) of herpes simplex virus type 1 (HSV-1) strain 17 have been determined by dideoxynucleotide sequence analysis. The analysis was undertaken to discover whether the three variants had arisen from the same initial event and the extent of the deletions with respect to the latency-associated transcripts (LATs) and the proposed LAT promoter region. It is not possible from the deletion boundaries to determine unequivocally whether the three variants had arisen from the same recombination event although 1706 could be descended from 1705 by illegitimate recombination. The results demonstrate that spontaneous deletions can occur at random within R l , the extent of the deletions in U l is constrained by the essential nature of U l genes in vitro but is otherwise arbitrary and deletions in 1704 completely remove both copies of the LAT promoter region and in IR l extend into the 5′ end of the LAT sequence.
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Amplification of the Moloney murine leukaemia virus genome and its possible role in facilitation of chemical carcinogenesis in normal rat kidney cells
More LessIn a previous study we have shown that a single infectious particle of Moloney murine leukaemia virus per cell is sufficient to facilitate chemical carcinogenesis in normal rat kidney cells. When these cells are exposed to the carcinogen after a low number of passages post-infection (p.i.), cell transformation becomes apparent only after many subsequent passages. On the other hand, when exposure is done after a high number of passages p.i., cell transformation can be detected in the treated culture or at the next passage. It is thus evident that whereas the carcinogenic effect is rapid, the viral effect becomes apparent only after a long period of latency. Here we provide evidence that this viral effect requires multiple proviruses and that the long latent period reflects the time needed for a sufficient accumulation of proviruses in some of the cells. This accumulation may result from multiple rounds of superinfection by virions released into the culture medium, although we cannot exclude other mechanisms of provirus amplification. Our data also suggest that this amplification enhances virus production.
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A synthetic peptide elicits antibodies reactive with the external glycoprotein of human T cell lymphotropic virus type I
More LessA synthetic peptide derived from the external glycoprotein of human T cell lymphotropic virus type I (HTLV-I) (Env-5; amino acids 242 to 256) reacted with IgG antibodies in serum specimens from HTLV-I-infected individuals. C-terminal residues of Env-5 were crucial for the antibody reactivity. Polyclonal rabbit antibodies to Env-5 did not inhibit syncytium formation but such antibodies reacted specifically with gp68env and gp46env glycoproteins of HTLV-I in an immunoblot analysis. Immunoprecipitation of the surface-labelled MT-2 (HTLV-I-infected) cell line with anti-Env-5 precipitated the gp68env precursor protein. It was concluded that peptide Env-5 mimics a surface-exposed epitope on the HTLV-I external glycoprotein.
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The entire nucleotide sequence of foxtail mosaic virus RNA
More LessThe nucleotide sequence of the RNA genome of foxtail mosaic virus (FMV), a member of the potexvirus family, is 6151 nucleotides long, exclusive of a poly(A) tail. The RNA contains five principal open reading frames (ORFs), designated from the 5′ terminus as encoding proteins with M r values of 152.3K (ORF1), 26.4K (ORF2) which overlaps an 11.3K (ORF3) product, 5.8K (ORF4) which overlaps a 28.8K readthrough protein (ORF5A) which leads into the coat protein cistron of 23.7K (ORF5). The sizes and composition of the proteins encoded by the ORFs are generally similar to those found in other potexviruses; the least similar is the coat protein which nonetheless retains apparently critical consensus regions. The 5′ terminus of the previously reported 0.9 kb subgenomic (sg) RNA was determined by S1 nuclease mapping and shown to begin with the sequence GAAGA, 43 nucleotides upsteam from the first nucleotide of the coat protein initiation codon. The positions of the 5′ end of this sgRNA and of that deduced from the nucleotide sequence for a 1.9 kb sgRNA are entirely consistent with the previously published sizes of these sgRNAs.
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Nucleotide sequence of raspberry bushy dwarf virus RNA-2: a bicistronic component of a bipartite genome
More LessNorthern blot analysis with cDNA probes to RNA-3 (1 kb) of raspberry bushy dwarf virus (RBDV) revealed extensive sequence homology with RBDV RNA-2 (2.2 kb). Nucleotide sequencing showed that RNA-2 contains two large open reading frames (ORFs), of 1074 (5′ ORF) and 822 (3′ ORF) bases. The 3′ ORF is virtually identical in sequence to RNA-3, which encodes the M r 30509 (30K) coat protein. The 5′ ORF encodes an M r 38860 (39K) protein which slightly resembles the 32K protein encoded by RNA-3 of alfalfa mosaic virus (AIMV). RBDV RNA-2 resembles AIMV RNA-3 in being bicistronic and encoding a coat protein at the 3′ end. Comparing RNA-1 and RNA-2 of RBDV, only the 18 3′-terminal nucleotides are identical in sequence but the 3′-terminal 71 nucleotides of each RNA species have the potential to form similar stem-loop structures.
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Evolutionary relationships in the cucumoviruses: nucleotide sequence of tomato aspermy virus RNA 1
More LessRNA 1 of the V strain of tomato aspermy virus (TAV) consists of 3410 nucleotides and contains one open reading frame (ORF) of 2982 nucleotides, resembling RNA 1 of cucumber mosaic virus (CMV) strains Q and Fny (68% and 66% identical, respectively) and of brome mosaic virus (BMV) (41% identical). In comparisons between amino acid sequences, three conserved regions (N-terminal, C-terminal and central) between TAV and each CMV were found. The N- and C-terminal regions were also conserved with BMV, and contained, respectively, consensus motifs for methyltransferases and for nucleic acid helicases. The 5′ and 3′ non-coding sequences were highly similar to those of TAV RNA 2. When the sequences for the genomic RNAs of the V and C strains of TAV, and of their encoded products, are compared with those reported for CMV strains representing either subgroup I (Fny-CMV) or subgroup II (Q-CMV) of CMV, it was found that the different virus-encoded proteins are conserved differently between these three viruses. Also, the divergence between TAV and both CMV subgroups has proceeded at different rates for the different ORFs. On the whole, the divergence between TAV and CMV is of the same order as that found between CMV subgroups I and II, which suggests that TAV, Q-CMV and Fny-CMV could be considered as representing three equivalent subgroups of a taxonomic entity.
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The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses
More LessRepresentative amino acid sequences of the RNA-dependent RNA polymerases of all groups of positive-strand RNA viruses were aligned hierarchically, starting with the most closely related ones. This resulted in delineation of three large supergroups. Within each of the supergroups, the sequences of segments of approximately 300 amino acid residues originating from the central and/or C-terminal portions of the polymerases could be aligned with statistically significant scores. Specific consensus patterns of conserved amino acid residues were derived for each of the supergroups. The composition of the polymerase supergroups was as follows. I. Picorna-, noda-, como-, nepo-, poty-, bymo-, sobemoviruses, and a subset of luteoviruses (beet western yellows virus and potato leafroll virus). II. Carmo-, tombus-, dianthoviruses, another subset of luteoviruses (barley yellow dwarf virus), pestiviruses, hepatitis C virus (HCV), flaviviruses and, unexpectedly, single-stranded RNA bacteriophages. III. Tobamo-, tobra-, hordei-, tricornaviruses, beet yellows virus, alpha-, rubi-, furoviruses, hepatitis E virus (HEV), potex-, carla-, tymoviruses, and apple chlorotic leaf spot virus. An unusual organization was shown for corona- and torovirus polymerases whose N-terminal regions were found to be related to the respective domains of supergroup I, and the C-terminal regions to those of the supergroup III polymerases. The alignments of the three polymerase supergroups were superimposed to produce a comprehensive final alignment encompassing eight distinct conserved motifs. Phylogenetic analysis using three independent methods of tree construction confirmed the separation of the positive-strand RNA viral polymerases into three supergroups and revealed some unexpected clusters within the supergroups. These included the grouping of HCV and the pestiviruses with carmoviruses and related plant viruses in supergroup II, and the grouping of HEV and rubiviruses with furoviruses in supergroup III.
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Tomato spotted wilt virus L RNA encodes a putative RNA polymerase
The complete nucleotide sequence of the large (L) genome segment of tomato spotted wilt virus (TSWV) has been determined. The RNA is 8897 nucleotides long and contains complementary 3′ and 5′ ends, comprising 62 nucleotides at the 5′ end and 66 nucleotides at the 3′ end. The RNA is of negative polarity, with one large open reading frame (ORF) located on the viral complementary strand. This ORF corresponds to a primary translation product of 2875 amino acids in length, with a predicted M r of 331500. Comparison with the polymerase proteins of other negative-strand viruses indicates that this protein most likely represents the viral polymerase. The genetic organization of TSWV L RNA is similar to that of the L RNA segments of Bunyamwera and Hantaan viruses, animal-infecting representatives of the Bunyaviridae.
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Heterologous encapsidation in mixed infections among four isolates of barley yellow dwarf virus
F. Wen and R. M. ListerWe used immunohybridization and ELISA to investigate heterologous encapsidation (transcapsidation and phenotypic mixing) between paired isolates of barley yellow dwarf virus (BYDV) in doubly infected oat plants, Avena sativa L. cv. Clintland 64. Virions in samples extracted from plants doubly infected with two viruses were trapped with an antibody specific to one virus, and the nucleic acids of the trapped virions were identified with a cDNA probe specific to the other. Heterologous encapsidation was found in mixed infections between isolates NY-RPV and NY-MAV-PS1, NY-RPV and P-PAV, NY-RMV and NY-MAV-PS1, P-PAV and NY-MAV-PS1, and NY-RPV and NY-RMV. Heterologous encapsidation between NY-RPV and P-PAV, and between NY-RPV and NY-MAV-PS1, occurred in one direction, while the heterologous encapsidation between P-PAV and NY-MAV-PS1 occurred in both directions. Further analysis by heterologous ELISA and immunohybridization assays with immunoprecipitated samples demonstrated that trans-capsidation was the predominant type of heterologous encapsidation in mixed infections of NY-RPV and P-PAV, NY-RPV and NY-MAV-PS1, and NY-RMV and NY-MAV-PS1; phenotypic mixing was the predominant type of heterologous encapsidation in mixed infections of P-PAV and NY-MAV-PS1. Phenotypic mixing was also detected in mixed infections of NY-RPV and NY-RMV. These results suggest that among BYDV isolates transcapsidation is more common between distantly related isolates than between more closely related isolates, and phenotypic mixing is more common between more closely related isolates than distantly related isolates.
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Influence of the C terminus of the small protein subunit of bean pod mottle virus on the antigenicity of the virus determined using monoclonal antibodies and anti-peptide antiserum
More LessMiddle component particles of bean pod mottle virus (BPMV) containing small protein subunits with a cleaved C terminus were used to produce monoclonal antibodies (MAbs). All MAbs were specific for cryptotopes, i.e. epitopes present only on dissociated BPMV protein. The MAbs reacted more strongly with virus protein preparations containing the cleaved form of the small subunit than with preparations containing only the uncleaved form. It seems that the presence of additional residues at the C terminus of the intact small subunit interferes with antibody binding. Antibodies raised against synthetic peptides corresponding to the C terminus of the uncleaved small subunit reacted with both intact virions and dissociated subunits. This C-terminal region seems to play a dominant role in the antigenicity of the virus.
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Molecular analysis of rice dwarf phytoreovirus segment S11 corresponding to wound tumour phytoreovirus segment S12
More LessThe complete nucleotide sequence of rice dwarf phytoreovirus (RDV) genome segment S11 was determined. S11 is 1067 nucleotides long. There is an inverted repeat of 10 bp adjacent to the conserved 5′-terminal hexanucleotide (5′ GGUAAA 3′) and 3′-terminal tetranucleotide (5′ UAGU 3′) sequences. A single large open reading frame found in the plus strand of S11 begins with the first AUG codon (bases 6 to 8) and extends for 567 bases. Evolutionary relatedness between RDV S11 and wound tumour phytoreovirus S12 based on amino acid sequence similarity (25.8%) was found. In addition to the first AUG triplet, RDV S11 possesses a second in-phase AUG triplet (positions 30 to 32) nearby, which conforms to the Kozak consensus sequence. Two forms of the protein were identified by using an in vitro transcription and translation system in which a tailored full-length cDNA was the initial template. The abolition of the first AUG codon by site-directed mutagenesis resulted in disappearance of the larger translation product. These results strongly suggest that the two products are translated from the first and second AUG codons. Whether the two proteins are expressed in vivo is at present unclear.
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Outer capsid protein heterogeneity of rice dwarf phytoreovirus
More LessThe 46K outer capsid protein encoded by RNA segment S8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELISA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of S9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.
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Effect of recombinant beet necrotic yellow vein virus with different RNA compositions on mechanically inoculated sugarbeets
More LessBeet necrotic yellow vein virus (BNYVV) inocula with different RNA compositions were prepared from infectious transcripts of RNAs 3 and 4 and the Rg 1 isolate, which has a genome consisting only of RNAs 1 and 2. The recombinant viruses were inoculated on 6- to 8-day-old sugarbeet seedlings by ‘vortexing’. Inocula containing RNAs 1 and 2 or 1, 2 and 4 produced some growth reduction, but the most dramatic effects, with yield reductions of about 95% in a highly susceptible variety, were seen when RNA 3 was also present in the inoculum. Under these conditions the side roots were brown and brittle and often deteriorated, but ‘root beardedness’ was not observed. This might be due to the fact that our experiments were done in the absence of Polymyxa betae. Alternatively, the heavy inoculation at a very young age may either have weakened the plants to such an extent that extensive root proliferation was impaired or it may have led to rapid deterioration of the proliferating rootlets, which would therefore be lost prior to or during removal of the tap roots from the soil. In the presence of RNA 3 the virus concentrations in tap roots were markedly increased suggesting that this RNA facilitates the multiplication and/or spread of the virus in root tissues.
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Molecular cloning, sequencing and expression in Escherichia coli of the odontoglossum ringspot virus coat protein gene
The sequence of the 3′-terminal 1865 nucleotides of the genome of the tobamovirus odontoglossum ringspot virus (ORSV) was determined. This sequence contained two open reading frames (ORFs), 912 and 477 nucleotides long. The 912 nucleotide ORF has been identified as the cell-to-cell transport protein gene. The 477 nucleotide ORF was expressed in Escherichia coli, and the product was detected by antibodies specific for the coat protein of ORSV. The amino acid sequence of protein encoded by this ORF shares 84% similarity with the tobacco mosaic virus (TMV) (vulgare) coat protein. The 3′-terminal untranslated region of ORSV comprises 414 nucleotides, 210 nucleotides more than that of TMV (vulgare) RNA.
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