- Volume 72, Issue 2, 1991
Volume 72, Issue 2, 1991
- Animal
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Production of polyclonal antibodies against the S and preS2 regions of woodchuck hepatitis virus: lack of detectable low glycosylated preS2 protein (GP33) in sera from infected animals
Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.
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Formation in vitro of the pTP-dCMP initiation complex of human adenovirus type 12
More LessWe report the covalent addition of [32P]dCMP to a protein from group A adenovirus 12 (Ad12)-infected human (KB) cells in vitro, using crude extracts. Synthesis of the 60K protein-dCMP complex required a DNA template containing a terminally located adenovirus replication origin; the protein-dCMP bond was alkali-labile but acid-stable. We therefore conclude that this product is the Ad12 terminal protein precursor (pTP)-dCMP initiation complex for DNA replication. Synthesis of Ad12 pTP-dCMP was specific for dCTP but was stimulated by dATP. In contrast to Ad2, the Ad12 initiation reaction required ATP. Antipeptide antiserum targeted to Ad DNA polymerase inhibited Ad12 pTP-dCMP synthesis in vitro, providing evidence that Ad DNA polymerase catalyses dCMP addition to pTP during initiation.
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Stabilization of human rhinovirus serotype 2 against pH-induced conformational change by antiviral compounds
More LessFour WIN compounds with anti-picornavirus activities were tested for their ability to stabilize human rhinovirus serotype 2 (HRV-2) against low pH-induced conformational changes in vitro, as determined by specific immunoprecipitation. These results were compared to the minimal inhibitory concentration (MIC) as measured in a plaque reduction assay. A direct relationship was observed between the concentration of the compound that prevented the low pH-induced conformational changes and the MIC, indicating that stabilization is an important element in the mode of action of these drugs against HRV-2.
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Comparison of the nucleotide sequence of the SH gene and flanking regions of mumps vaccine virus (Urabe strain) grown on different substrates and isolated from vaccinees
More LessThe small hydrophobic (SH) protein gene and flanking regions of the Urabe Am9 vaccine strain of mumps virus were amplified by the polymerase chain reaction and sequenced directly by the dideoxynucleotide chain termination method. The 434 bp sequence was identical for the Urabe strain isolated from vaccines produced by three manufacturers and for virus isolated following post-vaccination parotitis. No changes were detected for coding, non-coding or intergenic regions between virus grown on different substrates. The Urabe virus SH coding region differed from the published sequence for strain SBL-1 by 14.4% at the nucleotide level and 24.6% at the amino acid level. The 5′ non-coding SH region was strongly conserved between the two strains (2% different), whereas the other non-coding regions were not.
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Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion
More LessVaccinia-measles recombinant viruses were used to examine the contribution of the individual measles virus glycoproteins in fusion. Although vaccinia virus recombinants expressing either the haemagglutinin or fusion proteins did not induce fusion in the cell lines examined, a double recombinant expressing both measles virus glycoproteins gave extensive syncytia in cells of human and simian origin. No fusion was observed in mouse, hamster or chicken cells. The fusion induced by the double recombinant could be specifically inhibited with either anti-fusion or anti-haemagglutinin monoclonal antibodies.
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The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus
More LessThe sequence of the H gene and flanking sequences in the F and L genes of canine distemper virus (CDV) have been determined. The H gene of CDV (1946 nucleotides) contains one large open reading frame starting at position 21 and terminating at position 1835, encoding a protein of 604 amino acid residues. This protein contains three potential glycosylation sites in the extracellular domain and, like all other paramyxoviruses, a N-terminal membrane-spanning hydrophobic anchor domain. The deduced H protein sequence shows an identity of 36% with rinderpest virus (RPV) and measles virus (MV). The identities at the nucleotide level are higher (RPV 52% and MV 53%). The amino acid sequence shows conservation of all the structural determinants with the H proteins of MV and RPV. The data also show that CDV is evolutionarily equidistant to RPV and MV with respect to the H gene.
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Sequence conservation of the outer capsid protein, VP5, of bluetongue virus, a contrasting feature to the outer capsid protein VP2
S. Oldfield, T. Hirasawa and P. RoyThe complete nucleotide sequence of a cDNA clone representing the segment 5 RNA of bluetongue virus (BTV) United States serotype 13 was determined. The comparison of the predicted amino acid sequence of the encoded protein (VP5) with the sequences of VP5 from two BTV United States serotypes, 10 and 2, and two isolates of BTV serotype 1 (Australian and South African), revealed that the protein is highly conserved among the different serotypes despite their geographical separation.
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Antibodies to type D retrovirus in talapoin monkeys
More LessSera from 154 African non-human primates were screened for the presence of antibodies to type D retrovirus proteins. Four of five talapoin monkeys (Miopithecus sp.) captured in western Africa were positive for antibodies to type D retrovirus by ELISA and by immunoblot reactivity. Talapoins are the only African non-human primates that have so far shown evidence for type D retrovirus infection. Thus, talapoin monkeys appear to be a reservoir of type D retrovirus infection.
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Chemoprophylaxis of scrapie in mice
More LessThree applications of the polyanion pentosanpolysulphate about 2 months before infection of mice with scrapie completely protected animals infected with up to 100 LD50, and considerably prolonged the lifespan of those infected with 100 to 10000 LD50. The clinical diagnosis was confirmed by immunoblot analysis for the protein of scrapie-associated fibrils.
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- Plant
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Purification, characterization and serological detection of virus-like particles associated with banana bunchy top disease in Australia
More LessIsometric virus-like particles, 18 nm in diameter, have been isolated from banana (Musa spp.) affected by bunchy top disease in Australia. Banana bunchy top disease-associated virus-like particles (BBTV) banded as a single component with buoyant density of 1.28 to 1.29 g/ml in Cs2SO4 and sedimented at about 46S in isokinetic sucrose density gradients. The A 260/A 280 of purified preparations was about 1.33. A single coat protein of M r 20500 identified with antibodies to BBTV particles from Australia. Single-stranded DNA of about 1 kb as well as ssRNA smaller than 0.45 kb was also associated with the particles. A polyclonal antiserum to BBTV, suitable for use in ELISA, was prepared. Stability and antigenicity of purified BBTV was impaired by storage at pH ≥ 8.5 and freezing at -20 °C without protectants. BBTV was detected by double antibody sandwich-ELISA with monoclonal and polyclonal antibodies, in field-infected banana plants, single aphids from an infective colony, and in experimentally aphid-inoculated banana plants. After transmission of BBTV particles by aphids from a banana bunchy top disease-affected to an uninfected banana plant, the disease was induced and BBTV was detected by ELISA in symptomatic leaves only. BBTV isolates from Australia, Taiwan, People’s Republic of China, Tonga, Western Samoa and Hawaii were found to be serologically related, which suggests a common aetiology for the disease.
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Virus-like particles associated with banana bunchy top disease contain small single-stranded DNA
More LessVirus-like particles were purified from banana plants with banana bunchy top disease. These particles were isometric with a diameter of 18 to 20 nm and a density of 1.28 to 1.30 g/ml in caesium sulphate. Associated with these particles were an ssDNA of about 1 kb and one major protein of M r 20100. DsDNA was synthesized from nucleic acid extracts from these particles and cloned. One clone, pBT338, hybridized specifically (i) with sap extracts from plants infected with banana bunchy top virus (BBTV) but not with sap extracts from healthy plants and (ii) with the small ssDNA in nucleic acid extracts from infected plants and virus-like particles. Banana bunchy top disease was transmitted from infected to healthy bananas by aphid inoculation and it was demonstrated that the small ssDNA was transmitted with the disease. It is probable that these particles represent the virions of BBTV containing small ssDNA and that the virus resembles subterranean clover stunt virus more than any other known virus.
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Resistance in Solanum brevidens to both potato virus Y and potato virus X may be associated with slow cell-to-cell spread
More LessA series of experiments was carried out to investigate the nature of the resistance of the wild potato species, Solanum brevidens, to potato virus X (PVX) and potato virus Y (PVY). In vitro inoculation of leaf protoplasts of S. brevidens and the virus-susceptible dihaploid S. tuberosum genotype PDH40 with PVX or PVY using polyethylene glycol showed that protoplasts of both species were similar in susceptibility. However, examination of protoplasts prepared from the leaves of S. tuberosum and S. brevidens inoculated 2 to 5 weeks earlier showed that the percentage of PVX- and PVY-infected leaf cells of S. tuberosum were, respectively, 45- to 100-fold and about 100-fold greater than the percentage of infected leaf cells of S. brevidens. These results suggest that resistance in S. brevidens to both PVX and PVY could be associated with slow cell-to-cell spread rather than with slow virus replication.
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Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus
The sequence of the 3′-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3′ terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3′ non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3′ non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3′ non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.
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Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter
More LessFull-length biologically active cDNAs of brome mosaic virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5′ ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower mosaic virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5′ end were infectious but RNAs transcribed from cDNAs with similar sequences were not.
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In vitro processing of the RNA-2-encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus
More LessIn vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, M r values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.
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Alfalfa mosaic virus RNA3 mutants do not replicate in transgenic plants expressing RNA3-specific genes
More LessThe RNA3 of alfalfa mosaic virus (AIMV) encodes the P3 protein and the virual coat protein (CP). RNA3 molecules transcribed in vitro replicated in protoplasts and plants when inoculated in mixtures with AIMV RNA1, RNA2 and CP. Transcripts with a deletion or inversion in the P3 gene replicated well in protoplasts but not in transgenic plants transformed with the P3 gene. Transgenic plants expressing the CP gene became infected after inoculation with a mixture of RNA1, RNA2 and wild-type RNA3 transcripts without addition of CP to the inoculum. Transcripts with a deletion in the CP gene replicated at a reduced level in protoplasts but not in CP-transformed plants. This suggests that P3 and CP are both required for cell-to-cell spread of AIMV and that mutations in the inoculum RNA could not be complemented in trans by the wild-type chimeric nuclear genes.
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Shortened forms of beet necrotic yellow vein virus RNA-3 and -4: internal deletions and a subgenomic RNA
More LessBeet necrotic yellow vein virus RNA-3 and RNA-4, produced as full-length biologically active transcripts in vitro, can undergo spontaneous internal deletions when inoculated onto Chenopodium quinoa leaves along with RNA-1 and -2. The deletion process is specific, giving rise to only a few major species, and can be rapid; deleted forms appear after only one or two passages in leaves. In one of the shortened forms of RNA-4, the deletion precisely eliminated one copy of a 15 nucleotide (nt) direct sequence repeat from the full-length prototype sequence, suggesting that ‘copy-choice’ switching of the replicase-template complex from one repeat to the other during RNA replication was responsible for the generation of this deletion. The deletion found in a major shortened form of RNA-3, on the other hand, did not occur near sequence repeats but began with GU and ended with AG like a nuclear intron sequence. Thus it is possible that the deleted sequence has been removed by splicing. However, two other deletions that were characterized were not associated with either of these types of sequence feature. An approximately 600 nt 5′-terminally truncated non-encapsidated form of RNA-3 was also detected in infected plant tissue. The evidence suggests that it is a subgenomic RNA derived from RNA-3.
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Cloning and sequencing of the S RNA from a Bulgarian isolate of tomato spotted wilt virus
E. Maiss, L. Ivanova, E. Breyel and G. AdamLibraries of cloned cDNA were prepared from complete genomic RNA and isolated S RNA of the Bulgarian L3 isolate of tomato spotted wilt virus (TSWV-L3). Northern blotting of TSWV genomic RNA detected clones specific for the L, M and S RNAs in the library from complete RNA. S RNA-specific clones selected from both libraries covered approximately 2.8 kb (about 95%) of the S RNA. Sequencing of these clones showed TSWV-L3 S RNA to be ambisense. It contains two open reading frames (ORFs); one of 1401 nucleotides located on the viral RNA encodes an M r 52400 (52K) protein, and the other of 774 nucleotides on the complementary strand encodes an M r 28900 (29K) protein. Expression of the 29K ORF in bacteria and immunological analysis of the fusion protein synthesized confirmed that the 29K protein is the N protein of TSWV-L3. Comparison with the published sequence for the S RNA of a Brazilian TSWV isolate, CNPH1, revealed almost complete identity in the amino acid sequences for the 29K protein, but several clustered amino acid exchanges in the putative 52K protein. In addition, the separating non-translated intergenic region of the S RNA of the Bulgarian isolate is 81 nucleotides longer than that of CNPH1.
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Ambisense segment 3 of rice stripe virus: the first instance of a virus containing two ambisense segments
More LessThe complete nucleotide sequence of rice stripe virus (RSV) segment 3 shows that it has two open reading frames, one in the viral-complementary sequence, which codes for the nucleocapsid protein, and the other in the viral-sense sequence. The non-coding region between the ambisense genes in RSV segment 3 contains several U and A tracts, as do the ambisense S segments of phleboviruses and uukuviruses. As we have previously shown that RSV segment 4 has an ambisense nature, this is the first instance of a virus containing two ambisense segments.
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Nucleotide sequence of raspberry bushy dwarf virus RNA-3
More LessA nucleotide sequence is reported for RNA-3, the smallest of the three major RNA species found in particles of raspberry bushy dwarf virus (RBDV). The sequence of 946 nucleotides contains a single large open reading frame which encodes an M r 30509 polypeptide. In vitro translation of RNA-3 yielded an M r 30000 product that reacted specifically with antiserum to RBDV particles and we conclude that the amino acid sequence deduced from the sequence of RNA-3 is that of the RBDV coat protein, or an immediate precursor of it. No affinities were detected by comparing the nucleotide sequence of RNA-3 with the sequences of other plant viruses.
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