- Volume 74, Issue 11, 1993
Volume 74, Issue 11, 1993
- Animal
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Comparison of 10 influenza A (H1N1 and H3N2) haemagglutinin sequences obtained directly from clinical specimens to those of MDCK cell- and egg-grown viruses
More LessPCR was used to amplify and sequence the complete HA1 region of the haemagglutinin (HA)-encoding genes of 10 clinical isolates of influenza virus of the H1N1 or H3N2 subtypes. These sequences were compared to those obtained from viruses isolated from the same specimens after passage in eggs and MDCK cells. Amino acid substitutions in the egg-derived HA sequences were found in nine out of the 10 specimens analysed, whereas seven out of eight of the MDCK-derived HA sequences were identical to those in the corresponding original specimens. Changes in the H1 HA occurred at residues 77a, 196 (also found in the corresponding HA from the MDCK isolate), 225, 226 and 227; changes in the H3 HA occurred at residues 137, 156, 186, 248 and 276. In addition, we have shown that an amino acid change at residue 145 in the HA of the H3 subtype that was previously demonstrated to be egg-selected is now present in circulating strains.
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Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein
More LessThree isolates of feline calicivirus (FCV) designated NADC, KCD and CFI/68 were compared for biochemical, serological and genetic variation within the capsid protein gene. The M r of the capsid protein from purified virions was approximately 66000 for the NADC virus isolate, which differed slightly from the relative mobilities of the purified capsid proteins of the KCD and CFI/68 isolates. Polyclonal antisera from either cats infected or rabbits hyperimmunized with the CFI/68 isolate cross-reacted with all three isolates by Western blot analysis. However, these polyclonal antisera to CFI/68 varied considerably in their virus-neutralization titres to the KCD and NADC isolates. Nucleotide sequence data confirmed the genetic variability among these FCV isolates. Comparison of the predicted amino acid sequence of the capsid protein among isolates revealed two regions of sequence divergence that probably contain the antigenically variable determinants. These hypervariable regions may vary by as much as 55% among isolates of FCV. The amino acid sequence diversity in the hypervariable regions of the KCD and NADC isolates correlated well with the virus-neutralization data and suggests that polyvalent vaccines may be more protective than the commonly used monovalent vaccines.
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A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies
More LessA panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.
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Complete sequence conservation of the human T cell leukaemia virus type 1 tax gene within a family cluster showing different pathologies
More LessWe have amplified, through PCR, the full-length tax gene of human T cell leukaemia virus type 1 (HTLV-1) derived from proviral DNA of peripheral blood lymphocytes of five first degree relatives of Afro-Caribbean origin. One patient (the father) had adult T cell leukaemia (ATL), one (the mother) tropical spastic paraparesis (TSP), and three (children) were healthy asymptomatic carriers. All five family members had identical tax nucleotide sequences as determined by direct sequencing of PCR products. This sequence was compared with tax gene sequences of an unrelated TSP patient of Afro-Caribbean origin, and of C8166 cells, and found to have one and seven nucleotide differences, respectively. At the amino acid level these three sequences differed from the HTLV-1 prototype Japanese strain (ATK-1). All sequence changes were clustered towards the 3′ end of the gene. These data demonstrate the complete conservation of an HTLV-1 gene following, presumably, horizontal and vertical transmission of the virus. Clones of this gene showed more sequence variation within the TSP patient than the ATL patient, mostly consisting of point mutations; there was no conservation of mutations between the two individuals. These mutations occurred only in individual clones of the ATL patient whereas those of the TSP patient were found to be repeated in different clones. A tax-specific cytotoxic T lymphocyte response was observed in two asymptomatic carriers with low antibody titres, whereas none was detected in an individual with a high antibody level. No tax-specific sequence was identified which may have contributed to the apparently high degree of transmission from mother to children (three of five children tested) nor account for the differences between disease symptoms in the parents.
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- Plant
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Isolates of citrus exocortis viroid recovered by host and tissue selection
More LessIsolates of citrus exocortis viroid (CEV) from a single sweet orange citrus source have been selected by sequential passage through the alternative hosts citron, Gynura aurantiaca, a hybrid tomato Lycopersicon esculentum × L. peruvianum, and from disorganized callus culture of the hybrid tomato. The distinctions in symptom expression, titre and electrophoretic mobility among the CEV isolates, operationally termed CEVc (citron), CEVg (Gynura), CEVt (tomato) and CEVcls (callus) are supported by characteristically different nucleotide sequences. The nucleotide sequence of full-length cDNA clones of CEVc purified from citron shows exchanges not reported for any previously described CEV variant. An unusual number of exchanges have been localized in the terminal domains of all the isolates analysed here. A common pattern of nucleotide exchanges, described as a ‘tomato signature’, can be detected in all of the isolates derived from hybrid tomato tissues.
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Nucleotide sequence evidence for the occurrence of three distinct whitefly-transmitted geminiviruses in cassava
More LessThe complete nucleotide sequence of the DNA of Indian cassava mosaic virus (ICMV) and a key part of that of a group B isolate of African cassava mosaic virus from Malawi (ACMV-M) were determined and compared at the nucleotide and encoded amino acid levels with the published sequences of an ACMV group A isolate (ACMV-K) and other whitefly-transmitted geminiviruses (WTGs). The DNA of ICMV consists of two circular single-stranded molecules, DNA-A [2815 nucleotides (nt)] and DNA-B (2645 nt), which differ substantially in sequence from the genome components of ACMV-K (DNA-A 70%, DNA-B 47% sequence identity) and other WTGs. ICMV DNA-A contains eight open reading frames (ORFs) encoding proteins of > 100 amino acid residues, of which four ORFs (one genome sense, three complementary sense) are comparable to those of other WTGs. DNA-B contains one ORF in each sense, as in other WTGs. None of the putative viral proteins are more similar in amino acid sequence to the proteins of ACMV-K than to those of another WTG. The coat protein of ACMV-M is more like that of tomato yellow leaf curl virus from Sardinia (86% sequence identity) than those of ICMV or ACMV-K. The intergenic regions of ACMV-K, ACMV-M and ICMV DNAs differ in size, and largely in sequence, except for two 30 to 40 nt sequences which are also conserved in other WTGs and can form stem—loop structures. The intergenic region of ICMV DNA contains three copies of a 41 nt sequence, and that of ACMV-M DNA contains an imperfect repeat of a 34 nt sequence which resembles the repeated sequence in ICMV DNA. The differences between ACMV-K, ACMV-M and ICMV are considered great enough to justify their separation as isolates of three distinct WTGs: African cassava mosaic virus, East African cassava mosaic virus and Indian cassava mosaic virus.
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Interference with brome mosaic virus replication by targeting the minus strand promoter
More LessSense and antisense strategies for interfering with the replication of brome mosaic virus (BMV) were examined. The effects of 200 nucleotide-long sense and antisense transcripts, corresponding to the viral 3′ end (-) strand promoter, on the accumulation of progeny viral RNAs were studied by co-inoculation with wild-type BMV RNAs. Progeny accumulation in barley protoplasts transfected with either sense or antisense transcripts of the (-) strand promoter and BMV RNAs-1 and -2 was decreased by more than 90%, and by 60 to 80% when RNA-3 was also present. This trans interference was concentration-dependent, and reduced both (+) and (-) strand progeny accumulation to a similar extent. The appearance of complementary (-) strands indicated that sense interfering transcripts could serve as templates for (-) strand synthesis, and the use of deletion mutants revealed that the observed interference was in part mediated by this template activity. The reproducibility of the protoplast assay used here allows rapid evaluation of interference strategies and comparisons to be made of alternative approaches to engineered resistance. The results presented here suggest that targeting viral (-) strand promoters with sense and antisense transcripts may be an effective method for engineering plant resistance to viral infection.
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Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus
More LessMutant movement proteins of red clover necrotic mosaic dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were produced in Escherichia coli using expression vectors. The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retardation. The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain.
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Effect of the alfalfa mosaic virus movement protein expressed in transgenic plants on the permeability of plasmodesmata
More LessSymplastic transport of different sized fluorescent probes has been assessed in leaf epidermal cells of transgenic Nicotiana plants expressing the movement protein (MP) of alfalfa mosaic virus (AMV). In both N. tabacum and N. benthamiana, the size exclusion limit (SEL) of plasmodesmata increased from M r 1000, which represents the commonly accepted limit, to over 4.4K. However, in control plants, movement of a 3K probe was seen in 11 to 22% of the injections, indicating that plasmodesmata may on occasion allow the passage of molecules larger than was previously thought. The increase of SEL due to the presence of the AMV MP, although significant, remains insufficient to permit the passage of viral particles and the possibility of other mechanisms involved in viral cell-to-cell spread is discussed.
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Complete nucleotide sequence and coding strategy of rice hoja blanca virus RNA4
More LessThe complete sequence of rice hoja blanca virus (RHBV) RNA4 has been determined, based on the sequence of the corresponding cDNA clones. RNA4 consists of 1991 nucleotides with two open reading frames (ORFs). One putative ORF is located in the 5′-proximal region of the viral RNA4; it encodes a protein of predicted M r 20076 which corresponds to the major non-structural protein that accumulates in RHBV-infected rice plants, and which bears limited sequence identity with the helper component of tobacco vein mottling potyvirus. The other ORF is located in the 5′-proximal region of the viral complementary RNA4 and encodes a protein of predicted M r 32469. Between the two ORFs is an intergenic region of 524 nucleotides, part of which can theoretically adopt a stable stem-loop structure; the 5′ and 3′ ends can potentially base-pair over 16 nucleotides, producing a pan-handle configuration. These characteristics are in favour of an ambisense coding strategy for RHBV RNA4.
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Reasons for the low accumulation level of aphid transmission factor protein in infected leaves with an aphid-non-transmissible cauliflower mosaic virus isolate, CM1841
More LessThe synthesis and accumulation of aphid transmission factor protein (p18) in cauliflower mosaic virus (CaMV)-infected turnip protoplasts were examined in time course and pulse-labelling experiments, comparing an aphid-non-transmissible isolate (CM1841) with an in vitro recombinant aphid-transmissible CaMV (CMBX) generated from the CM1841 isolate. There was little difference in the synthesis and accumulation of p18 between CM1841- and CMBX-infected protoplasts. When the accumulation of p18 in infected leaves was monitored from 3 to 28 days post-symptom emergence (p.e.) by Western blotting, the amount of p18 accumulated in CM1841-infected leaves continuously decreased from 3 days p.e. throughout the experimental period, whereas the amount of p18 in CMBX-infected leaves was lowest at 3 days p.e. and increased thereafter. These results suggested that CM1841 differed from CMBX not in the synthesis of p18 but in the stability of p18 in infected leaves.
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