- Volume 74, Issue 11, 1993
Volume 74, Issue 11, 1993
- Review Article
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Toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily
More LessGeneral introduction. Based on their morphological and physicochemical characteristics, the Toroviridae were initially proposed to constitute a new family of enveloped RNA viruses (Horzinek & Weiss, 1984; Horzinek et al., 1987). However, recent analysis of the genetic information and replication strategy of the prototype Berne virus (BEV) (Snijder et al., 1988, 1990a, c) has revealed that toroviruses are not unique: they are clearly related to the Coronaviridae and, more distantly, to the arteriviruses (den Boon et al., 1991b). This information has led to the reclassification of the toroviruses as a new genus in the coronavirus family (Pringle, 1992) and to the introduction of the unofficial term ‘coronavirus-like superfamily’ to indicate the evolutionary ties between the three virus groups mentioned above.
The history of torovirus research not only illustrates the taxonomic consequences present-day molecular analysis may have; the BEV genome has also turned out to be a showcase for the two driving forces in RNA virus evolution: divergence from a common ancestor and RNA recombination (Snijder et al., 1991a).
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- Bacterial
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Characterization of a new bacteriophage which infects bacteria of the genus Acidiphilium
More LessA novel bacteriophage, termed φAc1, that infects strains of the genus Acidiphilium (acidophilic, heterotrophic, aerobic, Gram-negative eubacteria) most commonly isolated from acidic mine drainage environments, has been discovered and several of its properties have been determined. This is the first report of a bacteriophage infecting such cells. The virion has a lambdoid morphology and is larger than λ, as shown by electron microscopy and sucrose gradient centrifugation. The sedimentation coefficient of the virion is approximately 615S. The nucleic acid of φAc1 is dsDNA, approximately 102 kb in length. Several experimental results show that φAc1 is a temperate phage. The plaques are turbid, and most cells isolated from plaques produced on sensitive cells by filter-sterilized phage preparations contain the phage and are resistant to further phage infection. Southern blot analysis shows that φAc1 prophage DNA is integrated into the bacterial genome during the temperate growth phase.
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- Animal
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Mapping and sequence of the gene encoding the African swine fever virion protein of M r 11500
More LessThe gene encoding the African swine fever virus protein of M r 11500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V. A serum raised against virion proteins of M r 12000 to 13000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to β-galactosidase. Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment. Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an M r of about 12000. A specific serum was raised against the E. coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle. Immunofluorescence experiments have shown that the protein localizes in virus factories.
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Mapping the genetic region coding for herpes simplex virus resistance to mouse interferon α/β
More LessHerpes simplex virus (HSV) ocular virulence has been associated with strain sensitivity to mouse interferon (IFN)-α/β. To identifiy the region of the virus genome associated with heightened resistance to this cytokine, intertypic recombinants were constructed using the intact genome of avirulent, IFN-sensitive HSV type 1 (strain 35) and XbaI-digested DNA from virulent, IFN-resistant HSV type 2 (strain 186). An intertypic recombinant, designated HSV-R4, was isolated which grew to titres 10- to 100-fold higher than HSV-1(35) in mouse ocular tissue in vivo, and induced stromal keratitis. The recombinant which was several orders of magnitude more resistant to mouse IFN-α/β than HSV-1(35) had a genome composed of HSV-1(35) DNA except for a 12 kb fragment (0.15 to 0.23 map units) derived from HSV-2(186). To define the IFN resistance locus further, three overlapping subclones of this 12 kb fragment were constructed from the HSV-2(186) genome and subjected to marker rescue experiments. The cloned BamHI D fragment was the only subclone that promoted HSV-1(35) ocular growth in vivo. An intertypic recombinant, designated HSV-R(BD), was isolated from the 35 × 186 BamHI D transfection progeny pool. This recombinant, in contrast to HSV-1(35), was several orders of magnitude more resistant to mouse IFN-α/β inhibition in vitro, grew 10- to 100-fold better in mouse ocular tissue in vivo, and caused severe necrotizing stromal keratitis in BALB/c mice. Analysis of the recombinant genome indicated that the HSV-2 genetic information responsible for IFN resistance of HSV-R(BD) was located within the BamHI D fragment, most likely mapping to that region containing three partial open reading frames designated UL14, UL15 and UL16. The products encoded by this region remain to be identified.
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Tumour necrosis factor α stimulates the activity of the human cytomegalovirus major immediate early enhancer/promoter in immature monocytic cells
More LessBoth tumour necrosis factor α (TNF-α) and phorbol 12-myristate 13-acetate (PMA) stimulated human cytomegalovirus (HCMV) major immediate early (IE) enhancer/promoter activity in the HL-60 granulocyte/monocyte progenitor cell line when added to transfected cells. In U-937 monocytic cells, by contrast, TNF-α had no stimulatory effect and the addition of PMA produced only marginal stimulation. In the mature THP-1 monocytic cell line and in differentiated HL-60 cells, addition of TNF-α caused inhibition of the IE enhancer/promoter activity. The stimulating effect of PMA, as observed in the other cell lines, however, remained. Thus the effect of TNF-α on the major IE enhancer/promoter activity is determined by the degree of differentiation of the infected cells. Unlike TNF-α and PMA, the interleukins IL-1, IL-3, IL-6 as well as the cytokine GM-CSF were found to have no detectable influence on the activity of the IE enhancer/promoter activity which, likewise, was not affected by the presence of the modulator sequence. Since premonocytic cells are suggested to be sites of HCMV latency, the stimulation by TNF-α could be of potential pathophysiological significance.
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Interactions between equine herpesvirus type 1 and equine herpesvirus type 4: T cell responses in a murine infection model
More LessInteractions involving the immune responses to equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) were studied in a murine infection model. When mice were inoculated intranasally with EHV-1, virus replication occurred in the respiratory tract and clinical signs were produced. In contrast, mice that were similarly inoculated with EHV-4 produced no evidence of virus replication and showed no clinical signs. When mice that had been inoculated with live EHV-4 were challenged 1 month later with EHV-1 they were partially protected. Although clinical signs were apparent on reinfection, virus replication in the respiratory tract was reduced in these mice compared with control mice that had not been previously immunized. Mice primed with heat-inactivated EHV-4, however, were not so protected. Live EHV-4-primed mice developed very low levels of antibody to EHV-1 and the humoral response could not account for this protection. However, the infected mice did give a strong delayed-type hypersensitivity reaction in a skin test using either EHV-1 or EHV-4 antigen. Spleen cells from EHV-4-primed donors provided a source of immune cells, including T cells which were used for transfer to recipient mice which were then challenged with EHV-1. The cells were protective; there was a reduction of virus replication on challenge with EHV-1 which correlated with the number of cells transferred. Modulation of the protective effect of primed cell populations was tested after depletion in vivo by means of complement-mediated lysis. The depletion of CD4-bearing cells produced the least effect on the protection afforded by cell transfer. In contrast, depletion of CD8-bearing cells markedly reduced the protection in recipients. EHV-1 and EHV-4 are wide-spread in horses and cross-infections are common. These results gained from a murine model indicate that important interactions occur at the level of T cell immunity between the two virus types which warrant further investigation in the natural host.
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Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells
The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.
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Measles virus antigens induce both type-specific and canine distemper virus cross-reactive cytotoxic T lymphocytes in mice: localization of a common Ld-restricted nucleoprotein epitope
More LessWe have studied the induction of the cytotoxic T lymphocyte (CTL) response to measles virus (MV) antigens expressed as vaccinia virus (VV) recombinants in a murine model. In C3H mice (H-2k) only the nucleoprotein (NP) induced a CTL response and this was shown to be cross-reactive with the closely related canine distemper virus (CDV). The presentation of this antigen was shown to be Kk-restricted. In BALB/c mice (H-2d), both the haemagglutinin (HA) and the NP induced a strong CTL response, the former being serotype-specific, whereas the latter cross-reacted with CDV. Both responses were found to be Ld-restricted. Based on the prediction for Ld T cell motifs, we tested a number of MV NP-derived nonapeptides for their capacity to sensitize P815 cells (H-2d) for lysis by spleen cells from VV-NP-immunized mice. One of these peptides, comprising amino acids 281 to 289 (Tyr-Pro-Ala-Leu-Gly-Leu-His-Glu-Phe) was as effective as cells expressing the complete NP protein. This motif is conserved in the CDV NP.
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Measles virus glycoproteins: studies on the structure and interaction of the haemagglutinin and fusion proteins
More LessWe have investigated the structure and interaction of the measles virus (MV) glycoproteins expressed at the cell membrane. Cross-linking studies with a variety of chemicals stabilized dimeric forms of the haemagglutinin (HA) or fusion (F) proteins, although by sucrose density gradient analysis, oligomers corresponding to tetramers and larger were observed for both proteins. In cells in which both HA and F were expressed at the surface, their close association was shown by cross-linking and co-immunoprecipitation.
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Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system
More LessThe spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S1 (with an N-terminal signal peptide) and C-terminal S2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFS1utt), the S1 protein with a C-terminal transmembrane domain derived from S2 (pSFS1tmd) or the S2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and S2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these VV-TVs. In cells transfected with the pSFS1utt and pSFS1tmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic S2 protein. The S1utt and S1tmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The S1tmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the S1utt protein was not. However, when the S1utt protein was expressed together with the ssS2 protein, the S1utt was detected on the cell membrane. This suggested that the S1utt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and S2 proteins associated in a similar manner to the authentic S1 and S2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or S2 nor in cells coexpressing both S1 and S2, although the whole S protein expressed by VV-TV did induce fusion.
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Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection
We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5′ untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1a, 1b, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3′ end of 5′ UTR, C, E1 and 5′ end of E2/NS1) and 8276 to 9394 (3′ end of NS5 and 3′ UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96%, compared to 73 to 74%, 73%, 70% or 69 to 70% against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported ‘type V (3a)’ isolates was 88 to 100%; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3′ UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5′ CGTAAAACTTCT GAACGGTC, sense and no. 339: 5′ GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.
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Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region
Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79% overall sequence similarity to one another. Phylogenetic analysis of nucleotide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylogenetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5′ non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the two-tiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered.
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Hexamethylene bisacetamide activates the human immunodeficiency virus type 1 provirus by an NF-κB-independent mechanism
More LessExpression of the human immunodeficiency virus type 1 (HIV-1) provirus in T lymphocytic and monocytic cells can be induced by treatment with hexamethylene bisacetamide (HMBA). The induction occurs at the transcriptional level within 1 to 3 h after the addition of the drug, and is not associated with detectable changes in the binding of transcription factors to the enhancer, TATA box or other regulatory regions of the HIV-1 long terminal repeat (LTR). Using the 5′ deletion mutants of HIV-1 LTR controlling the expression of the chloramphenicol acetyltransferase gene, we found that the deletion of the κB enhancer did not affect HIV-1 inducibility, whereas the deletion of the Sp1 binding sites abolished transcriptional activation. However, the presence of the HIV-1 LTR Sp1 binding sites in the context of the heterologous promoter did not induce responsiveness to HMBA. We conclude that HMBA increases transcription through the secondary modification of the basal transcription complex suggesting the existence of a regulatory pathway that circumvents the requirement for the induction of NF-κB or other DNA-specific binding proteins.
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Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and the monocistronic rev mRNA
More LessThe transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (92 kb), at least five FIV-specific transcripts [52, 44 (doublet), 17 and 14 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 92, 52 and 44 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev − mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3′ region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.
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Characterization of RNA-binding domains of hepatitis delta antigen
Hepatitis delta antigen (HDAg), the only protein encoded by the hepatitis delta virus (HDV), binds specifically genomic and antigenomic strands of the HDV RNA. In a previous study, three recombinant HDAg subdomains were synthesized, covering residues 11 to 78, 79 to 163 and 164 to 212, and only the middle domain was shown to be responsible for the binding to HDV RNA. To investigate HDAg sequences involved in HDV RNA binding, we synthesized five peptides, 15 to 29 residues in length, and tested their ability to bind HDV RNA using a simple non-radioactive ELISA with digoxigenin-labelled HDV genomic or antigenomic RNA probes. The specificity of interactions was demonstrated by comparison with control peptides and non-HDV RNA probes, and with an inhibition assay using recombinant HDAg. The HDAg-binding domain found within the middle region (79 to 163) of HDAg was more finely mapped: it is located between residues 79 and 107. In addition, another domain (residues 2 to 27) of HDAg was also found to bind specifically to HDV RNA. These two peptides share sequence similarities at residues 2 to 10 and 97 to 107 with other RNA-binding domains.
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Trans-activation of the adenovirus E2 promoter by human papillomavirus type 16 E7 is mediated by retinoblastoma-dependent and -independent pathways
More LessIn common with the adenovirus E1A and simian virus 40 large T oncoproteins, the E7 protein of human papillomavirus (HPV) type 16 interacts with the retinoblastoma (Rb) tumour suppressor protein (pRb). The functional importance of this interaction for HPV-16 E7 protein was investigated by analysis of the trans-activating function of E7 at the adenovirus E2 promoter in a set of breast tumour cell lines. Trans-activation by HPV-16 E7 in two pRb-deficient cell lines demonstrated that pRb is not essential for E7-mediated trans-activation, but reconstitution of Rb expression indicated the existence of an Rb-mediated pathway of E7 trans-activation. This pathway results from suppression by E7 of a trans-repressing function encoded by the Rb gene. The E7 protein is shown to be capable of interacting in vivo with the Rb-related protein p107. Furthermore, analysis of a fusion construct between the amino terminus of Rb and the carboxy terminus of p107 suggests that, in common with pRb, the p107 protein trans-represses the adenovirus E2 early promoter. Therefore it is proposed that the pRb-independent pathway of E7 trans-activation is a consequence of the suppression of trans-repression by p107.
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Characterization of an in vivo reactivation model of herpes simplex virus from mice trigeminal ganglia
More LessHerpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. Viral gene expression includes the latency-associated transcripts (LATs) which have been linked to the ability of the virus to resume replication and reactivate. However, the molecular basis of reactivation and the mechanisms of action of these transcripts are unknown. In order to study these parameters, an in vivo reactivation model is needed. We investigated use of the mouse as the experimental animal, modifying the route of infection, the viral strain and the reactivation protocol. Following administration of human immunoglobulin 1 day prior to corneal infection, no infectious virus was detected in trigeminal ganglia (TG). However, latency was established in all infected animals as indicated by explant reactivation of TG, and in vivo reactivation was achieved in 30 to 40% of them. DNA quantification revealed that TG of immunized mice contained more HSV-1 DNA than did those of non-immunized mice. By in situ hybridization twice as many neuronal cells in TG of immunized mice were positive for LATs, compared with infected but non-immunized, mice. These findings suggest that suppression of primary infection facilitates reactivation by increasing HSV-1 copy number in latently infected nervous tissue.
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The herpes simplex virus type 1 strain 17 open reading frame RL1 encodes a polypeptide of apparent M r 37K equivalent to ICP34.5 of herpes simplex virus type 1 strain F
More LessThe region between the ‘a’ sequence and the 5′ end of the IE1 gene within the long repeat sequence of the herpes simplex virus (HSV) genome plays an important role in the neurovirulence of both HSV-1 strain F and HSV-1 strain 17. However, there has been controversy over the protein-coding potential of this region. Although an open reading frame (ORF) was predicted in HSV-1(F) and shown to encode a polypeptide called ICP34.5, only recently has a corresponding ORF, designated RL1, been recognized in HSV-1(17). To determine whether the HSV-1(17) ORF is expressed, we raised antipeptide sera against predicted amino acid sequences from RL1; one serum specifically recognized a 37K protein in HSV-1(17)-infected cell extracts. Compared with the corresponding HSV-1(F) polypeptide the HSV-1(17) protein has a lower apparent M r, shows similar kinetics of accumulation and intracellular localization but may accumulate to lower levels than the HSV-1(F) protein. The non-neurovirulent HSV-1(17) deletion variant 1716 fails to synthesize detectable levels of ICP34.5. Thus we have established that HSV-1(17), like HSV-1(F), expresses ICP34.5, a protein important for HSV neurovirulence.
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Sequence variation within neutralizing epitopes of the envelope glycoprotein B of human cytomegalovirus: comparison of isolates from renal transplant recipients and AIDS patients
More LessThe envelope glycoprotein B of human cytomegalovirus (CMV) is a major target of the neutralizing antibody response against this virus, and hence has importance as a potential subunit vaccine. PCR was utilized to amplify DNA encoding the dominant antigenic determinant on this molecule, AD-1 (codons 552 to 635), and DNA sequencing was carried out in order to compare nucleotide variation in AD-1 between clinical isolates of CMV and the laboratory strain AD169. Wild-type CMV strains isolated from AIDS patients were not only more likely to possess nucleotide substitutions (19/24 compared to 5/25, P < 0·0001) than those from renal transplant recipients, but they also exhibited a greater degree of nucleotide sequence divergence (6·94 versus 0·82 substitutions/1000 bp, P < 0·0001; 96·0 to 100% versus 99·4 to 100% similarity). Increased sequence variation in the AIDS patients did not correlate with absolute peripheral blood CD4+ T cell level (r = 0·33, P > 0·1). Only two strains from AIDS patients and one strain from the renal transplant recipients possessed nucleic acid substitutions that resulted in codon changes, indicating that AD-1 is relatively well conserved amongst clinical isolates of CMV. The demonstration of strains with codon changes within neutralizing epitopes, however, highlights the importance of taking into consideration the presence of these strains within the wildtype virus population when preparing subunit vaccines.
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Murine cytotoxic T cell response specific for human cytomegalovirus glycoprotein B (gB) induced by adenovirus and vaccinia virus recombinants expressing gB
A murine model of the cytotoxic T lymphocyte (CTL) response to glycoprotein B (gB) of human cytomegalovirus (HCMV) was developed based on the use of adenovirus (Ad) and vaccinia virus (Vac) recombinants expressing gB. Mice of different major histocompatibility haplotypes [CBA (H-2k), BALB/k (H-2k) and BALB/c (H-2d)] infected with the Ad-gB recombinant developed an Ad-specific CTL response. However, only the H-2k mice developed a significant HCMV gB-specific CTL response, as indicated by the major histocompatibility complex class I-restricted lysis of Vac strain Copenhagen (VacC)–gB recombinant-infected target cells by H-2k mouse immune spleen cells. The VacC-gB recombinant elicited only a weak gB-specific CTL response in these mice, indicating that the observed gB-specific CTL response in mice is dependent on the expression vector used for immunization. The gB-specific cytotoxicity observed in H-2k mice was mediated by the CD8 lymphocyte subset.
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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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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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