- Volume 79, Issue 1, 1998
Volume 79, Issue 1, 1998
- Articles
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Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats
Feline calicivirus (FCV) is a respiratory pathogen of cats that is capable of causing persistent infections. This study examined the evolution of a hypervariable region of the FCV capsid gene both during 90 passages in cell culture and during replication in persistently infected cats. This region of the capsid protein is known to contain neutralization epitopes and may be a target for immune evasion during virus persistence in the host. Sequence analysis showed that FCV exists as a quasispecies which evolved both in cell culture and in persistently infected cats. Changes involved both loss of sequence present in the infecting isolate and a gain of both synonymous and non-synonymous nucleotide substitutions to generate sequences not detected within earlier isolates. Overall, these changes led to a reduction in population heterogeneity over time. Where virus populations were highly homogeneous allowing a consensus sequence to be determined, evolution rates for the consensus sequence ranged from 0·10 – 1·07 substitutions per nucleotide per year. Marked changes in virus neutralization profiles were seen in isolates obtained sequentially from a persistently infected cat. This was not the case with cell culture passaged virus, suggesting that the individual amino acid changes found only in virus from persistently infected cats may significantly alter the antigenic profile of FCV, and may be the result of immune selection.
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Molecular epidemiology of rabbit haemorrhagic disease virus outbreaks in France during 1988 to 1995
More LessIn order to evaluate genetic variation between rabbit haemorrhagic disease virus (RHDV) isolates and to derive phylogenetic relationships, 56 virus isolates collected from various parts of France over a 7 year period (1988 to 1995) were examined. Analyses were carried out by direct nucleotide sequencing of PCR fragments of three genomic regions encoding the capsid protein (VP60) (regions A and B) and a non-structural protein (region C). Multiple sequence alignments revealed maximum nucleotide divergence of 7·6, 9·4 and 8·7% for regions A, B and C, respectively, indicating a high level of conservation between isolates. Irrespective of the genomic region analysed, phylogenetic analyses carried out using various methods allowed the identification of three genogroups; distribution of isolates within these genogroups appears to be more related to the year of their collection than to their geographical origin. The possible evolution of RHDV is discussed.
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Genetic analysis of mengovirus protein 2A: its function in polyprotein processing and virus reproduction
More LessTo examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2Ais replaced either byTheiler’s murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro junction was highly efficient. Although the resulting L-P1 precursor was cleaved at the L-VP4 junction, further processing of the P1 precursor was abrogated. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro construct did not result in viable virus. Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was reduced to 25%. Although inhibition of hostcell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged. The results also confirm that the processing cascade of L-P1-2A occurs sequentially and is probably regulated by subsequent conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.
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Evidence for biotype-specific effects of bovine viral diarrhoea virus on biological responses in acutely infected calves
The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biological responses they induce was studied in 3- to 6-month-old calves inoculated intranasally with a homologous pair of non-cytopathic and cytopathic strains. Marked differences in virological and serological events occurred following exposure to a specific BVDV strain. The non-cytopathic biotype was frequently recovered from nasal secretions and blood cells during the first 28 days post-inoculation whereas the cytopathic counterpart was detected infrequently in nasopharyngeal swabs only. There was no correlation of the recovery of infectious virus in vivo with the biotype-specific neutralizing humoral immune response. Furthermore, seroconversion did not correlate with resistance to reinfection as judged by the transient viraemia and/or shedding of virus observed in a challenge experiment.
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Infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine T lymphocytes
More LessT lymphocytes obtained from pigs infected with a lethal dose of classical swine fever virus were analysed for phenotypic changes in the composition of T-cell subpopulations and for alterations in their immune responsiveness in vitro during the course of disease. Viral antigen detected in all subpopulations and the selective depletion of CD4 CD8 γ/δ T cells showed that peripheral blood T lymphocytes were affected in the terminal stage (14–19 days postinfection) of classical swine fever whereas no implications for T lymphocytes were obvious during the first 10 days after infection. Furthermore, a depletion of CD1 CD4 CD8 ‘common thymocytes’ was characteristic for the infected animals. Studies on immune functions of peripheral T lymphocytes revealed an abrogation of cellular immune responses as early as 3–5 days after infection and thus before detection of viral antigens in these cells. The data suggest that early immunosuppression represents a crucial event for the manifestation of classical swine fever.
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Genomic analysis of two GB virus A variants isolated from captive monkeys
The recent isolation of GB viruses A and B from GB agent infected tamarins and their lack of involvement in human hepatitis has sparked interest in the origin of these viruses. Several healthy non-human primate species have been shown to harbour sequences 52–79% identical to the GBV-A 5′ nontranslated region. In this paper we report the near genome length sequence of GBV-Amx 70047 and GBV-A,ri 1122. These sequences support previous observations about the genomic organization of GBV-A and provide insight into the genomic variability within this virus genus. Although the GBV-A variant polyproteins possess many motifs conserved between other members of the Flavi- viridae, they do not encode a basic core-like protein. Amino acid sequence comparisons and phylogenetic analysis demonstrate variability within the GBV-A genus similar to that observed between hepatitis C virus (HCV) types. However, genomic organization and disease association demonstrate a closer evolutionary relationship to GBV-C than to HCV.
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An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein
More LessThe gene encoding the nucleoprotein (N) and PCR- derived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in Escherichia coli BL21(DE3) transformed by recombinant expression vector pET-14b containing N and PCR-generated subfragment cDNAs under the control of the T7 RNA polymerase promoter. Following induction with IPTG, recombinant His-tagged proteins were expressed, purified by affinity metal chelation chromatography under denaturing conditions and re- natured. Protein blots were hybridized with various radiolabelled nucleic acid probes. Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain. To confirm this observation, two more accurate approaches were undertaken: (i) a gel retardation assay of RNA and purified protein complexes was done; and (ii) RNA-protein complexes were cross-linked by UV light and analysed on a denaturing polyacrylamide gel. All these experiments led us to conclude that the middle part of N is the domain which interacts with RNA, despite the absence of homology with known consensus amino acid sequences of other RNA-binding proteins.
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Characterization of a simian T-lymphotropic virus from a wild-caught orang-utan (Pongo pygmaeus) from Kalimantan, Indonesia
In a recent serological survey among 143 excaptive orang-utans two individuals were found that reacted positive in an ELISA detecting antibodies which cross-react with human T-lympho- tropic virus type I (HTLV-I) antigens. Infection of both animals with an HTLV-I or simian T-lympho- tropic virus (STLV)-like virus was confirmed by Western blot analysis. A third wild-caught animal, which was not part of the original serological survey, was also found to be infected with an HTLV- related virus in a diagnostic PCR assay and Western blot assay. Nucleotide sequence analysis of the 709 bp PCR fragment from the tax/rex region of the HTLV/STLV genome confirmed infection of orangutans with an STLV similar to but clearly distinct from other Asian STLVs.
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Antibodies to the C-terminal dipeptide of mouse mammary tumor virus [MMTV(SW)] superantigen effectively inhibit T-cell activation in vivo
More LessBALB/c mice were immunized against the 19 C- terminal amino acids of the mouse mammary tumour virus [MMTV(SW)] superantigen, either actively with the recombinant ORF19 protein or passively by subcutaneous implantation of 6E1.8 hybridoma cells. Protection against the MMTV(SW) superantigen-induced activation of specific T cells was obtained in both immunizations. Whereas the monoclonal antibody provided complete protection, similar antibody titres showed less effective protection in actively immunized mice. This was due to the presence of a histidine tag on the recombinant ORF19 protein preventing immune recognition of the C-terminal amino acids during immunization. Preincubation of the 6E1.8 monoclonal antibodies with modified ORF19 peptides at their C terminus established specific recognition of the last two amino acids of MMTV(SW) superantigen by this antibody, and showed that these amino acids are critical in the interaction between the MMTV(SW) superantigen and the Vβ element of the T-cell receptor.
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Evolutionary relationships within a subgroup of HERV-K-related human endogenous retroviruses
More LessThe prototype endogenous retrovirus HERV-K10 was identified in the human genome by its homology to the exogenous mouse mammary tumour virus. By analysis of a short 244 bp segment of the reverse transcriptase (RT) gene of other HERV-K10-like sequences, it has become clear that these elements represent an extended family consisting of multiple groups (the HML-1 to HML-6 subgroups). Some of these elements are transcriptionally active and contain an intact open reading frame for the RT protein, raising the possibility that this family is still expanding through retrotransposition. To better define the relationship of these endogenous retroviruses, we identified ten new members of the HML-2 subgroup. PCR was used to amplify reverse- transcribed RNA of a 595 bp region of the RT gene in a variety of human cell samples, including normal and leukaemic bone marrow and peripheral blood, placenta cells and a transformed T cell line. We provide an extensive phylogenetic analysis of the relationships for this cluster of HERV-K-related endogenous retroviral elements. Nucleotide diversity values for nonsynonymous versus synonymous codon positions indicate that moderately strong selection is or was operating on these retroviral RT gene segments. The evolution of this class of endogenous retroelements is discussed.
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CD34+ cells and their derivatives contain mRNA for CD4 and human immunodeficiency virus (HIV) co-receptors and are susceptible to infection with M- and T-tropic HIV
More LessHighly purified (> 98%) CD34 cells directly after isolation (D0) or 2 weeks in culture (D14) were CD4 and contained mRNA for the T-tropic HIV coreceptor, CXCR-4, and minor co-receptor, CCR-2B. D14 but not D0 cells were RT-PCR positive for mRNA for the major M-tropic human immunodeficiency virus (HIV) co-receptor, CCR-5, and potential coreceptor, CCR-1. D14 and D0 cells were susceptible to T- (HXB2) and M-tropic HIV (Bal), showing greater virus production with Bal than HXB2, and with higher virus production levels in D14 compared to D0 cells. Seven days post-infection of D0 cells Bal DNAwas present in CD14brightand CD14 fractions, suggesting D0 infection of diverse progenitor types. HXB2 DNA was detected in CD14bright cells alone indicating D0 infection of monocyte progenitors only. It is concluded that CD34 cells and cultured derivatives are susceptible to M- and T-tropic HIV and this correlates in part with co-receptor expression at the mRNA level.
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Neutralization of primary and T-cell line adapted isolates of human immunodeficiency virus type 1: role of V3-specific antibodies
The role of the third variable domain (V3) of gp120 in the neutralization of primary and T-cell line adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1) by serum from HIV-1-infected individuals was investigated. A primary virus isolate, M2424/4, when adapted to H9 cells, was more sensitive to neutralization on MT2 cells than the same stock passaged in PBMC. Neutralization of the PBMC-passaged stock by V3-specific MAbs was abrogated by addition of V3 (MN) peptide. However, exogenous V3 (MN) peptide failed to reduce the neutralization of this isolate on PBMC, or MT2 cells, by high titre anti-HIV-1 polyclonal human sera in contrast to the extensive reduction of neutralization by the same sera on MT2 cells using the prototype MN strain (4- to ≥ 24-fold) and the TCLA M2424/H9 isolate (2- to 8-fold). These results indicate that the neutralization of primary virus isolates by serum from HIV-1-infected individuals is not significantly mediated by V3- specific antibodies.
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Immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus Ankara-based multi-CTL epitope vaccine for human immunodeficiency virus type 1 in mice
A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8 T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8 T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 106 p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-γ upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.
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Expansion of CD8alpha+beta- cells in cats infected with feline immunodeficiency virus
CD8 lymphocytes have been subdivided into CD8αβ and CD8αα populations in the peripheral blood lymphocytes (PBL) of humans and in several animal species but have not yet been investigated in cats. Feline immunodeficiency virus (FIV) causes progressive immunological disorders similar to human AIDS. In this study, we analysed CD8 cells in PBL of FIV-infected or uninfected cats by two-colour flow cytometric analysis. In specific pathogen-free adult cats, feline CD8α β high cells were observed but CD8α β cells were not found in significant numbers. On the other hand, not only CD8α β high but also CD8α β and CD8α β low cell populations were observed in cats chronically infected with FIV. The expansion of the CD8β low or CD8β subpopulations resulted in the apparent differences in CD4/CD8 ratios depending on the anti-CD8 MAb used. These findings suggest a need to reconsider the CD4/CD8 ratio in studies of FIV infection. Furthermore, we found that the CD8α β cell population expressed CD5 at a low level.
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Protein-binding properties of the putative AP-1 and ATF sequences in the feline immunodeficiency virus long terminal repeat
Electrophoresis-mobility-shift assays with nuclear extracts from a feline renal cell line and a T-lymphoblastoid cell line revealed that the AP-1 and ATF sites of feline immunodeficiency virus (FIV) TM2 strain had similar protein-binding properties to those of FIV Petaluma strain and consensus sequences of AP-1 and ATF sites, and that nuclear factors binding to these sites differed between the two cell lines. Cross-competition and gel-supershift assays demonstrated that the AP-1 and ATF sites had similar protein-binding properties. The effects of internal deletions of AP-1 and/or ATF sites on the basal promoter activity were also examined. Although deletion of either site moderately reduced activity, a mutant deleted in both sites had dramatically reduced activity. Therefore, we suggest that these two sites co-operatively regulate transcriptional activity of the promoter.
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Detection of Jembrana disease virus in spleen, lymph nodes, bone marrow and other tissues by in situ hybridization of paraffin-embedded sections
More LessJembrana disease virus (JDV) is a lentivirus that causes an acute, severe disease syndrome in infected Bali cattle in Indonesia. An in situ hybridization technique was developed that detected JDV genomic RNA in formalin-fixed paraffin-embedded tissue sections, using a digoxigenin-labelled ribo- probe. Large numbers of JDV-infected cells were demonstrated in many tissue sections from experimentally infected animals early in the disease course, which was consistent with the extremely high circulating viraemia previously reported to occur during the febrile phase. The number of infected cells was consistently highest in sections of spleen, followed by many other tissues including lymph nodes, lungs, bone marrow, liver and kidney. Infected cells were also identified in the general circulation and within unusual intravascular lesions in lung sections. The relatively high level of infection found in bone marrow suggested that its involvement may be important in the disease pathogenesis, as it is with other lentiviruses.
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Disruption of the 5' and 3' splice sites flanking the major latency-associated transcripts of herpes simplex virus type 1: evidence for alternate splicing in lytic and latent infections
More LessThe herpes simplex virus type 1 (HSV-1) latency- associated transcripts (LATs) are the only viral gene products expressed within latently infected neurones. The most abundant (major) LATs consist of twocollinear nuclear polyA RNAsof2 kb and 1·5 kb which it has been suggested represent stable in- trons derived from a less abundant primary transcript (minor LAT). Consistent with this proposition is the identification of consensus splice donor and acceptor sites flanking major LATs which are conserved between HSV types 1 and 2. Here we test the functionality of the predicted splice sites within the context of the virus genome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were con-structed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation during productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice donor site significantly reduces levels of 2 kb major LAT during productive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in situ hybridization analyses of latently infected neuronal tissue. From these data it can be concluded that the proposed splice sites flanking the major LAT region are dispensable for 2 kb major LAT production in neurones latently infected with HSV-1 but constitute functional splicing signals in productively infected non-neuronal cells.
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Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis
More LessHerpes simplex virus type 1 (HSV-1) transcription can be arrested at the immediate early (IE) stage by continuous treatment of cells with inhibitors of protein synthesis, usually cycloheximide, from the time of infection. We have analysed the effect of cycloheximide on IE gene expression with HSV-1 mutants deficient in the production of functional levels of the three major transactivators, the virion protein (VP16) and two IE proteins (ICP0 and ICP4). Expression from the HSV-1 IE promoters that control synthesis of ICP0 and ICP27 was, unexpectedly, stimulated by inhibition of protein synthesis. The effect was observed for the ICP0 promoter in its normal genome location and also when cloned upstream of the Escherichia coli lacZ coding sequences and inserted into the viral thymidine kinase locus. Expression from the human cytomegalovirus major IE promoter, when cloned into the genome of HSV-1 mutants, was also increased by inhibition of protein synthesis. Cycloheximide did not affect the intracellular stability of lacZ-specific RNA, suggesting that the response represented an increase in mRNA production. Activation of the ICP0 promoter was observed when protein synthesis was blocked by alternative agents. Since inhibitors of protein synthesis are known to activate cellular signal transduction pathways, our findings demonstrate new mechanisms for the regulation of HSV-1 IE gene expression which may be important during latency and reactivation. The results also highlight previously unrecognized difficulties in analysing the intrinsic activities of promoters when cloned into the HSV-1 genome.
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An efficient selection system for packaging herpes simplex virus amplicons
More LessDue to their simplicity and flexibility of genomic construction, herpes simplex virus (HSV) amplicon- based vectors are attractive vehicles for gene delivery. However, a significant problem faced in the generation of amplicon stocks is the low amplicon to helper virus (A/H) ratio. In order to improve the proportion of amplicons generated, a selection system for amplicon production was developed in which the HSVthymidine kinase (TK) gene is inserted into an amplicon plasmid and an HSV mutant with both TK and glycoprotein H (gH) genes deleted is used as a helper virus. Using a protocol in which amplicon stocks are passaged 2–3 times in BHK cells of TK and gH genotype in the presence of selection medium containing methotrexate, stock preparations with high A/H ratio (up to 5:1) and high amplicon titre (> 1 × 109 infectious units/ml) were generated. In vitro characterization demonstrated that a high level of biologically functional products can be efficiently produced from these amplicon constructs.
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Characterization of the bovine herpesvirus 1 UL8 gene and gene products
More LessThe bovine herpesvirus 1 (BHV-1) strain Schön-böken UL8 gene and the 5′ flanking region were sequenced. Comparison of the UL8 ORF with the previously reported UL8 ORF of BHV-1 strain Cooper revealed significant differences that were mainly due to three frame-shifted segments. Reanalysis of the Cooper sequence after isolation of the respective segments from genomic DNA by PCR did not confirm the discrepancies; on the contrary, our results indicate a high degree of sequence conservation between the UL8 proteins of different BHV-1 isolates. A monospecific antiserum, raised against a bacterially expressed TrpE-UL8 fusion protein, identified the 80 kDa apparent molecular mass UL8 polypeptide which is localized in the nucleus of infected cells. Analysis of transcripts and time-course studies demonstrated that the UL8 protein is translated from a delayed-early expressed 3·1 kb polyadenylated mRNA which initiates within the UL9 ORF.
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Apoptosis of CD4+ T lymphocytes in human herpesvirus-6 infection
More LessThe present authors have recently reported that inoculation with human herpesvirus-6 (HHV-6) renders CD4 T lymphocytes susceptible to apoptosis in vitro. In order to confirm that apoptosis of CD4 T lymphocytes also occurs in HHV-6 infection in vivo, apoptosis of lymphocytes isolated from nine patients with exanthem subitum and from an adult patient with severe HHV-6 infection was examined. Peripheral blood mononuclear cells were cultured for 3 days and apoptosis of lymphocytes was then examined by flow cytometry of propidium iodide- stained DNA. The percentages of hypodiploid DNA, indicating apoptosis, in lymphocytes from 10 patients with HHV-6 infection were significantly higher than those from five infant patients with noninfectious diseases and five healthy adults (P < 0·0002). DNA fragmentation was also detected by agarose gel electrophoresis in lymphocytes from patients with HHV-6 infection. Apoptosis appeared to occur predominantly in CD4 T lymphocytes and HHV-6 was isolated from the CD4 T lymphocyte fraction. These data demonstrate that HHV-6 renders CD4 T lymphocytes susceptible to apoptosis in vivo.
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Four tRNA-like sequences and a serpin homologue encoded by murine gammaherpesvirus 68 are dispensable for lytic replication in vitro and latency in vivo
More LessExperimental infection of inbred strains of laboratory mice with murine herpesvirus 68 (MHV-68), a natural pathogen of wild rodents, results in acute productive infection of the lung followed by a latent infection of B lymphocytes. We have previously shown that MHV-68 encodes an open reading frame with similarity to poxvirus serpins, designated ORF1, and eight novel tRNA-like sequences. The latter are processed into mature, uncharged tRNAs and are abundantly expressed during both lytic and latent infection. In this study it is demonstrated that deletion of four of the tRNA-like sequences and ORF1 from the virus genome does not affect the ability of MHV-68 to replicate in vitro or to establish, and reactivate from, latency in vivo.
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Molecular characterization of the garlic virus X genome
More LessThe complete nucleotide sequence of the cDNA genome for garlic virus X (GVX), one of the major viruses infecting garlic plants, was determined. GVX is a single-stranded positive-sense RNA virus consisting of 8106 nucleotides excluding the 3 ′-end poly(A) tail and contains six open reading frames (ORFs) which encode putative proteins of 174 kDa (ORF1), 26 kDa (ORF2), 12 kDa (ORF3), 32 kDa (ORF4), 26 kDa (ORF5) and 15 kDa (ORF6). The putative viral proteins show similarity to those of carlaviruses and potexviruses but show the highest homology to shallot virus X (ShVX). Even though the GVX genome contains most of the structural elements common to carlaviruses and potexviruses, it is distinguished from them by the presence of an ORF4 which encodes an unusual protein. These results suggest that GVX may belong to an unassigned group of ShVX and GarV-type viruses rather than to the carlaviruses or potexviruses.
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Charge changes near the N terminus of the coat protein of two potyviruses affect virus movement
More LessMutants of tobacco vein mottling virus (TVMV) with substitutions of Lys or Arg for Asp in the DAG motif at position 5 in the coat protein (CP) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. Occasional systemic infections occurred when Nicotiana bentha- miana or transgenic tobacco plantsexpressing wild- type TVMV CP were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or substitutions of Glu for Lys at position 8. The compensatory nature of these mutations was demonstrated by recreating one of the most common alterations. Tobacco etch virus (TEV) mutants with substitutions of Lys for Asp in the two DAG motifs near the CP N terminus also failed to infect tobacco plants systemically, and in situ histochemical analysis showed limited movement. A certain net charge evidently must be maintained near the CP N terminus for systemic movement to occur.
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A hypersensitive response-like mechanism is involved in resistance of potato plants bearing the Rysto gene to the potyviruses potato virus Y and tobacco etch virus
More LessPotato plants carrying the Rysto gene from Solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. The resistance maybe due to Rysto or to a closely linked gene. In this investigation, we used potato virus Y (PVY) and a tobacco etch virus construct that encodes β-glucuronidase (TEV-GUS) to monitor virus infections of potato plants. Systemic spread of either virus in resistant potato plants was not detectable by serology, RT-PCR, GUS assay or bioassay although each replicated in the initially infected cells of leaves from resistant potato cultivars and was transported into neighbouring cells. However, 3 days post-inoculation (p.i.) a necrotic reaction set in that stopped movement and accumulation of both viruses by 7 days p.i. The resistance reaction (probably a hypersensitive reaction) became visible as necrotic streaks on veins on the lower leaflet surfaces of some potato cultivars carrying the Rysto gene and may be elicited by a common potyviral gene product.
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Differential interactions among isolates of peanut stunt cucumovirus and its satellite RNA
More LessThe interactions of seven isolates of peanut stunt cucumovirus (PSV) originating from North America, Europe and Africa, and two variants of PSV satellite RNA (sat RNA) were analysed. Electrophoretic and immunoblot analyses of the coat protein (CP) and Northern blot hybridization analyses of the viral RNAs showed that isolates PSV F352, 1339 and 1507 belonged to subgroup I, and isolates PSV W, Su and B to subgroup II. The seventh isolate, robinia mosaic virus (RoMV) clustered with subgroup I isolates by CP analysis, but was related to both subgroups by RNA hybridization analysis. The ability to support the accumulation of two newly described sat RNA variants, P4 and P6 sat RNAs, was not related to PSV isolate classification: neither PSV W nor RoMV were helper viruses for these PSV sat RNAs. Symptom modulation by both sat RNAs was the same: the presence of sat RNA did not modify the symptoms induced by subgroup I isolates but exacerbated the symptoms induced by subgroup II isolates in both tobacco and cowpea. Sat RNAs P4 and P6 contained 393 nucleotides, and differed only in three nucleotide substitutions. This resulted in marked differences in infectivity, level of accumulation and relative encapsidation between both the sat RNAs. Accumulation levels and relative encapsidation of sat RNAs was also affected by the isolate of helper virus.
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Interference with Physalis mottle tymovirus replication and coat protein synthesis by transcripts corresponding to the 3′-terminal region of the genomic RNA—role of the pseudoknot structure
More LessThe role of the 3′ noncoding (NC) region of Physalis mottle tymovirus genomic RNA in the multiplication of the virus was examined using an in vivo protoplast assay system. Coat protein (CP) synthesis was specifically inhibited by sense 3′ NC region transcripts. To establish the role of the pseudoknot structure present in the NC region in virus multiplication, four site-specific mutants, two of which disrupted the pseudoknot structure while the other two restored the structure, were constructed. Interestingly, none of the four sense mutant transcripts inhibited CP synthesis, suggesting that the specific sequence representing the 3′ terminal pseudoknot structure may play an important role in virus multiplication. However, the wild-type antisense 3′ NC transcript as well as the four antisense mutant transcripts inhibited CP synthesis, suggesting that the inhibitions by antisense transcripts could be due to the formation of RNA-RNA hybrids at the 3′ end of the genomic RNA.
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The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family
More LessThe complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5′ ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequi-virus families. The 3′ ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.
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Identification of an early positive regulatory gene of mycobacteriophage L1
More LessAmong 14 temperature-sensitive, growth-defective mutants of mycobacteriophage L1 showing a lysis-defective phenotype at 42 °C, six are, in addition, defective in phage DNA synthesis at 42 °C. In the present study, we show that one of the latter six mutants, L1G27ts901, is also defective in the synthesis of both an L1-specific exonuclease (a representative delayed early protein), and of RNA in both the delayed early and late periods but not in the immediate early period. The results of a temperature-shift experiment suggest that the synthesis of L1 exonuclease is regulated by G27 at the level of transcription. Furthermore, the temperature-sensitive defect in delayed early and late RNA synthesis could be largely overcome when the L1G27ts901-infected culture was shifted from 32 to 42 °C at 10 min but not at zero time post-infection. These results suggest that the primary effect of the G27ts901 mutation is to make the phage defective in transcription of delayed early genes at 42 °C, and the defect in late RNA synthesis by this mutant is a secondary effect which is caused by its inability to express regulatory gene products. We conclude that G27 is involved in the positive regulation of expression of the delayed early genes of L1 at the transcriptional level.
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Volumes and issues
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Volume 105 (2024)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 72 (1991)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)