- Volume 75, Issue 8, 1994
Volume 75, Issue 8, 1994
- Articles
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- Animal
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Purification and properties of virus particles, infectious subviral particles, cores and VP7 crystals of African horsesickness virus serotype 9
Methods were developed for the purification, at high yield, of four different particle types of African horse-sickness virus serotype 9 (AHSV-9). These products included virus particles purified on CsCl gradients which contain proteins apparently directly comparable to those of bluetongue virus (VP1 to VP7); virus particles purified on sucrose gradients which also contain, as a variable component, protein NS2; infectious subviral particles (ISVPs), containing chymotrypsin cleavage products of VP2; and cores, obtained by treating purified ISVPs with 1 m-MgCl2 to remove the components of the outer capsid layer (VP5 and VP2 cleavage products). Additional protein bands migrating with apparent M rs lower than that ofVP5 were detected during SDS-PAGE analysis of virus particles. These appear to be conformational variants of VP5 and are identified as VP5″ and VP5 BHK-21 cells infected with this strain of AHSV-9 produce large quantities of flat, usually hexagonal crystals of VP7, a major group antigen and core protein; these were also purified. Either 20 mg of virus particles, 20 mg of ISVPs or 10 mg of cores as well as 20 mg of VP7 crystals could be purified from approximately 8 × 109 infected cells. None of the preparations of particles or crystals showed any detectable contamination with BHK-21 cell proteins or antigens, as determined by SDS-PAGE or indirect ELISA. Virus particle and ISVP preparations had similar specific infectivities for BHK-21 cells (approximately 1 × 109 TCID50/A 260 unit) but the infectivity of cores was approximately 105-fold lower.
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Recognition of rotavirus antigens by mouse L3T4-positive T helper cells
More LessA lymphocyte proliferation assay was used to examine the helper T cell response to rotavirus in mice following parenteral immunization with the UK strain of bovine rotavirus. Mixed populations of lymphocytes prepared from spleen or peripheral lymph nodes were tested for proliferation in the presence of UK strain rotaviruses, prepared as cell culture lysates, ultracentrifuged (pelleted) lysates, sucrose-purified virus and caesium chloride-purified virus. Live rotavirus induced non-specific stimulation of lymphocytes, which was not observed in response to inactivated virus. Putative helper T cells of the L3T4+ phenotype were prepared as an enriched population from UK strain-immunized mice or grown in vitro as a polyclonal T cell line. The response of L3T4+-enriched cells from mice immunized with inactivated virus was dependent on antigen-presenting cells (APCs). Cells obtained following immunization with live virus did not require further addition of APCs. The response of the L3T4+ T cell line was wholly dependent on APCs. UK strain-specific L3T4+ cells responded to whole UK rotavirus and to isolated VP6 of both UK and C486 rotavirus strains. The results indicate that virus-specific L3T4+ T cells are induced following rotavirus immunization and can respond to epitopes on VP6. UK strain-primed L3T4+ cells also responded to an avian rotavirus strain, Ch2, which shares only minimal serological cross-reactivity with the UK strain. T cell recognition of rotavirus may thus be broadly cross-reactive.
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Characterization of neutralization epitopes on the VP7 surface protein of serotype G11 porcine rotaviruses
More LessRotavirus strain A253, isolated from the faeces of a diarrhoeic piglet in Venezuela, was classified as serotype G11 by cross-neutralization studies and by comparison of the deduced amino acid sequence of the VP7 surface protein. The epitopes involved in neutralization of the two G11 porcine rotavirus strains A253 and YM were analysed using neutralization-resistant mutants selected with seven neutralizing monoclonal antibodies (MAbs), monotype-specific (M-) MAbs and serotype-specific (S-) MAbs, produced against VP7 of strain A253. Crossneutralization tests and sequence analysis of the escape mutants selected from strains A253 and YM indicated the presence of two antigenic sites, one common to both M-MAbs and S-MAbs in region A (positions 87, 91 and 96) and the other defined by one S-MAb in region C (position 223). All A253 variants selected with M-MAbs and two S-MAbs, although having different amino acid substitutions, had a change at amino acid position 87, whereas YM variants involved residues 91 and 96, part of the same antigenic site. Compared to strain A253, the YM strain presents an amino acid substitution at position 87 and was not recognized by M-MAbs. These results suggest that in the VP7 of G11 serotype specificity, the amino acid at position 87 is an important component of a neutralization site associated with region A and the intraserotypic variation between strains A253 and YM may account for the selection of mutations at different positions by a single MAb.
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Cleavage site of the poliovirus receptor signal sequence
More LessWe have shown recently that the human poliovirus receptors (hPVRs) expressed on the surface of cultured cells are 80K glycoproteins, whereas the previously reported 67K forms are partially glycosylated intermediate glycoforms. Both the membrane-bound 80K and 67K forms of hPVR are glycosylated derivatives of the two isoforms hPVRα and hPVRδ, where the latter two can be resolved only by SDS-PAGE upon enzymatic deglycosylation. Here we report the N-terminal sequence analysis of the mature 80K as well as the intermediate 67K glycoforms of hPVR which has allowed us to identify the signal peptidase cleavage site of the unprocessed hPVR. The signal sequence that directs translocation of hPVR across the membrane of the endoplasmic reticulum on its route to the glyco-processing pathway has thus been defined. We compare this signal sequence with those of the putative monkey poliovirus receptor and the mouse poliovirus receptor homologue.
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Human enteric Caliciviridae: a new prevalent small round-structured virus group defined by RNA-dependent RNA polymerase and capsid diversity
More LessSequence comparison of the RNA-dependent RNA polymerases of small round-structured viruses (SRSVs) from 10 recent U.K, outbreaks of gastroenteritis revealed significant genetic variation. Computer analyses indicated that these viruses can be divided into two discrete groups. SRSV group I contains the previously characterized antigenic type 1 Norwalk and type 3 Southampton viruses. The amino acid sequences of the RNA polymerase, capsid and ORF3 of these two viruses are relatively similar (about 92%, 69% and 72% amino acid identity, respectively). A representative member of group II SRSVs, Bristol virus, was subjected to a detailed genetic analysis. Bristol virus is a recent antigenic type 2 isolate from a U.K. hospital outbreak of gastroenteritis. Using a single clinical sample the 3′-terminal 3881 nucleotide cDNA sequence [excluding the poly(A) tail] of this virus was determined. Analysis of the sequence revealed significant differences from those of group I viruses with the RNA polymerase region, capsid and ORF3 showing only about 62%, 43% and 30% amino acid identity respectively with the equivalent proteins of the Norwalk and Southampton viruses. These data suggest that the morphologically identical SRSVs belong to at least two genetically distinct groups.
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Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus
More LessThe nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3′ terminus to the end of the nucleoprotein (N) gene. The 3′ leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3′-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus. The N gene comprises 1328 nucleotides from the transcription initiation consensus sequence (AACAGG) to the conserved transcription termination-poly(A) sequence [CATG(A)7] and encodes a polypeptide of 431 amino acids with an estimated M r of 49159 and a pI of 5·4. The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Piry, Chandipura) than to lyssaviruses (rabies and Mokola). An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from 5′-terminal (559 bp) and 3′-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C-terminal fragment, but not the N-terminal fragment. Two of these antibodies also reacted with baculovirus-expressed rabies virus N protein. Polyclonal mouse ascitic fluids derived from BEFV, rabies virus and several other related viruses were also shown to cross-react in immunoblots with purified preparations of rabies virus and BEFV N proteins.
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Comparison of cDNAs encoding the gibbon ape leukaemia virus receptor from susceptible and non-susceptible murine cells
More LessThe gibbon ape leukaemia virus (GaLV) family of type C retroviruses consists of five closely related viral isolates, GaLV SF, GaLV SEATO, GaLV Br, GaLV H and simian sarcoma-associated virus. The cDNA encoding the human receptor for GaLV SEATO had previously been isolated. We now demonstrate that all of the above GaLVs can use the human form of the GaLV receptor to infect cells. All murine cells analysed to date have been found to be resistant to infection by GaLVs owing to the absence of a functional GaLV receptor. We have now identified a murine cell line which is unique in its susceptibility to GaLV infection. This cell line was established from a Japanese feral mouse, Mus musculus molossinus. We cloned and sequenced the cDNA for the receptor expressed in these cells and compared it to the cDNA for the GaLV receptor expressed in resistant murine cells such as NIH 3T3 (derived from M. m. musculus) and MDTF (derived from M. dunni tail fibroblasts). The crucial region for GaLV infection (the fourth extracellular domain) from the functional M. m. molossinus GaLV receptor is quite divergent from the same region of the M. m. musculus and M. dunni proteins, but similar to that of the functional human GaLV receptor. These results confirm the importance of the amino acids of this region in GaLV receptor function.
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Critical involvement of human T cell leukaemia virus type I virions in mediating the viral mitogenic effect
More LessHuman T cell leukaemia virus type I (HTLV-I) is a direct activator of human resting T lymphocytes. The present study was undertaken to delineate further the role of viral particles and to define the involvement of envelope glycoproteins in the induction of T cell mitogenic stimulation. Virus-producing cells treated with paraformaldehyde (PFA) were found to be unable to induce the formation of syncytia, but still able to trigger the proliferation of resting T cells. Likewise, PFA-treated virus particles were still mitogenic. These results suggest that the mitogenic event is triggered before the fusion of the envelope with the cell membrane. Furthermore, HTLV-I envelope-expressing cells obtained after infection of C8166/45 cells (HTLV-I- transformed, but defective in virion production) with an HTLV-I envelope recombinant vaccinia virus were unable to activate normal T cells. Human immunodeficiency virus type 1 particles produced by C8166/45 cells were also devoid of mitogenic ability. However, when HTLV-I viral preparations were purified by chromatography, only the virion-containing fractions were found to be mitogenic for human resting T lymphocytes. This mitogenic activity was partially abolished by preincubating the purified virus with a monoclonal antibody directed to the surface envelope glycoprotein. Finally, treatment of HTLV-I-transformed cells by tunicamycin, an inhibitor of N-linked glyco-sylation, led to the production of virus particles with a decreased mitogenic activity. Collectively, these observations suggest that the HTLV-I mitogenic activity is triggered by the contact of HTLV-I virions with T cells.
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Initiation of reverse transcription during cell-to-cell transmission of human immunodeficiency virus infection uses pre-existing reverse transcriptase
H3B cells, a laboratory clone of H9 cells persistently infected with the HTLV-IIIB strain of human immunodeficiency virus (HIV), contained significant levels of cell-associated reverse transcriptase (RT) activity measured by in vitro assays using either exogenous or endogenous templates. The cell-associated RT activity detected using exogenous template was almost wholly in a soluble (non-sedimentable) form whereas endogenous activity sedimented as a particulate structure associated with viral RNA. Despite this, H3B cells did not contain episomal HIV DNA detectable by Southern blot, indicating that in vivo reverse transcription was not occurring to any significant extent in these cells. However, when susceptible HUT 78 cells were infected by co-cultivation with H3B cells, dramatic synthesis of episomal HIV DNA occurred. Concurrently with this de novo initiation of reverse transcription, however, we found no detectable change in intracellular levels or cleavage profiles of immunoprecipitable RT polypeptides. Finally, actinomycin D pre-treatment of H3B cells to prevent de novo transcription from donor cell proviral DNA after co-cultivation did not affect the initiation of in vivo reverse transcription following cell-to-cell HIV infection. These results demonstrated that cells persistently infected with HIV contained significant fully cleaved cell-associated RT in a form that was active in vitro but not in vivo and that following cell-to-cell transmission of HIV infection to susceptible cells, de novo reverse transcription was initiated without detectable evidence of further synthesis or proteolytic processing of HIV RT. The nature of this initiation process requires further study.
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Human immunodeficiency virus type 1 Tat activity in human neuronal cells: uptake and trans-activation
Neurological dysfunction in AIDS occurs in the absence of productive infection of neurons, and may involve modulation of neuronal cell function by viral or cellular products released from surrounding infected cells. The human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat may be one such factor, as it can act as a neurotoxin, induces marked morphological changes in neurons and astrocytes in primary embryonic rodent brain cultures, and is released by certain HIV-1-infected cells. In addition, Tat can alter expression of cellular genes in several non-neuronal cell types. To explore the possibility that Tat may also mediate neuronal dysfunction in AIDS through non-lethal effects on neurons, we determined the trans-activating ability of Tat in human neuronal cells. We generated human neuronal cell lines stably expressing several HIV-1 tat genes, and also tested human neuronal cells exposed to extracellular recombinant Tat protein. Both endogenously expressed Tat as well as exogenous recombinant Tat protein up-regulated HIV-1 long terminal region (LTR)-driven gene expression by several hundred-fold. Only brief exposure to recombinant Tat was necessary and no toxic effects were seen at levels sufficient for trans-activation. Furthermore, Tat significantly enhanced virus expression in neuronal cells transfected with molecular clones of HIV-1. These results show that Tat is trans-activationally active in human neuronal cells, and can be taken up from the extracellular compartment by these cells in a biologically active form. Neurons represent an important potential target for Tat-mediated cellular dysfunction.
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Comparison of biological properties of feline immunodeficiency virus isolates using recombinant chimeric viruses
The biological properties of homogeneous populations of feline immunodeficiency viruses derived from infectious molecular clones of the TM1, TM2 and Petaluma strains were compared. Differences in in- fectivity for Crandell feline kidney (CRFK) cells, and in syncytium formation and replication kinetics in a feline T lymphoblastoid cell line (MYA-1 cells) were observed. To investigate the basis of these differences between the TM2 and Petaluma strains, we first compared the basal promoter activity of the long terminal repeat which is a highly divergent region, but no significant difference in activities was found in CRFK cells. We then constructed two recombinant chimeric clones which carry gag, pol, vif and ORF A from the heterologous virus. From analyses using the chimeric clones, it was revealed that efficient virus growth in CRFK cells and MYA-1 cells was regulated by the gag, pol, vif and ORF A regions, whereas viral determinants of infectivity for CRFK cells, and syncytium formation and cytopathogenicity in MYA-1 cells, were located in the env region.
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Down-regulation of human adenovirus E1a by E3 gene products: evidence for translational control of E1a by E3 14·5K and/or E3 10·4K products
The mechanism for down-regulation of E1a expression by products encoded in the E3 transcription unit of human adenovirus types 2 and 5, that occurs in infected L929 cells, has been investigated further. We show that the phenomenon occurs in different mouse cells and also in some human cells suggesting that the observations have relevance to natural human infections. We also provide evidence that probably all viral proteins are down-regulated by E3 products, although to different extents, but that host proteins are unaffected. Whereas E1a protein levels and synthesis are reduced in the presence of E3 products, E1a protein half-life and polysomal Ela RNA levels and size distribution are not. These data suggest that E3 products down-regulate E1a protein levels by interfering with the translation of E1a- specific mRNA. Studies were additionally carried out with mutant adenoviruses containing different defects in the E3 transcription unit. Based on these studies it seems likely that the E3 14·5K and 10·4K proteins are crucially involved in Ela down-regulation. Our data are discussed in terms of strategies for immune evasion by group C human adenoviruses.
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E5a gene of human papillomavirus type 11 is required for initiation but not for maintenance of transformation in NIH 3T3 cells
More LessWe have previously shown that the E5a gene of human papilloma virus type 11 (HPV-11/HPV-6c) is a transforming oncogene. In order to dissect the biological consequences of E5a gene expression we utilized the lac operator/repressor system to manipulate E5a gene expression. Cells were cotransfected with the lac repressor gene and the E5a gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of E5a gene could therefore be repressed by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region. The transfected cells were cultured in the presence of the inducer IPTG and under G418 selection. IPTG derepressed E5a gene expression by binding to the repressor and reducing its affinity for the lac operator sequence. In these studies, we found that E5a-transformed cells still maintained the transformed phenotype as judged by growth density, cell morphology and anchorage-independent growth when E5a gene expression was repressed. We also found that c-jun expression was induced 3 h after E5a expression was induced by IPTG and c-jun expression was not shut down after repression of E5a expression. This is the first demonstration that the E5a gene of HPV-11 initiates transformation of NIH 3T3 cells but is dispensable for maintenance of the transformed phenotype.
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Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of four lepidopteran insects
More LessEnhancin, an infectivity-enhancing protein from Trichoplusia ni granulosis virus (TnGV) was tested for its ability to increase Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) infection in the larvae of four lepidopteran insects. Enhancin increased the mortality of AcMNPV infection in all the four insect species tested. Peritrophic membrane (PM) assays showed altered protein profiles in PMs treated with enhancin in all the four species. This supports the hypothesis that enhancin affects virus infection by altering the structural integrity of the PMs. The binding of enhancin to the midgut brush border membranes (BBMs) was determined and specific binding sites were found on the BBM of Pseudaletia unipuncta. No specific binding sites were found on the BBMs of T. ni, Helicoverpa zea or Spodoptera exigua. Therefore, specific binding of enhancin to the midgut cell membrane may not be necessary for the enhancement of baculovirus infection in insects.
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Analysis of the C-polyhedrin genes from different geographical isolates of a type 5 cytoplasmic polyhedrosis virus
More LessThe C-polyhedrin genes of two different geographic isolates of a type 5 cytoplasmic polyhedrosis virus (CPV) were cloned. A CPV infecting Orgyia pseudotsugata (OpCPV), isolated in the Pacific Northwest of the U.S.A., and a CPV infecting Heliothis armigera, isolated in South Africa, were studied. Both genes were found to be 883 nucleotides in length and encoded a predicted protein of 246 residues (M r of 28890). Comparison of the nucleotide sequences of these two viruses with another type 5 geographic isolate, infecting Euxoa scandens (EsCPV; isolated in Eastern Canada), showed that there were only 17 nucleotide differences among the three genes. The only nucleotide variation that had an effect on the encoded protein was a deletion of nucleotide 774 in the gene of EsCPV. The deletion introduces a frameshift mutation resulting in the alteration of the carboxyl-terminal amino acid sequence. Sequence alignment of the OpCPV C-polyhedrin showed little homology to a type 1 CPV (infecting Bombyx mori) or with analogous proteins (N-polyhedrins) from two baculoviruses infecting O. pseudotsugata. Interestingly, most of the conserved residues between the N- and C-polyhedrins were either basic or aromatic amino acids.
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Sequence alterations within and downstream of the A-type inclusion protein genes allow differentiation of Orthopoxvirus species by polymerase chain reaction
More LessA PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. This assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox virus. The primer pair flanked a region exhibiting distinct and specific DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fragments of different sizes. The presented PCR protocol, combined with BglII restriction digests, allowed the unequivocal assignment of 42 orthopoxvirus (OPY) strains and isolates to the correct OPY species. The resulting classification corresponded exactly with known biological data for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small BglII fragment. DNA sequencing showed that the lack of this BglII fragment was caused by a deletion of 72 nucleotides.
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Swine-reconstituted SCID mice as a model for African swine fever virus infection
More LessInjection of swine peripheral blood mononuclear cells into mice with severe combined immunodeficiency (SCID), resulted in the stable long-term establishment of a functional swine immune system (SCID-sw). Swine immunoglobulins were present in the serum of SCID-sw mice and swine cells were detected in the blood as well as in lymph nodes and spleen using monoclonal antibodies raised against cell subpopulations. Swine lymphocytes from reconstituted SCID mice responded in vitro to specific antigens or mitogens. When SCID-sw mice were challenged with African swine fever (ASF) virus, ASF virus-infected cells were detected in blood and spleen, and antiviral antibodies and virus-specific T cells were generated.
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Presence of human cytomegalovirus (HCMV) immediate early mRNA but not ppUL83 (lower matrix protein pp65) mRNA in polymorphonuclear and mononuclear leukocytes during active HCMV infection
During an active human cytomegalovirus (HCMV) infection, leukocytes harbouring the HCMV lower matrix protein pp65 (ppUL83) are present in the peripheral blood and can be detected with the HCMV antigenaemia assay. In the present study, it was investigated whether the presence of pp65 in these cells was due to transcription of the virus genome or might be the result of uptake of this viral protein. Peripheral blood leukocytes of transplant recipients and AIDS patients with an active HCMV infection were investigated for the presence of HCMV immediate early (IE) antigen and pp65 using well characterized monoclonal antibodies, and for the presence of the corresponding mRNAs using non-radioactive in situ hybridization. Both mononuclear and polymorphonuclear cells were found to contain IE antigen and pp65. However, only mRNAs encoding IE antigen were found in these cells, whereas mRNAs encoding pp65 were not detected. In contrast, both IE antigen and pp65, as well as their corresponding mRNAs, were detected in the circulating late-stage HCMV-infected endothelial cells that were also present in the leukocyte fractions. These findings demonstrate that a restricted viral gene expression (transcription of IE genes) does occur in mononuclear and polymorphonuclear leukocytes. However, the abundant presence of the early antigen pp65 without detectable presence of the corresponding mRNA in these cells strongly indicates uptake of this protein by the phagocytic leukocytes, rather than de novo synthesis.
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Negatively cis-acting elements in the distal part of the promoter of Epstein-Barr virus trans-activator gene BZLF1
Epstein-Barr virus (EBV) replicates in a latent or a lytic way in the infected organism, depending on the type and level of differentiation of the host cell. The switch between latency and lytic replication was previously shown, for Burkitt’s lymphoma cell lines, to depend on the viral BZLF1 gene product. Protein-DNA assays were used to identify the cis-acting elements that represent the link between regulating signal transduction pathways and the viral cascade of gene expression. Specific binding of proteins to several sites of the BZLF1 promoter during latency was shown. Induction of the lytic cycle by stimulation with 12-O-tetradecanoyl- phorbol 13-acetate abolished the binding of these proteins to the distal promoter (positions −227 to −551), suggesting a functional role for the down- regulation of promoter activity during latency. Computer analysis identified a multiply repeated sequence motif, HI, in this region and exonuclease III footprints confirmed that these sites act as specific protein recognition sites. Using a set of reporter plasmids we were able to demonstrate a negative regulatory effect of the HI motif in some B lymphoid cell lines, in contrast to epithelial HeLa cells. The HI silencer elements are different from other silencer elements described so far in respect of their sequence and protein-binding pattern during the activation of BZLF1.
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The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse
More LessFour specific pathogen-free ponies were infected intranasally with equine herpesvirus 1 (EHV-1) and two were similarly infected with an EHV-1 thymidine kinase deletion mutant. The primary infections were characterized by a transient fever accompanied by virus shedding into nasal mucus and viraemia. No virus was detected in clinical specimens after 15 days post-infection. Two months later a reactivation stimulus was administered to all six ponies and only the four that had been previously inoculated with wild-type EHV-1 shed virus into nasal mucus (for 10 days), proving the presence of a latent infection. No recurrence of viraemia was observed. The animals were monitored for a further 6 weeks and were consistently shown to be free from infectious virus. Tissues were then obtained postmortem. Co-cultivation of explanted trigeminal ganglia from two out of the four ponies that carried the wild-type virus yielded cultures positive for infectious virus. Apart from nasal epithelium, no infectious virus was recovered from any other tissue. PCR confirmed the presence of virus DNA in the ganglia from all six ponies. Lymphoid tissues also yielded positive signals using this technique. The relevance of virus detection by PCR in lymphoid and neural tissues is discussed in relation to the potential for reactivation of latent virus in the host. However, evidence is presented to show that EHV-1 is neurotropic and, in common with other members of the alpha- herpesvirus subfamily, establishes latency in sensory ganglia from which virus can be reactivated.
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Reactivation of latent herpes simplex virus from explanted dorsal root ganglia
More LessReactivation was induced by explantation of dorsal root ganglia (DRG) from mice that were latently infected with herpes simplex virus type 1. Reactivation was first detected, using combined in situ hybridization and immunocytochemistry, at 2 or 3 days post-explantation (p.e.). Evidence of reactivation was found primarily in neurons that did not also contain latency-associated transcripts (LATs). Occasionally, mRNA of immediate early gene 2 (IE2) or IE4/5, in the absence of other viral mRNAs or antigen, was found in LAT+ neurons. Thus, if reactivation was occurring in LAT+ neurons, the LATs must have been lost as an early consequence; however we could detect neither a decrease in the percentages of LAT+ neurons nor a reduction in the intensity of the LAT signal during the period of reactivation. However, the number of foci of reactivation was generally less than 2·9% of the estimated number of LAT+ cells in the DRG; this may account for our failure to see such changes. A redistribution of the LATs into the cytoplasm was found in some cells but this could reflect the poor survival and consequent death of the explanted neurons. We conclude that the majority of LAT+ neurons did not reactivate on explantation and that if reactivation occurred only in LAT+ neurons, the LATs must have been removed from the nucleus as an early consequence of reactivation. Alternatively, there may be a population of latently infected cells that do not express LATs.
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Nucleotide sequence of the genes encoding the canine herpesvirus gB, gC and gD homologues
More LessThe nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB, gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses.
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Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses
More LessThe extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gpl4, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein of M r 135K which was composed of the truncated gpl4 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of M r 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M r 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis. The gpl4 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gpl4 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that posttranslational modification of the EHV-1 gpl4 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.
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The herpes simplex virus type 1 UL37 gene product is a component of virus particles
More LessThe herpes simplex virus type 1 UL37 gene encodes a protein with an M r of 120K that is produced at late times after infection. To study the properties of this protein we have linked a 10 amino acid epitope derived from a human cytomegalovirus protein to the UL37 polypeptide coding sequences by inserting an oligonucleotide at a SpeI site that is unique in the virus genome and lies close to the 3′ end of the open reading frame. From studies on the resultant virus recombinant using a monoclonal antibody that recognizes the inserted epitope we find that, contrary to a previous report, the UL37 protein is a structural component of both virions and L particles and is present in the tegument of virus particles. Indirect immunofluorescence analysis revealed that the protein is distributed throughout infected cells but is more abundant in the cytoplasm than the nucleus.
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Specific binding of herpes simplex virus type 1 (HSV-1) strain HSZP to host cell surface can provoke interference with early shutoff of host protein synthesis induced by HSV-1 strain KOS
More LessThe HSZP strain of herpes simplex virus type 1 (HSV-1) is defective with respect to the early shutoff of host protein synthesis but is effective at interfering with the early shutoff function of the HSV-1 KOS strain, even when heat-inactivated or neutralized by antibody. The interference was not due to exclusion of strain KOS by HSZP at the level of adsorption or penetration. The component responsible for the interference is an integral part of HSZP virions. Strain HSZP inactivated with zinc ions failed to interfere with the early shutoff function of the superinfecting strain KOS. The same effect was also found with strain HSZP purified from cells treated with 2-deoxy-d-glucose. This finding supports the idea that a specific interaction between HSZP virions and the cell surface can be responsible for the interference phenomenon.
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The herpes simplex virus type 1 strain 17+ γ34.5 deletion mutant 1716 is avirulent in SCID mice
Laboratory animal models are important tools for the identification of avirulent herpes simplex virus type 1 (HSV-1) strains which have potential for use in humans as vaccine strains or gene therapy vectors. We have studied an HSV-1 17+ variant, 1716, that has a deletion in the γ34.5 gene and which replicates poorly in the footpads of mice and is unable to grow in the mouse central nervous system or dorsal root ganglia (DRG) of the peripheral nervous system following peripheral inoculation. However, 1716 is known to be capable of establishing latent infections in the DRG of mice. Here we show that 1716 is avirulent after ocular infection and has low virulence after intracranial inoculation in SCID mice. Since SCID mice are much more sensitive to HSV-1 infection than immunocompetent mice, our results clearly demonstrate the drastically reduced virulence of the variant 1716 and provide additional support for the hypothesis that this variant would be avirulent in humans.
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Identification of a target protein of US3 protein kinase of herpes simplex virus type 2
More LessHerpes simplex virus type 2 (HSV-2) gene US3 has been shown to encode a serine-threonine protein kinase. In this study, we have tried to identify target proteins of the US3 protein kinase using a US3 lacZ insertion mutant of HSV-2. When permeabilized cells were labelled with [γ-32P]ATP under the optimum conditions for the US3 enzyme, the most striking difference between wild-type HSV-2 strain 186- and mutant-infected cells was observed in the phosphorylation of proteins ranging in M r values from 14K to 22K. Studies of in vitro phosphorylation with purified virions and with cells infected with a US9-defective HSV-1 mutant suggested that a tegment phosphoprotein encoded by the US9 gene may be a target of HSV-2 US3 protein kinase.
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The fibre of bovine adenovirus type 3 is very long but bent
More LessThe sequence of the fibre of bovine adenovirus type 3 (BAd3) predicts an extremely long structure due to a large number of 15-residue repeats in the fibre shaft, the tail and head domains being similar in size to the human adenovirus fibres. The length of the fibre was confirmed using negative-strain electron microscopy of BAd3 pentons (fibre plus penton base). The fibre was found to be bent in several discrete places and the bending sites appear to correspond with irregular repeats in the shaft. We suggest that bending of the fibre is needed for the interaction of the penton base with the secondary receptors on the cell surface.
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Human papillomavirus (HPV) type 11 recombinant virus-like particles induce the formation of neutralizing antibodies and detect HPV-specific antibodies in human sera
More LessRecombinant human papillomavirus type 11 (HPV-11) virus-like particles (VLPs) were tested for their ability to induce the formation of neutralizing antibodies, and were also tested for serodiagnostic capabilities in an ELISA in comparison with HPV-11 whole virions. VLPs, purified by CsCl density gradient centrifugation from the cell-free supernatant of Ac 11 LI-infected Sf9 suspension cell cultures, were used to immunize rabbits and anti-VLP antibodies were tested in the athymic mouse model of HPV-11 infection. Pretreatment of infectious HPV-11 virions with the immune serum of VLP-treated animals caused a marked reduction of graft growth (P < 10−4) and viral gene expression (P < 10 −4), similar to the effects obtained using whole virion postimmune serum, and consistent with immune neutralization. To assess the serodiagnostic capabilities of VLPs, a VLP ELISA was developed and used to analyse sera that were tested previously in an HPV-11 whole virion ELISA. Specific antibodies were detected in 49% of patients’ sera (P = 2 × 10−4), and individual VLP sero- reactivities correlated with those previously obtained using whole virions as the antigen (r = 0·87; P < 10−6). These results indicate that recombinant VLPs can be used to elicit a neutralizing antibody response, and can substitute faithfully for native virions in the development of HPV-serodiagnostic immunoassays.
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Woodchuck hepatitis virus surface antigen produced in vitro fails to bind polymerized woodchuck serum albumin
More LessUsing a plasmid (pSWS) similar to one that has been successfully used for large-scale production of hepatitis B virus (HBV) envelope protein particles (pSVS) but containing the corresponding woodchuck hepatitis virus (WHV) envelope gene sequences, we have stably transformed the rodent dihydrofolate reductase-deficient cell line CHO dhfr −. Although production of WHV envelope particles in CHO/pSWS cell lines was low, it was sufficient to test whether these particles could bind to polymerized serum albumin. Whereas binding of HBV particles produced in CHO/pSVS cells to polymerized human serum albumin could readily be detected, we found no evidence that the WHV envelope protein particles produced in vitro bind to either human or woodchuck polymerized serum albumin.
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The p10 gene of natural isolates of Bombyx mori nuclear polyhedrosis virus encodes a truncated protein with an M r of 7700
More LessSequence analysis of the p10 genes of three Bombyx mori nuclear polyhedrosis virus (BmNPV) isolates collected in Taiwan (Ta) and Japan (T3 and D1) showed that all possessed a deletion of an adenine residue, 210 bp downstream from the first base of the initiation codon when compared to the p10 gene of Autographa califomica (multinucleocapsid) NPV (AcMNPV). This deletion caused a downstream termination codon to come inframe with the coding sequence of p10, so that the p10 gene of BmNPV encoded a protein of 70 amino acid residues with an M r of 7700. This is considerably shorter than the 10000 M r protein encoded by the closely related AcMNPV.
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Analysis of murine antibody responses to baculovirus-expressed human immunodeficiency virus type 1 envelope glycoproteins
More LessAn analysis of the humoral immunogenicity of a candidate AIDS vaccine (VaxSyn) in a murine model system is presented. Sera taken from a panel of mice immunized with the immunogen were analysed for their ability to bind a panel of gpl20-representing peptides and limited reactivity to known sites of immunological interest was observed. Monoclonal antibodies (MAbs) were characterized as binding to a more restricted variety of regions on gpl20 including Cl, V2, V4 and the C terminus. A tetrazolium-based cytocidicity assay was used and shown to be an effective and objective method for the screening of human immunodeficiency virus (HlV)-neutralizing activity in large numbers of samples. None of the MAbs characterized in this study neutralize HIV-1 reference strains. The significance of these findings in view of previous publications is discussed.
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Removal of the cleavage site of recombinant feline immunodeficiency virus envelope protein facilitates incorporation of the surface glycoprotein in immune-stimulating complexes
Recombinant vaccinia viruses were constructed that expressed the complete env gene of feline immunodeficiency virus with or without the nucleotide sequence encoding the cleavage site between the surface (SU) protein and the transmembrane (TM) protein. The removal of this cleavage site resulted in the expression of a 150K protein that was processed into a 130K protein and was not cleaved into the SU and the TM proteins. Removal of the cleavage site also facilitated incorporation of the SU protein in immune-stimulating complexes (iscoms). Antibody responses to both an SU and a TM peptide representing two immunodominant B cell epitopes were measured. These were higher in cats immunized with iscoms prepared from the cleavage site-deleted envelope protein than in cats immunized with iscoms prepared from the native envelope protein or immunized with the envelope protein and the adjuvant Quil A.
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Genetic relatedness between influenza A (H1N1) viruses isolated from humans and pigs
More LessComplete nucleotide sequences were obtained from the nucleoprotein genes of three influenza A viruses and partial nucleotide sequences were obtained from the polymerase, neuraminidase, matrix, and non-structural protein genes of four influenza A viruses that had been isolated between 1931 and 1939 from clinically sick pigs in the United States or Europe. A phylogenetic analysis of the open reading frames of nine nucleoprotein genes showed that the U.S. swine influenza virus isolates from 1931 and 1937 originated from the classic swine viral nucleoprotein lineage, whereas the European swine strains from 1938 and 1939 were placed among the early human influenza virus nucleoprotein lineage. All the partial gene sequences obtained from the two European swine strains from 1938 and 1939 were also more closely related to early human H1N1 reference strains than to the U.S. swine isolates from 1931 and 1937, indicating that none of the four viruses isolated from swine had acquired genes from a heterologous lineage through reassortment.
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Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system
More LessAn in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3′ end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3′ end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.
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Effect of maternal antibody on IgA antibody response in nasopharyngeal secretion in infants and children during primary respiratory syncytial virus infection
The IgA antibody response to respiratory syncytial virus (RSV) was determined in nasopharyngeal secretions (NPS) of 22 infants and children infected with RSV group A strains, employing an ELISA. The antibody activity observed during the convalescent phase against whole virus, fusion glycoprotein (F) and large glycoprotein (G) was examined in young infants (under 6 months) and compared with that of older individuals (6 to 16 months). Both groups showed similar degrees of IgA antibody activity to whole virus in NPS; however, older individuals showed a significantly higher activity of IgA F antibody than that of IgA G antibody in the NPS. On the other hand, in the NPS of young infants, IgA F antibody was somewhat suppressed and IgA G antibody activity predominated over that of IgA F. Pre-existing (maternal) serum IgG anti-RSV F antibody activity was higher than that of antibody to G. A significant reverse correlation was observed between the activity of preexisting serum IgG F antibody and NPS IgA F antibody in the convalescent phase after primary infection with RSV. These observations suggest that maternally derived RSV IgG antibody, which contains abundant anti-F activity, may suppress the development of IgA F antibody response at infection sites in the respiratory tract in young infants during primary RSV infection. These changes may be related to the severity of acute infection and longer convalescence often observed in young infants during RSV infection.
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Synthesis of the tomato golden mosaic virus AL1, AL2, AL3 and AL4 proteins in vitro
More LessTranscripts derived from the leftward region of tomato golden mosaic virus DNA A were translated in wheat-germ and rabbit reticulocyte lysate systems. The largest protein (M r 40K) produced from transcripts encompassing open reading frame (ORF) AL1 was identified as the AL1 protein by immunoprecipitation with AL1-specific antibodies. However the main product was a protein of M r 10K, that was shown by in vitro mutagenesis to be the product of AL4, an ORF contained within AL1 DNA in a different reading frame. Translation of transcripts containing ORF AL2 or ORF AL3 gave the AL2 and AL3 proteins respectively; both proteins were also efficiently produced from transcripts containing both ORFs which overlap over about two-thirds of their length. Translation of a transcript containing the four ORFs gave all four proteins, consistent with a previous report that three of these (AL1, AL2 and AL3) can be translated from a single polycistronic RNA in transgenic tobacco plants. It is suggested that the leftward region of DNA A of the whitefly-transmitted geminiviruses may be expressed by two principal messenger RNAs, one encoding the AL1 and AL4 proteins and the other encoding the AL2 and AL3 proteins, and that the AL4 and AL3 proteins may be translated from these messenger RNAs by a leaky scanning mechanism.
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Restriction fragment length polymorphism analysis of reverse transcription-PCR products reveals the existence of two major strain groups of beet necrotic yellow vein virus
Beet necrotic yellow vein virus (BNYW)-infected sugarbeets were obtained from many parts of Europe and also from some sites in Asia and the U.S.A. Reverse transcription (RT)–PCR products of more than 1 kbp were obtained for four different regions of the viral genome which may be particularly important with respect to the pathogenic properties of the virus, i.e. for the coat protein and the 42K protein-encoding regions on RNA 2 and for major parts of RNAs 3 and 4. Restriction fragment length polymorphism (RFLP) patterns obtained with these PCR products revealed the existence of two major strain groups of BNYW, named type A and type B. The A type was detected in Greece, the former Yugoslavia, Slovakia, parts of Austria, Italy, Spain, parts of France, Belgium, The Netherlands and England as well as in Asia (Turkey, Kazachstan, China and Japan) and the U.S.A. The B type occurs in Germany and parts of France. Mixed infections were detected at the borderline regions between areas of the A and B types. Comparisons of published and newly determined nucleotide sequences of the respective parts of the BNYW genome indicate that the percentage of nucleotide differences between the A and the B type is approximately 3 % for the respective regions of RNAs 2 and 3 and approximately 1·5% for RNA 4. Nucleotide sequences appear to be remarkably stable within each of the two strain groups. The majority of the nucleotide differences between the A and B types occur in the third triplet position. The amino acid changes in the coat protein area are outside the four previously determined antigenic regions that are accessible on the surface of the virus particles and are involved in the formation of continuous and presumably also discontinuous epitopes. This may explain why serological differences between the two strain groups have not been found.
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Genomic heterogeneity of rice dwarf phytoreovirus field isolates and nucleotide sequences of variants of genome segment 12
Electrophoretic profiles of the dsRNAs of field isolates of rice dwarf virus (RDV) were compared with those of an isolate maintained at Hokkaido University (RDV-H). Unexpectedly, the genomic dsRNAs of most of the field isolates showed distinct electrophoretic mobility profiles. This was the case even among isolates from the same region. Genome segment 12 (SI2) from some variants migrated faster than S12 from RDV-H. These RNAs were converted to full-length cDNAs and sequenced. S12 from all the variants had the same length of 1066 nucleotides with nucleotide sequence identities of 96 to 99%. Three open reading frames previously reported were present in all the variants, and the sequence identities were 95 to 99% for P12, 98 to 100% for P120Pa, and nearly 100% for P120Pb. A comparison of the nucleotide and amino acid sequences of the variants with sequences of the RDV-H and Akita isolate showed that there are two genomic types, one represented by RDV-H and the other by the Akita isolate.
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Nucleotide sequence and infectivity of cucumber mosaic cucumovirus (strain K) RNA2 involved in breakage of replicase-mediated resistance in tobacco
More LessReplicase-mediated resistance represents a genetically engineered form of resistance against viral infections and is established by using an intact or modified viral polymerase (replicase) gene for the transformation of the host. The K strain of cucumber mosaic cucumovirus (K-CMV) has been shown to break replicase-mediated resistance generated using a modified RNA2 of the subgroup I strain Fny-CMV. A full-length cDNA clone of K-CMV RNA2 was generated and RNA transcripts were shown to be infectious in combination with Fny-CMV RNAs 1 and 3. We further demonstrate that the ability of K-CMV to overcome resistance in the transgenic plants maps to RNA2. The complete nucleotide sequence of this RNA was determined and sequence differences from Fny-CMV RNA2 are discussed with regard to functional domains and their possible involvement in resistance breakage.
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Sequence analysis of rice hoja blanca virus RNA 3
More LessRNA 3 of rice hoja blanca tenuivirus (RHBV) has 2299 nucleotides and resembles RNA 3 of other tenuiviruses such as maize stripe (MStV) and rice stripe (RStV) viruses in potentially coding with an ambisense strategy for two proteins. Both the viral-sense protein of 23K and the complementary-sense protein of 35K have about 46% amino acid identity with the analogous proteins encoded by RNA 3 of MStV and RStV. As the proteins of MStV and RStV have about 65 % identity between themselves, RHBV cannot be a South and Central American strain of the Asian RStV. The intergenic region resembles those of other tenuiviruses, being rich in A and U residues, but its predicted folding pattern is unlike those of other tenuiviruses. Instead, the predicted folding of the intergenic region was indistinguishable from that of the coding regions and there was no evidence for a distinct hairpin-loop structure. The significance to the evolution of tenuiviruses of the similarities that the two proteins have with their analogues in other tenuiviruses is discussed.
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Selection versus recombination: what is maintaining identity in the 3′ termini of blueberry leaf mottle nepovirus RNA1 and RNA2?
More LessThe 3′ non-coding regions (NCR) of RNA1 and RNA2 of blueberry leaf mottle nepovirus (BBLMV) are nearly identical with differences occurring at only four positions. The presence of this 1·4 kb duplication indicates that recombination has occurred at least once in the evolutionary history of BBLMV. Since high mutation rates are common in RNA viruses, strong selection pressure and/or high frequency of recombination must be operating in order to maintain identity in this duplicated region. The possible involvement of high frequency RNA recombination in maintaining identity was investigated. The four conserved differences between the 3′ NCR of RNA1 and RNA2 were used as markers to detect recombinants in a viral population. Nucleotide sequences of BBLMV cDNA clones were compared to the 3′ consensus sequence and deviations were examined to determine whether they were due to single base mutations or recombinational events. No evidence of recombination was found in any of the cDNA clones sequenced and all differences were attributed to mutations. If recombination occurred in the 3′ NCR of BBLMV, the frequency was below 1·1% between markers. The data indicate that identity in the 3′ NCR of RNA1 and RNA2 of BBLMV was maintained without high levels of recombination. The high number of mutations observed in a BBLMV population and lack of observable recombination indicate that other mechanisms, such as selection, play an important role in the conservation of identity in the 3′ NCR.
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Volumes and issues
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Volume 105 (2024)
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Volume 2 (1968)
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Volume 1 (1967)