- Volume 78, Issue 9, 1997
Volume 78, Issue 9, 1997
- Articles
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Protection of pigs against Aujeszky's disease by DNA vaccination
More LessVaccination with DNA constructs encoding viral antigens has been shown to induce antiviral immunity in various model hosts. However, relevant natural virus-host systems have so far been analysed to only a very limited extent. To test the efficacy of DNA vaccination in an economically important large animal, pigs were immunized against Aujeszky’s disease, a serious virus infection caused by the alphaherpesvirus pseudorabies virus (PrV), which is characterized by severe central nervous and respiratory symptoms. After vaccination with plasmid vectors containing genes for immunogenic envelope glycoproteins C or D (gC or gD) of PrV under control of the major immediate early promotor of human cytomegalovirus, animals developed serum antibodies which recognized the respective antigen in immunoblot and exhibited neutralizing activity. Animals vaccinated with the gC expression plasmid were fully protected against a lethal challenge with PrV strain 75V19,and showed partial protection against the highly virulent NIA-3 strain. In contrast, protection was not observed after vaccination with the gD plasmid. Three intramuscular or intradermal immunizations with as little as 1 μg of gC plasmid DNA resulted in seroconversion and partial protection against lethal NIA- 3 infection. Specific antibodies were detected until at least 9 months after vaccination. In addition, a cellular immune response specific for gC could be demonstrated in proliferation assays of peripheral mononuclear lymphocytes. Our results thus demonstrate the potency of DNA vaccination for protection of large animals against a lethal virus infection.
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Deletion of glycoprotein gM of pseudorabies virus results in attenuation for the natural host
More LessGlycoprotein M (gM) is one of the very few nonessential glycoproteins conserved throughout the herpesvirus family. Despite this conservation little is known about its function in virus replication. To test for the importance of gM in vivo in a natural virus-host system, 6-week-old piglets were intranasally infected with a gM mutant of the alphaherpesvirus pseudorabies virus (PrV). Following infection virus excretion from the nasal mucosa was decreased ca. 100-fold compared to wild-type or revertant virus. Clinical signs were limited to transiently elevated temperature. In contrast, animals infected by wild-type or revertant virus exhibited high fever, severe respiratory symptoms and affliction of the central nervous system. Prior infection with gM PrV conferred protection against challenge infection and animals mounted an antibody response against gM after wild-type virus infection. Thus, gM is important for efficient virus replication in vivo and deletion of gM may contribute to development of live attenuated, genetically marked vaccines.
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Characterization of the proteinase specified by varicella-zoster virus gene 33
More LessVaricella-zoster virus (VZV) genes 33 and 33·5 are predicted to encode the VZV proteinase and its substrate (theassembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33·5 was co-expressed with the VZV proteinase, processing of the VZV33·5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33·5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.
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Bovine herpesvirus-1 infects activated CD4+ lymphocytes
More LessAcute virus infections can induce immune deficiencies, as shown by immunosuppression to a variety of antigens and mitogens. Previously we observed that live bovine herpesvirus-1 (BHV-1) induced considerable lymphocyte death in culture, suggesting that the virus infected one or more cell populations. Our goal was to identify the cells infected by BHV-1 and the mechanism resulting in cell death. ConA activated cells were cultured with BHV-1 and stained with monoclonal antibodies specific for virus envelope glycoproteins (gB, gC and gD) and lymphocyte surface proteins (CD2, CD4 and CD8) and a molecule associated with γ/δ cells. Two- colour immunofluorescence revealed that virus glycoproteins were preferentially expressed on T lymphocytes of the CD4Mphenotype. Live virus was required for virus glycoprotein expression, and by 48 h considerable loss of CD4 expression was observed. To confirm virus replication, RNA was isolated from cells, reverse transcribed and amplified using primers to a 342 bp region of immediate- early and early genes (IER2.9/ER2.6) or a 392 bp region of an early gene (gD). Immediate-early/early gene products were detected in CD4 T lymphocytes but not in infectious virions. Lymphocyte apoptosis was observed by 7 h post-infection with increasing levels of cell death at 24–48 h after infection. These findings suggest that the loss of proliferating CD4 T cells during infection or vaccination with modified live vaccines provides the opportunity for secondary infections that commonly occur following BHV-1 infection.
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The pathogenesis of ED71, a defined deletion mutant of equine herpesvirus-1, in a murine intranasal infection model for equine abortion
More LessA series of mutants of equine herpesvirus-1 (EHV-1) which contain deletions in non-essential genes was previously characterized in a murine intranasal infection model. One mutant, ED71 which was shown to be attenuated in the model, was further characterized by inoculation into pregnant mice. Despite the attenuation previously reported, intranasal inoculation of pregnant mice resulted in premature parturition and the birth of dead or dying foetuses. Furthermore, mice inoculated before pregnancy with the same mutant, and subsequently challenged 14 days after conception with wild-type virus, were not protected from abortion.
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Identification of a variant B-specific neutralizing epitope on glycoprotein H of human herpesvirus-6
We have identified the human herpesvirus-6 variant B (HHV-6B)-specific neutralizing epitope on glycoprotein H (gH) which is recognized by monoclonal antibody (MAb) OHV3, with complement-independent neutralizing activity. HHV-6 gHs from HHV-6A (strain U1102) and HHV-6B (strain HST) were expressed in a T7-vaccinia virus transient expression system. OHV3 reacted with HST gH, but not with U1102 gH, in an immunoprecipitation assay and an indirect immunofluorescence assay. In addition, OHV3 reacted with chimeric gHs, formed between U1102 gH and HST gH, containing amino acids 272 to 422 of HST gH. Sequence comparison between U1102 and HST showed seven amino acid differences in this region. Site-specific mutations were introduced into these positions and then reactivity against OHV3 was investigated. The arginine at position 389 of HST gH was shown to be a determinant of the HHV-6B-specific reactivity of OHV3.
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The processing, transport and heterologous expression of Epstein-Barr virus gp110
Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.
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Marek’s disease virus EcoRI-Q gene (meq) and a small RNA antisense to ICP4 are abundantly expressed in CD4+ cells and cells carrying a novel lymphoid marker, AV37, in Marek’s disease lymphomas
Mature lymphomas produced in Rhode Island Red (RIR) chickens infected with the RB1B strain of Marek’s disease virus (MDV) were examined for the presence of viral DNA and RNA and expression of viral antigens. In situ hybridization showed that all tumours examined contained viral DNA in areas of lymphoid infiltration. In 3/5 tumours, there was a correlation between the number and distribution of cells expressing the Marek’s disease EcoRI-Q gene (meq) and those that carried the lymphoid cell marker AV37. Expression of the MDV-specific phos- phoprotein pp38 was infrequent in lymphomas but abundant in a splenic tumour which also expressed the viral glycoprotein gB. Northern blot analysis of lymphocyte fractions purified by immunoaffinity showed that CD4 andAV37 fractions from lymphomas expressed meq and the small RNA antisense to ICP4 (SAR). The results are consistent with the notion that transformed cells are CD4Mcells, carrying the AV37 marker and expressing meq and SAR.
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Sequence variations and viral genomic state of human papillomavirus type 16 in penile carcinomas from Ugandan patients
Sequence variations in the E6/E7 (nt 34–880) and the L1 (nt 6584–7035) ORFs, and in the long control region (LCR) (nt 7289–93) of human papillomavirus type 16 (HPV-16) were analysed in five penile carcinoma biopsies obtained from Ugandan patients. Uganda is a country with a high incidence of genital cancers. All five isolates were classified as members of African-1 lineage (Af1) by phylogenetic analysis based on LCR sequences. The E6 gene phylogenetic analysis, however, showed that four isolates fell into a new subclass designated Af1-u. This subclass, characterized by three point mutations located at the 5′ end of the E6 gene with resulting changes in amino acids at positions 10 and 14, is distinguishable from the Af1 class by the absence of synonymous mutations at nt 286 and 289. The nonsynonymous substitution at nt 335 was present in three out of five samples. The E6 Af1 mutation pattern was present in only a single Ugandan HPV-16 isolate. Nucleotide sequence analysis of the E7 and L1 regions did not allow any Af1 subclass identification. The physical state of the viral DNA in these samples was characterized by PCR and Southern blot analysis. Oligonucleotides which enable amplification of the full length E2 region (nt 2734–3872) failed to amplify the target sequence in four out of five samples, suggesting disruption of the E2 ORF and integration of the HPV genome into the human DNA. Southern blot analysis confirmed the virus integration status. Our results contribute to the characterization of the HPV-16 ‘African lineages’ with the identification of the Af1- u subclass; furthermore, this is also the first report showing that in male genital cancers HPV-16 is integrated into the human genome with disruption of the E2 ORF.
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A monoclonal antibody against intact human papillomavirus type 16 capsids blocks the serological reactivity of most human sera
More LessA type-specific and neutralizing mouse MAb (V5) against human papillomavirus (HPV) type 16 capsids was found to block the serological reactivity of human sera with the corresponding capsids. Out of 352 human serum samples tested for the presence of IgG against HPV-16, more than 75% of reactive sera were completely blocked by the V5 antibody. Type-specific MAbs against HPV-6, −18 and −33 were also found to block serological reactivity with capsids of the corresponding HPV types for the majority of reactive human sera. The results suggest that most antibodies in human sera that are reactive with intact HPV capsids recognize the same or closely related major antigenic determinant(s).
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Primary induction of human cytotoxic lymphocytes against a synthetic peptide of the human immunodeficiency virus type 1 protease
More LessIdentification of in vitro immunogenicT-cell epitopes is important for the design of immunotherapeutics targeted to specific antigenic sites. To identify candidate cytotoxic T-lymphocyte (CTL) epitopes in the protease of human immunodeficiency virus type 1 (HIV-1) strain MN, we synthesized 9-mer and 10- mer peptides containing the HLA-A*0201 binding motif. Binding affinity of the peptides was measured by HLA-A*0201 up-regulation on T2 cells. Peptides with high binding-affinity were tested for their ability to stimulate primary CTLs from healthy HIVnegative blood donors. Peptide-specific CTLs were obtained from five out of six donors by stimulation with a 9-mer (LVGPTPVNI) or a 10-mer (VLVGPTPVNI) peptide derived from a highly conserved amino acid stretch in the C-terminal region of the protease. Addition of peptide-specific CTLs to acutely HIV-infected lymphocytes resulted in inhibition of p24 gag production. In conclusion, a highly conserved HIV protease peptide regularly elicits peptide-specific CTLs. Targeting immune responses against defined epitopes in non-variable regions may be a feasible way to minimize the risk of virus escape from immune surveillance.
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Evolution of human immunodeficiency virus subtype A in women seroconverting post partum and in their offspring post-natally infected by ingestion of breast milk
The evolution of genomic RNA of human immunodeficiency virus type 1 (HIV-1), subtype A, was studied in three Rwandan mother-child pairs over a period of 12–30 months. In two pairs a homogeneous subtype A V3 sequence population was observed at seroconversion and the virus populations in the children resembled those in the mothers. One of these mother-child pairs was infected with an A/C recombinant virus (Ap17/Cp24). In the third pair, a heterogeneous V3 sequence population was observed in the maternal seroconversion sample but the V3 sequence population in the child’s sample was homogeneous. In each individual the intra- and intersample variation (between the seroconversion and follow-up samples) increased over time in both the V3 region and p179ag . Independent evolution for 1–2 years did not abolish the epidemiological relationship between virus populations in mother and child.
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Biochemical and functional interaction of the human immunodeficiency virus type 1 Tat transactivator with the general transcription factor TFIIB
More LessTat strongly stimulates transcription of the human immunodeficiency type 1 (HIV-1) provirus by interacting with various cellular transcription factors, including TFIID. The results presented in this report indicate that the effect exerted by Tat also involves an interaction with TFIIB. A direct protein-protein interaction between Tat and TFIIB was observed in vitro. Detailed analysis of this interaction showed that the cysteine-rich and core domains of Tat bind to the N-terminal moiety of the general transcription factor. The role of the interaction between Tat and TFIIB in the activation of the entire HIV-1 promoter was analysed. Transfection experiments performed using a reporter construct containing the HIV-1 long terminal repeat fused to a reporter gene showed that overexpression of TFIIB progressively suppressed Tat-induced transcription. This effect was weakened by an increase in the intracellular con centration of Tat. A similar consequence of TFIIB overexpression was observed in a HeLa cell line stably transformed with a construct corresponding to the lacZ gene under the control of the HIV-1 promoter. Mutants of TFIIB which differed in their ability to interact with Tat and to function in basal transcription were analysed. The ability of TFIIB mutants defective for basal transcription to inhibit Tat-induced activity of the HIV-1 promoter depended on their capacity to interact with Tat. Mutants of TFIIB functional for basal transcription, but defective for the interaction with Tat, exhibited a dominant negative effect. From these data we propose a model in which interaction between Tat and both general transcription factors TBP and TFIIB maintains the transcriptional initiation complex in an active configuration.
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Analysis of the genetic diversity and phylogenetic relationship of Italian isolates of feline immunodeficiency virus indicates a high prevalence and heterogeneity of subtype B
The genetic diversity of 32 Italian isolates of feline immunodeficiency virus (FIV) was studied. Isolates were obtained from domestic cats living in different areas. Sequence data were obtained from a 308 bp fragment of the p25 region of the gag gene. Phylogenetic relationships among these sequences and previously published sequences were determined. All the Italian isolates could be assigned to subtype B; however, four isolates formed two separate clusters and may represent genetic out- liers. The reliability of classification results was confirmed by repeating the phylogenetic analysis with DNA sequences from the entire gag genes of two isolates and from the surface glycoprotein domain of the env gene of four isolates. It is concluded that the short segment of gag used permits reliable genotyping of FIV isolates. The study also shows that subtype B is largely prevalent in Italy.
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Augmentation of human T cell leukaemia virus type I Tax transactivation by octamer binding sites
More LessThe human T cell leukaemia virus type I (HTLV-I) Tax protein is an activator of viral and cellular gene expression. Tax does not bind DNA directly, but does interact with cellular DNA binding proteins. These interactions bring Tax to a specific group of promoters and may help to determine the specificity of Tax transactivation. Previous studies have demonstrated that the activity of Tax, when tethered to a given promoter, is enhanced by the presence of adjacent transcription factor binding sites. To examine the specificity of this augmentation, a series of transcription factor binding sites was tested for the ability to enhance the activity of a Gal-Tax fusion protein. The greatest increase in Gal-Tax activity was observed when an octamer binding site was placed adjacent to the Gal4 binding sites. However, the octamer binding site failed to independently function as a Tax responsive element in the absence of an adjacent Tax-tethering element. Oct-2 was not required for augmentation of Gal-Tax activity as this enhancement was observed in BHK- 21 cells, which lack Oct-2. The ability of octamer binding sites to augment transcription was specific for Gal-Tax, as compared to other transactivators. Taken together, these results demonstrate that the degree of Tax transactivation can be influenced by the elemental composition of the target promoter.
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Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus
Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50–66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.
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Molecular evolution and phylogeny of dengue-4 viruses
More LessNucleotide sequences of the envelope protein genes of 19 geographically and temporally distinct dengue (DEN)-4 viruses were determined. Nucleic acid sequence comparison revealed that the identity among the DEN-4 viruses was greater than 92%. Similarity among deduced amino acids was between 96 and 100%; in most cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis generated phylogenetic trees, which indicated that geographically independent evolution of DEN-4 viruses had occurred. DEN-4 viruses were separated into two genetically distinct subtypes (genotypes). Genotype-1 contains viruses from the Philippines, Thailand and Sri Lanka; genotype-2 consists of viruses from Indonesia, Tahiti, the Caribbean Islands (Puerto Rico, Dominica) and Central and South America.
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Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells
The complete nucleotide sequences of the genomes of dengue-1 virus virulent 45AZ5 PDK-0 and attenuated vaccine candidate strain 45AZ5 PDK-27 have been determined and compared with the dengue-1 virus Western Pacific (West Pac) 74 parent strain from which 45AZ5 PDK-0 was derived. Twenty-five (0·23%) nucleotide and 10 (0·29%) amino acid substitutions occurred between parent strain dengue-1 virus West Pac 74 and virulent strain 45AZ5 PDK-0, which was derived from the parent by serial passage in diploid foetal rhesus lung (FRhL-2) and mutagenized with 5-azacytidine.These substitutions were preserved in the 45AZ5 PDK-27 vaccine. 45AZ5 PDK-0 and PDK-27 strains, which differ by 27 passages in primary dog kidney (PDK) cells, show 25 (0·23%) nucleotide and 11 (0·32%) amino acid divergences. These comparative studies suggest that the changes which occurred between the West Pac 74 and 45AZ5 PDK-0 strains may alter the biological properties of the virus but may not be important for attenuation. Important nucleotide base changes responsible for attenuation accumulated between 45AZ5 PDK-0 and 27.
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Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African West Nile viruses
We compared the sequence of an envelope protein gene fragment from 21 temporally distinct West Nile (WN) virus strains, isolated in nine African countries and in France. Alignment of nucleotide sequences defined two groups of viruses which diverged by up to 29%. The first group of subtypes is composed of nine WN strains from France and Africa. The Austral-Asian Kunjin virus was classified as a WN subtype in this first group. The second group includes 12 WN strains from Africa and Madagascar. Four strains harboured a 12 nucleotide in-frame deletion. The loss of the corresponding four amino acids resulted in the loss of the potential glycosylation site present in several WN strains. The distribution of virus subtypes into two lineages did not correlate with host preference or geographical origin. The isolation of closely related subtypes in distant countries is consistent with WN viruses being disseminated by migrating birds.
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Characterization of truncated forms of hepatitis C virus glycoproteins.
Hepatitis C virus (HCV) glycoproteins (E1 and E2) both contain a carboxy-terminal hydrophobic region, which presumably serves as a membrane anchor. When they are expressed in animal cell cultures, these glycoproteins, in both mature complexes and misfolded aggregates, are retained in the endoplasmic reticulum. The effect of carboxy-terminal deletions on HCV glycoprotein secretion and folding was examined in this study. Sindbis and/or vaccinia virus recombinants expressing truncated forms of these glycoproteins ending at amino acids 311,330, 354 and 360 (truncated E1), and 661, 688, 704 and 715 (truncated E2) were constructed. When expressed using Sindbis virus vectors, only truncated forms of E1 and E2 ending at amino acids 311 (E1t311) and 661 (E2t661), respectively, were efficiently secreted. Analysis of secretion of truncated forms of E2 glycoprotein expressed by vaccinia viruses indicated that significant secretion was still observed for a protein as large as E2t715. However, only secreted E2t661 appeared to be properly folded. Secreted HCV glycoprotein complexes were also detected in the supernatant of cell culture when E1t311 and E2t661 were coexpressed. Nevertheless, these secreted complexes, as well as E1t311 expressed alone, were misfolded. The effect of coexpression of E1 and E2 glycoproteins on each other’s folding was evaluated with the help of a conformation-sensitive monoclonal antibody (for E2) or by analysing intramolecular disulfide bond formation (for E1). Our data indicate that the folding of E2 is independent of E1, but that E2 is required for the proper folding of E1.
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The sequence and genomic organization of a GB virus A variant isolated from captive tamarins.
More LessRecently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to neargenome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-trans-lationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.
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A single dose immunization with rabbit haemorrhagic disease virus major capsid protein produced in Saccharomyces cerevisiae induces protection
More LessThe gene coding for the major capsid protein (VP60) from rabbit haemorrhagic disease virus was expressed in Saccharomyces cerevisiae under the phosphoglycerate kinase promoter. The recombinant VP60 produced in yeast was antigenically similar to the viral polypeptide as determined with a polyclonal serum. Electron microscopic observation of the recombinant yeast-derived antigen revealed the presence of virus-like particles similar in size and appearance to native capsids. Subcutaneous vaccination of rabbits with a single dose of this antigen in the absence of commercial adjuvants conferred complete protection against the haemorrhagic disease.
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Evolution of European bat lyssaviruses.
More LessForty-seven European bat lyssaviruses (EBL) and two African insectivorous bat lyssaviruses (Duvenhage viruses) were selected for a comparison to be made of their evolutionary relationships. Studies were based on direct sequencing of the PCR-amplified products of the 400 nucleotides coding for the amino terminus of the nucleoprotein. Phylogenetic relationships were analysed after bootstrap resampling using the maximum parsimony and the neighbour-joining methods. Analyses of both the nucleotide and amino acid sequences placed these viruses in three separate clusters, namely genotype 4 (Duvenhage), genotype 5 (EBL1) and genotype 6 (EBL2). Evolutionary analysis of the nucleoprotein gene of EBL1 and EBL2 indicated low intrinsic heterogeneity mainly due to synonymous substitutions. In addition, both EBL1 and EBL2 evolved into at least two genetically distinguishable lineages (a and b) following geographical drifting. We can speculate that subsequently the lineages EBL1a and EBL1b were introduced into parts of northern Europe from two different geographical directions; EBL1b was probably introduced most recently and was from North Africa. Eptesicus serotinus appears to be the principal reservoir for EBL1 and Myotis dasycneme and M. daubentonii the reservoirs for EBL2.
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Inhibition of influenza viral polymerases by minimal viral RNA decoys
More LessAll gene segments of influenza virus share a common feature at their respective termini. Both the 5′- and 3′-terminal sequences are highly conserved and possess partial inverted complementarity. This allows for the formation of a double-stranded duplex, which plays a major role in transcription, replication and packaging of the viral genome. In vitro studies have shown that the viral polymerase binds to short RNA molecules containing these termini. In this study, attempts were made to test whether mini-RNA decoys containing either or both termini can inhibit the activity of the viral polymerase in vivo. RNA molecules containing either the 5′ or the 3′ noncoding sequences were unable to inhibit NS-CAT RNA replication, while mini-RNA decoys consisting of both the 5′ and 3′ noncoding sequences of vRNA or cRNA were able to efficiently inhibit the activity of the viral polymerases expressed from vaccinia virus vectors.
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Assembly of recombinant Newcastle disease virus nucleocapsid protein into nucleocapsid-like structures is inhibited by the phosphoprotein
More LessA recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.
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Three major alleles of rotavirus NSP4 proteins identified by sequence analysis.
More LessComparison of nonstructural glycoprotein NSP4 gene sequences from 22 rotavirus strains originating from six host species and of 14 different combinations of G and P types revealed the presence of three distinct NSP4 alleles, represented by strains Wa, KUN and AU-1. Genetic distances between any of these alleles (18·0%) were significantly greater than those within each allele (5·5%) and phylogenetic analysis suggested that divergence into three distinct alleles had occurred at about the same time during evolution. While amino acid variation among strains was minimal in the amino-terminal two-thirds of the protein (aa 1–130), variability increased toward the carboxy terminus of the enterotoxic peptide region (aa 114–135) and was greatest between residues 135 and 141. Comparison of the amino acid sequences corresponding to the enterotoxic peptide region between strains isolated from asymptomatic neonates and those from children with diarrhoea failed to identify any conserved changes that correlated with the capacity of the virus to cause disease. Amino acids were relatively conserved in the domains important for viral morphogenesis.
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Infectious cDNA clone used to identify strawberry mild yellow edge-associated potexvirus as causal agent of the disease
More LessA full-length in vivo infectious cDNA clone of strawberry mild yellow edge-associated potexvirus (SMYEaV) was constructed and used to inoculate Fragaria vesca ‘Alpine’ seedlings and Rubus rosifolius. Both host plants could be infected using particle bombardment or agroinoculation, but not by mechanical inoculation. A method that used potted strawberry plants for particle bombardment resulted in high survival and infection rates. The plants developed systemic infection and virus particles were detected by ELISA and immuno-electron microscopy. Mechanical inoculation of Chenopodium quinoa and C. foetidum with the 35S construct resulted in localized infections. F. vesca ‘Alpine’ indicator plants produced symptoms that were indistinguishable from control plants inoculated with a naturally occurring isolate of strawberry mild yellow edge by graft or aphid transmission. These results suggest that SMYE potexvirus is the causal agent of strawberry mild yellow edge disease. As this virus is capable of causing the disease, we propose the name strawberry mild yellow edge potexvirus, with the acronym SMYEPV, to replace the name strawberry mild yellow edge-associated potexvirus.
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The proteins encoded by rice grassy stunt virus RNA5 and RNA6 are only distantly related to the corresponding proteins of other members of the genus Tenuivirus
More LessThe genome of rice grassy stunt virus (RGSV) consists of six RNA segments. The nucleotide (nt) sequences of the two smallest segments, RNAs 5 and 6, were determined and found to comprise 2704 and 2584 nt, respectively. The 5′ - and 3′ -terminal sequences of both RNAs were identical over a length of 21 nt and could potentially form a panhandle-like structure due to intramolecular complementarity. Each RNA segment contained a virus (v) sense open reading frame (ORF) in the 5′-proximate region, and a virus complementary (vc) ORF in the 3′-proximate region, indicating an ambisense coding strategy. The protein encoded by the ORF on the vc strand of RNA5 was identified as the viral nucleocapsid protein (M r 35927). The ORF on the v strand of RNA6 encoded a protein of M r 20581 which represented the major nonstructural protein, previously shown to be produced in RGSV-infected rice tissues. The predicted proteins encoded by RGSV RNAs 5 and 6 were only distantly similar in sequence to the four proteins encoded by RNAs 3 and 4 of other viruses belonging to the genus Tenuivirus. These low sequence similarities, together with the apparently distinct number of genome segments, set RGSV apart from the other tenuiviruses and indicate that it should be placed in a taxonomically separate genus.
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Baculoviruses contain a gene for the large subunit of ribonucleotide reductase
More LessIn the genomes of two baculoviruses, Spodoptera exigua and S. littoralis multicapsid nucleopoly-hedroviruses (SeMNPV and SpliMNPV, respectively), an open reading frame (ORF) encoding the large subunit of ribonucleotide reductase (RR1) was identified. The predicted amino acid sequences of SeMNPVand SpliMNPV RR1 showed high homology to RR1 proteins from eukaryotes (ca. 70% and 80% similarity, respectively). The amino acid residues thought to be involved in catalytic function were conserved in the baculoviral RR1 ORFs. The RR1 ORFs in SeMNPV and SpliMNPV were located in different genomic positions. In SeMNPV, the RR1 ORF was located upstream of the polyhedrin gene, in an anti-genomic orientation. In SpliMNPV, the RR1 ORF preceded the p74 gene. By searching databanks, sequences homologous to the N terminus of RR1 were also detected upstream of the polyhedrin gene of three other baculoviruses, Mamestra brassicae multicapsid NPV, Panolis flammea multicapsid NPV and Orgyia pseudotsugata single nucleocapsid NPV. The baculovirus type species, Autographica californica multicapsid NPV, however, does not encode RR. A 2·7 kb transcript could be detected throughout infection with SeMNPV, classifying SeMNPV rr1 as an early gene. Primer extension analysis revealed several early and late start sites. None of the major start sites showed similarity to previously characterized baculoviral transcriptional start motifs. Phylogenetic analysis of prokaryotic, eukaryotic and viral RR1 proteins suggested that SeMNPV and SpliMNPV acquired the gene for RR1 from a eukaryotic source, but independently from each other.
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Expression of the Drosophila retrovirus gypsy as ultrastructurally detectable particles in the ovaries of flies carrying a permissive flamenco allele
More LessThe endogenous retrovirus gypsy is controlled by the Drosophila gene flamenco (flam). New insertions of gypsy occur in any individual Drosophila if its mother is homozygous for the flam 1 permissive allele and contains functional gypsy proviruses. The ovaries of flam 1 females also contain high amounts of gypsy RNAs. Unexpectedly however, gypsy derepression does not occur in the flam 1 female germline proper but in the somatic follicular epithelium of the ovary. Since extracts from these females are able to efficiently infect the germ-line of a strain devoid of active gypsy proviruses, we assume that a similar kind of germ-line infection, which would occur inside the flam 1 females themselves, could be required for gypsy insertions to occur in their progeny. This hypothesis was confirmed by electron microscopy observations showing that non-enve-loped intracytoplasmic particles containing gypsy RNAs accumulate in the apical region of the flam 1 follicle cells, close to specific membrane domains to which the gypsy envelope proteins are targeted, whereas both are absent in the flam controls. Low amounts of similar virus-like particles were also observed in flam 1 oocytes, but it is not yet known whether they entered passively or as a result of membrane fusion. This is the first report of the beginning of a retrovirus cycle in invertebrates and these observations should be taken into account when explaining the maternal effect of the flamenco gene on the multiplication of gypsy proviruses.
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Immunodetection of PrPSc in spleens of some scrapie-infected sheep but not BSE-infected cows
The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.
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Volumes and issues
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