- Volume 80, Issue 1, 1999
Volume 80, Issue 1, 1999
- Articles
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Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641.
New variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) are caused by the same strain of pathogen and, as sheep can develop experimental BSE, this has raised concern that humans may be at risk from eating mutton if BSE has naturally transmitted to sheep. Biochemical typing of abnormal prion proteins (PrPsc) has been suggested to detect BSE in sheep. Although this approach is ingenuous, we can now report biochemical evidence of strain variation in contemporary and archival brain tissue from cases of experimental BSE or experimental and natural scrapie in sheep. Interestingly, we found at least one isolate of natural scrapie (CH 1641) with a very similar, but not identical, PrPsc profile to BSE but which differs from BSE in its transmission characteristics to mice.
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Scrapie strain-specific interactions with endogenous murine leukaemia virus.
More LessThe finding that a senescence-accelerated mouse (SAMP8) shows early brain ageing, with histopathological changes resembling those seen in scrapie, combined with the discovery of high levels of endogenous murine leukaemia virus (MuLV) in brains of SAMP8 mice prompted us to examine the effect of scrapie infection on MuLV titres in this strain and in one of its progenitors, the AKR strain. Three scrapie strains (ME7, 22L and 139A) that had a comparatively short incubation period in SAMP8 and AKR mice caused an increase in brain MuLV titres that was scrapie strain-specific: in each mouse strain, the greatest effect was with 1 39A, and the least with ME7. The 22A scrapie strain, which has a long incubation period in SAMP8 mice, did not affect MuLV titres in brains of this mouse strain. Previous analyses of scrapie incubation periods in AKR, SAMP8 and another strain derived from an AKR cross (SAMR1) showed an inverse relationship between brain MuLV titres and scrapie incubation periods. This finding, combined with the effect of scrapie on MuLV titres, suggests an interaction between the scrapie infectious process and MuLV replication.
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Protease-resistant prion protein produced in vitro lacks detectable infectivity.
More LessThe ‘protein-only’ hypothesis of prion propagation argues that infectious prions consist of PrP(Sc), a conformational isomer of host-derived prion protein (PrP(C)), which can be distinguished from PrP(C) by its partial resistance to proteases. While protease-resistant PrP has been produced by mixing PrP(Sc) and recombinant-derived PrP(C) in vitro, bioassay of any new infectivity has been precluded by the need to use a large molar excess of same species PrP(Sc). Transgenic mice expressing a chimaeric hamster-mouse PrPC (MH2M PrP(C)) are, unlike conventional mice, highly susceptible to Sc237 hamster scrapie. In addition, they produce MH2M PrP(Sc) and infectivity which is pathogenic for conventional mice. We have therefore attempted to produce MH2M PrP(Sc) in vitro as any infectivity produced could be distinguished from the hamster PrP(Sc) used to promote the conversion by bioassay in conventional mice. Although protease-resistant MH2M PrP was produced, no infectivity was detected on bioassay. These results argue that acquisition of protease resistance by PrP(C) is not sufficient for the propagation of infectivity.
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Construction and characterization of murine neuroblastoma cell clones allowing inducible and high expression of the prion protein.
More LessA tetracycline-inducible expression system has been established for the prion protein (PrP) in murine neuroblastoma cells (N2a). For this purpose, N2a cells were first stably transfected with either the tetracycline-controlled transactivator or the reverse transactivator. After selection of N2a clones which carried one of these transactivators, the murine PrP gene (Prnp) was introduced under the control of the transactivator-responsive promoter in a second round of stable transfection. Stably double-transfected N2a clones carrying the reverse type but not the normal transactivator were found to be fully inducible, giving a low background of Prnp expression before induction and high expression after induction. Stably double-transfected N2a cells were at least as productive as N2a cells over-expressing Prnp permanently under the control of a strong viral promoter. Furthermore, the selected N2a clones allowed the Prnp expression level to be quantitatively controlled by varying the level of the effector substance, the tetracycline-derivative doxycycline. The clones were fully controllable, as over-expression could be switched on and off as desired. These N2a clones may become an important tool for elucidation of the cellular function of PrP and may pave the way for the tetracycline-inducible expression of many genes in this neuroblastoma cell line.
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The bovine papillomavirus type 4 long control region contains an epithelial specific enhancer.
More LessBovine papillomavirus type 4 (BPV-4) is a mucosal epitheliotropic virus that is a causative agent in alimentary carcinoma of cattle. The long control region (LCR) of this virus controls expression of the transforming proteins, E8 and E7. Deletion mutants of the LCR were prepared and assayed for their ability to activate transcription from the LCR promoter in primary bovine palate keratinocytes (the natural target cell for BPV-4) and fibroblasts. The LCR was at least an order of magnitude more active in keratinocytes than in fibroblasts. An epithelial specific enhancer was identified that activated transcription from the SV40 promoter to levels identical to the full-length LCR. One of the active sites in the enhancer is 100% conserved in the LCR of human papillomavirus type 16. The results demonstrate that the BPV-4 LCR has an epithelial specific enhancer, which offers the opportunity to study epithelial specific transcriptional regulation of papillomavirus promoters.
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The E2 protein of human papillomavirus type 16 is translated from a variety of differentially spliced polycistronic mRNAs
More LessThe major regulation protein of human papillomavirus (HPV) transcription is the viral E2 protein. Previous studies have identified a variety of alternatively spliced mRNAs containing multiple open reading frames (ORFs) encoding the E2 protein of HPV type 16. In these mRNAs the E2 ORF is contained as an internal ORF. In the present study, the translational capacities of three mRNA species starting at the p97 promoter and containing the 880/2581, 880/2708 and 226/2708 splice junctions upstream of the E2 ORF were investigated. Partial cDNAs spanning the E2 ORF and the related upstream ORFs were synthesized and assessed for E2 protein translation in vivo, in COS cells, and in vitro, in cell-free systems. Results of these analyses indicated that E2 protein was translated from all three mRNAs. Translation efficiency of E2 from the natural polycistronic templates was lower compared with that from a synthetic monocistronic control. Translation from the d-type bicistronic template (226/2708) was more efficient than that from the a-type (880/2708) and a′-type (880/2561) polycistronic templates. Further investigation of the translation of proteins encoded by the ORFs preceding the E2 ORF showed that a- and a′-type templates served for translation mainly of E7 but also of E61, while the d-type template served for translation of E6IV. Overall, the translation data support the suggestion that the corresponding mRNAs may function as polycistronic transcripts.
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The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.
The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.
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Adenovirus core protein VII displays a linear epitope conserved in a range of human adenoviruses.
More LessA monoclonal antibody (MAb) which recognized a linear epitope on polypeptide VII of human adenovirus (Ad) serotype 4 also interacted with polypeptides VII of Ad serotypes 2, 5, 7 and 10, but not with 12 and 40, in Western blotting. Utilizing a hexapeptide phage display library, the MAb was found to recognize the consensus sequence RXYXPX. A peptide based on a similar sequence from Ad2, viz. VEEARNYTPTPPPV, was synthesized and shown to inhibit binding of the MAb to polypeptide VII. Direct sequencing of the Ad4 polypeptide VII gene validated these observations, the sequence RNYTPA being detected. Comparison with gene sequences from other Ads indicates that this sequence is preserved in polypeptide VII of types 2 and 5 but in types 12 and 40 insertion of another residue disrupts this motif.
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US3 protein kinase of herpes simplex virus type 2 plays a role in protecting corneal epithelial cells from apoptosis in infected mice.
To clarify the biological role of US3 protein kinase of herpes simplex virus type 2 (HSV-2) in vivo, the expression of the viral antigen, the appearance of apoptotic bodies and DNA fragmentation were examined immunohistologically after corneal infection of mice with three different kinds of HSV-2 strain 186: the wild-type virus, a US3-deficient mutant (L1BR1) and its revertant (L1B-11). In both wild-type 186- and L1B-11-infected mice, viral antigen was diffusely found in the corneal epithelium; no apoptotic changes were detected in the epithelial cells. Whereas, in L1BR1-infected mice, HSV-immunoreactivity was localized around the virus-inoculated sites, and a large number of apoptotic bodies were observed in the corneal epithelium with dual-positive reactions for both HSV-immunostaining and TUNEL staining. These results suggest that the US3 protein kinase plays an important role in protecting HSV-2-infected cells from apoptotic death in vivo.
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Bovine herpesvirus 1 requires glycoprotein H for infectivity and direct spreading and glycoproteins gHW450 and gB for glycoprotein D-independent cell-to-cell spread.
More LessBy analogy with glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), gH may also be essential for penetration and cell-to-cell spread of bovine herpes-virus 1 (BHV-1). This was verified with a gH-negative BHV-1 mutant (gH-BHV-1), which replicated normally on gH-expressing cells but was unable to form plaques and infectious progeny on non-complementing cells. The block in entry could be overcome by polyethylene glycol-induced membrane fusion, demonstrating that gH is not essential for egress. Propagation of gH-BHV-1 on cell lines expressing wild-type gH or gHW450, which complements the function of BHV-1 gD for cell-to-cell spread, indicated that gHW450 is more efficient than wild-type gH in mediating direct spread of BHV-1. This was supported by the plaque sizes induced by rescued gH-BHV-1 that expressed wild-type gH and gHW450. Infection of cell lines expressing gH of BHV-1, HSV-1 and PRV with gH-BHV-1, HSV-1 and PRV mutants demonstrated that heterologous gH molecules could not complement gH function in penetration or cell-to-cell spread.
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Biphasic translocation of a 70 kDa heat shock protein in human cytomegalovirus-infected cells.
More LessHuman cytomegalovirus (HCMV) reportedly induces the expression of a 70 kDa heat shock protein (hsp70) with no known function in the virus replication cycle. We report here a remarkably specific translocation pattern of hsp70 during HCMV infection of human diploid fibroblasts. Immunofluorescent observation and Western blotting of subcellular fractions revealed nuclear localization of hsp70 early in infection and predominantly cytoplasmic localization of hsp70 late in infection. Treatment of HCMV-infected cells with cycloheximide followed by treatment with actinomycin D allowed virus immediate-early gene expression but inhibited hsp70 nuclear localization. Phosphonoacetic acid and tunicamycin, both of which reportedly inhibit HCMV DNA replication, did not inhibit HCMV-induced nuclear localization of hsp70 but inhibited hsp70 translocation from the nucleus to the cytoplasm. These results indicate a correlation between HCMV multiplication and hsp70 localization, suggesting that hsp70 may play a role in HCMV multiplication.
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Identification of transactivator and nuclear localization domains in the Epstein–Barr virus DNA polymerase accessory protein, BMRF1.
More LessThe Epstein–Barr virus (EBV) BMRF1 gene product is an essential component of the viral DNA polymerase and is absolutely required for lytic virus replication. In addition to its polymerase accessory protein function, we recently demonstrated that BMRF1 is a transactivator, inducing expression of the essential oriLyt promoter, BHLF1. However, the regions of BMRF1 required for transactivation of BHLF1 are unknown. Here we demonstrate that the carboxy-terminal portion of the BMRF1 protein (amino acids 378–404), although not required for DNA binding or polymerase processivity function, is required for transactivator function as well as nuclear localization. Site-directed mutagenesis of this region allowed us to separate the transactivator and nuclear localization motifs of BMRF1. The two DNA-binding domains of BMRF1 are also required for efficient transactivation of the BHLF1 promoter.
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Analysis of murine gammaherpesvirus-68 transcription during lytic and latent infection.
More LessMurine gammaherpesvirus-68 (MHV-68) is a gamma2-herpesvirus that upon experimental infection of laboratory mice establishes a latent infection in B lymphocytes. To date, no virus-encoded gene products have been reported to be expressed during latent infection. In this study, viral transcription has been analysed in a persistently infected B-cell line and abundant and preferential transcription of open reading frame M3 has been identified. Significantly, in situ hybridization analysis of latently infected mouse spleens with probes corresponding to 20 MHV-68 ORFs demonstrated active transcription of a single ORF, corresponding to M3. The kinetics and pattern of transcription of M3 were compared with that of the virally encoded tRNAs (vtRNAs), previously demonstrated to constitute a marker for latent infection in the spleen. Transcription of vtRNAs in splenic tissue could be first detected at 7 days post-inoculation (p.i.) in scattered cells in periarteriolar lymphoid sheaths (PALS). At 10 days p.i., vtRNA transcription was widespread and localized not only to cells in PALS but also to cells within developing germinal centres and from 21 days p.i. expression was detected exclusively within lymphoid follicles. Transcription of vtRNAs could be detected as late as 70 days p.i. In contrast, the histological localization of M3 transcription, which was first detected at 7 days p.i. in scattered cells in PALS, never changed and transcription could not be detected beyond 21 days p.i. These results suggest that M3 is an ORF that is expressed early during the establishment of latency in vivo.
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Induction of human herpesvirus-8 DNA replication and transcription by butyrate and TPA in BCBL-1 cells.
Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mm) are used, whereas reduction of butyrate concentration to 0.3 mm decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20% of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIPI, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0.3 mm delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mm butyrate could be diminished and delayed by reduction of butyrate concentration to 0.3 mm, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.
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Localization of isoprenylated antigen of hepatitis delta virus by anti-farnesyl antibodies.
More LessHepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.
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Characterization of the major nuclear localization signal of the Borna disease virus phosphoprotein.
More LessBorna disease virus (BDV) replicates and transcribes its negative-sense RNA genome in the nucleus. The BDV phosphoprotein (P) is localized in the nucleus of infected cells and cells transfected with P expression constructs. To identify the nuclear localization signal (NLS) of P, COS-7 cells were transfected with wild-type or mutant forms of P fused with green fluorescent protein (GFP). Whereas GFP alone was exclusively cytoplasmic, P or P-GFP were nuclear. Analysis of carboxy- and amino-terminal truncation mutants of P indicated that amino acids (aa) 20-37 are sufficient to promote efficient nuclear accumulation of the fusion protein. Residual nuclear import of GFP was observed with portions of P including aa 33-134 or aa 134-201, suggesting the presence of additional NLS motifs. The major NLS of P appears to be bipartite. It consists of two basic aa domains, R22RER25 and R30PRKIPR36, separated by four non-basic aa, S26GSP29.
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A recombinant measles virus expressing biologically active human interleukin-12.
More LessSuppression of cell-mediated immunity (CMI) is well-documented during and after measles. This immunosuppression is suggested to result from decreased production of interleukin-12 (IL-12), a key interleukin for CMI. In an attempt to clearly discern the role of IL-12 in measles-induced immunosuppression, a measles virus (MV) that expresses biologically active human IL-12 was generated. This was achieved by inserting the coding sequences of the two subunits (p35 and p40) of human IL-12 separated by an internal ribosome entry site in an additional transcription unit between the H and the L genes of MV. Although the IL-12-expressing MV grew slightly slower than the normal MV, it stably maintained the inserted sequences (3.2 kb) and uniformly expressed the foreign genes after 10 passages in cell culture. These findings suggest that MV is a well-suited vector for delivery of proteins of immunogenic and therapeutic importance.
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Alternative mechanisms of interaction between homotypic and heterotypic parainfluenza virus HN and F proteins.
More LessCell fusion by human parainfluenza virus (HPIV) type 2 or type 3 requires the coexpression of both the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins from the same virus type, indicating that promotion of fusion requires a type-specific interaction between F and HN. In this report we have further investigated the interaction of the ectodomains of the F and HN glycoproteins from HPIV2 and HPIV3. We constructed mutants of the HPIV2 F and HPIV3 F proteins (F′-KDEL) lacking a transmembrane anchor and a cytoplasmic tail, and containing a C-terminal signal for retention in the endoplasmic reticulum (ER). The P12 and P13 F′-KDEL proteins were both found to be retained intracellularly, and neither could induce cell fusion when co-expressed with homotypic HN proteins. Qualitative and quantitative cell-fusion assays also showed that both the P12 F′-KDEL and P13 F′-KDEL proteins have inhibitory effects on P12 F- and HN-induced cell fusion. However, the F-KDEL mutants were found to inhibit cell fusion by two distinct mechanisms. An interaction between P12 F′-KDEL and P12 HN results in intracellular retention of HN, and a block in its transport to the cell surface. In contrast, P13 F′-KDEL was found to suppress the steady-state intracellular expression levels of HPIV2 HN. These results support the conclusion that fusion involves an interaction between the HN and F proteins, and suggest that an association between F and HN may occur in the ER.
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RNA editing-like phenomenon in paramyxovirus V gene mRNA observed in insect cells infected with a recombinant baculovirus.
More LessThe V gene of the paramyxovirus human parainfluenza virus type 2 (hPIV2) is transcribed into both V and P mRNA. The V mRNA is a faithful transcript of the V gene; however, the P mRNA is transcribed by an RNA-editing mechanism in hPIV2-infected mammalian cells. Recombinant baculoviruses (rBV) were constructed containing the wild-type V gene, which has seven G residues at its editing site, and a manipulated V gene with ten G residues at its editing site. A small amount of the P protein was synthesized, in addition to the V protein, when the wild-type V gene was expressed in rBV-infected insect cells. Furthermore, synthesis of the P protein increased when rBV containing the manipulated V gene was used to infect insect cells. Both the P and V proteins were detected after in vitro translation of mRNA from rBV-infected cells. Moreover, G-residue insertions and a deletion were detected in mRNA. Since the P protein was not detected after in vitro translation of V RNA that had been transcribed in vitro by T7 RNA polymerase, these results suggest that the non-encoded G residues were inserted and deleted during transcription in insect cells. This RNA editing-like phenomenon and the implications of the length of the G cluster are discussed.
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Evolutionary pattern of the G glycoprotein of human respiratory syncytial viruses from antigenic group B: the use of alternative termination codons and lineage diversification.
More LessPartial sequences of the G protein gene of 33 isolates from antigenic group B of human respiratory syncytial virus were determined. Phylogenetic analysis indicated that the evolutionary pattern of group B viruses is similar to that previously described for isolates of antigenic group A, including worldwide distribution of related viruses and co-circulation of viruses from different lineages during the same epidemic. Dominance of AG+GA over UC+CU transitions was observed when G sequences of group B viruses were compared, as previously found in viruses from antigenic group A. Interestingly, differences in protein length, determined by the usage of alternative termination codons, were more pronounced in group B than in group A viruses. Changes in protein length correlated with the classification of viruses in different lineages. Thus, mutations that determined termination codon usage seem to have played an important role in the diversification of group B viruses.
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Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae.
More LessWe have completely sequenced the genome of Newcastle disease virus (NDV) vaccine strain LaSota. The sequences of the 3′- and 5′-terminal ends of the RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends. The entire genomic sequence, which was established by sequencing cDNA fragments generated by high-fidelity RT-PCR, consists of 15186 nt. Comparison of the 5′-terminal sequence of NDV LaSota with the 5′-terminal sequences of ten members of the Paramyxovirinae showed that NDV LaSota has an unusually long 5′ untranscribed region. Comparison of the entire genomic sequences showed that NDV is only distantly related to the other members of the genus Rubulavirus, to which NDV has been assigned. In this paper we present data which suggest that NDV should not be classified in the genus Rubulavirus, but instead should be considered as a member of a new genus within the subfamily Paramyxovirinae.
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Role of neuraminidase in influenza virus-induced apoptosis.
The virulent influenza virus clone 7a produced a greater level of apoptosis in MDCK cells compared with the attenuated strain A/Fiji. In both cases, apoptosis could be partially blocked by treatment with three anti-neuraminidase compounds [4-amino-(GR121158A) and 4-guanidino-(GG167; Zanamivir) 2,3-dehydro-N-acetylneuraminic acid and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA)] when they were given to cells during the virus attachment/entry phase, but not subsequent to this phase. In contrast, GG167, which does not enter cells, did not affect the numbers of infected cells and, in addition, acted late in the infection cycle to inhibit virus yields. Clone 7a neuraminidase was more active than A/Fiji neuraminidase when fetuin was used as the substrate. Similar differences in activity between the two viruses were seen when alpha-2,6 sialyl lactose was used as a substrate, but not with alpha-2,3 sialyl lactose. No sequence differences in the enzyme active site of the two neuraminidases were observed, indicating that differences in neuraminidase specificity and activity may be dictated by other residues. These results suggest that neuraminidase plays some role in the induction of apoptosis and that it acts prior to or during virus entry. However, apoptosis was considerably reduced when UV-irradiated virus, which retains >75% of its neuraminidase activity, was used. In addition, ammonium chloride, used to prevent virus entry, reduced virus-induced apoptosis. Amantadine, which inhibits virus uncoating, also inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2), but not the amantadine-resistant clone 7a. Hence, one or more intracellular processes are also involved in influenza virus-induced apoptosis.
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Involvement of the cytoskeleton in Junin virus multiplication.
More LessThe role of the cellular cytoskeleton in Junin virus (JV) infection was explored in two ways. Firstly, the action of inhibitors that affect individual cytoskeletal systems (microtubules or microfilaments) selectively was analysed. It was found that perturbations of microtubule or microfilament networks caused by colchicine, nocodazole, nifedipine, EGTA or DMSO strongly affected virion production and viral protein expression at non-cytotoxic concentrations. Secondly, the extent of association of viral proteins and infectious virus particles with the cytoskeletal fraction of monkey Vero cells was determined by using three non-ionic detergents, Triton X-100 (TX-100), NP-40 and octyl glucoside (OG). The cytoskeleton retained nearly 70% of the external JV envelope glycoprotein GP38 and about 40% of the JV nucleoprotein NP, according to TX-100 and OG insolubility results. Furthermore, 1% of the total cell-bound infectivity was detected in the detergent-insoluble fraction, suggesting that cytoskeletal components are involved in the initiation of the assembly and budding processes of JV particles at the plasma membrane.
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Isolation and biological characterization of 3(2H)-isoflavene-resistant and -dependent poliovirus type 2 Sabin mutants.
More LessPoliovirus type 2 Sabin mutants were selected for drug resistance and dependence by plating on HeLa cell monolayers in the presence of 3(2H)-isoflavene, a compound related to dichloroflavan, which prevents the shut-off of host translation and poliovirus RNA and protein synthesis. The drug-resistant mutants grew equally well in the presence and in the absence of the drug, while the drug-dependent mutants only grew in the presence of the compound. One dependent and one resistant mutant were characterized biologically in more detail. The resistant mutant did not exhibit thermolability. The mild thermolability exhibited by the dependent mutant was not affected by the addition of 3(2H)-isoflavene, indicating that the substance does not bind the poliovirus type 2 Sabin capsid. The translation of viral proteins and the shut-off of host protein translation during cell infection were not inhibited in either mutant. In the absence of the drug, the cleavage of the precursor VPO, a step in virus protein processing, was affected in the dependent mutant. The dependence of the mutant on the drug was due to the inability of 75S empty particles to reach maturation: our results strongly suggest that this phenomenon is strictly dependent on the reduction of RNA synthesis, confirming the existence of a dynamic equilibrium between RNA production and genome encapsidation during the poliovirus replication cycle.
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A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis.
Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A-E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT-PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92% nucleotide identity across ORF1 and 88% in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83% nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40% of the HEV-infected patients in this study were genotype 4 positive.
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Sequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and Asia
More LessThe epidemiology of tick-borne encephalitis virus was investigated by comparative sequence analysis of virus strains isolated in endemic areas of Europe and Asia. Phylogenetic relationships were determined from the nucleotide and amino acid sequences of the major envelope (E) protein of 16 newly sequenced strains and nine previously published sequences. Three genetic lineages could be clearly distinguished, corresponding to a European, a Far Eastern and a Siberian subtype. Amino acids characteristic for each of the subtypes (‘signature’ amino a cids) were identified and their location in the atomic structure of protein E was determined. The degree of variation between strains within subtypes was low and exhibited a maximum of only 2.2% at the amino acid level. A maximum difference of 5.6% was found between the three subtypes, which is in the range of variation reported for other flaviviruses.
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Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against hepatitis C virus.
The capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) was assessed. BALB/c mice were immunized with peptides based on the CTL and helper T cell (Th) epitopes of the HCV core, with a mixture of CTL and Th peptides (CTL+Th) or with a conjugated Th-CTL peptide. Mice immunized with CTL, CTL+Th and Th-CTL peptides, but not those immunized with Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic activity induced by immunization with Th-CTL was much higher than that induced by immunization with CTL+Th or CTL alone. However, rapid and high cytotoxic activities against HCV core were not only detected after immunization with peptides containing the CTL epitope but also as a result of infection with recombinant vaccinia virus carrying the HCV core gene after immunization with the Th epitope alone. Immunization with peptides containing the Th epitope also elicited spleen cell proliferation. This study demonstrates the capacity of both Th and CTL activated peptide vaccines to elicit CD8+, MHC class I-restricted CTLs. The capacity of such CTLs to contribute towards a protective and/or pathogenic immune response against HCV can now be assessed in mouse models.
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Molecular characterization and expression of the S3 gene of muscovy duck reovirus strain 89026.
Although reovirus infection is one of the major virus diseases of muscovy ducks in France, no vaccine is available and nothing is known about the structure and function of the genes and proteins of the reovirus involved. The complete S3 genome segment of the muscovy duck reovirus strain 89026 has been cloned and the nucleotide and deduced amino acid sequences are reported here. The S3 genome segment is 1201 bp long and possesses the same terminal motifs (5′ GCTTTTT and TATTCATC 3′) as the S3 genome segment of known chicken reovirus strains. It contains one open reading frame that encodes a protein of 367 amino acids with a molecular mass of 40.8 kDa. The gene, encoding the sigmaB major outer-capsid protein, was cloned into two different baculovirus transfer vectors and expressed in insect cells as a glutathione S-transferase fusion protein or a non-fused protein. The antigenicity of the two recombinant proteins was demonstrated by immunoblot assay. The potential immunogenic role of sigmaB protein was studied in a protection assay against reovirus infection of specific-pathogen-free muscovy ducks. No antibodies could be detected by ELISA or immunoblot in ducks immunized with the recombinant proteins and no significant protection was noted after the challenge. However, whereas the weights of wild-type baculovirus-infected and challenge-control ducks were significantly lower than those of unchallenged ducks, the weights of male ducks previously immunized with the sigmaB recombinant proteins did not differ significantly from males of either group. This work is the first to provide molecular data for a duck reovirus.
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Molecular characterization of double-stranded RNA segments encoding the major capsid proteins of a Palyam serogroup orbivirus that caused an epizootic of congenital abnormalities in cattle.
More LesscDNA cloning of the double-stranded RNA genome of Chuzan virus, a member of the Palyam serogroup orbiviruses, was carried out and the complete nucleotide sequences of RNA segments 2, 3, 6 and 7, encoding the major capsid proteins VP2, VP3, VP5 and VP7, respectively, were determined. The individual segments had single open reading frames and short inverted repeats adjacent to the conserved terminal sequences. Comparative sequence analysis with other serogroups of the genus Orbivirus suggested that VP2 is the principal determinant of serotype specificity and the neutralizing antigen of the Palyam serogroup. VP5 is also considered to be associated with antigenic variability. Both VP3 and VP7 probably contain serogroup-specific epitopes. Phylogenetic profiles demonstrated that the Palyam serogroup virus is more closely related to African horsesickness virus than to bluetongue virus and epizootic haemorrhagic disease virus.
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Role of class I major histocompatibility complex-restricted and -unrestricted suppression of human immunodeficiency virus type 1 replication by CD8+ T lymphocytes.
CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (ACs) are capable of suppressing HIV-1 replication in CD4+ peripheral blood mononuclear cells (PBMC) by a variety of known and unknown mechanisms. In the present study, cell contact-dependent, major histocompatibility complex type I (MHC I)-unrestricted, CD8+ cell-mediated suppression of HIV-1 LAI replication was detected. CD8+ PBMC of ACs suppressed HIV-1 replication more efficiently in MHC I-matched CD4+ PBMC than in mismatched cells. However, even when MHC I was totally mismatched, CD8+ cells still suppressed replication to a considerable extent in CD4+ PBMC. This MHC I-unrestricted, CD8+ cell-mediated HIV-1 suppression required cell contact and was not effective against cells of the established T cell line ILT-KK. In contrast, MHC I-restricted HIV-1 suppression by CD8+ T cells was detected when ILT-KK cells were used as a target. By using these systems, we examined MHC I-restricted and -unrestricted suppressive activities of CD8+ cells in various donors in more detail. Although both types of CD8+ cell-mediated HIV-1 suppression diminished at the advanced stage of the infection, MHC I-unrestricted suppression diminished earlier than MHC I-restricted suppression, in parallel with the decline in CD4+ T cells. These results suggest that suppression by the MHC I-restricted mechanism alone may fail to protect against CD4+ T-cell loss at the late stage of infection.
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A human immunodeficiency virus type 1 Env-granulocyte-macrophage colony-stimulating factor fusion protein enhances the cellular immune response to Env in a vaccinia virus-based vaccine.
Vaccinia virus (VV) infection induces protective T- and B-cell responses, making recombinants based on VV good candidates for the development of effective vaccines to other viruses. VV recombinants expressing the human immunodeficiency virus (HIV) envelope protein (Env) have been generated in several laboratories and shown to induce anti-HIV cellular and humoral immune responses in vaccinated humans and in chimpanzees. To increase the immunogenicity of the Env antigen, a VV recombinant was generated that expresses a chimeric antigen consisting of the Env protein fused to an immunostimulatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The chimeric protein retained GM-CSF biological activity when expressed by this recombinant virus (VV-GM-gp120) in cells infected in vitro. Infection of BALB/c mice with VV-GM-gp120 triggered a higher HIV-specific cellular immune response, as measured by interferon-gamma production, than that induced by a VV recombinant expressing the native Env protein. Moreover, although anti-gp120 antibody titres were similar in sera from mice inoculated with either of the VV recombinants, immunization with the recombinant expressing the fusion protein elicited antibodies against a broader spectrum of Env epitopes. These results indicate that HIV Env antigen fusion to GM-CSF provides a means to improve the anti-HIV immune response.
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Properties and mechanism of action of a 17 amino acid, V3 loop-specific microantibody that binds to and neutralizes human immunodeficiency virus type 1 virions.
More LessOnly two virus-neutralizing peptide microantibodies (MicroAbs) have been described and little is known about their mode of action. This report concerns a 17 amino acid cyclized MicroAb, derived from the third complementarity-determining region of the heavy chain of MAb F58 (IgG1), that recognizes the same minimum epitope in the V3 loop of the gp120 envelope protein of human immunodeficiency virus type 1 (HIV-1) as the MAb. The MicroAb was able to bind to and neutralize free virus particles. It was up to 5-fold more efficient in mass terms than F58 IgG and its neutralization rate on a molar basis was only 32-fold lower. The mechanism of neutralization of the MicroAb was also investigated. A high level of neutralization (99%) occurred without any significant decrease in attachment of virus to target C8166 cells. Neutralized virus attached to CD4, the HIV-1 primary receptor. Fusion of virions to cells was partially inhibited by the MicroAb, whereas F58 IgG has been shown to inhibit fusion significantly. Thus, neutralization by the MicroAb appears to be mediated, at least in part, by inhibition of fusion. Control peptides, in which the tyrosine at position 5 or 6 was deleted or changed to phenylalanine, showed no antiviral activity, attesting to the specificity of interaction of the MicroAb with the virion. It therefore appears that the MicroAb acts like an immunoglobulin. The data also show that the MicroAb/MAb F58 epitope on the V3 loop is not involved in attachment of virus to CD4 but is required for subsequent events in early infection.
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The antibody response of cattle infected with bovine immunodeficiency virus to peptides of the viral transmembrane protein.
More LessThe development of the antibody response to peptides of the transmembrane glycoprotein of bovine immunodeficiency virus (BIV) was followed over a period of 50 weeks in six cattle experimentally infected with the BIV(FL112) isolate. Antibody was detected by an enzyme immunoassay using either a linear or a cyclized peptide with structural features common to an immunodominant region of other lentiviruses. The assay was specific for BIV, detecting antibody in bovine sera to BIV(FL112) or BIV(R29) but not to six other common viruses of cattle. Antibody was present in the sera of all cattle inoculated with BIV(FL112) within 4 weeks of infection, peaked between 10 and 30 weeks and persisted in most cattle during the 50 weeks of observation. These features indicate that this assay may be useful in identifying cattle infected with other strains of BIV in the field.
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Comparative sequence analysis and predictions for the envelope glycoproteins of foamy viruses.
More LessThe foamy viruses (FVs) are a genus of complex retroviruses that has recently been found to possess several novel molecular features. There is increasing interest in the development of FVs as novel vectors for gene delivery. As there are remarkably few published studies of FV proteins, these recent findings prompted us to predict the structural features of FV glycoproteins with the aid of computer programs. We analysed all seven available FV Env sequences, a greater number of sequences than in previously published analyses. The relative rates of change for FV structural proteins were Pol < Env < Gag in increasing order, which differs from all other retroviruses. We determined that this difference is primarily caused by a higher relative rate of change for FV Gag proteins. We analysed the functional domains of FV glycoproteins and found that their structural organization was generally similar to other retroviruses. Putative structures were identified for the signal peptide, cleavage site, fusion peptide, membrane-spanning domain and the unique endoplasmic reticulum retrieval signal. Based on the predicted secondary structure of the transmembrane glycoprotein (TM) subunit, gp47, we also identified a unique prolonged central ‘sheets and loops’ region as the dominant feature of an unusually lengthy TM ectodomain. This lengthy central domain was flanked at each end by alpha-helices. The predictions reported here will stimulate and facilitate experimental approaches to better understand the structure and function of FV glycoproteins, and should assist in the planning and development of FV vectors.
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Diversity of human endogenous retrovirus class II-like sequences.
Class II human endogenous retroviruses (HERVs), often referred to as mouse mammary tumour virus (MMTV)-like or HERV-K elements, have similarities to several animal infectious retroviruses. Single clones from each of nine class II HERV groups (NMWV 1 to NMWV 9), isolated from a human breast cancer cell genomic library, were sequenced over a 244 bp stretch of the conserved reverse transcriptase region. These sequences were aligned to related exogenous and endogenous retroviruses and a phylogenetic tree was constructed. Sequences with more than 80% identity were considered as members of one group and we report here that the class II HERV family consists of at least ten groups. Three of the sequenced clones, from groups NMWV 3, 7 and 9, could not be related to any other previously identified elements and constituted their own groups. NMWV 8 had no similarity to any retroviral sequences in the sequenced region and is so far considered to be non-retroviral.
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Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus.
More LessHPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, is a recently identified retrovirus associated with myeloid leukosis in meat-type chickens. Although this virus shows high sequence identity to other ALV subgroups within the gag and pol genes, its env gene is highly diverged (with only about 40% sequence identity) from other ALV subgroups. On the other hand, the sequence of the env gene of HPRS-103 was 75% identical to that of E51, a member of the EAV family of endogenous avian retroviruses. It is reported here that the chicken genome also contains another EAV-related element, EAV-HP, showing much greater sequence identity (over 97%) to the HPRS-103 env gene. Southern blotting analysis showed that EAV-HP-related sequences were distinct from EAV-O and were present in all lines of chicken examined and in grey jungle fowl, but were absent from several other avian species. The potential role of these endogenous sequences in the evolution of ALV subgroup J viruses is discussed.
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The minor coat protein of beet yellows closterovirus encapsidates the 5′ terminus of RNA in virions.
More LessFilamentous particles of beet yellows closterovirus (BYV) are built of two related capsid proteins, of which the minor species, p24, forms a 75 nm tail at one end of the virion. In the present work, we used polyclonal antibodies against p24 for isolating the ‘tailed’ virion segments from sonicated BYV particle preparations. The [gamma-32P]ATP-labelled RNA obtained from the antibody-selected particle segments consistently showed stronger hybridization with the 5′-terminal BYV cDNA clones than with the 3′-terminal cDNA clones. These data clearly indicate that it is the 5′-terminal portion of the closterovirus RNA genome that is encapsidated by p24.
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An analysis of herpes simplex virus gene expression during latency establishment and reactivation
More LessIn order to facilitate an analysis of the pattern of herpes simplex virus gene expression during latency establishment and reactivation, recombinant viruses containing the lacZ reporter gene under control of either the immediate early 110 (IE110) promoter or the latency-associated promoter have been constructed. Histochemical staining of ganglia taken from mice infected with these viruses allows for the rapid identification and quantification of sensory neurones in which these two promoters are active. Using the mouse ear model, this study demonstrates that, during the establishment of latency in vivo, IE110 promoter activity is only detectable in ganglia which provide innervation to the site of virus inoculation. Latency, however, is efficiently established not only in these ganglia, but also in adjacent ganglia whose neurones do not innervate the ear, and in which there was no evidence of IE110 expression during the acute phase of infection. This implies that replication-competent virus can efficiently establish latency in the absence of detectable IE110 expression. In addition, it has been possible to investigate viral gene expression in neurones following ganglionic explant culture by monitoring IE110 promoter-driven lacZ expression within reactivating neurones. This study shows that virus can be reactivated from all latently infected ganglia, but that reactivation appears to be more efficient from ganglia which provide innervation to the site of infection. The implications of these results for the mechanisms involved in latency establishment and reactivation are discussed.
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Similarities in the genome organization of tobacco rattle virus and pea early-browning virus isolates that are transmitted by the same vector nematode.
More LessAlthough sequence data have been obtained for several tobravirus isolates, only two of these isolates are nematode-transmissible. Tobacco rattle virus (TRV) PpK20 is transmitted by Paratrichodorus pachydermus, whereas pea early-browning virus (PEBV) TpA56 is transmitted by Trichodorus primitivus. To clarify whether differences in the genome structure of these isolates are relevant to the specificity of interactions with particular vector nematodes, or merely reflect a taxonomic difference between TRV and PEBV, we have sequenced RNA2 of a new isolate of TRV (TpO1) that is transmitted by the same vector nematode as PEBV TpA56 but is not transmitted by the nematode vector of TRV PpK20. TRV TpO1 RNA2 encodes, in 5′ to 3′ order, a coat protein (CP), a 9K protein, a 2b (29K) protein and a 2c (18K) protein. Amino acid sequence comparison shows that both the CP and 2b proteins of TRV TpO1 resemble more closely the analogous proteins from PEBV TpA56 than those from TRV PpK20. Also, the TRV TpO1 9K protein has similarities with the PEBV 9K protein whereas this protein is lacking in TRV PpK20.
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Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus
More LessThe genome of the nucleopolyhedrovirus (NPV) (T3 strain) pathogenic for Bombyx mori (Bm) was sequenced and analysed. The BmNPV genome was 128413 nucleotides long with a GMC content of 40% and contained 136 open reading frames (ORFs) encoding predicted proteins of over 60 amino acids. Although phenotypically different, the genome organizations of BmNPV and Autographa californica multinucleocapsid NPV (AcMNPV) were closely related. The BmNPV genome was over 90% identical to about three-quarters of the genome of AcMNPV. The relatedness of predicted amino acid sequences of corresponding ORFs between BmNPV and AcMNPV was about 90%. However, the BmNPV genome lacked homologues of the following AcMNPV ORFs: Ac3 (conotoxin), Ac7 (orf603), Ac48 (etm), Ac49 (pcna), Ac70 (hcf-1), Ac86 (pnk/pnl) and Ac134 (p94). In addition, BmNPV contained five ORFs related to Ac2. A high frequency of multiple 3 bp insertions was also found within BmNPV and AcMNPV coding sequences.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 100 (2019)
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Volume 99 (2018)
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Volume 98 (2017)
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Volume 97 (2016)
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Volume 96 (2015)
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Volume 95 (2014)
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Volume 94 (2013)
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Volume 93 (2012)
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Volume 92 (2011)
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Volume 91 (2010)
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Volume 90 (2009)
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Volume 89 (2008)
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Volume 88 (2007)
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Volume 87 (2006)
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Volume 86 (2005)
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Volume 85 (2004)
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Volume 84 (2003)
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Volume 83 (2002)
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Volume 78 (1997)
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Volume 77 (1996)
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Volume 76 (1995)
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Volume 75 (1994)
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Volume 74 (1993)
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Volume 73 (1992)
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Volume 72 (1991)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)