- Volume 75, Issue 9, 1994
Volume 75, Issue 9, 1994
- Review Article
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- Bacterial
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Restriction map of the genomic DNA of Lactobacillus casei bacteriophage PL-1 and nucleotide sequence of its cohesive single-stranded ends
More LessA restriction map of the genomic DNA of Lactobacillus casei phage PL-1 was constructed using the restriction endonucleases BamHI EcoRI, HindIII, KpnI, NruI and XhoI. The PL-1 genome was 42·2 kb in size and had complementary cohesive ends forming a ring-like monomer. The cohesive ends, analysed with exonuclease III and SI nuclease, were 3 -terminated single strands protruding from both ends. The nucleotide sequence of the cohesive ends, determined by the dideoxynucleotide method, comprised four A + T and 10 G + C pairs: 5′ GAGGCCGACCGTTC 3′ /3′ CTCCGGCTGGCA- AG 5′. Thus, the cohesive ends of PL-1 DNA were higher in G + C content than those of other known bacteriophage DNAs.
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Characterization of two novel filamentous phages of Xanthomonas
Two filamentous phages of Xanthomonas campestris pv. vesicatoria and Xanthomonas oryzae pv. oryzae were isolated and designated ϕXv and ϕXo, respectively. They were similar to other filamentous phages of Xanthomonas in (i) shape, (ii) restrictive host specificity, (iii) high stability, (iv) an ssDNA genome, (v) a dsDNA as the replicative form (RF), (vi) propagation without lysis of host cells and (vii) ability to integrate into the host chromosome. These phages showed sequence homology to filamentous phage ϕLf of X. c. pv. campestris. ϕXv was inactivated by antisera against ϕXv, ϕXo and ϕLf, whereas ϕ Xo and ϕLf were inactivated only by their respective antisera and the anti-ϕXv serum. Both the single-stranded phage DNAs and the RF DNAs of ϕXv, ϕXo and ϕLf were able to transfect X. c. pv. vesicatoria, X. o. pv. oryzae and X. c. pv. campestris. Physical maps of ϕXv and ϕXo were constructed for the RF DNAs. Genome sizes were estimated, based on mapping data, to be 6·8 kb for ϕXv and 7·6 kb for ϕXo, larger than that of the ;ϕLf genome (6·0 kb). The difference in genome sizes appeared to result from insertions of large DNA fragments. These fragments and the regions mediating integration were localized in the physical maps.
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- Animal
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Experimental infection of mink with bovine spongiform encephalopathy
To determine whether the aetiological agent of bovine spongiform encephalopathy (BSE) is pathogenic for mink, standard dark mink were inoculated with coded homogenates of bovine brain from the U.K. Two homogenates were from cows affected with BSE. The third was from a cow that came from a farm with no history of having had BSE or having been fed ruminant- derived, rendered by-products, the proposed vehicle for introduction of the BSE agent. Each homogenate was inoculated intracerebrally into separate groups of mink and a pool of the three was fed to a fourth group. Signs of neurological disease appeared in mink an average of 12 months after intracerebral inoculation and 15 months after feeding. Decreased appetite, lethargy and mild to moderate pelvic limb ataxia were the predominant clinical signs, quite unlike the classic clinical picture of transmissible mink encephalopathy (TME). Microscopic changes in brain sections of most affected mink were those of a scrapie-like spongiform encephalopathy. Vacuolar change in grey matter neuropil was accompanied by prominent astrocytosis. Varying greatly in severity from one mink to another, the degenerative changes occurred in the cerebral cortex, dorsolateral gyri of the frontal lobe, corpus striatum, diencephalon and brainstem. Although resembling TME, the encephalopathy was distinguishable from it by less extensive changes in the cerebral cortex, by more severe changes in the caudal brainstem and by sparing of the hippocampus. The results of this study extend the experimental host range of the BSE agent and demonstrate for the first time the experimental oral infection of mink with a transmissible spongiform encephalopathy agent from a naturally infected ruminant species.
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Detection of human antibodies to Crimean-Congo haemorrhagic fever virus using expressed viral nucleocapsid protein
More LessDiagnosis of Crimean-Congo haemorrhagic fever (CCHF) virus infections is hampered by the problems of handling this human pathogen, which requires the highest levels of biological containment. Recombinant antigens were examined for their potential as nonhazardous diagnostic reagents. The nucleocapsid (N) gene of the Greek AP92 isolate of CCHF virus was sequenced from cloned PCR products and the open reading frame was identified by homology to the N protein of a Chinese isolate of CCHF virus. The N protein was expressed to high levels in a baculovirus expression system. Three N protein-derived peptides were expressed in Escherichia coli as fusions with glutathione S-transferase and the antigenicities of these proteins and the baculovirus-expressed protein were tested by ELISA. When tested with laboratory animal sera representing all seven serogroups of nairoviruses, the only reactive sera were those raised to CCHF virus (Greek, Nigerian and Chinese isolates) and, more weakly, Hazara virus. When tested with a panel of known positive and negative human sera, the baculovirus-expressed N protein, and the peptide derived from the central region of the N protein, proved to be the best for identifying CCHF virus-specific IgG.
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Measles virus receptor properties are shared by several CD46 isoforms differing in extracellular regions and cytoplasmic tails
Human CD46, a member of the family of regulators of complement activation, has been shown recently to act as a measles virus (MV) receptor, interacting with the virus envelope glycoprotein haemagglutinin (HA). Owing to alternative RNA splicing, several CD46 isoforms are co-expressed in all tissues except erythrocytes. The optional exons encode extracellular serine-, threonine- and proline-rich regions of CD46 (designated STP-A, -B and -C) which are located proximal to the plasma membrane, and alternative cytoplasmic tails (CYT1 or CYT2). The ability of the BC-CYT2, B-CYT2 and BC-CYT1 CD46 isoforms, expressed in rodent Chinese hamster ovary (CHO) cells, to mediate MV infection was tested. Every isoform was recognized by a monoclonal antibody (MAb), MCI20.6, which recognizes the MV-binding site on CD46. CHO cells expressing any of these CD46 isoforms were able to bind MV, the level of binding correlating with the CD46 expression level. Likewise, MV infection induced the cell-cell fusion of all CD46-expressing CHO cells but not of the parental CHO cells. Accordingly, MV replication was observed after infection of CHO cells expressing each CD46 isoform but not after infection of parental CHO cells. Finally, cell surface expression of every isoform was decreased after infection by MV. Altogether these data showed that the specific STP regions of CD46 played no major role in HA-mediated MV binding to CD46, virus infection and virus-induced down-regulation of CD46. Moreover, the CYT1 and CYT2 cytoplasmic tails of CD46 are either functionally similar although having distinct amino acid sequences or are dispensable for interaction with HA of MV.
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Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein
More LessThe haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome, (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties, (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface, (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.
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Potential for transmission of avian influenza viruses to pigs
Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. Pigs have been suggested to generate such reassortants as intermediate hosts. In order for pigs to serve as ‘mixing vessels’ in genetic reassortment events, they must be susceptible to both human and avian influenza viruses. The ability of avian influenza viruses to replicate in pigs, however, has not been examined comprehensively. In this study, we assessed the growth potential of 42 strains of influenza virus in pigs. Of these, 38 were avian strains, including 27 with non-human-type haemagglutinins (HA; H4 to H13). At least one strain of each HA subtype replicated in the respiratory tract of pigs for 5 to 7 days to a level equivalent to that of swine and human viruses. These results indicate that avian influenza viruses with or without non-human-type HAs can be transmitted to pigs, thus raising the possibility of introduction of their genes into humans. Sera from pigs infected with avian viruses showed high titres of antibodies in ELISA and neutralization tests, but did not inhibit haemagglutination of homologous viruses, cautioning against the use of haemagglutination-inhibition tests to identify pigs infected with avian influenza viruses. Co-infection of pigs with a swine virus and with an avian virus unable to replicate in this animal generated reassortant viruses, whose polymerase and HA genes were entirely of avian origin, that could be passaged in pigs. This finding indicates that even avian viruses that do not replicate in pigs can contribute genes in the generation of reassortants.
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Persistent influenza C virus possesses distinct functional properties due to a modified HEF glycoprotein
More LessA model of long term viral persistence has been established by selecting a spontaneous mutant strain of influenza C/Ann Arbor/1/50 virus in a permanent carrier culture of MDCK cells. Infectivity and cell tropism are mainly determined by the multifunctional viral membrane glycoprotein (HEF). HEF analysis was aimed at identifying a putative correlation between sequence and function, i.e. receptor binding, enzymatic activity, antigenicity and rate of infection. The current experimental picture is summarized by the following findings: (i) C/Ann Arbor/1/50 persistent virus carries a modified receptor-binding sequence, (ii) receptor-binding activity is altered, as indicated by a higher efficiency in recognizing low amounts of the receptor determinant N-acetyl-9-O-acetylneuraminic acid, (iii) direct attachment to cell surfaces differs from that of wild-type virus, as measured by slower kinetics of viral elution, (iv) receptor-destroying enzymatic activity is diminished, (v) characteristic features of virion surface morphology are altered or unstable, (vi) persistent-type HEF epitopes are distinguishable by monoclonal antibodies from wild- type and (vii) viral infectivity is intensified for cells bearing a low number of receptors. The sum of these changes highlights a structurally and functionally modified HEF glycoprotein that allows long term viral persistence. In order to clarify which of the described points are required for the persistent viral phenotype, a working concept is presented.
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Action of brefeldin A on translation in Semliki Forest virus-infected HeLa cells and cells doubly infected with poliovirus
More LessBrefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFA-treated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.
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Sequence analysis of two porcine rotaviruses differing in growth in vitro and in pathogenicity: distinct VP4 sequences and conservation of NS53, VP6 and VP7 genes
More LessThe VP4, VP7, NS53 and VP6 genes of two porcine rotavirus variants which differ in their in vitro growth properties and pathogenicity have been cloned and sequenced. The VP4 genes show only 67·2% nucleic acid and 70·6% amino acid identity. The VP4 gene of one variant (4S) is closely related to that of the bovine UK rotavirus strain, whereas the VP4 gene of the other variant (4F) is only distantly related to known VP4 genes and is likely to represent a new P serotype. In contrast the NS53 (VP5), VP6 and VP7 genes of the 4F and 4S variants show greater than 99 % nucleotide and amino acid identity, indicating that the two viruses are genetically related by a reassortment event. The implications for the role of VP4 in the determination of in vitro growth characteristics and pathogenicity are discussed.
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Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus
More LessProcessing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rWs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rW carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rW carrying the gag and pro gene or a rW expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-,pro- andpol- expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rWs expressing gag and pro or gag, pro and pol formed mature particles.
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T cell proliferative response to bovine leukaemia virus (BLV): identification of T cell epitopes on the major core protein (p24) in BLV-infected cattle with normal haematological values
More LessPeripheral blood mononuclear cells (PBMCs) from bovine leukaemia virus (BLV) -seronegative cattle and from BLV-seropositive cows either with normal haematological values or persistent lymphocytosis were tested for their proliferative response to BLV antigens. Cells from only BLV-infected cattle with normal lymphocyte counts were stimulated to a detectable level by the fetal lamb kidney cell supernatant containing BLV antigens. Proliferation assays performed with the purified major core protein p24 indicated that this protein has to be processed through a chloroquine-sensitive compartment before being recognized by CD4+ T lymphocytes. Forty-one 15-mer overlapping peptides spanning the entire p24 sequence were synthesized and analysed for their stimulating potential. It appeared that two regions included T cell epitopes recognized by PBMCs from three of five animals tested. These regions were represented by amino acids 31 to 55 (PGSQVWIQTL-RLAILQADPTPADLE) and 141 to 165 (AESYVE-FVNRLQISLADNLPDGVPK). The possible implication of this cell-mediated immune response in BLV pathogenesis and vaccine development is discussed.
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Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding
More LessHuman T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration. A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14. Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used. Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides. Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation. This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases. As the N- and C-terminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel β-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PR14. Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective. An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14.
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Persistent infection of MT-4 cells by human immunodeficiency virus type 1 becomes increasingly likely with in vitro serial passage of wild-type but not nef mutant virus
More LessOur previous studies have shown that human immunodeficiency virus type 1 (HIV-1), with mutations in accessory genes such as vif vpr or vpu, can generate persistent infection of MT-4 cells, whereas infection by wild-type or nef mutant HIV-1 causes extensive cell death. The possibility of generating a naturally attenuated form of HIV-1 with reduced cytopathogenicity in MT-4 cells was examined by in vitro serial passage of the wild-type and a nef mutant form of HIV-1, each derived from the infectious molecular clone pNL432. The ability to cause persistent infection was observed after four passages of wild-type HIV-1 with the frequency of persistence becoming progressively higher with serial passage. In contrast, persistent infection was not observed even after 50 passages of the nef mutant virus. Sequence analysis of the accessory gene loci in genomes recovered from the persistent infections caused by passaged virus revealed mutations in vif and vpr, but not in vpu. The processing of the Env precursor to mature forms was not modified in any of the passages of either wild-type or nef mutant HIV-1. However, when compared with acute infections caused by similarly passaged virus of both wild-type and ne/mutant HIV-1, persistent infections by passaged wild-type HIV-1 showed a significant decrease in the cell surface expression and function of Env. Cell surface CD4 was only partially down-regulated on cells acutely infected with the passaged viruses, whereas on cells persistently infected with passaged wild-type HIV-1 it was completely down- regulated. These results suggest that, during serial passage of HIV-1, mutations accumulate at least in the accessory genes vif and vpr in parallel with a lesser interaction between cell surface Env and CD4 molecules, and lead to the generation of less cytopathogenic viruses capable of persistent infection. Our results also suggest an important role for the nef gene product in the generation of HIV-1 strains that are less cytopathogenic.
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Conditional regulatory elements of human immunodeficiency virus type 2 long terminal repeat
More LessMutational analysis of the human immunodeficiency virus type 2 (HIV-2) long terminal repeat (LTR) revealed a novel cis-acting positive and a negative regulatory element in the U3 region, located upstream of the enhancer-promoter region. These elements acted in a cell type-specific manner, being most active in human lymphocytic CEM cells, more active in Jurkat cells than in human monocytic U937 cells and least active in epithelioid HeLa cells. The down-modulatory effect of the negative regulatory element was abolished by HIV-2 Tat, suggesting the involvement of upstream DNA elements in optimal Tat-mediated trans-activation. The sequence elements that respond to T cell activation signals were also located in the upstream U3 region. Notably, the magnitude of the effect of the upstream regulatory elements depended on the basal activity of the LTR, which was also cell type-dependent. This emphasizes the importance of the cell-specific transcriptional factors and other effectors in regulating HIV gene expression. These observations may be relevant to the cell type-specific restriction of virus replication in vivo.
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Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3
More LessThe three avian polyomaviruses budgerigar fledgling disease virus types 1 to 3 (BFDV−1 to −3) contain genomes of identical size, 4981 bp. With differences of up to only 15 bp between the three genomes, these viruses show distinct tropism for cultured cells of various avian species: infection of chicken embryo (CE) cells with BFDV−1 and −2 results in virus propagation, whereas BFDV-3 is not replicated; all three viruses replicate, with different efficiencies, in infected Muscovy duck cells. Transfection of CE cells with BFDV-3 DNA results in a single productive cycle. As shown by construction of hybrid virus genomes and site-directed mutagenesis, a single amino acid difference (glycine instead of valine or alanine) within the common region of the minor structural proteins VP2/VP3 is responsible for this type of abortive infection of CE cells. Further experiments indicate a defect in one of the early steps during infection, at or prior to uncoating.
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Assembled baculovirus-expressed human papillomavirus type 11 L1 capsid protein virus-like particles are recognized by neutralizing monoclonal antibodies and induce high titres of neutralizing antibodies
Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (LI) virus-like particles (VLPs) were produced in insect cells and purified on CsCl density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 LI
VLPs were variable in size with a surface topography similar to that of infectious HPV-11. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.
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Cell-mediated immune responses to E7 peptides of human papillomavirus (HPV) type 16 are dependent on the HPV type infecting the cervix whereas serological reactivity is not type-specific
Forty-two women attending a colposcopy clinic for evaluation of abnormal cervical cytology and 13 normal controls were studied for the presence of lymphocyte proliferation (LP) cell-mediated immune (CMI) responses and serological reactivity to E7 peptides of human papillomavirus type 16 (HPV-16). HPV was typed by Southern blot hybridization of exfoliated cervicovaginal cell DNA. Positive LP responses (stimulation index ⩾ 5·0) to one or more E7 peptides were observed in 28·6% (12 of 42) of patients and 23·1 % (three of 13) of controls. Of patients infected with HPV-16,-31 or -33, 63·6% (seven of 11) showed a positive LP response compared with 14-3 % (two of 14) of women infected with other HPV types (P = 0·02), 17·6 % (three of 17) negative for HPV (P = 0·02) and 23·1 % (three of 13) of controls (HPV status unknown) (P = 0·05). C-terminal peptide 109 (amino acids 72 to 97) elicited positive LP responses in 45-4% (five of 11) of patients infected with HPV -16, -31 or -33 compared with 71 % (one of 14) patients infected with other HPVs (P = 0·04), 5·9 % (one of 17) of women negative for HPV (P = 0·02) and 7·7% (one of 13) of controls (P = 0·05). HPV-16 group-specific LP responses of borderline significance were also observed against E7 peptides 103,105 and 108 (17−37, 37−54 and 62−80) (P = 0·07). ELISA reactivity (IgG) to E7 peptide 109 (72−97) was present in 7·7% (one of 13) of controls, 35·3 % (six of 17) of HPV-negative patients, 42·9 % (six of 14) of patients infected with other HPVs, and only 9·1 % (one of 11) of patients infected with HPV-16, -31 or -33. CMI responses to C-terminal HPV-16 E7 peptide 109 (72−97) were thus significantly related to ongoing cervical infection with HPV-16 and closely related types, whereas serological reactivity to E7 peptides was not HPV type-specific.
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Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin
More LessThe wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).
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Herpesvirus saimiri small RNA and interleukin-4 mRNA AUUUA repeats compete for sequence-specific factors including a novel 70K protein
More LessA highly oncogenic strain of the lymphotropic tumour virus herpesvirus saimiri (HVS; strain 484-77) expresses four small RNAs (HSUR1 to 4) in high copy numbers in transformed T cells. In HSUR1 and HSUR2 the 5′ terminal regions contain conserved AUUUA sequence repeats. The same AUUUA repeats occur in the 3′ noncoding regions of growth factor, lymphokine and protooncogene mRNAs, and the sequence is involved in rapid mRNA degradation. We report here that by using a highly specific u.v. cross-linking method we identified a novel 70K binding factor with AUUUA sequence specificity. Non-radiolabelled competition and V8 protease analysis show that the protein can form a complex with the 3′ non-coding region of interleukin-4 mRNA and bind the AUUUA repeats of a HVS small RNA. We also detected an AUUUA-specific minor 32K human protein with the same electrophoretic mobility as a marmoset factor implicated in growth factor mRNA destabilization. The findings are consistent with the hypothesis that the viral small RNAs can compete for factors involved in rapid degradation of growth factor mRNAs and may contribute to viral oncogenesis.
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Identification of a putative cellular receptor for bovine herpesvirus 1
More LessRecognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.
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A glycoprotein E deletion mutant of bovine herpesvirus 1 is avirulent in calves
A marker vaccine elicits an antibody response in the host that can be distinguished from the antibody response induced by a wild-type strain. To obtain a bovine herpesvirus 1 (BHV-1) marker vaccine, we constructed a glycoprotein E (gE) deletion mutant. This was obtained by removing the complete gE coding region from the BHV-1 genome. To attenuate the gE deletion mutant further, we also introduced a small deletion in the thymidine kinase (TK) gene. We selected three mutants: the gE deletion mutant, a TK deletion mutant and a gE/TK double deletion mutant, and examined their virulence and immunogenicity in calves.After intranasal inoculation, the TK deletion mutant showed some residual virulence, whereas the gE and gE/TK deletion mutants were avirulent. The calves inoculated with the deletion mutants were protected against disease after challenge exposure and shed significantly less virus than control calves. Deleting the gE gene, therefore, has little effect on the immunogenicity of BHV-1, but is sufficient to reduce the virulence of BHV-1 in calves. These findings led us to conclude that the gE deletion mutant is a good candidate for a modified live BHV-1 marker vaccine.
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Role of envelope glycoproteins gI, gp63 and gIII in the invasion and spread of Aujeszky’s disease virus in the olfactory nervous pathway of the pig
More LessOne-week-old pigs were infected intranasally with the Ka strain of Aujeszky’s disease virus (ADV) or with mutants that were lacking the non-essential envelope glycoproteins gI, gp63 or gill. The invasion and spread of these strains in the olfactory nervous pathway were examined by assessing virus levels and by localizing viral antigens in the olfactory mucosa representing the first neuronal level, in the olfactory bulb representing the second neuronal level and in the lateral olfactory gyrus, the rostral perforated substance and the piriform lobe, all representing the third neuronal level. The Ka parental strain invaded and spread up to the third neuronal level. The extent of invasion and spread of the gIII− mutant were similar to those of the parental strain. The gp63− mutant replicated normally in the olfactory mucosa, but its spread to all the other levels was limited as compared with that of the parental strain. The gI− mutant showed a defect in infection at all neuronal levels. These results indicate that, of the non-essential envelope glycoproteins, gl plays the major role in neural invasion and spread of ADV in its natural host. The pattern of invasion and spread of these mutants in the olfactory pathway of pigs was similar to that previously observed in the trigeminal pathway. The type of nervous pathway therefore appears not to influence the neuropathogenesis of ADV or mutants deleted in non-essential envelope glycoproteins in the pig.
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The establishment of cytomegalovirus latency in organs is not linked to local virus production during primary infection
More LessRecovery from primary cytomegalovirus (CMV) infection is associated with resolution of the productive infection without clearance of the virus genome from affected organs. The presence of latent CMV genome in multiple organs provides the molecular basis for recurrence of CMV within multiple organs, and explains the diversity in the organ manifestations of recrudescent CMV disease during states of immunodeficiency. As a part of a unifying concept of multifocal CMV latency and recurrence, previous work has demonstrated the importance of primary virus replication for the overall load of latent CMV in organs and the risk of recurrence. In the present report, the establishment of CMV latency was studied in a murine model in which the course of primaiy infection in the immunocompromised host after syngeneic bone marrow transplantation was modulated by a CD8+ T cell immunotherapy. The antiviral CD8+ effector cells limited virus replication in all organs and protected the recipients from lethal CMV disease, but after resolution of the productive infection virus DNA remained. Interestingly, the copy number of latent virus DNA in tissue did not quantitatively reflect the preceding virus production in the respective organ. Specifically, in contrast to the case in the lungs and the salivary glands, virus replication in the spleen was suppressed by CD8+ T cells to below the limit of detection; yet, virus DNA was also detected in the spleen during latency and accordingly, virus recurrence in the spleen could be induced. These findings demonstrate that the control of virus replication in a particular organ does not prevent the establishment of latency in that organ.
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Human cytomegalovirus late protein encoded by ie2: a trans-activator as well as a repressor of gene expression
More LessIn order to study the function of human cytomegalovirus (HCMV) immediate early gene 2 (ie2) (UL122) gene products made at late times during infection, cDNA clones were isolated from an expression library made with 74 h post-infection mRNA. Based on screening of the library, 1 % of transcripts in infected cells at this time were ie2 region-specific, and transcripts encoding γIE2338aa, a 40K late gene product, were more abundant than those encoding IE2 579aa, an a gene product made throughout infection. As expected, the cDNA capable of directing the expression of γIE2338aa was derived from a contiguous genomic region within exon 5 of the ie1/ie2 region. The cDNA clones encoding γIE2338aa and IE2579aa were compared for their ability to trans-activate viral and cellular promoters and to repress expression from the ie1/ie2 promoter via the ie2 cis-repression signal. Unexpectedly, γIE2338aa trans-activated a variety of test promoters when cotransfected with the major a gene product, IE1491aa. Promoters derived from the cellular β-actin gene, the simian virus 40 early region and the human immunodeficiency virus were all responsive to γIE2338aa plus IE1491aa, although several β promoters derived from the HCMV genome were unresponsive. Thus, this abundant late product from the ie2 region may play a role in trans-activation in addition to its role as a repressor of α gene expression.
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Identification of human herpesvirus 6 uracil-DNA glycosylase gene
More LessUracil-DNA glycosylase encoded in many species functions as a DNA repair enzyme that removes uracil residues from DNA. To investigate the potential function of uracil-DNA glycosylase encoded by human herpesvirus 6 (HHV-6), we sequenced a DNA clone (pSTY09), identified an open reading frame of 765 bp and compared the putative amino acid sequence with other uracil-DNA glycosylases, by computer analysis. The amino acid sequence of HHV-6 had similarities to other uracil-DNA glycosylases, with the highest degree of similarity to those of human cytomegalovirus and Epstein-Barr virus. Two strongly conserved regions in uracil-DNA glycosylase of other species also existed in HHV-6. The gene product which was expressed in Escherichia coli demonstrated uracil-DNA glycosylase activity. This is the first report to identify and characterize the uracil-DNA glycosylase gene in HHV-6.
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The herpes simplex virus gene UL26 proteinase in the presence of the UL26.5 gene product promotes the formation of scaffold-like structures
More LessThe herpes simplex virus type 1 (HSV-1) polypeptides encoded by genes UL26 and UL26.5 are thought to form a scaffold around which the capsid shell assembles. The UL26 gene specifies a proteinase that cleaves both itself and the UL26.5 gene product. To study the structure and function of the UL26 and UL26.5 gene products, the proteins were expressed in ceils infected with recombinant baculoviruses containing the genes under the control of the polyhedrin promoter. Both polypeptides were made in large amounts, approaching the levels of polyhedrin protein expressed in wild-type baculovirus. The UL26 polypeptide behaved in a similar manner to the protein made in HSV-l-infected cells, cleaving itself rapidly into the capsid proteins VP21 and VP24 and converting the UL26.5 product more slowly into the capsid protein VP22a. The results of immuno-blot analysis using antisera specific for the amino-terminal region of the UL26 polypeptide suggested that both the first and second ATGs in the UL26 open reading frame were recognized as translational start signals but the first ATG was the preferred initiation codon as is the case in HSV-l-infected cells. Electron microscopic examination of thin section preparations of cells infected with both the UL26.5- and UL26- recombinant baculoviruses revealed the presence of large numbers of small spherical particles, often arranged in a semi-crystalline array. These clusters of scaffold-like particles were not present in cells infected with UL26- recombinant baculovirus but were observed occasionally in UL26.5-recombinant baculovirus-infected cells. The results suggest that the proteinase, in the absence of other HSV capsid proteins, stimulates the formation of large numbers of scaffold-like particles present either as semi-crystalline arrays or as dispersed structures.
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Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2
More LessThe herpes simplex virus (HSV) gene RL1 encodes the protein ICP34·5, which is a specific neurovirulence factor. Null mutants in RL1 fail to replicate in the central nervous system of mice and are therefore totally non-neurovirulent. Additionally, they fail to replicate in neurons of the peripheral nervous system, although they are capable of establishing and reactivating from a latent infection. As the precise function of ICP34·5 in HSV-neuronal interactions is unknown, we have studied the role of ICP34·5 in vitro by examining in detail the phenotypes of RL1-negative viruses in two defined tissue culture systems. The first was mouse embryo fibroblast 3T6 cells, in which RLl-negative mutants are impaired and the in vivo phenotype is mimicked. This impairment is amplified when the cells are in the stationary state. The second was mouse embryo testicular carcinoma F9 cells which, in the undifferentiated state, provide a reversal of phenotype; wild-type virus fails to grow but RLl-negative virus replicates efficiently. Differentiation results in the ability to support wild-type virus growth. The stage at which the replication cycle is blocked plus the role of cellular factors is addressed in both tissue culture systems. Evidence is provided that cell type and cell state are crucial to ICP34·5-cellular interaction and hence, based on these parameters, ICP34·5 can be defined as a host-range determinant. Identification of cellular proteins that specifically interact with or are homologues of ICP34·5 may lead to the identification of neuron-specific proteins that have a similar role.
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Oral-oesophageal inoculation of mice with herpes simplex virus type 1 causes latent infection of the vagal sensory ganglia (nodose ganglia)
More LessHerpes simplex virus type 1 (HSV-1) gingivostomatitis during childhood is known to result in a latent infection of the trigeminal ganglion neurons, which innervate the oral mucosa. During latency the viral genome is maintained in a non-infectious state. However, stimuli such as stress, fever or localized trauma can cause HSV-1 to reactivate in neurons and produce recrudescent disease in the peripheral tissues. Recently, HSV-1 proteins and nucleic acids have been detected in biopsies from human duodenal and gastric ulcers, raising the possibility that HSV-1 latency within the enteric nervous system is involved in this chronic recurrent gastrointestinal disorder. The studies in mice described here were done to determine whether HSV-1 latency could be established in neurons that innervate the murine gut. We found that after either intraperitoneal or oral-oesophageal inoculation of mice, HSV-1 establishes a latent infection in nodose ganglia of the vagus nerve, whose sensory neurons project to the gastrointestinal tract. This animal model of HSV-1 latency in the vagal sensory ganglia will be useful to examine the possible relationship between HSV-1 and recurrent gastrointestinal disease.
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The differentiation state of monocytic cells affects their susceptibility to infection and the effects of infection by dengue virus
More LessThis study describes the susceptibility to dengue virus infection of a monocytic cell line at different states of differentiation. Infectious virus titres increased in undifferentiated U937 cells following infection with clinical isolates but only when the cells were infected via their Fc receptors. No c.p.e. was observed and virus was not secreted into supernatant fluid. Once differentiated, cells were susceptible to infection either with virus alone or with virus-antibody complexes. Infection was cytolytic and virus was released into the supernatant fluid. Similar results were obtained with freshly isolated peripheral blood monocytes. Increased blood vessel permeability, which occurs in dengue haemorrhagic fever and dengue shock syndrome patients, has been correlated with secondary heterotypic infections and has been postulated to arise from antibody-enhanced infection of monocytes. The data presented suggest a possible mechanism whereby infected monocytes undergoing diapedesis through blood vessel walls might differentiate sufficiently during the process to release virus and cytokines at localized sites on blood vessels.
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Hepatitis C virus genotype 4 is highly prevalent in central Africa (Gabon)
More LessFollowing a survey of hepatitis C virus (HCV) infection recently carried in central Africa (Gabon), we cloned and sequenced PCR products of the 5′ non-coding and capsid-encoding regions of HCV RNA from three randomly selected HCV RNA-positive Gabonese subjects. In the capsid-encoding region, the identity between the three Gabonese isolates was 91 to 98%. The three Gabonese sequences showed a divergence of 11 to 17 % from published HCV genotypes I to IV (1a, 1b, 2a and 2b) isolates and of 6 to 11 % from HCV genotype 4 isolates. Thus the Gabonese isolates, termed HC-G, belong to HCV genotype 4. Based on the sequences of the three isolates, a specific probe (cpsG) was designed to detect the HC-G genotype in 30 randomly selected anti-HCV-positive Gabonese subjects, 14 of whom were HCV RNA-positive. Analysis with cpsG showed that 10 of 14 of the HCV RNA-positive subjects were infected by the HC-G genotype. HC-G is therefore highly prevalent in the HCV RNA-positive Gabonese population. The availability of these Gabonese sequences should facilitate the design of specific serological tests for African HCV isolates.
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Protection elicited by a replication-defective adenovirus vector expressing the tick-borne encephalitis virus non-structural glycoprotein NS1
More LessTick-borne encephalitis virus (TBEV) encodes a conserved, immunogenic, non-structural protein NS1 that is glycosylated and secreted from infected cells in an oligomeric form. An adenovirus recombinant, RAd51, expressing high levels of TBEV NS1 has previously been demonstrated to protect mice against a lethal challenge with TBEV. We show here that BALB/c mice infected with TBEV experienced a transient viraemia between days 3 to 5 post-inoculation that was detectable prior to the encephalitic phase of infection. Mice vaccinated with RAd51 were protected against both the viraemic and encephalitic infections associated with the TBEV challenge. Protection was demonstrated to be due to NS1 synthesized de novo from RAd51 in the vaccinated mice. Since TBEV NS1 is expressed on the cell surface, antibody-dependent complement-mediated cytolysis (CMC) of infected cells was considered as a possible mechanism of protection. Vaccination with the recombinant adenovirus proved to be effective in a mouse strain carrying a genetic deletion in the complement receptor C5. CMC is therefore not an essential component of the observed protective immune response.
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The biological characterization of field isolates of canine distemper virus from Japan
Eight isolates of canine distemper virus (CDV) were obtained from seven dogs suffering from distemper by co-cultivation of their mononuclear cells with a marmoset B lymphoblastoid cell line, B95a. Six of the seven dogs had received one or more vaccinations. All of the isolates readily proliferated in B95a cells, but were not completely neutralized by anti-CDV canine plasma, which had high neutralizing activity against the Onderstepoort laboratory strain of CDV. Furthermore, different reactivities of monoclonal antibodies (MAbs) against CDV were observed between the field isolates and laboratory or vaccine strains of CDV in immunofluorescence studies. Immunoprecipitation analysis using MAbs detected the haemagglutinin protein of each new field isolate as 69K, 75K and 155K forms, and the fusion protein as 64K and 65K forms; the corresponding proteins of the Onderstepoort strain were detected as 75K and 61K proteins respectively. It is apparent from these results that the new field isolates of CDV have very different antigenic properties from the Onderstepoort vaccine strain.
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Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89)
More LessWe describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). The sequence of the 3′ -terminal 2483 nucleotides of the genome was found to be 95·4 % identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence. The gene coding for this structural polypeptide has been expressed in bacteria as a β-galactosidase fusion protein or using a T7 RNA polymerase-based system. The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity. The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies. When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV.
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Sequences of VP6 genes of human rotavirus strain RV3 and its vaccine derivative
More LessThe nucleotide sequence of the genomic segment encoding the major antigen, VP6, of human rotavirus strain RV3 was determined for the virus isolated from faeces. This was compared with the sequence of the cognate gene of tissue culture-adapted candidate vaccine virus that was derived from RV3 and had been passaged 30 times. A single silent nucleotide difference was detected between the two genes and the deduced amino acid sequence for both showed the highest degree of identity (⩾ 97·5%) with the VP6 proteins of rotavirus strains belonging to the same subgroup. The amino acid residues that might affect VP6 subgroup epitope type from RV3 and other viruses of the same or different subgroups were compared. The commonality of particular amino acids among strains of different subgroups suggests that the presence of all subgroup-specific amino acids may be necessary for subgroup determination.
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Major core protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes
Full-length cDNA of the RNA genome segment coding for the major core protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.
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Growth ability of human immunodeficiency virus type 1 auxiliary gene mutants in primary blood macrophage cultures
A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.
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CD3-dependent lymphocyte activation by human T cell leukaemia virus type I-producing T cells
More LessHuman T cell leukaemia virus type I-(HTLV-I) transformed cells are capable of stimulating the proliferation of normal T lymphocytes, or stimulating interleukin 2 expression in Jurkat T lymphoid cells. This effect is mediated by the CD2/lymphocyte function-associated antigen 3 (LFA-3) adhesion/signalling pathway. The current work demonstrates that CD3 is also required for this effect, suggesting that the T cell receptor (TCR)/CD3 complex is mediating this effect. However, this effect does not appear to be due to a superantigen since no change in TCR expression was found after HTLV-I-mediated proliferation, nor was proliferation inhibited by an antibody against the specific TCR expressed on Jurkat cells (TCR Vβ8).
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Monoclonal antibodies to equine arteritis virus proteins identify the GL protein as a target for virus neutralization
Monoclonal antibodies (MAbs) to equine arteritis virus (EAV) proteins were produced and characterized. The protein specificities of eight MAbs were determined definitively by immunoprecipitation of EAV proteins expressed from vaccinia virus recombinants (WRs). Included were two new WRs produced for this study, expressing the M and the GL proteins, respectively. Three MAbs were determined to be N-specific and five MAbs recognized the GL protein. One GL-specific MAb, 17F5, of the IgA class, efficiently neutralized EAV infectivity. In competitive binding assays (CBAs), the N- specific MAbs defined a single antigenic domain on this protein. Four GL-specific MAbs, including MAb 17F5, demonstrated strong reciprocal competition in binding to the GL protein but differed in their virus-neutralizing ability. Thus the antigenic domain defined by these MAbs is probably composed of overlapping or closely adjacent epitopes. The fifth GL-specific MAb, a nonneutralizing antibody, may define an epitope adjacent to this antigenic domain as reciprocal CBAs demonstrated lower competition.
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Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles
More LessThe L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.
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Detection of E5 oncoprotein in human papillomavirus type 16-positive cervical scrapes using antibodies raised to synthetic peptides
More LessPolyclonal antibodies were raised to partial and full-length synthetic peptides of human papillomavirus type 16 (HPV-16) E5. Antisera specificity for HPV-16 E5 was demonstrated by their ability to recognize not only their peptide immunogens but also full-length peptide and a glutathione S-transferase-E5 fusion protein. The most reactive antiserum, PE-6, raised to a full-length peptide, was used in Western blot analysis to identify HPV-16 E5 protein from exfoliated cervical cells. A strong, single band at approximately 20K was detected in two of six HPV-16-positive samples from women with a history of low-grade cervical intraepithelial neoplasia. The apparent M r by SDS-PAGE suggests that HPV-16 E5 forms homodimers in vivo, but not through cysteine linkage.
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Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados
The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.
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Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-β-galactosidase fusion proteins
More LessThe 605 amino acid E1 protein of bovine papillomavirus type 1 (BPV-1) is a multifunctional nuclear protein required for viral DNA replication. A nuclear localization signal (NLS) sequence was previously defined by point mutations in three short adjacent clusters of basic amino acids located in the amino-terminal region of the E1 protein. In this study, we used a fusion protein approach to evaluate the contribution of other regions of the E1 protein to nuclear transport. The nearly full- length E1 gene and six non-overlapping sub fragments were each fused in-frame with the lacZ gene in a eukaryotic expression vector. Each clone was electroporated into COS-1 cells, and the intracellular location of the E1-β-galactosidase fusion proteins was deter mined by immunofluorescence. Only the constructs containing the full-length E1 or a single subregion (E1 -259; amino acids 84 to 166) produced fusion proteins that entered the nucleus. Point mutations in the NLS sequences of the E1-259-lacZ construct prevented nuclear translocation of the corresponding fusion protein. This confirms the previous result that the cluster of basic amino acids is critical for nuclear transport. Furthermore, the data obtained in this investigation indicated that the region of E1 containing the NLS sequence was not only necessary, but was also sufficient for nuclear localization. No other region of E1 contained independent nuclear localization activity.
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Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains
More LessThe relationship between herpes simplex virus (HSV) DNA replication and establishment of latent infection was examined using an experimental model that makes use of the segmental sensory innervation of mouse flanks (T7 to T12). Ganglia from consecutive thoracic segments of C57BL/10 mice latently infected with a virulent strain of HSV-1 (SC16) were compared with respect to (i) HSV DNA levels, (ii) latency-associated transcripts (LATs) and (iii) numbers of LAT+ neurons. In concordance with previous results, two patterns of virus persistence were detected distinguished by either a low (10 to 23) or high (approx. 200) number of viral genomes/LAT+ neuron. The high copy pattern was associated, anatomically, with ganglia directly innervating inoculated skin (T7/8). Paradoxically, the highest number of LAT+ neurons and the highest concentrations of LATs were detected in spinal segments (e.g. T10) containing the lowest number of viral genomes, implying that most of the latent SC 16 DNA detected at T7 and T8 was transcriptionally repressed. When neuronal amplification of HSV DNA during the establishment phase was prevented by infecting mice with a viral thymidine kinase deletion mutant (TKDM21), the high copy pattern was eliminated and each LAT+ neuron contained, on average, 22 TKDM21 genomes. We conclude that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with SC 16. Structurally, latent TKDM21 DNA lacked free genomic termini, consistent with persistence of input DNA in an integrated or circular episomal configuration.
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A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae
The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactiva- table using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV- 1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5 %) mice infected with KOS, 20 of 42 (48 %) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.
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Long term herpes simplex virus type 1 infection of nerve growth factor-treated PC12 cells
More LessThe behaviour of herpes simplex virus type 1 (HSV-1) strain 17 in tissue cultures of PC12 cells treated with nerve growth factor (NGF) was studied. PC12 cells respond to NGF by ceasing to proliferate and extending long neurites. After differentiation with NGF, cultures were infected with HSV-1 and maintained in the presence of the hormone for several weeks. These longterm infected cultures were tested for HSV DNA, transcripts and the ability to produce virus, before and after NGF removal. Before NGF removal, the cultures were characterized by little or no virus production and the presence of HSV-1 DNA in a predominantly endless form. In situ analysis of long-term infected cultures revealed latency-associated transcript expression in only a portion of the cells. However, as shown by an infectious centre assay, virus was present in almost all cells in the population. Moreover, removal of NGF from long-term cultures resulted in the appearance of significantly increased amounts of virus in the media. The degree to which this system resembles HSV latency in vivo is discussed.
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Five new cytotoxic T cell epitopes identified within Epstein-Barr virus nuclear antigen 3
More LessEpstein-Barr virus (EBV) CD8+ cytotoxic T lymphocyte (CTL) epitopes are currently being considered for inclusion into subunit vaccines. Here we describe the characterization of live new CTL epitopes within EBV nuclear antigen 3 (EBNA3), confirming EBNA3 as a major target for CTL recognition.
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Deletion of fowlpox virus homologues of vaccinia virus genes between the 3β-hydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes
A fragment of 4156 bp of fowlpox virus (FPV) genomic DNA contains homologues of vaccinia virus 3β- hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β -HSD; A44L) and DNA ligase (A50R) genes. The FPV locus has clearly been rearranged relative to that of vaccinia virus as homologues of genes A45R to A49R, including the thymidylate kinase and a gene with homology to superoxide dismutase, are deleted. The deleted genes are replaced by two open reading frames: for a serine proteinase inhibitor with homology to vaccinia virus gene K2L and for a protein with no significant homology to proteins in the databases. In addition, the FPV homologues of A44L and A50R are in the same polarity in FPV whereas they are in opposite polarities in vaccinia virus. Increased 3β -HSD activity has been demonstrated in cells infected with either of two different strains of FPV or with canarypox virus.
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Pharmacological studies of a new derivative of amphotericin B, MS-8209, in mouse and hamster scrapie
Transmissible subacute spongiform encephalopathies (TSSE) are neurodegenerative diseases characterized by the presence of a modified, partially proteinase-resistant host protein, PrPSc, which accumulates in the brains of infected individuals. Recently it has been reported that amphotericin B (AmB) treatment of hamsters infected with scrapie strain 263K prolongs the incubation period of the disease, and dissociates in vivo replication of the scrapie agent from PrPSc accumulation. We report here on data obtained after treatment with AmB and one of its derivatives, MS-8209, in experimental scrapie of mouse and hamster. Treatment was carried out by the intraperitoneal route 6 days per week, at three different dosages initiated at the time of infection. Two regimens were used: during the early time of infection or throughout the experimental infection. Results indicate that MS-8209 was as efficient as AmB in prolonging the incubation time and decreasing PrPSc accumulation in the hamster scrapie model. A dose-dependent response was observed in mice treated early after experimental infection. At a dose of 2·5 mg/kg, MS-8209 significantly prolonged the incubation period (by 11·9%). In longterm treatment of mice, MS-8209 and AmB markedly reduced PrPSc levels in the preclinical stage of the disease. These data demonstrate that the effect of AmB is not restricted to one model (hamster-263K). This regimen leads to an inversion of the PrPSc to proteinase- sensitive protein (prpSens) ratio, suggesting PrPSens (presumably cellular PrPc) accumulation occurs before its conversion into PrPSc. As it has been shown that AmB does not modify the infectivity titre, we conclude that the drugs could act by inhibiting either the interaction of the scrapie agent with PrPSens during the early times of infection or the conversion of PrPSens into PrPsc.
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- Plant
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Coat protein of cucumber necrosis virus is not required for efficient generation or accumulation of defective interfering RNAs
More LessIt is generally assumed that defective interfering (DI) forms of viruses are encapsidated in structural proteins encoded by the helper virus. Virion RNA extracts from cucumber necrosis virus (CNV) infections showing high levels of cellular DI RNAs contain barely detectable levels of DI RNAs, suggesting that DI RNAs are encapsidated very inefficiently. In addition, accumulation of CNV DI RNAs occurs at equal efficiency in coinoculated plants using either synthetic wild-type CNV genomic RNA as helper or a mutant of CNV which lacks the coat protein-coding sequence. Together this shows that the CNV coat protein is not required for efficient accumulation of CNV DI RNA in plants. Factors that could account for the high level of CNV DI RNAs in plants are discussed.
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Synthesis of infectious transcripts of blueberry scorch carlavirus in vitro
More LessBlueberry scorch carlavirus (BBScV) is a filamentous virus with a polyadenylated, positive-sense RNA genome. A near full-length cDNA clone of BBScV was constructed by assembly of clones from a cDNA library. To generate a full-length cDNA clone, the 5′ terminus was mutagenized by PCR to introduce nucleotides present in the wild-type virus and not in the near full- length clone, and then fused directly to the T7 bacteriophage RNA polymerase promoter at the 5′ terminus. Capped and uncapped BBScV transcripts were synthesized in vitro from the full-length cDNA clone. Capped transcripts were infectious, producing systemic symptoms identical to those caused by the wild-type virus following inoculation onto Chenopodium quinoa leaves. Uncapped transcripts were substantially less infectious than capped transcripts. This represents the first report of infectious transcripts for a member of the carlavirus group.
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Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus
More LessThe complete nucleotide sequence of the genomic RNA of bamboo mosaic virus (BaMV) was determined by sequencing a set of overlapping cDNA clones and by direct sequencing of the viral RNA. The RNA genome of BaMV is 6366 nucleotides long [excluding 3′ poly(A) tail] and contains six open reading frames (ORFs 1 to 6) coding for polypeptides with M r values of 155K, 28K, 13K, 6K, 25K and 14K, respectively. The genome organization and sizes of the encoded proteins are very similar to those of other potexviruses which have been sequenced except that ORF 6 lies completely within ORF 1. The first five putative proteins of the BaMV genome show identities ranging between 44 to 59%, 26 to 49%, 30 to 53%, 15 to 35% and 20 to 30%, respectively, to the corresponding ORFs of other members of the potexvirus group. However the putative product ORF 6 shows no significant similarity to those of other potexvirus ORF products.
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Nucleotide sequence and genome organization of peanut stripe potyvirus
More LessA blotch isolate of peanut stripe virus (PStV) was cloned, sequenced and compared with other full-length potyvirus sequences. The viral genome was 10059 nucleotides (nt) in length excluding the poly(A) tail. Two potential AUG start codons were identified in the 5′non-translated region. Analysis of in vitro translation products from transcripts containing the first 600 nt of the PStV genome indicated that the first AUG (nt 134 to 136) was preferred over the second AUG (nt 146 to 148) for initiation of translation. Within the single large open reading frame, eight processed proteins were predicted. In general, motifs conserved in other potyviral sequences were also found in the PStV genome. The presence of a 6K protein between the P3 and Cl proteins was predicted. An altered amino acid motif, from FI(V)VRG to FMIIRG, within the carboxyl terminus of the PI protein, separates the PStV sequence from the majority of potyvirus sequences. Based on comparisons with available full-length potyvirus genome sequences, PStV was found to be most closely related to soybean mosaic virus.
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Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus
More LessThe nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98·8K. The N- terminal amino acid sequences of virion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97 % identical to that of SLRSV-H. Except in the 3′ and 5′ terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.
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- Fungal
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Double-stranded RNAs and proteins associated with the 34 nm virus particles of the cultivated mushroom Agaricus bisporus
More LessAgaricus bisporus fruit bodies affected by La France disease contain a specific set of nine dsRNA molecules. A method was developed to isolate intact virus particles containing these dsRNAs. Using precautions to limit proteolysis, virus particles 34 nm in diameter were banded in a Nycodenz gradient together with the nine disease-associated dsRNAs and three proteins of Mr 120K, 115K and 90K. Two of these viral proteins were easily cleaved by proteases present in the fruit bodies, without affecting the morphological appearance, migration in Nycodenz density gradients or dsRNA content of the virus.
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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 82 (2001)
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Volume 81 (2000)
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Volume 80 (1999)
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Volume 79 (1998)
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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 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)