- Volume 87, Issue 4, 2006
Volume 87, Issue 4, 2006
- Animal
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- DNA viruses
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An essential role of ERK signalling in TPA-induced reactivation of Kaposi's sarcoma-associated herpesvirus
More LessKaposi's sarcoma-associated herpesvirus (KSHV) is implicated causally in the development of several human malignancies, including primary effusion lymphoma (PEL). PEL cells serve as tools for KSHV research, as most of them are latently infected and allow lytic virus replication in response to various stimuli. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is the most potent inducer of lytic KSHV reactivation; nevertheless, the exact mechanism by which it induces reactivation remains unknown. It has previously been reported by our group that the protein kinase C (PKC) δ isoform plays a crucial role in TPA-mediated KSHV reactivation. Here, the activation pathway was dissected and it was demonstrated that TPA induces KSHV reactivation via stimulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. Western blot analysis revealed a rapid phosphorylation of ERK1/2. Cells treated with MAPK/ERK inhibitors before TPA addition demonstrated repression of ERK1/2 phosphorylation, which was associated with a block of KSHV lytic-gene expression. This inhibition prevented c-Fos accumulation, yet increased c-Jun phosphorylation. Similar results were obtained in response to rottlerin, a selective PKCδ inhibitor. Notably, the PKC inhibitor GF 109203X reduced ERK1/2 phosphorylation, c-Fos accumulation, c-Jun phosphorylation and KSHV reactivation. It is proposed that TPA induces KSHV reactivation through at least two arms. The first involves PKCδ, ERK phosphorylation and c-Fos accumulation, whilst the second requires another PKC isoform that induces the phosphorylation of c-Jun. c-Fos and c-Jun jointly form an active AP-1 complex, which functions to activate the lytic cascade of KSHV.
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The M4 gene of murine gammaherpesvirus 68 modulates latent infection
Murine gammaherpesvirus 68 (MHV-68) encodes a set of unique genes, M1, M2, M3 and M4, and eight non-translated tRNA-like molecules that are thought to be important in virus–host interactions and latent infection. The M4 gene is predicted to encode a novel secreted protein. To investigate the role of M4 in viral pathogenesis, a mutant MHV-68 that did not express M4 was constructed and its replication was characterized in vitro and in vivo. Virus replication was identical to the wild type in vitro and no difference could be detected in virus replication in the lung following intranasal infection. However, in the spleen, virus deficient in M4 expression was severely attenuated in the establishment of latency. These results indicate a critical role for M4 in MHV-68 pathogenesis.
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Yaba-like disease virus chemokine receptor 7L, a CCR8 orthologue
More LessYaba-like disease virus (YLDV) gene 7L encodes a seven-transmembrane G protein-coupled receptor with 53 % amino acid identity to human CC chemokine receptor 8 (CCR8). Initial characterization of 7L showed that this 56 kDa cell-surface glycoprotein binds human CCL1 with high affinity (K d=0·6 nM) and induces signal transduction by activation of heterotrimeric G proteins and downstream protein kinases. Further characterization of YLDV 7L is presented here and shows that murine CC chemokines can induce G-protein activation via the 7L receptor, despite having a low binding affinity for this receptor. In addition, when expressed by recombinant vaccinia virus (VACV), YLDV 7L was found on the outer envelope of VACV extracellular enveloped virus. The contribution of 7L to poxvirus pathogenesis was investigated by infection of mice with a recombinant VACV expressing 7L (vΔB8R-7L) and was compared with the outcome of infection by parental and revertant control viruses. In both intranasal and intradermal models, expression of 7L caused attenuation of VACV. The role of this protein in viral virulence is discussed.
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293T cells expressing simian virus 40 T antigen are semi-permissive to bovine adenovirus type 3 infection
More LessHuman cells do not normally support productive bovine adenovirus type 3 (BAdV-3) infection. Here, the outcome of BAdV-3 infection of both 293 cells and 293 cells modified to constitutively express the simian virus 40 (SV-40) T antigen (293T cells) was studied. Whereas BAdV-3 could efficiently infect 293 cells, there was a block in virus DNA replication, late-gene expression and virus production. In contrast, replication and efficient virus production could be detected in 293T cells infected with BAdV-3 or transfected with a replication-competent genomic BAdV-3 clone (pFBAV304). Early-phase gene expression was detected readily in both BAdV-3-infected 293 and 293T cells. However, the progression to efficient viral DNA synthesis and late-phase protein synthesis occurred only in 293T cells. Electron microscopy and virus growth kinetics demonstrated the formation of progeny virus in 293T cells. The SV-40 T antigens act to overcome a barrier in BAdV-3 DNA replication in 293 cells.
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Site-directed mutagenesis of the VP2 gene of Chicken anemia virus affects virus replication, cytopathology and host-cell MHC class I expression
More LessChicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.
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Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain
More LessThe present study was designed to investigate the prevalence of swine Torque teno virus (TTV) in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-affected Spanish swine. Nested PCR (nPCR) assays to detect two distinct TTV genogroups were applied. A significantly higher prevalence of TTV infection was found in sera from PMWS-affected animals (97 %) than in sera from non-PMWS-affected animals (78 %). Whilst PMWS-affected pigs (91 %) were more likely to be infected with TTV from genogroup 2 than non-PMWS-affected swine (72 %), no such difference was observed with genogroup 1. Nucleotide sequences of nPCR products were 91–99 % identical between strains within a genogroup. In contrast, inter-genogroup sequence identities were 49–58 %. Phylogenetic analyses demonstrated that genogroups form different clusters without association with PMWS or porcine circovirus type 2 infection status of the animals. These results indicate a high prevalence of both swine TTV genogroups in Spain, being present more frequently in PMWS-affected animals than in non-PMWS-affected animals.
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Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein
More LessIn the family Baculoviridae, two distinct envelope fusion proteins are identified in budded virions (BVs). GP64 is the major envelope fusion protein of group I nucleopolyhedrovirus (NPV) BVs. An unrelated type of envelope fusion protein, named F, is encoded by group II NPVs. The genome of Helicoverpa armigera (Hear) NPV, a group II NPV of the single nucleocapsid or S type, also encodes an F-like protein: open reading frame 133 (Ha133). It was demonstrated by N-terminal sequencing of the major 59 kDa protein present in HearNPV BV that this protein is one of the two F subunits: F1 (transmembrane subunit of 59 kDa) and F2 (surface subunit of 20 kDa), both the result of cleavage by a proprotein convertase and disulfide-linked. The HearNPV F protein proved to be a functional analogue of GP64, as the infectivity of an AcMNPV gp64-deletion mutant was rescued by the introduction of the HearNPV F gene. It was also demonstrated by chemical cross-linking that HearNPV F is present in BVs as an oligomer whereby, unlike GP64, disulfide bonds are not involved. Deglycosylation assays indicated that both F1 and F2 possess N-linked glycans. However, when F was made in Hz2E5 cells, these glycans did not have an α-1-3 core fucose modification that usually occurs in insect cells. As α-1-3 core fucose is a major inducer of an allergic response in humans, the present observation makes the HearNPV–Hz2E5 system an attractive alternative for the production of recombinant glycoproteins for therapeutic use in humans.
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Characterization of white spot syndrome virus replication in in vitro-cultured haematopoietic stem cells of freshwater crayfish, Pacifastacus leniusculus
More LessReplication of White spot syndrome virus (WSSV) was investigated in haematopoietic cells (hpt cells) derived from haematopoietic tissue (hpt) of freshwater crayfish, Pacifastacus leniusculus. Temperature and type of inoculum for virus replication were studied. The cell culture remained viable at a wide range of temperatures ranging from 4 to 25 °C. WSSV replicated in cells, as evidenced by in situ hybridization, RT-PCR and by the presence of virions visualized with an electron microscope. Moreover, the results showed that the infectivity of WSSV to hpt cells is dependent on temperature and a supplemented growth factor (cytokine) astakine. WSSV replicated more rapidly at higher temperatures than at lower temperatures. No virus replication was observed at 4 °C. Detectable WSSV-infected cells were present as early as 36 h post-inoculation, demonstrated by in situ hybridization or RT-PCR of VP28 expression at 25 °C. Hpt cells can survive a few weeks at 25 or 16 °C and longer than several months at 4 °C.
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- Plant Viruses
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Corchorus yellow vein virus, a New World geminivirus from the Old World
More LessA bipartite begomovirus infecting Jute mallow (Corchorus capsularis, Tilliaceae) in Vietnam was identified using novel degenerate PCR primers. Analysis of this virus, which was named Corchorus yellow vein virus (CoYVV), showed that it was more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. Evidence is provided that CoYVV is probably indigenous to the region and may be the remnant of a previous population of New World begomoviruses in the Old World.
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Tobacco mosaic virus (TMV) and potato virus X (PVX) coat proteins confer heterologous interference to PVX and TMV infection, respectively
More LessReplication of Potato virus X (PVX) was reduced in transgenic protoplasts that accumulated wild-type coat protein (CPWT) of Tobacco mosaic virus (TMV) or a mutant CP, CPT42W, that produced highly ordered states of aggregation, including pseudovirions. This reaction is referred to as heterologous CP-mediated resistance. However, protoplasts expressing a CP mutant that abolished aggregation and did not produce pseudovirions, CPT28W, did not reduce PVX replication. Similarly, in transgenic tobacco plants producing TMV CPWT or CPT42W, there was a delay in local cell-to-cell spread of PVX infection that was not observed in CPT28W plants or in non-transgenic plants. The results suggest that the quaternary structure of the TMV CP regulates the mechanism(s) of heterologous CP-mediated resistance. Similarly, transgenic protoplasts that produced PVX CP conferred transient protection against infection by TMV RNA. Transgenic plants that accumulated PVX CP reduced the cell-to-cell spread of infection and resulted in a delay in systemic infection following inoculation with TMV or TMV RNA. Heterologous CP-mediated resistance was characterized by a brief delay in systemic infection, whilst homologous CP-mediated resistance conferred reduced or no systemic infection.
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- Other Agents
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Functional disruption of the prion protein gene in cloned goats
The cellular prion protein (PrPC), a membrane glycoprotein anchored to the outer surface of neurons, lymphocytes and other cells, is associated directly with the pathogenesis of the transmissible spongiform encephalopathies (TSEs) occurring mainly in humans, cattle, sheep and goats. Although mice lacking PrPC develop and reproduce normally and are resistant to scrapie infection, large animals lacking PrPC, especially those species in which TSE occurs naturally, are currently not available. Here, five live PRNP +/− goats cloned by gene targeting are reported. Detailed RNA-transcription and protein-expression analysis of one PRNP +/− goat showed that one allele of the caprine PRNP gene had been disrupted functionally. No gross abnormal development or behaviour could be seen in these PRNP +/− goats up to at least 3 months of age. These heterozygous PRNP +/− goats are ready to be used in producing homozygous PRNP −/− goats in which no PrPC should be expressed.
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Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks
Susceptibility to scrapie in sheep is influenced by polymorphisms of the prion protein (PrP) gene, whereas no strong association between genetics and scrapie has yet been determined in goats due to the limited number of studies on these animals. In this case–control study on 177 goats from six Italian scrapie outbreaks, the association between PrP alleles and the occurrence of scrapie was studied. Three silent mutations and 11 PrP polymorphisms were identified, of which two polymorphisms (L133Q and M137I) and one silent mutation (T202T) have not been reported previously. Twelve alleles were determined by cloning. Statistical analysis suggested a possible protective role against scrapie for the glutamine to lysine mutation at codon 222.
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PrPSc accumulation in fetal cotyledons of scrapie-resistant lambs is influenced by fetus location in the uterus
More LessPlacentae from scrapie-infected ewes have been shown to accumulate PrPSc when the genotype of the fetus is of a susceptible genotype (VRQ/VRQ, ARQ/VRQ or ARQ/ARQ). Cotyledons from fetuses of genotypes ARR/ARR, ARQ/ARR and ARQ/VRR have previously been shown to be resistant to PrPSc accumulation. By using ewes from a naturally infected scrapie flock, cotyledons from fetuses of multiple births of different genotypes were examined. PrPSc was detected in fetal cotyledons of genotype ARQ/ARQ, but not in cotyledons from their dizygotic twin of genotype ARQ/ARR. This confirms earlier reports of single fetuses of these genotypes, but is the first description of such a finding in twin fetuses, one of each genotype. It is also demonstrated that cotyledons from sibling fetuses of genotypes ARQ/VRQ and ARQ/ARQ have different patterns of PrPSc accumulation depending on whether the dam is of genotype ARQ/ARQ or ARQ/VRQ. Lastly, it is shown that cotyledons from fetuses with resistant genotypes are weakly positive for PrPSc when they have shared the same pregnant uterine horn with a fetus of a susceptible genotype with cotyledons positive for the detection of PrPSc. Additionally, a PCR product for the Sry gene, a product specific to males, was found in cotyledons from female fetuses that had shared a uterine horn with a male fetus. This indicates that some sharing of fetal blood occurs between placentomes and fetuses residing in the same uterine horn, which can result in PrPSc accumulation in cotyledons with resistant genotypes.
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Bovine spongiform encephalopathy agent in spleen from an ARR/ARR orally exposed sheep
Olivier Andréoletti, Nathalie Morel, Caroline Lacroux, Virginie Rouillon, Céline Barc, Guillaume Tabouret, Pierre Sarradin, Patricia Berthon, Philippe Bernardet, Jacinthe Mathey, Séverine Lugan, Pierrette Costes, Fabien Corbière, Juan-Carlos Espinosa, Juan Maria Torres, Jacques Grassi, François Schelcher and Frédéric LantierOral contamination with bovine spongiform encephalopathy (BSE) agent in susceptible PRNP genotype sheep results in widespread distribution of prion in the host. Because ARR homozygous sheep are considered to be resistant to transmissible spongiform encephalopathies, they have been selected to eradicate scrapie from sheep flocks and to protect the human food chain from small ruminant BSE risk. However, results presented here show that several months after an oral challenge with BSE agent, healthy ARR/ARR sheep can accumulate significant amounts of PrPSc in the spleen.
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- Phage
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Origin and evolution of overlapping genes in the family Microviridae
More LessThe possibility of creating novel genes from pre-existing sequences, known as overprinting, is a widespread phenomenon in small viruses. Here, the origin and evolution of gene overlap in the bacteriophages belonging to the family Microviridae have been investigated. The distinction between ancestral and derived frames was carried out by comparing the patterns of codon usage in overlapping and non-overlapping genes. By this approach, a gradual increase in complexity of the phage genome – from an ancestral state lacking gene overlap to a derived state with a high density of genetic information – was inferred. Genes encoding less-essential proteins, yet playing a role in phage growth and diffusion, were predicted to be novel genes that originated by overprinting. Evaluation of the rates of synonymous and non-synonymous substitution yielded evidence for overlapping genes under positive selection in one frame and purifying selection in the alternative frame.
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- Jgv Direct
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Isolation of a new strain of the flavivirus cell fusing agent virus in a natural mosquito population from Puerto Rico
The genus Flavivirus contains approximately 70 single-stranded, positive-sense RNA viruses that are mosquito-borne, tick-borne or have no known vector. Two discoveries support previous suggestions of the existence of a large number of unsampled flaviviruses: (i) a new flavivirus, Kamiti River virus, was recently isolated from Kenyan mosquitoes, and (ii) sequences with high similarity to those of flaviviruses have been found integrated into the genome of Aedes mosquitoes, suggesting a past infection with a virus (or viruses) that has yet to be discovered. These sequences were related most closely to a flavivirus that infects insects alone, cell fusing agent virus (CFAV). CFAV was originally isolated in the laboratory from an Aedes aegypti cell line. To date, this virus had not been found in the wild. In the present study, over 40 isolates of a novel strain of CFAV were discovered from mature mosquitoes sampled from the wild in Puerto Rico. The viral strain was present in a range of mosquito species, including Aedes aegypti, Aedes albopictus and Culex sp., from numerous locations across the island and, importantly, in mosquitoes of both sexes, suggesting vertical transmission. Here, results from viral screening, and cell culture and molecular identification of the infected mosquitoes are presented. Experimental-infection tests were also conducted by using the original CFAV strain and a highly efficient reverse-transcription mechanism has been documented, in which initiation of copying occurs at the 3′ terminus of either the genomic RNA or the intermediate of replication, potentially elucidating the mechanism by which flaviviral sequences may have integrated into mosquito genomes.
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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)