- Volume 87, Issue 8, 2006
Volume 87, Issue 8, 2006
- Animal
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- DNA viruses
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Identification of Hepatitis B virus putative intergenotype recombinants by using fragment typing
More LessEight hundred and thirty-seven human Hepatitis B virus (HBV) genomes were categorized into pure genotypes and potential intergenotypes, according to their fragment types which were determined based on similarity and phylogenetic analyses of 13 contrived fragments of 250 bp against the corresponding fragments of the consensus sequences of genotypes A–H. Twenty-five intergenotypes, including 171 genomes, were revealed from the potential intergenotype recombinants by phylogenetic analysis of the precisely derived mosaic fragments. Among these, four new intergenotypes were discovered. Many genomes were revealed as putative intergenotype recombinants for the first time. About 87 % of the putative recombinants were B/C (120) and A/D (29) hybrids. The other recombinants comprised A/B/C, A/C, A/E, A/G, C/D, C/F, C/G, C/U (U for unknown genotype) and B/C/U hybrids. Genotypes A and C showed a higher recombination tendency than did other genotypes. The results also demonstrated region priority and breakpoint hot spots in the intergenotype recombination. Recombination breakpoints were found to be concentrated mainly in the vicinity of the DR1 region (nt 1640–1900), the pre S1/S2 region (nt 3150–100), the 3′-end of the C gene (nt 2330–2450) and the 3′-end of the S gene (nt 650–830). These results support the suggestion that intergenotype recombinants may result from co-infection with different genotypes.
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Response of immunocompetent and immunosuppressed Spodoptera littoralis larvae to baculovirus infection
The Mediterranean lepidopteran pest Spodoptera littoralis is highly resistant to infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via the oral route, but highly sensitive to infection with budded virus (BV) via the intrahaemocoelic route. To study the fate of AcMNPV infection in S. littoralis, vHSGFP, an AcMNPV recombinant that expresses the reporter green fluorescent protein gene under the control of the Drosophila heat-shock promoter, and high-resolution fluorescence microscopy were utilized. S. littoralis fourth-instar larvae infected orally with vHSGFP showed melanization and encapsulation of virus-infected tracheoblast cells serving the midgut columnar cells. At 72 h post-infection, the viral foci were removed during the moult clearing the infection. Thus, oral infection was restricted by immune responses to the midgut and midgut-associated tracheal cells. By contrast, injection of BV into the haemocoel resulted in successful infection of tracheoblasts, followed by spread of the virus through the tracheal epidermis to other tissues. However, in contrast to fully permissive infections where tracheoblasts and haemocytes are equally susceptible to infection, a severe limitation to vHSGFP infection of haemocytes was observed. To investigate the resistance of S. littoralis haemocytes to BV infection with AcMNPV, the larval immune system was suppressed with the Chelonus inanitus polydnavirus or a putatively immunosuppressive polydnavirus gene, P-vank-1. Both treatments increased the susceptibility of S. littoralis larvae to AcMNPV. It is concluded that the resistance of S. littoralis to AcMNPV infection involves both humoral and cellular immune responses that act at the gut and haemocyte levels. The results also support the hypothesis that tracheolar cells mediate establishment of systemic baculovirus infections in lepidopteran larvae. The finding that polydnaviruses and their encoded genes synergize baculovirus infection also provides an approach to dissecting the responses of the lepidopteran immune system to viruses by using specific polydnavirus immunosuppressive genes.
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Spliced mRNAs detected during the life cycle of Chicken anemia virus
More LessThe existence of spliced mRNA in Chicken anemia virus (CAV) was investigated, as three proteins appeared to be derived from a single 2.0 kb mRNA species. Human Torque teno virus (TTV), which displays a number of genomic similarities to CAV, is known to transcribe three mRNA species, suggesting that CAV may also have multiple mRNAs. Northern analysis of infected chicken MDCC-MSB1 cells revealed a 2.0 kb mRNA 3 h post-infection (p.i.) and additional 1.6, 1.3 and 1.2 kb bands visible at 48 and 72 h p.i. MDCC-MSB1 or COS1 cells transfected with a CAV clone showed similar results. The poly(A)+ RNA of infected cells was subjected to RT-PCR using a suite of CAV-specific primers. The major 2.0 kb RNA reacted with every primer, but the 1.3 and 1.2 kb RNAs only annealed to certain primers. The 2.0 kb mRNA had no deletions or mutations and was capable of encoding all three known CAV proteins. The 1.3 kb RNA had a splice site joining nt 1222 to nt 1814 and encoded head/tail viral protein 1 (VP1) without a frameshift. In addition, the 1.2 kb RNA possessed a splice site joining nt 994 to nt 1095 and encoded several putative, novel proteins with frameshift mutations. These splice sites conformed to the previously described GT–AG splicing rule. One further 0.8 kb RNA species appeared to be derived from a homologous recombination event. Discovery of the presence of spliced mRNA in CAV strengthens the similarity between CAV and TTV.
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- Plant Viruses
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Functional analysis of the five melon necrotic spot virus genome-encoded proteins
More LessFunction of the melon necrotic spot virus (MNSV) genome-encoded proteins (p29, p89, p7A, p7B and p42) has been studied. Protein-expression mutants of an infectious, full-length cDNA clone of a Spanish MNSV-Al isolate and a recombinant green fluorescent protein (GFP)-expressing virus were used in infection bioassays on melon plants. Results revealed that p29 and p89 are both essential for virus replication, whereas small proteins p7A and p7B are sufficient to support viral movement between adjacent cells operating in trans. It is also demonstrated that, in addition to its structural role as coat protein, p42 is an important factor controlling symptoms and is required for systemic transport. Moreover, both p42 and p7B, among all of the MNSV-encoded proteins, were able to delay RNA silencing in transient-expression assays on GFP-transgenic Nicotiana benthamiana plants. Finally, the presence of p42 also produced an enhancing effect on local spread similar to that of potyviral helper component proteinase (HC-Pro), probably due to its RNA silencing-suppression ability.
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Detection of 6K1 as a mature protein of 6 kDa in plum pox virus-infected Nicotiana benthamiana
More LessThe RNA genome of Plum pox virus (PPV) encodes one large polyprotein that is subsequently cleaved into mature viral proteins. One of the products of proteolytic processing, the 6K1 protein, has not yet been identified in vivo for any member of the genus Potyvirus. In this study, 6K1-specific polyclonal antiserum was raised against PPV 6K1 expressed in Escherichia coli as a translational fusion with the N terminus of avian troponin C and an unusual metal-binding cluster of troponin T-1. For detection of 6K1 in vivo, a pPPV-H6K1-NAT infectious clone was constructed, enabling concentration of histidine-tagged 6K1 by affinity chromatography. Affinity-purified 6K1 was detected in locally infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) and, in addition, in systemically infected leaves at 14 d.p.i., 6K1 was detected exclusively as a protein of 6 kDa and no polyprotein precursors were identified with the raised anti-6K1 antiserum.
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Identification of an RNA-silencing suppressor in the genome of Grapevine virus A
More LessHigher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.
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Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication
More LessThe TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.
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Barley stripe mosaic virus-encoded proteins triple-gene block 2 and γb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication
More LessReplication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo- and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and γb fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare ‘Black Hulless’). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and γb protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and γb proteins play a previously unknown functional role at the site of virus replication.
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Orchid fleck virus is a rhabdovirus with an unusual bipartite genome
More LessOrchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21–38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3′-UGUGUC---GACACA-5′), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.
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- Other Agents
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No abnormal prion protein detected in the milk of cattle infected with the bovine spongiform encephalopathy agent
Milk specimens were collected from lactating cows that had previously been challenged with bovine spongiform encephalopathy (BSE)-infected brain at 4–6 months of age. One group of 10 animals received a single oral dose of 100 g, a second group received 1 g and the third was made up of unexposed controls. The cows were inseminated artificially, and calved at approximately 2 years of age and annually thereafter. Milking was done within the first week following calving and at 10-weekly intervals during the lactation period. Specimens were centrifuged to obtain a fraction enriched for somatic cells and these fractions were analysed for disease-associated, abnormal prion protein (PrPBSE) by using a modified commercial BSE ELISA and a different confirmatory assay. No abnormal prion protein has so far been identified in the cell fraction of milk from cattle incubating BSE by using these methods at their limits of detection.
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Codon 129 polymorphism of the human prion protein influences the kinetics of amyloid formation
The human prion protein (PrP) has a common polymorphism at residue 129, which can be valine or methionine. This polymorphism has a strong influence on susceptibility to prion diseases and on prion-strain properties. Previous work has shown that this amino acid variation has no measurable effect on the native structure of cellular PrP (PrPC). Here, it is shown that the polymorphism does not change the efficiency of conversion to the β-PrP conformation or affect the binding of copper(II) ions. However, in a partially denatured conformation, the polymorphic variation has a profound influence on the ability of the protein to form amyloid fibrils spontaneously.
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- Phage
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Expression of phage P4 integrase is regulated negatively by both Int and Vis
More LessPhage P4 int gene encodes the integrase responsible for phage integration into and excision from the Escherichia coli chromosome. Here, the data showing that P4 int expression is regulated in a complex manner at different levels are presented. First of all, the Pint promoter is regulated negatively by both Int and Vis, the P4 excisionase. The N-terminal portion of Int appears to be sufficient for such a negative autoregulation, suggesting that the Int N terminus is implicated in DNA binding. Second, full-length transcripts covering the entire int gene could be detected only upon P4 infection, whereas in P4 lysogens only short 5′-end covering transcripts were detectable. On the other hand, transcripts covering the 5′-end of int were also very abundant upon infection. It thus appears that premature transcription termination and/or mRNA degradation play a role in Int-negative regulation both on the basal prophage transcription and upon infection. Finally, comparison between Pint–lacZ transcriptional and translational fusions suggests that Vis regulates Int expression post-transcriptionally. The findings that Vis is also an RNA-binding protein and that Int may be translated from two different start codons have implications on possible regulation models of Int expression.
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- Jgv Direct
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Genomic characterization of a novel poxvirus contributing to the decline of the red squirrel (Sciurus vulgaris) in the UK
The genome of a virulent squirrelpox virus (SQPV) isolate was characterized in order to determine its relationship with other poxviruses. Restriction enzyme analysis suggested a genome length of approximately 158 kb, whilst sequence analysis of the two ends of the genome indicated a G+C composition of approximately 66 %. Two contiguous stretches of 23 and 37 kb at the left-hand and right-hand ends of the genome, respectively, were sequenced allowing the identification of at least 59 genes contained therein. The partial sequence of a further 15 genes was determined by spot sequencing of restriction fragments located across the genome. Phylogenetic analysis of 15 genes conserved in all the recognized genera of the subfamily Chordopoxvirinae confirmed that the SQPV does not group within the family Parapoxvirinae, but instead partitions on its own in a separate clade of the poxviruses. Analysis of serum from British woodland rodents failed to find any evidence of SQPV infection in wood mice or bank voles, but for the first time serum samples from grey squirrels in the USA were found to contain antibody against SQPV.
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Prolonged survival of Puumala hantavirus outside the host: evidence for indirect transmission via the environment
The capability of rodent-borne viruses to survive outside the host is critical for the transmission dynamics within rodent populations and to humans. The transmission of Puumala virus (PUUV) in colonized bank voles (Clethrionomys glareolus) was investigated and additional longevity studies in cell culture with PUUV and Tula (TULV) hantaviruses were performed. Wild-type PUUV excreted by experimentally infected donor bank voles was shown to be transmitted indirectly between rodents through contaminated beddings, and maintained its infectivity to recipient voles at room temperature for 12–15 days. In cell culture supernatants, PUUV and TULV remained infectious for 5–11 days at room temperature and up to 18 days at 4 °C, but were inactivated after 24 h at 37 °C. Interestingly, a fraction of dried virus was still infectious after 1 h at 56 °C. These results demonstrated that hantavirus transmission does not require direct contact between rodents, or between rodents and humans, and that the indirect transmission of PUUV through contaminated environment takes place among the rodents for a prolonged period of time. The results also have implications for safety recommendations for work with hantaviruses and for preventive measures.
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Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells
Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (>5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.
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Replication of Bovine respiratory syncytial virus in murine cells depends on type I interferon-receptor functionality
Bovine respiratory syncytial virus (BRSV) is able to counteract the alpha/beta interferon (IFN-α/β)-mediated antiviral response for efficient replication in a host-specific manner. Mice models have been developed for experimental infection with human, but not bovine, respiratory syncytial virus strains. Here, it is shown that BRSV can replicate efficiently on primary cell cultures derived from type I IFN receptor-deficient, but not from wild-type IFN-competent, mice. However, BRSV infection was not enhanced in mice devoid of the type I IFN receptor. These results show that type I IFN is a major host-range determinant for infection at the cellular level, but that other factors control virus replication and pathology in vivo.
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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)