- Volume 85, Issue 6, 2004
Volume 85, Issue 6, 2004
- Review
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The cell cycle and how it is steered by Kaposi's sarcoma-associated herpesvirus cyclin
More LessA timely coordination of cellular DNA synthesis and division cycles is governed by the temporal and spatial activation of cyclin-dependent kinases (Cdks). The primary regulation of Cdk activation is through binding to partner cyclin proteins. Several gammaherpesviruses encode a viral homologue of cellular cyclin D, which may function to deregulate host cell cycle progression. One of these is encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and is called K cyclin or viral cyclin (v-cyclin). v-Cyclin is expressed in most of the malignant cells that are associated with KSHV infection in humans, labelling v-cyclin as a putative viral oncogene. Here are described some of the major structural and functional properties of mammalian cyclin/Cdk complexes, some of which are phenocopied by v-cyclin. In addition, the molecular events leading to orderly progression through the G1/S and G/M cell cycle phases are reviewed. This molecular picture serves as a platform on which to explain v-cyclin-specific functional properties. Interesting but largely speculative issues concern the interplay between v-cyclin-mediated cell cycle deregulation and molecular progression of KSHV-associated neoplasms.
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- Animal
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- RNA viruses
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Nef from a primary isolate of human immunodeficiency virus type 1 lacking the EE155 region shows decreased ability to down-regulate CD4
The human immunodeficiency virus (HIV) nef gene encodes a 27 kDa myristoylated cytosolic protein that has an important role in the pathogenesis of AIDS. One function of Nef is the down-regulation of CD4 and MHC class I surface molecules in HIV-infected cells. Nef directly isolated from an infected individual (KS2), who could be defined as a long-term non-progressor, was compared with Nef from a standard laboratory strain, HIV-1 NL4-3. KS2 Nef protein was characterized by its lowered ability to down-regulate CD4, while still maintaining the ability to down-regulate MHC class I. The ability of KS2 Nef to down-regulate CD4 was more prominent when CD4 was measured 2–3 days after transfer of the nef gene to the target cells, and also when the effect was measured in CD4+-enriched primary T cells. The amino acid sequence analysis indicated that the most notable feature of KS2 Nef was lack of the two glutamic acids: the EE155 region. When the EE155 region was added to KS2 Nef, the CD4 down-regulation ability was increased almost to the level of NL4-3 Nef. Conversely, when the EE155 region was deleted from NL4-3, its CD4 down-regulation ability was dramatically impaired. These data suggested that the EE155 region plays an important role(s) in the down-regulation of CD4 by Nef protein and also that primary nef sequences could be very useful in identifying the original biological functions of Nef in vivo.
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Multiple human immunodeficiency virus type 1 Nef functions contribute to efficient replication in primary human macrophages
More LessThe human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1SF162, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.
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CXCR4-mediated T cell apoptosis in human immunodeficiency virus infection
More LessMechanisms of CXCR4-mediated T lymphocyte apoptosis in human immunodeficiency virus (HIV) infection are poorly understood. The authors used peripheral blood mononuclear cells isolated from HIV type 1-infected subjects and assessed both CD4+ and CD8+ T cell apoptosis in the presence and absence of CXCR4 blockade by AMD3100. Both CD4+ and CD8+ T cell apoptosis could be inhibited by CXCR4 blockade, mostly in acquired immunodeficiency syndrome subjects and more weakly in asymptomatic HIV-positive subjects, and depended only partially on the syncytium-inducing/non-syncytium-inducing viral envelope phenotype. Immune activation of CD8+, but not CD4+, T cells was CXCR4-dependent, resulting in increased T cell apoptosis. In the presence of monocyte-derived macrophages, CXCR4-mediated apoptosis targeted mostly CD8+ T cells, with CD4+ T cells being more weakly affected. Several immune and viral factors thus play a role in CXCR4-mediated T cell apoptosis in HIV infection: CD4/CD8 phenotype, viral envelope phenotype, T cell activation and T cell–macrophage intercellular contacts.
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Development of a homology model for clade A human immunodeficiency virus type 1 gp120 to localize temporal substitutions arising in recently infected women
More LessThe virus population transmitted by a human immunodeficiency virus type 1 (HIV-1) infected individual undergoes restriction and subsequent diversification in the new host. However, in contrast to men, who have limited virus diversity at seroconversion, there is measurable diversity in viral envelope gene sequences in women infected with clade A HIV-1. In this study, virus sequence diversity in three unrelated, clade A infected women preceding and shortly after seroconversion was evaluated. It was demonstrated that there is measurable evolution of envelope gene sequences over this time interval. Furthermore, in each of the three individuals, amino acid substitutions arose at five or six positions in sequences derived at or shortly after seroconversion relative to sequences obtained from the seronegative sample. Presented here is a model of clade A gp120 to determine the location of substitutions that appeared as the virus population became established in three clade A HIV-1 infected women.
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Unusually long target site duplications flanking some of the long terminal repeats of human endogenous retrovirus K in the human genome
More LessHuman endogenous retroviruses (HERVs) make up a substantial part of the human genome. HERVs and solitary long terminal repeats (solo LTRs) are usually flanked by 4–6 nt short direct repeats through the well-known mechanism of their integration. A number of solo LTRs flanked by unusually long direct repeats were detected in the human genome. These unusual structures might be a product of an alternative virus insertion mechanism.
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Characterization of a mobilization-competent simian immunodeficiency virus (SIV) vector containing a ribozyme against SIV polymerase
More LessExploitation of the intracellular virus machinery within infected cells to drive an anti-viral gene therapy vector may prove to be a feasible alternative to reducing viral loads or overall virus infectivity while propagating the spread of a therapeutic vector. Using a simian immunodeficiency virus (SIV)-based system, it was shown that the pre-existing retroviral biological machinery within SIV-infected cells can drive the expression of an anti-SIV pol ribozyme and mobilize the vector to transduce neighbouring cells. The anti-SIV pol ribozyme vector was derived from the SIV backbone and contained the 5′- and 3′LTR including transactivation-response, Ψ and Rev-responsive elements, thus requiring Tat and Rev and therefore limiting expression to SIV-infected cells. The data presented here show an early reduction in SIV p27 levels in the presence of the anti-SIV pol ribozyme, as well as successful mobilization (vector RNA constituted ∼17 % of the total virus pool) and spread of the vector containing this ribozyme. These findings provide direct evidence that mobilization of an anti-retroviral SIV gene therapy vector is feasible in the SIV/macaque model.
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Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver
More LessIn the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5×109 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1·04 g ml−1 with 73 % of virus below 1·08 g ml−1. Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn2+ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.
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Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice
To assess the effects of constitutive hepatitis C virus (HCV) gene expression on liver, transgenic mice carrying the entire HCV open reading frame inserted in the α1 antitrypsin (A1AT) gene were generated. Expression of A1AT/HCV mRNA was found to be mainly limited to perivascular areas of the liver as indicated by in situ hybridization analysis. HCV core protein was detected in Western blots of liver extracts, whereas the expression of E2, NS3 and NS5 proteins was revealed by immunostaining of liver samples using HCV-specific antisera. Histological analysis of HCV transgenic mice showed that these animals develop extensive steatosis, but very little necrosis of liver tissue. Moreover, a consistent T cell infiltrate and a slight hepatocyte proliferation were observed. Phenotypic analysis of cells infiltrating the liver indicated that recruitment and/or expansion of residing CD8+, NK, NKT and γ δ T cells occurred in transgenic animals. Among these cells, a large fraction of CD8+ T lymphocytes released mainly IL-10 and, to a lesser extent, IFN-γ upon mitogenic stimulation in vitro. Furthermore, both intrahepatic lymphocytes and splenocytes did not produce cytokines in response to HCV antigens. Thus, these data indicate that constitutive expression of HCV proteins may be responsible for intrahepatic lymphocyte recruitment in absence of viral antigen recognition. This response is likely to be driven by virus-induced cellular factors and may play a significant role in the immunopathology of chronic HCV infection and liver disease.
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Identification of novel hepatitis C virus-specific cytotoxic T lymphocyte epitopes by ELISpot assay using peptides with human leukocyte antigen-A*2402-binding motifs
The human leukocyte antigen (HLA)-A*2402 is common in Asians. The authors attempted to identify epitopes for HLA-A*2402-restricted, hepatitis C virus (HCV)-specific CD8+ T cells by an enzyme-linked immunospot (ELISpot) assay using peripheral blood CD8+ T cells from HLA-A*2402-positive hepatitis C patients and synthetic HCV peptides based on HLA-A*2402-binding motifs and the amino acid sequence of type 1b HCV. Ten novel epitopes were identified in five of seven HLA-A*2402-positive patients with acute or short-term chronic HCV infection (<3 years), but in none of four with longer-term chronic infection (>10 years). Only one of the ten epitopes proved to be definitely HLA-A*2402-restricted. Another epitope was identified in one of two HLA-A*2402-negative acute hepatitis C patients. In two of the six patients with positive CD8+ T cell responses, the targeted epitopes were multiple. The same epitope was targeted in two patients. When patients with unresolved acute HCV infection were treated with alpha interferon, peripheral blood HCV-specific CD8+ T cells decreased with resolution of the hepatitis. In conclusion, CD8+ T cell responses to HCV infection are heterogeneous. One definite HLA-A*2402-restricted and ten probably non-HLA-A*2402-restricted epitopes were identified. Patients with short-term HCV infection are suitable for searching for novel HCV epitopes, but peripheral blood HCV-specific CD8+ T cells decrease markedly after loss of antigenic stimulation.
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Priming with CpG-enriched plasmid and boosting with protein formulated with CpG oligodeoxynucleotides and Quil A induces strong cellular and humoral immune responses to hepatitis C virus NS3
More LessCell-mediated immune responses to hepatitis C virus (HCV) proteins play a key role in recovery from infection. The NS3 protein of HCV is of special interest, since it is one of the most conserved proteins and NS3-specific immune responses are stronger and more frequently observed in patients resolving the infection than in chronically infected patients. Since these characteristics make NS3 an attractive vaccine candidate, the objective of this study was to optimize NS3-specific immune responses. Results from this group first demonstrated that a plasmid enriched with 24 CpG motifs (pBISIA24-NS3) tends to induce the strongest and most consistent Th1-biased immune response. Subsequently, it was shown that NS3 formulated with CpG oligodeoxynucleotide and Quil A (rNS3+CpG+Quil A) adjuvants induces a balanced immune response in mice, whereas rNS3 combined with either CpG or Quil A elicits a Th2-biased response. To further enhance NS3-specific cell-mediated immune responses, a vaccination regime consisting of priming with pBISIA24-NS3, followed by boosting with rNS3+CpG+Quil A, was explored in mice and pigs. When compared to immunization with rNS3+CpG+Quil A, this regime shifted the immune response to a Th1-type response and, accordingly, enhanced MHC I-restricted killing by cytotoxic T lymphocytes in mice. Although immunization with pBISIA24-NS3 also induced a Th1-biased response, including cytotoxicity in the mice, the humoral response was significantly lower than that induced by the DNA prime–protein boost regime. These results demonstrate the advantage of a DNA prime–protein boost approach in inducing a strong NS3-specific cell-mediated, as well as humoral, immune response, in both inbred laboratory and outbred large animal species.
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Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes
More LessMice were immunized intramuscularly with free recombinant hepatitis C virus (HCV) NS3 (non-structural protein 3) protein, liposomes encapsulating rNS3 or rNS3 and CpG mixture, liposomes co-encapsulating rNS3 and CpG or liposomes co-encapsulating rNS3 and GpC. Liposomes co-encapsulating rNS3 and CpG induced a much higher titre of anti-HCV NS3 IgG and the dominant IgG subtype was IgG2a. Liposomes co-encapsulating rNS3 and GpC also induced high levels of anti-HCV NS3 IgG antibody, but the dominant IgG subtype was still IgG1, the same as in free HCV/NS3 immunized mice. Liposomes encapsulating rHCV NS3 and the mixture of rHCV NS3 and CpG did not increase the antibody response but switched the IgG subtype. A cytokine profile analysis revealed that the levels of Th1 cytokines in the mice immunized with liposomes co-encapsulating rHCV NS3 and CpG were significantly higher than in other mice while the levels of Th2 cytokine were significantly lower than in the mice immunized with naked rNS3. IL-12 in the mice immunized with liposome-NS3-CpG was significantly higher than in other mice. In conclusion, liposomes co-encapsulating HCV NS3 and CpG are a good candidate vaccine to induce strong Th1 immune responses against hepatitis C viruses.
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Susceptibility of mouse primary cortical neuronal cells to coxsackievirus B
Coxsackievirus B (CVB) is often associated with aseptic meningitis and encephalitis, but the six serotypes of CVB vary in their relative disease severity. To elucidate the detailed mechanisms of CVB-induced cytopathological effects, the morphological and biochemical characteristics caused by the CVB serotypes in mouse primary cortical neuronal cells were investigated. By 24 h post-infection, all CVB serotypes except CVB2 induced severe cytotoxic alterations, including a loss of neurites. Both fluorescence and transmission electron microscopy revealed CVB-induced morphological changes indicative of apoptosis, including heavily condensed nuclei, subsequent chromatin condensation into the periphery of the nuclei and oligonucleosomal DNA fragmentation. It was also found that infection with all six CVB serotypes led to productive virus replication, which was completed prior to an apoptotic signal. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone significantly inhibited nuclear changes associated with virus-induced apoptosis, but had less effect on virus-associated cytopathic effects and no effect on virus production. In contrast, the transcription inhibitor actinomycin D profoundly inhibited all three virus-induced events. Taken together, these findings demonstrate that all six CVB serotypes can efficiently replicate in mouse cortical neuronal cells and that productive replication of these CVBs, except for CVB2, induces multiple cytopathological effects, including apoptotic alterations.
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The Rhopalosiphum padi virus 5′ internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system
Cap-independent internal initiation of translation occurs on a number of viral and cellular mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously, the activity of the RhPV 5′ UTR IRES has been demonstrated in mammalian, Drosophila and wheat germ in vitro translation systems. It is now shown that this IRES also functions within Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system, and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic reporter mRNA transcript increased translation of the second cistron 23-fold within Sf21 cells. In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES therefore has the potential to be utilized in insect cell expression systems.
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Gut-homing (α 4 β 7 +) Th1 memory responses after inactivated poliovirus immunization in poliovirus orally pre-immunized donors
More LessMucosal infections are prevented by a specialized local immune system. Immune cells of this compartment can also be found in the blood and are characterized by the expression of mucosa-specific homing molecules. Here, the cellular immune responses after inactivated poliovirus immunization (IPV) in poliovirus orally pre-immunized donors were investigated. Subcutaneous IPV induced a transient increase in the proliferative response against poliovirus antigen and in the number of poliovirus-specific CD4+ T cells in the blood of the vaccinees. These cells were characterized to be of the effector memory type (CD45RA−/CD45RO+/CCR7−/CD27+) and expressed the homing molecule α 4 β 7, indicating their origin from the gut. Together these data show the recurrence of gut-derived poliovirus-specific cells upon IPV and evaluate the whole-blood assay as a powerful tool for monitoring the success of a vaccination.
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Poliovirus binding to its receptor in lipid bilayers results in particle-specific, temperature-sensitive channels
More LessPoliovirus (PV) infection starts with binding to its receptor (PVR), followed by a receptor-aided, temperature-sensitive conformational change of the infectious particle (sedimenting at 160S) to a particle which sediments at 135S. Reported in this communication is the successful incorporation into lipid bilayers of two forms of the receptor: the full-length human receptor and a modified clone in which the extracellular domains of the receptor were fused to a glycosylphosphatidylinositol tail. Addition of virus (160S) to receptor-containing bilayers leads to channel formation, whereas no channels were observed when the receptor-modified viral particle (135S) was added. Increasing the temperature from 21 to 31 °C led to a 10-fold increase in the magnitude of the single channel conductance, which can be interpreted as a conformational change in the channel structure. A mutant PV with an amino acid change in VP4 (one of the coat proteins) which is defective in genome uncoating failed to produce channels, suggesting that VP4 might be involved in the channel architecture. These studies provide the first electrophysiological characterization of the interactions between poliovirus and its receptor incorporated into a lipid bilayer membrane. Furthermore, they form the foundation for future studies aiming at defining the molecular architecture of the virus–receptor complex.
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Phylogenetic analysis of wild-type 1 polioviruses isolated during the final period of transmission in Turkey
The last poliomyelitis case associated with a wild poliovirus in Turkey occurred in November 1998. This was the last known case of paralytic poliomyelitis caused by indigenous wild poliovirus in the World Health Organization's European Region. This study investigated the genetic relationships of wild-type 1 polioviruses at the latest period of transmission. A phylogenetic tree was constructed on the basis of the VP1/2A sequence from 14 wild-type 1 polioviruses isolated from Turkey in 1994–1998, along with those from other areas of the world. The Turkey isolates in the latest period of transmission were closely related to each other, forming a cluster distinct from other strains. The results showed that these viruses had been spreading indigenously in the eastern and south-eastern parts of Turkey, and ceased transmission there during 1998. This finding serves as a reference for future poliovirus surveillance both in Turkey and worldwide.
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Complete genome sequences of all members of the species Human enterovirus A
More LessThe species Human enterovirus A (HEV-A) in the family Picornaviridae consists of coxsackieviruses (CV) A2–A8, A10, A12, A14 and A16 and enterovirus 71. Complete genome sequences for the prototype strains of the 10 serotypes whose sequences were not represented in public databases have been determined and analysed in conjunction with previously available complete sequences in GenBank. Members of HEV-A are monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5′ non-translated region (NTR), where they are known to cluster with members of HEV-B. The HEV-A prototype strains were about 66 to 86 % identical to one another in deduced capsid amino acid sequence. Antigenic cross-reactivity has been reported between CVA3-Olson and CVA8-Donovan, between CVA5-Swartz and CVA12-Texas-12 and between CVA16-G-10 and EV71-BrCr. Similarity plots, individual sequence comparisons and phylogenetic analyses demonstrate a high degree of capsid sequence similarity within each of these three pairs of prototype strains, providing a molecular basis for the observed antigenic relationships. In several cases, phylogenies constructed from the structural (P1) and non-structural regions of the genome (P2 and P3) are incongruent. The incongruent phylogenies and the similarity plot analyses imply that recombination has played a role in the evolution of the HEV-A prototype strains. CVA6-Gdula clearly contains sequences that are also present in CVA10-Kowalik and CVA12-Texas-12, suggesting that these three strains have a shared evolutionary history despite their lack of similarity in the capsid region.
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Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV
Avian hepatitis E virus (avian HEV), recently identified from a chicken with hepatitis–splenomegaly syndrome in the United States, is genetically and antigenically related to human and swine HEVs. In this study, sequencing of the genome was completed and an attempt was made to infect rhesus monkeys with avian HEV. The full-length genome of avian HEV, excluding the poly(A) tail, is 6654 bp in length, which is about 600 bp shorter than that of human and swine HEVs. Similar to human and swine HEV genomes, the avian HEV genome consists of a short 5′ non-coding region (NCR) followed by three partially overlapping open reading frames (ORFs) and a 3′NCR. Avian HEV shares about 50 % nucleotide sequence identity over the complete genome, 48–51 % identity in ORF1, 46–48 % identity in ORF2 and only 29–34 % identity in ORF3 with human and swine HEV strains. Significant genetic variations such as deletions and insertions, particularly in ORF1 of avian HEV, were observed. However, motifs in the putative functional domains of ORF1, such as the helicase and methyltransferase, were relatively conserved between avian HEV and mammalian HEVs, supporting the conclusion that avian HEV is a member of the genus Hepevirus. Phylogenetic analysis revealed that avian HEV represents a branch distinct from human and swine HEVs. Swine HEV infects non-human primates and possibly humans and thus may be zoonotic. An attempt was made to determine whether avian HEV also infects across species by experimentally inoculating two rhesus monkeys with avian HEV. Evidence of virus infection was not observed in the inoculated monkeys as there was no seroconversion, viraemia, faecal virus shedding or serum liver enzyme elevation. The results from this study confirmed that avian HEV is related to, but distinct from, human and swine HEVs; however, unlike swine HEV, avian HEV is probably not transmissible to non-human primates.
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Molecular determinants of antigenicity of two subtypes of the tick-borne flavivirus Omsk haemorrhagic fever virus
In 1964, D. H. Clarke defined two antigenic subtypes of Omsk haemorrhagic fever virus (OHFV) based on polyclonal antibody absorption and haemagglutination assays. The current report defines the molecular basis for these antigenic subtypes by comparison of the complete genomes of OHFV strains Kubrin (subtype I) and Bogoluvovska (subtype II). There were six nucleotide differences between these two strains throughout the entire genome and they encoded four amino acid changes including three in the viral envelope (E) protein. Two of these changes were in solvent-exposed regions of domain 3 of the E protein, one of which lies in a region that could easily function in virus–host cell or virus–antibody interactions. These results demonstrate the minimal changes that are required to significantly alter the antigenicity of flaviviruses and also demonstrate the tremendous genetic stability of the tick-borne flaviviruses.
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Shift in Japanese encephalitis virus (JEV) genotype circulating in northern Vietnam: implications for frequent introductions of JEV from Southeast Asia to East Asia
This study analyses the evolutionary relatedness of 16 Japanese encephalitis virus (JEV) isolates (nine from Vietnam and seven from Japan) to previously published JEV strains using E gene sequence data. Vietnamese and Japanese strains isolated between 1986 and 1990 were found to cluster in genotype 3. However, more recent Vietnamese and Japanese strains isolated between 1995 and 2002 grouped within genotype 1, now a dominant though previously unreported genotype in Vietnam. In addition, in this study, strains isolated between 1995 and 2002 were more closely related to those isolated in the 1990s than to the older genotype 1 strains. Recently, the introduction of JEV genotype 1 into Japan and Korea has also been reported. Hence this genotype shift phenomenon may be occurring throughout all East Asia. Further studies on JEV ecology are needed to clarify the mechanism of JEV genotype 1 spread to new territories.
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Interaction of classical swine fever virus with dendritic cells
Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-α/TNF-α or polyinosinic–polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-α responses in these DCs and even suppressed pIC-induced IFN-α induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-α were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation.
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Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain
The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase–transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P–P and N–P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120–150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P–P and N–P complexes, and P controls the RNA binding activity of N.
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Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design
Human metapneumovirus (hMPV), a recently identified paramyxovirus, is the causative agent of respiratory tract disease in young children. Epidemiological studies have established the presence of hMPV in retrospective as well as current clinical samples in Europe, USA, Canada, Hong Kong and Australia. The hMPV disease incidence rate varied from 7 to 12 %. This rate of disease attack places hMPV in severity between respiratory syncytial virus and human parainfluenza virus type 3, two common respiratory pathogens of young children, the elderly and immunosuppressed individuals. To evaluate the effectiveness and safety of future hMPV antiviral drugs, therapeutic and prophylactic monoclonal antibodies (mAbs), and vaccine candidates, it was necessary to identify small-animal and primate models that efficiently supported hMPV replication in the respiratory tract and produced neutralizing serum antibodies, commonly a clinical correlate of protection in humans. In this study, various rodents (mice, cotton rats, hamsters and ferrets) and two primate species, rhesus macaques and African green monkeys (AGMs), were evaluated for hMPV replication in the respiratory tract. The results showed that hamsters, ferrets and AGMs supported hMPV replication efficiently and produced high levels of hMPV-neutralizing antibody titres. Hamsters vaccinated with subgroup A hMPV were protected from challenge with subgroup A or subgroup B hMPV, which has implications for hMPV vaccine design. Although these animal models do not mimic human hMPV disease signs, they will nevertheless be invaluable for the future evaluation of hMPV antivirals, mAbs and vaccines.
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Cell surface activation of the alternative complement pathway by the fusion protein of measles virus
More LessMeasles virus (MV)-infected cells are activators of the alternative human complement pathway, resulting in high deposition of C3b on the cell surface. Activation was observed independent of whether CD46 was used as a cellular receptor and did not correlate with CD46 down-regulation. The virus itself was an activator of the alternative pathway and was covered by C3b/C3bi, resulting in some loss in infectivity without loss of virus binding to target cells. The cell surface expression of MV fusion (F), but not haemagglutinin, envelope protein resulted in complement activation of the Factor B-dependent alternative pathway in a dose-dependent manner and F–C3b complexes were formed. The underlying activation mechanism was not related to any decrease in cell surface expression of the complement regulators CD46 and CD55. The C3b/C3bi coating of MV-infected cells and virus should ensure enhanced targeting of MV antigens to the immune system, through binding to complement receptors.
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Mapping of domains responsible for nucleocapsid protein–phosphoprotein interaction of henipaviruses
More LessHendra virus (HeV) and Nipah virus (NiV) are members of a new genus, Henipavirus, in the family Paramyxoviridae. Each virus encodes a phosphoprotein (P) that is significantly larger than its counterparts in other known paramyxoviruses. The interaction of this unusually large P with its nucleocapsid protein (N) was investigated in this study by using recombinant full-length and truncated proteins expressed in bacteria and a modified protein-blotting protein-overlay assay. Results from our group demonstrated that the N and P of both viruses were able to form not only homologous, but also heterologous, N–P complexes, i.e. HeV N was able to interact with NiV P and vice versa. Deletion analysis of the N and P revealed that there were at least two independent N-binding sites on P and they resided at the N and C termini, respectively. Similarly, more than one P-binding site was present on N and one of these was mapped to a 29 amino acid (aa) C-terminal region, which on its own was sufficient to interact with the extreme C-terminal 165 aa region of P.
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Ser-123 of the large antigen of hepatitis delta virus modulates its cellular localization to the nucleolus, SC-35 speckles or the cytoplasm
More LessHepatitis delta virus (HDV) is a defective virus and requires hepatitis B virus (HBV) to supply envelope proteins (HBsAg) for maturation and secretion. It is known that two proteins produced by HDV, the small (SDAg) and large (LDAg) antigens, are located in the nucleolus, speckles and the cytoplasm and are involved in genome replication and virion packaging. However, little is known about how they are targeted to the specific sites where they act. A green fluorescence protein fused to LDAg (GFP–LD) has been shown previously to translocate from the nucleolus to SC-35 speckles in the presence of the casein kinase II inhibitor dichlororibofuranosyl benzimidazole. In this study, we determined which amino acids of GFP–LD were responsible for the translocation from the nucleolus to SC-35 speckles and created three GFP–LD derivatives, GFP–LDS2A, GFP–LDS123A and GFP–LDS2/123A. Fluorescence microscopy studies showed that Ser-123 mutants had a high tendency to target SC-35 speckles in both transfected HeLa and HuH-7 cells and suggested that Ser-123, but not Ser-2, plays a role in modulating LDAg translocation to the nucleolus or to SC-35 speckles. This study also demonstrated that HBsAg plays a role in facilitating the transportation of LDAg from the nucleus to cytoplasm. Compared with GFP–LD and GFP–LDS2A, mutants of Ser-123 were less efficiently transported to the cytoplasm and resulted in a lower level of secretion. In contrast, little or no isoprenylation mutant was observed in the cytoplasm of HuH-7 cells expressing HbsAg, suggesting that the isoprenylation of LDAg plays a role in export from the nucleus. Thus, the current study demonstrated that both cis and trans elements modulate HDAg translocation to various subcellular sites.
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During entry of alphaviruses, the E1 glycoprotein molecules probably form two separate populations that generate either a fusion pore or ion-permeable pores
More LessStudies using the alphavirus Semliki Forest virus have indicated that the viral E1 fusion protein forms two types of pore: fusion pores and ion-permeable pores. The formation of ion-permeable pores has not been generally accepted, partly because it was not evident how the protein might form these different pores. Here it is proposed that the choice of the target membrane determines whether a fusion pore or ion-permeable pores are formed. The fusion protein is activated in the endosome and for steric reasons only a fraction of the activated molecules can interact with the endosomal membrane. This target membrane reaction forms the fusion pore. It is proposed that the rest of the activated molecules interact with the membrane in which the protein is anchored and that this self-membrane reaction leads to formation of ion-permeable pores, which can be detected in the target membrane after fusion of the viral membrane into the target membrane.
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Cytokine production by virus-specific CD8+ T cells varies with activation state and localization, but not with TCR avidity
More LessThe ability of virus-specific CD8+ T cells to produce cytokines was studied in mice infected with lymphocytic choriomeningitis virus and vesicular stomatitis virus. Intracellular staining was used to visualize cytokine-producing CD8+ and CD4+ T cells. Overall, virus-specific CD8+ T cells produce a similar range of cytokines (IFN-γ, TNF-α, IL-2, GM-CSF, RANTES, MIP-1α and MIP-1β) as CD4+ T cells, but the relative distribution of cytokine-producing subsets is different. Moreover, cytokine-producing CD8+ T cells were found to dominate numerically at all time-points tested. Co-staining for more than one cytokine revealed that while all cytokine-producing CD8+ T cells synthesized IFN-γ, additional cytokines were produced by partly overlapping subsets of this population. The frequency of cells producing more than one cytokine was higher in a tertiary site (peritoneum) and generally increased with transition into the memory phase; however, GM-CSF producing cells were only present transiently. Concerning factors predicted to influence the distribution of cytokine-producing subsets, IFN-γ and IL-12 did not play a role, nor was extensive virus replication essential. Notably, regarding the heterogeneity in cytokine production by individual cells with similar epitope specificity, variation in TCR avidity was not the cause, since in vivo-activated TCR transgene-expressing cells were as heterogeneous in cytokine expression as polyclonal cells specific for the same epitope.
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Evidence of rotavirus intragenic recombination between two sublineages of the same genotype
More LessRotavirus G4 prevalence increased during the past decade, with one of the highest prevalences reported during rotavirus surveillance in Argentina. Intragenotype diversity analysis has led to its subdivision into lineages (I and II) and sublineages (Ia–Id). On analysis of Argentine and G4 VP7 sequences from other locations, one Argentine strain (ArgRes1723) appeared to be an intermediate between G4 sublineages Ib and Ic. Similarity and bootscanning analyses and Sawyer's test were carried out to demonstrate the recombinant nature of this strain. It was concluded that intragenic recombination occurred between sequences of sublineages Ib and Ic, with a crossover point between nucleotide positions 336 and 387. This study constitutes the first report of a mechanism of evolution in rotaviruses that is currently considered unusual – a recombination event between two strains of the same rotavirus genotype. These results will help increase current knowledge about rotavirus evolution and divergence, improving our understanding of the adaptation mechanisms used by these viruses.
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Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA
A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-α displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.
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- DNA viruses
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A naturally occurring C-terminal truncated isoform of the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus does not associate with viral episomal DNA
More LessThe latency-associated nuclear antigen (LANA) encoded by orf73 of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to viral episomal DNA and nuclear heterochromatin in infected cells. A 3·2 kb transcript in KSHV-positive primary effusion lymphoma (PEL) cells (BCP-1 and BC-3) encoding a C-terminal truncated form of LANA (LANA-Δ76) has been identified. This transcript has the addition of a poly(A) tail at nt 3264 of orf73 resulting in an in-frame stop codon (TAA) effectively truncating LANA by 76 aa (∼8 kDa). Examination of the coding region revealed the presence of a non-canonical polyadenylation signal (AGTAAA) 17 nt upstream of the poly(A) tail. The protein expressed from this transcript is representative of the faster migration of the LANA doublet bands observed by SDS-PAGE and Western blot. Mutation of the poly(A) signal from AGTAAA to TGTACA produced a protein that co-migrated with the larger LANA isoform. A C-terminal LANA-Δ76 EGFP fusion protein localized to the nucleus but did not co-localize with endogenous LANA in BCP-1 cells, or heterochromatin in HEK293 cells. Using an electrophoretic mobility shift assay (EMSA), the authors were able to show that LANA-Δ76 does not bind to the KSHV terminal repeat motif known to interact with LANA. These data provide evidence for the presence of an isoform of LANA that may perform alternative functions in KSHV-infected cells.
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Inhibition of the Epstein–Barr virus lytic cycle by Zta-targeted RNA interference
More LessEpstein–Barr virus (EBV) reactivation into the lytic cycle plays certain roles in the development of EBV-associated diseases, so an effective strategy to block the viral lytic cycle may be of value to reduce the disease risk or to improve the clinical outcome. This study examined whether the EBV lytic cycle could be inhibited using RNA interference (RNAi) directed against the essential viral gene Zta. In cases of EBV reactivation triggered by chemicals or by exogenous Rta, Zta-targeted RNAi prevented the induction of Zta and its downstream genes and further blocked the lytic replication of viral genomes. This antiviral effect of RNAi was not likely to be mediated by activation of the interferon pathway, as phosphorylation of STAT1 was not induced. In addition, novel EBV-infected epithelial cells showing constitutive activation of the lytic cycle were cloned; such established lytic infection was also suppressed by Zta-targeted RNAi. These results indicate that RNAi can be used to inhibit the EBV lytic cycle effectively in vitro and could also be of potential use to develop anti-EBV treatments.
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p16INK4A-independence of Epstein–Barr virus-induced cell proliferation and virus latency
Epstein–Barr virus (EBV) has the ability to promote cell cycle progression following the initial infection of primary resting B-lymphocytes and to cause cell cycle arrest at the onset of the viral replicative cycle. Various mechanisms have been proposed for the proliferative effects, including the up-regulation of cyclin D2 by the viral EBNA-2 and EBNA-LP proteins, direct binding of EBNA3C to the retinoblastoma protein (pRb), and down-regulation of the p16INK4A tumour suppressor by the viral LMP1 product. To try to gain insight into the relative importance of these mechanisms, the ability of EBV to immortalize lymphocytes from an individual who is genetically deficient for p16INK4A was examined. From detailed analyses of the resultant lymphoblastoid cell lines it is concluded that p16INK4A status has little bearing on EBV's ability to manipulate the cell cycle machinery and a model to accommodate the previously proposed routes taken by EBV to bypass the restriction point is presented.
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Single-nucleotide polymorphisms in two Marek's disease virus genes (Meq and gD): application to a retrospective molecular epidemiology study (1982–1999) in France
More LessMarek's disease virus (MDV) is a herpesvirus that causes a lymphoproliferative disease in chickens. Vaccines against MDV are available, but the virus is gradually becoming more virulent. A molecular epidemiology study of MDV was carried out by assessing nucleotide variation in two different genes, Meq and gD, in 68 French field isolates circulating from 1982 to 1999, compared with reference strains. Viral DNA was amplified by nested PCR and sequenced directly. Comparison of the nucleotide sequences revealed a high nucleotide sequence identity (98 %). Single-nucleotide polymorphisms were identified, leading to the identification of three gene alleles for gD and six for Meq. Nine combinations of alleles were identified. A majority of French isolates (60·5 %) clustered in the C1 type, which has been present for over 17 years. Waves of non-C1-type isolates appeared when vaccine efficacy decreased. Furthermore, specific discriminating sequences were obtained for the CVI-988 vaccine strain.
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Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology
Infection with the murine gammaherpesvirus MHV-68 has profound effects on splenic and mediastinal lymph node pathology in mice which lack the interferon-γ receptor (IFN-γ R−/−). In these mice MHV-68 infection causes fibrosis and loss of lymphocytes in the spleen and the mediastinal lymph node as well as interstitial pulmonary fibrosis and fibrotic changes in the liver. The changes are associated with transient elevated latent virus loads in the spleen. Four independent virus mutants with insertions and/or deletions in the left end of the genome fail to induce the pathological changes and establish latency at normal levels in the spleen. The data indicate that the pathology does not correlate with any of the known genes encoded within this region of the genome, genes M1–M4 and the eight vtRNAs. Northern analysis of mRNAs transcribed by wild-type and mutant viruses shows that at least two uncharacterized transcripts are encoded within this region. These transcripts are absent in the mutant viruses and are candidates for the virus genes responsible for the aberrant pathology in IFN-γ R−/− mice.
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A new member of the interleukin 10-related cytokine family encoded by a poxvirus
Poxviruses express numerous proteins involved in manipulating the host immune response. Analysis of the primary sequence and predicted structure of the 134R protein of Yaba-like disease virus (Y134R) indicated that it is similar to cellular proteins of the IL-10 family, specifically IL-19, IL-20 and IL-24. A flag-tagged Y134R was expressed from mammalian cells and identified as a secreted, monomeric glycoprotein that stimulated signal transduction from class II cytokine receptors IL-20Rα/IL-20Rβ (IL-20R type1) and IL-22R/IL-20Rβ (IL-20R type 2). Y134R induced phosphorylation of signal transducers and activators of transcription, their translocation to the nucleus and the induction of reporter gene expression. In contrast, Y134R was unable to induce similar responses from either the IL-22 or IFN-λ (IL-28A, IL-28B, IL-29) class II cytokine receptors. To examine the role Y134R plays during a poxvirus infection, a vaccinia virus recombinant expressing Y134R was constructed and tested in a murine intranasal infection model. Compared with control viruses, the virus expressing Y134R had a reduced virulence, manifested by reduced weight loss, signs of illness and virus titres in infected organs. These results demonstrate that Y134R is a new viral member of the IL-10-related cytokine family and that its activity in vivo affects virus virulence.
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Recent isolates of parapoxvirus of Finnish reindeer (Rangifer tarandus tarandus) are closely related to bovine pseudocowpox virus
Cases of papular stomatitis in Finnish reindeer have been reported for many years. The causative agent was thought to be Orf virus (ORFV), one of the Parapoxviridae, although this assumption was based mainly on clinical symptoms, pathology and electron microscopy. Here sequence analyses of the viral DNA isolated from a recent outbreak of disease in 1999–2000 are presented in comparison to that isolated from earlier outbreaks in 1992–1994. The results show that the virus isolated from the 1999–2000 outbreak is most closely related to Pseudocowpox virus, whereas those from previous years grouped with ORFV. The present study describes a method for genetic characterization and classification of parapoxviruses (PPVs) and provides for the first time an extended phylogenetic analysis of PPVs isolated from Finland, established members of the genus Parapoxvirus and selected members of the subfamily Chordopoxvirinae.
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Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18
Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types.
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Identification of membrane proteins differentially expressed in human papillomavirus type 16 E5-transfected human keratinocytes by nanoelectrospray ionization mass spectrometry
More LessMembrane proteins differentially expressed in human papillomavirus type 16 (HPV-16) E5-transfected HaCaT cells have been identified. Membrane proteins were isolated and separated by two-dimensional gel electrophoresis. Spots showing quantitative differences between E5-transfected and control cells were extracted and the proteins were identified by nanoelectrospray ionization mass spectrometry. A total of 24 spots was analysed. Among the proteins showing differential expression, a decreased amount of calnexin and increased expression of hsp70, proteins both involved in maturation and transport of MHC class I complexes to the plasma membrane, were noticed. These findings correlate with the decreased surface expression of MHC class I molecules described in E5-expressing cells, HPV-positive cervical lesions and cervical carcinomas. These results stress the value of the proteomic approach, as used here in the experimental design, which allows the correlation of changes in host gene expression with biological functions of viral genes.
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Enhanced oncogenicity of Asian-American human papillomavirus 16 is associated with impaired E2 repression of E6/E7 oncogene transcription
Asian-American (AA) variants of human papillomavirus 16 (HPV-16) are linked to a high incidence of cervical cancer in Mexico, with some evidence strongly suggesting that they are more oncogenic than European (E) variants, including their association with younger women and their higher associated risk of cervical cancer. Differences in the regulation of viral E6/E7 oncogene transcription by the E2 protein may be involved in the higher oncogenicity of AA variants. In E variants, E6/E7 oncogene transcription is repressed by the E2 protein and is frequently up-regulated by the destruction of the E2 gene during viral integration. In contrast, the E2 gene is retained in full in most AA-positive carcinomas, raising the possibility of alternative mechanisms for increasing viral oncogene transcription. The authors investigated whether the higher oncogenicity of AA variants is linked to differences in E6/E7 oncogene transcription and the mechanism of E2 deactivation. E6/E7 and E1/E2 transcripts were explored by RT-PCR in 53 HPV-16-positive cervical carcinomas, 39 retaining (20 European and 19 AA) and 14 having lost (12 European and 2 AA) the E1/E2 genes, and transcription repression activity of the AA E2 genes was tested in four cell lines that constitutively express the β-galactosidase reporter or E6/E7 genes driven by the viral long control region. E6/E7 oncogene transcripts were found in all carcinomas, but only those positive for AA variants with E1/E2 genes had complete E2 transcripts. E2 transcripts were down-regulated by splicing in E-positive carcinomas retaining E1/E2. AA E2 genes were impaired for repression of E6/E7 oncogene transcription in vivo. These results suggest that E6/E7 oncogene expression starts earlier in AA than E variant infections, since E variants need E2 to be destroyed or down-regulated.
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TT virus-derived apoptosis-inducing protein induces apoptosis preferentially in hepatocellular carcinoma-derived cells
TT virus (TTV) is widespread among the global population. Its pathogenic nature is still unclear but TTV seems to be more prevalent in cases of hepatitis than in healthy individuals. TTV harbours similarities to chicken anaemia virus (CAV). Here, the apoptotic potential of a putative TTV-derived 105 aa protein and of the main apoptosis-inducing agent of CAV, Apoptin, is compared. As the putative protein induced apoptosis in various human hepatocellular carcinoma (HCC) cell lines, it was named TTV-derived apoptosis-inducing protein (TAIP). The apoptotic activity of TAIP in HCC lines was comparable with that of Apoptin. Conversely, unlike Apoptin, TAIP induced only low-level apoptosis in several non-HCC human cancer cell lines. The data suggest that TAIP acts in a different way to Apoptin as it is selective to a certain degree for HCC lines. This activity of TAIP, coupled with the heterogeneity of TTV isolates, may help to explain the variable reports of TTV pathogenicity.
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- Plant Viruses
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Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter
More LessA replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3′-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3′ terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem–loop structures, which are followed by an enhancer region.
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Evidence for contribution of an internal ribosome entry site to intercellular transport of a tobamovirus
More LessPreviously, it has been shown that tobacco mosaic virus (TMV) U1 and crucifer-infecting TMV contain a 75 nt internal ribosome entry site (IRES) upstream of movement protein (MP) gene (IRESU1 MP,75 and IRESCR MP,75, respectively). A movement-deficient TMV mutant, KK6, has been constructed previously [ Lehto, K., Grantham, G. L. & Dawson, W. O. (1990). Virology 174, 145–157 ] by insertion of the second coat protein subgenomic promoter (CP SGP-2) upstream of the MP gene, in addition to the natural CP SGP-1. Here, the authors compare the efficiency of movement function expression by KK6 and a derivative, K86, obtained by insertion of IRESCR MP,75 between the CP SGP-2 and MP genes resulting in restoration of IRESCR MP,75 function in the 5′-untranslated sequence of the I2 subgenomic RNA of K86. The data indicate that the efficiency of K86 movement was largely restored by this insertion, which was apparently due to the translation-enhancing ability of IRESCR MP,75.
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Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes
To evaluate RNA silencing for the control of geminivirus infection, two classes of post-transcriptionally silenced (PTS) plants were tested using Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep-210-transgenic plants, a sense×antisense hybrid and two multicopy sense lines. In both classes, PTS plants accumulated low or undetectable amounts of Rep-210 protein and mRNA but high amounts of Rep-210 small interfering RNAs. PTS plants were susceptible to TYLCSV when challenged by agroinoculation or using high viruliferous whitefly (Bemisia tabaci) pressure, although some plants were resistant at low whitefly pressure. Delayed infections were also observed, indicating that TYLCSV could overcome transgene silencing of rep and of the nested C4 gene. TYLCSV infection boosted transgene silencing but this did not lead to recovery. The data suggest that if the virus reaches a threshold level of expression/replication in the initially infected cells then virus spreading can no longer be prevented.
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The C terminus of the movement protein of Brome mosaic virus controls the requirement for coat protein in cell-to-cell movement and plays a role in long-distance movement
More LessThe 3a movement protein (MP) plays a central role in the movement of Brome mosaic virus (BMV). To identify the functional regions in BMV MP, 24 alanine-scanning (AS) MP mutants of BMV were constructed. Infectivity of the AS mutants in the host plant Chenopodium quinoa showed that the central region of BMV MP is important for viral movement and both termini of BMV MP have effects on the development of systemic symptoms. A green-fluorescent-protein-expressing RNA3-based BMV vector containing a 2A sequence from Foot-and-mouth disease virus was also constructed. Using this vector, two AS mutants that showed more efficient cell-to-cell movement than wild-type BMV were identified. The MPs of these two AS mutants, which have mutations at their C termini, mediated cell-to-cell movement independently of coat protein (CP), unlike wild-type BMV MP. Furthermore, a BMV mutant with a truncation in the C-terminal 42 amino acids of MP was also able to move from cell to cell without CP, but did not move systemically, even in the presence of CP. These results and an encapsidation analysis suggest that the C terminus of BMV MP is involved in the requirement for CP in cell-to-cell movement and plays a role in long-distance movement. Furthermore, the ability to spread locally and form virions is not sufficient for the long-distance movement of BMV. The roles of MP and CP in BMV movement are discussed.
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Significance of the 3′-terminal region in minus-strand RNA synthesis of Hibiscus chlorotic ringspot virus
More LessRNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae. After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro. When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3′-terminal two C residues at the 3′ end in vitro and in vivo. The CCC-3′ terminal nucleotide sequence was optimal and nucleotide variations from CCC-3′ diminished minus-strand synthesis. In addition, two putative stem–loops (SLs) located within the 3′-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 3′ terminus and the two SLs structures located in the 3′UTR are essential for efficient minus-strand synthesis of HCRSV.
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- Other Agents
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Standards for the assay of Creutzfeldt–Jakob disease specimens
Assays for the agent of Creutzfeldt–Jakob disease (CJD) include measurement of infectivity in different animal systems, such as wild-type or transgenic mice, and detection of PrPSc by different methods and formats. The various assays could be best calibrated against each other by use of uniform readily available materials, and samples of four human brains, two from sporadic CJD patients, one from a variant CJD patient and one from a non-CJD patient, have been prepared as 10 % homogenates dispensed in 2000 vials each for this purpose. Results of in vitro methods, particularly immunoblot assays, were compared in the first collaborative study described here. While dilution end-points varied, the minimum detectable volume was surprisingly uniform for most assays and differences in technical procedure, other than the sample volume tested, had no detectable systematic effect. The two specimens from sporadic CJD cases contained both type 1 and type 2 prion proteins in approximately equal proportions. The materials have been given the status of reference reagents by the World Health Organization and are available for further study and assessment of other in vitro or in vivo assay procedures.
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Amyloid imaging probes are useful for detection of prion plaques and treatment of transmissible spongiform encephalopathies
Diagnostic imaging probes have been developed to monitor cerebral amyloid lesions in patients with neurodegenerative disorders. A thioflavin derivative, 2-[4′-(methylamino)phenyl] benzothiazole (BTA-1) and a Congo red derivative, (trans, trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) are representative chemicals of these probes. In this report, the two chemicals were studied in transmissible spongiform encephalopathies (TSE). Both BTA-1 and BSB selectively bound to compact plaques of prion protein (PrP), not only in the brain specimens of certain types of human TSE, but also in the brains of TSE-infected mice when the probes were injected intravenously. The chemicals bound to plaques in the brains were stable and could be detected for more than 42 h post-injection. In addition, the chemicals inhibited abnormal PrP formation in a cellular model of TSE with IC50 values of 4 nM for BTA-1 and 1·4 μM for BSB. In an experimental mouse model, the intravenous injection of 1 mg BSB prolonged the incubation period by 14 %. This efficacy was only observed against the RML strain and not the other strains examined. These observations suggest that these chemicals bind directly to PrP aggregates and inhibit new formation of abnormal PrP in a strain-dependent manner. Both BTA-1 and BSB can be expected to be lead chemicals not only for imaging probes but also for therapeutic drugs for TSEs caused by certain strains.
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Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie
Over the last 30 years, many drugs have been tested both in cell culture and in vivo for their ability to prevent the generation of prions and the development of transmissible spongiform encephalopathies. Among the compounds tested, dendrimers are defined by their branched and repeating molecular structure. The anti-prion activity of new cationic phosphorus-containing dendrimers (P-dendrimers) with tertiary amine end-groups was tested. These molecules had a strong anti-prion activity, decreasing both PrPSc and infectivity in scrapie-infected cells at non-cytotoxic doses. They can bind PrP and decrease the amount of pre-existing PrPSc from several prion strains, including the BSE strain. More importantly, when tested in a murine scrapie model, the dendrimers were able to decrease PrPSc accumulation in the spleen by more than 80 %. These molecules have a high bio-availability and therefore exhibit relevant potential for prion therapeutics for at least post-exposure prophylaxis.
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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)