- Volume 87, Issue 7, 2006
Volume 87, Issue 7, 2006
- Review
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Latency and reactivation of human cytomegalovirus
More LessHuman cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, maintained at least in part by its carriage in the absence of detectable infectious virus – the hallmark of latent infection. Reactivation from latency in immunocompromised individuals, in contrast, often results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and key to understanding their biology. However, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. This review will detail our current knowledge of where HCMV is carried in healthy individuals, which viral genes are expressed upon carriage of the virus and what effect this has on cellular gene expression. It will also address the accumulating evidence suggesting that reactivation of HCMV from latency appears to be linked intrinsically to the differentiation status of the myeloid cell, and how the cellular mechanisms that normally control host gene expression play a critical role in the differential regulation of viral gene expression during latency and reactivation.
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Kaposi's sarcoma-associated herpesvirus immune modulation: an overview
More LessKaposi's sarcoma-associated herpesvirus (KSHV) is the most recently discovered human herpesvirus. It is the aetiological agent of Kaposi's sarcoma (KS), a tumour frequently affecting AIDS patients not receiving treatment. KSHV is also a likely cause of two lymphoproliferative diseases: multicentric Castleman's disease and primary effusion lymphoma. The study of KSHV offers exciting challenges for understanding the mechanisms of virus pathogenesis, including those involved in establishing infection and dissemination in the host. To facilitate these processes, approximately one-quarter of KSHV genes encode cellular homologues or unique proteins that have immunomodulatory roles in cytokine production, apoptosis, cell signalling and the immunological synapse. The activities of these molecules are considered in the present review and the positions of their genes are mapped from a complete KSHV genome sequence derived from a KS biopsy. The understanding gained enables the significance of different components of the immune response in protection against KSHV infection to be evaluated. It also helps to unravel the complexities of cellular and immunological pathways and offers the potential for exploiting viral immunomodulators and derivatives in disease therapy.
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Unravelling the complexities of respiratory syncytial virus RNA synthesis
More LessHuman respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3′ ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.
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- Animal
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- RNA viruses
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Non-structural protein 4A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis
Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.
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Inhibition of dengue virus replication by mycophenolic acid and ribavirin
More LessDengue viruses (DEN), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. The effects of mycophenolic acid (MPA) and ribavirin (RBV) on DEN replication in monkey kidney (LLC-MK2) cells were examined. MPA (IC50=0.4±0.3 μM) and RBV (IC50=50.9±18 μM) inhibited DEN2 replication. Quantitative real-time RT-PCR of viral RNA and plaque assays of virions from DEN2-infected and MPA (10 μM)- and RBV (⩾200 μM)-treated cells showed a fivefold increase in defective viral RNA production by cells treated with each drug. Moreover, a dramatic reduction of intracellular viral replicase activity was seen by in vitro replicase assays. Guanosine reversed the inhibition of these compounds, suggesting that one mode of antiviral action of MPA and RBV is by inhibition of inosine monophosphate dehydrogenase and thereby depletion of the intracellular GTP pool. In addition, RBV may act by competing with guanine-nucleotide precursors in viral RNA translation, replication and 5′ capping.
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Interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells
More LessSevere acute respiratory syndrome (SARS) of humans is caused by a novel coronavirus of zoonotic origin termed SARS-associated coronavirus (SARS-CoV). The virus induces severe injury of lung tissue, as well as lymphopenia and destruction of the architecture of lymphatic tissue by as-yet-unknown mechanisms. In this study, the interaction of SARS-CoV with dendritic cells (DCs), the key regulators of immune responses, was analysed. Monocyte-derived DCs were infected with SARS-CoV and analysed for viability, surface-marker expression and alpha interferon (IFN-α) induction. SARS-CoV infection was monitored by quantitative RT-PCR, immunofluorescence analysis and recovery experiments. SARS-CoV infected both immature and mature DCs, although replication efficiency was low. Immature DCs were activated by SARS-CoV infection and by UV-inactivated SARS-CoV. Infected DCs were still viable on day 6 post-infection, but major histocompatibility complex class I upregulation was missing, indicating that DC function was impaired. Additionally, SARS-CoV infection induced a delayed activation of IFN-α expression. Therefore, it is concluded that SARS-CoV has the ability to circumvent both the innate and the adaptive immune systems.
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Transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells
More LessUnderstanding the orchestrated genome-wide cellular responses is critical for comprehending the early events of coronavirus infection. Microarray analysis was applied to assess changes in cellular expression profiles during different stages of two independent, highly controlled murine hepatitis virus (MHV) infections in vitro. Fibroblast-like L cells were infected at high multiplicity in order to study the direct effects of a synchronized lytic coronavirus infection. Total RNA was harvested from MHV- or mock-infected L cells at 3, 5 and 6 h post-infection and hybridized to Affymetrix microarrays representing approximately 12 500 murine genes and expressed sequences. The expression data were compared to their respective mock-infected controls. Quantitative RT-PCR of selected transcripts was used to validate the differential expression of transcripts and inter-experiment reproducibility of microarray analysis. It was concluded that MHV-A59 infection in fibroblast-like cells triggers very few transcriptional cellular responses in the first 3 h of infection. Later, after having established a productive infection, a chemokine response is induced together with other cellular changes associated with RNA and protein metabolism, cell cycle and apoptosis. Interferon responses are not triggered during infection, although the L cells can be readily stimulated to produce interferon by dsRNA, a known potent inducer of interferon. Possibly, the interferon response is actively counteracted by a virus-encoded antagonist as has been described previously for other RNA viruses.
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RNA signals in the 3′ terminus of the genome of Equine arteritis virus are required for viral RNA synthesis
More LessRNA virus genomes contain cis-acting sequences and structural elements involved in virus replication. Both full-length and subgenomic negative-strand RNA synthesis are initiated at the 3′ terminus of the positive-strand genomic RNA of Equine arteritis virus (EAV). To investigate the molecular mechanism of EAV RNA synthesis, the RNA secondary structure of the 3′-proximal region of the genome was analysed by chemical and enzymic probing. Based on the RNA secondary structure model derived from this analysis, several deletions were engineered in a full-length cDNA copy of the viral genome. Two RNA domains were identified that are essential for virus replication and most likely play a key role in viral RNA synthesis. The first domain, located directly upstream of the 3′ untranslated region (UTR) (nt 12610–12654 of the genome), is mainly single-stranded but contains one small stem–loop structure. The second domain is located within the 3′ UTR (nt 12661–12690) and folds into a prominent stem–loop structure with a large loop region. The location of this stem–loop structure near the 3′ terminus of the genome suggests that it may act as a recognition signal during the initiation of minus-strand RNA synthesis.
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Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells
More LessIt was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in different ways, the NS1 protein of Influenza virus, NSs of Tospovirus Tomato spotted wilt virus and HC-Pro of Zucchini yellow mosaic virus were expressed and investigated to determine if they antagonize induced RNAi. Using the recombinant SFV replicon expressing firefly luciferase, silencing was induced and the suppressor activity of these inhibitors during or after initiation of siRNA synthesis was tested, to determine which step of the RNAi pathway is impaired. It was found that these proteins, identified in mammalian or plant systems, also display activity in tick cells. These data suggest that ticks utilize a mechanism similar to the one found in other eukaryotes.
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Identification and characterization of a virus-inducible non-coding RNA in mouse brain
More LessInfection of mice with Japanese encephalitis virus or Rabies virus results in the activation of a gene encoding a novel, non-coding RNA (ncRNA) in the mouse central nervous system. This transcript, named virus-inducible ncRNA (VINC), is identical to a 3.18 kb transcript expressed in mouse neonate skin (GenBank accession no. AK028745) that, together with a number of unannotated cDNAs and expressed sequence tags, is grouped in the mouse unigene cluster Mm281895. VINC is expressed constitutively in early mouse embryo and several adult non-neuronal mouse tissues, as well as a murine renal adenocarcinoma (RAG) cell line. Northern blotting of nuclear and cytoplasmic RNAs revealed that VINC is localized primarily in the nucleus of RAG cells and is thus a novel member of the nuclear ncRNA family.
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Phylogeography of the deer mouse (Peromyscus maniculatus) provides a predictive framework for research on hantaviruses
Phylogeographical partitioning of Sin Nombre and Monongahela viruses (hantaviruses) may reflect that of their primary rodent host, the deer mouse (Peromyscus maniculatus). Lack of a comprehensive assessment of phylogeographical variation of the host has precluded the possibility of predicting spatial limits of existing strains of these viruses or geographical regions where novel viral strains might emerge. The complete cytochrome b gene was sequenced for 206 deer mice collected from sites throughout North America to provide a foundation for future studies of spatial structure and evolution of this ubiquitous host. Bayesian analyses of these sequences partitioned deer mice into six largely allopatric lineages, some of which may represent unrecognized species. The geographical distributions of these lineages were probably shaped by Quaternary climatic events. Populations of mice were apparently restricted to refugia during glacial advances, where they experienced genetic divergence. Expansion of these populations, following climatic amelioration, brought genetically distinctive forms into contact. Occurrence of parallel changes in virus strains can now be explored in appropriate regions. In New Mexico, for example, near the location where Sin Nombre virus was first discovered, there are three genetically distinctive lineages of deer mice whose geographical ranges need to be delineated precisely. The phylogeography of P. maniculatus provides a framework for interpreting geographical variability, not only in hosts, but also in associated viral variants and disease transmission, and an opportunity to predict the potential geographical distribution of newly emerging viral strains.
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Pathogenesis of Dugbe virus infection in wild-type and interferon-deficient mice
More LessIn 129 mice, infection with the nairovirus Dugbe virus (DUGV) was lethal following intracerebral but not intraperitoneal inoculation. Following both routes of inoculation, immunostaining of tissue sections demonstrated virus-positive cells in the brain, indicating that DUGV is neuroinvasive in mice. Many brain areas were affected and neurones were the main cell type infected. Infected cells showed punctate accumulations of viral nucleoprotein in the cytoplasm, indicative of virus replication sites. Immunostaining for activated caspase 3 demonstrated no evidence of apoptosis. The type I interferon (IFN) system plays a significant role in defence against DUGV, as 129 IFN-α/β R−/− mice died rapidly following both intraperitoneal and intracerebral inoculations. Studies were undertaken to determine whether the IFN-inducible proteins, protein kinase R (PKR) and MxA, were important for protection; neither PKR nor constitutively expressed human MxA played significant roles.
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A mouse model of persistent brain infection with recombinant Measles virus
Measles virus (MV) nucleocapsids are present abundantly in brain cells of patients with subacute sclerosing panencephalitis (SSPE). This invariably lethal brain disease develops years after acute measles as result of a persistent MV infection. Various rodent models for MV infection of the central nervous system (CNS) have been described in the past, in which the detection of viral antigens is based on histological staining procedures of paraffin embedded brains. Here, the usage of a recombinant MV (MV-EGFP-CAMH) expressing the haemagglutinin (H) of the rodent-adapted MV-strain CAM/RB and the enhanced green fluorescent protein (EGFP) is described. In newborn rodents the virus infects neurons and causes an acute lethal encephalitis. From 2 weeks on, when the immune system of the genetically unmodified animal is maturating, intracerebral (i.c.) infection is overcome subclinically, however, a focal persistent infection in groups of neurons remains. The complete brain can be analysed in 50 or 100 μm slices, and infected autofluorescent cells are readily detected. Seven and 28 days post-infection (p.i.) 86 and 81 % of mice are infected, respectively, and virus persists for more than 50 days p.i. Intraperitoneal immunization with MV 1 week before infection, but not after infection, protects and prevents persistence. The high percentage of persistence demonstrates that this is a reliable and useful model of a persistent CNS infection in fully immunocompetent mice, which allows the investigation of determinants of the immune system.
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Matrix protein and glycoproteins F and H of Peste-des-petits-ruminants virus function better as a homologous complex
More LessThe matrix (M) protein of paramyxoviruses forms an inner coat to the viral envelope and serves as a bridge between the surface glycoproteins (F and H) and the ribonucleoprotein core. Previously, a marker vaccine (RPV-PPRFH) was produced for the control of peste des petits ruminants (PPR) disease, where the F and H genes of Rinderpest virus (RPV) were replaced with the equivalent genes from Peste-des-petits-ruminants virus (PPRV); however, this virus grew poorly in tissue culture. The poor growth of the RPV-PPRFH chimeric virus was thought to be due to non-homologous interaction of the surface glycoproteins with the internal components of the virus, in particular with the M protein. In contrast, replacement of the M gene of RPV with that from PPRV did not have an effect on the viability or replication efficiency of the recombinant virus. Therefore, in an effort to improve the growth of the RPV-PPRFH virus, a triple chimera (RPV-PPRMFH) was made, where the M, F and H genes of RPV were replaced with those from PPRV. As expected, the growth of the triple chimera was improved; it grew to a titre as high as that of the unmodified PPRV, although comparatively lower than that of the parental RPV virus. Goats infected with the triple chimera showed no adverse reaction and were protected from subsequent challenge with wild-type PPRV. The neutralizing-antibody titre on the day of challenge was ∼17 times higher than that in the RPV-PPRFH group, indicating RPV-PPRMFH as a promising marker-vaccine candidate.
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Sequence analysis of the fusion protein gene from infectious salmon anemia virus isolates: evidence of recombination and reassortment
M. Devold, M. Karlsen and A. NylundStudies of infectious salmon anemia virus (ISAV; genus Isavirus, family Orthomyxoviridae) haemagglutinin–esterase (HE) gene sequences have shown that this gene provides a tool for genotyping and, hence, a tool to follow the dissemination of ISAV. The problem with using only the HE gene is that ISAV has a segmented genome and one segment may not tell the whole story about the origin and history of ISAV from outbreaks. To achieve a better genotyping system, the present study has focused on segment 5, the fusion (F) protein gene, which contains sequence variation at about the same level as the HE gene. The substitution rates of the HE and F gene sequences, based on 54 Norwegian ISAV isolates, are 6.1(±0.3)×10−6 and 8.6(±5.0)×10−5 nt per site per year, respectively. The results of phylogenetic analysis of the two gene segments have been compared and, with the exception of a few cases of reassortment, they tell the same story about the ISAV isolates. A combination of the two segments is recommended as a tool for future genotyping of ISAV. Inserts (INs) of 8–11 aa may occur close to the cleavage site of the precursor F0 protein in some ISAV isolates. The nucleotide sequence of two of these INs shows 100 % sequence identity to parts of the 5′ end of the F protein gene, whilst the third IN is identical to a part of the nucleoprotein gene. This shows that recombination is one of the evolutionary mechanisms shaping the genome of ISAV. The possible importance of the INs with respect to virulence remains uncertain.
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Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing
More LessThe proteolytic processing of human immunodeficiency virus (HIV) particles mediated by the viral pol-encoded protease (PR) is essential for viral infectivity. The pol coding sequence partially overlaps with the gag coding sequence and is translated as a Gag–Pol polyprotein precursor. Within Gag–Pol, the C-terminal p6 gag domain is replaced by a transframe peptide referred to as p6*, which separates the Gag nucleocapsid domain from PR. Several previous in vitro studies have ascribed a PR-suppression regulatory function to p6*. Here, it was demonstrated that an HIV-1 Gag–Pol lacking p6* is efficiently incorporated into virions when coexpressed with HIV-1 Gag precursor. However, the released virions are not processed appropriately and show a greatly reduced viral infectivity. This suggests that the p6* is indispensable during the process of PR-mediated virus particle maturation.
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1′S-1′-Acetoxychavicol acetate isolated from Alpinia galanga inhibits human immunodeficiency virus type 1 replication by blocking Rev transport
More LessAIDS remains a major global health concern. Despite a number of therapeutic advancements, there is still an urgent need to develop a new class of therapy for human immunodeficiency virus (HIV). Here, it was shown that 1′S-1′-acetoxychavicol acetate (ACA), a small molecular compound isolated from the rhizomes of Alpinia galanga, inhibited Rev transport at a low concentration by binding to chromosomal region maintenance 1 and accumulating full-length HIV-1 RNA in the nucleus, resulting in a block in HIV-1 replication in peripheral blood mononuclear cells. Additionally, ACA and didanosine acted synergistically to inhibit HIV-1 replication. Thus, ACA may represent a novel treatment for HIV-1 infection, especially in combination with other anti-HIV drugs.
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Reduced ability of newborns to produce CCL3 is associated with increased susceptibility to perinatal human immunodeficiency virus 1 transmission
The role of CC chemokines in protection against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission is not well understood. It was observed that mitogen-induced production of CCL3 and CCL4 by cord-blood mononuclear cells was increased among infants born to HIV-positive compared with HIV-negative mothers, and that a deficiency in production of CCL3 was associated with increased susceptibility to intrapartum HIV-1 infection. CCL3-L1 gene copy number was associated with CCL3 production and with vertical transmission. However, at equivalent CCL3-L1 gene copy numbers, infants who acquired HIV-1 infection relative to their exposed but uninfected counterparts had lower production of CCL3, suggesting that they may harbour some non-functional copies of this gene. Nucleotide changes that may influence CCL3 production were evident in the CCL3 and CCL3-L1 genes upstream of exon 2. Our findings suggest that infants who display a deficient-production phenotype of CCL3 are at increased risk of acquiring HIV-1, indicating that this chemokine in particular plays an essential role in protective immunity.
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Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations
More LessThe placenta is unique amongst normal tissues in transcribing numerous different human endogenous retroviruses at high levels. In this study, RT-PCR and immunohistochemistry were used to investigate the expression of syncytin in human trophoblast. Syncytin transcripts were found in first-trimester trophoblast cells with both villous and extravillous phenotypes and also in the JAR and JEG-3 choriocarcinoma cell lines. Syncytin protein was detected in villous trophoblast and in all extravillous trophoblast subpopulations of first- and second-trimester placental tissues. It was also present in ectopic trophoblast from tubal implantations. This study confirms that syncytin is expressed widely by a variety of normal human trophoblast populations, as well as choriocarcinoma cell lines.
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- DNA viruses
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Bovine herpesvirus 1 immediate-early protein (bICP0) interacts with the histone acetyltransferase p300, which stimulates productive infection and gC promoter activity
More LessThe immediate-early protein, bICP0, of Bovine herpesvirus 1 (BHV-1) transactivates viral promoters and stimulates productive infection. bICP0 is expressed constitutively during productive infection, as its gene contains an immediate-early and an early promoter. Like other ICP0 homologues encoded by members of the subfamily Alphaherpesvirinae, bICP0 contains a zinc RING finger located near its N terminus. Mutations that disrupt the bICP0 zinc RING finger impair its ability to activate transcription, stimulate productive infection, inhibit interferon-dependent transcription in certain cell types and regulate subnuclear localization. bICP0 also interacts with a cellular chromatin-remodelling enzyme, histone deacetylase 1 (HDAC1), and can relieve HDAC1-mediated transcriptional repression, suggesting that bICP0 inhibits silencing of the viral genome. In this study, it was shown that bICP0 interacted with the histone acetyltransferase p300 during productive infection and in transiently transfected cells. In addition, p300 enhanced BHV-1 productive infection and transactivated a late viral promoter (gC). In contrast, a CH3-domain deletion mutant of p300, which is a dominant-negative mutant, did not activate the gC promoter. bICP0 and p300 cooperated to activate the gC promoter, suggesting that there is a synergistic effect on promoter activation. As p300 can activate certain antiviral signalling pathways (for example, interferon), it was hypothesized that interactions between p300 and bICP0 may dampen the antiviral response following infection.
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Macrophage cultures are susceptible to lytic productive infection by endothelial-cell-propagated human cytomegalovirus strains and present viral IE1 protein to CD4+ T cells despite late downregulation of MHC class II molecules
The contribution of CD4+ T cells to control of human cytomegalovirus (HCMV) has been shown and infected tissue macrophages might contribute to this response by antigen presentation. As shown previously, CD4+ T cells recognize HCMV immediate-early antigen IE1 on glioblastoma cells manipulated to express MHC class II molecules. Here, the possible interference of virus-induced MHC class II downmodulation with the presentation of IE1 by natural target cells was analysed. The capacity of IE1-specific CD4+ T-cell clones to recognize HCMV-infected monocyte-derived macrophages was tested. Various HCMV strains were used to achieve efficient infection of macrophages. Activation of CD4+ T cells by infected macrophages was evaluated at different time points after infection. Endothelial-cell-adapted HCMV strains efficiently infected cultured human macrophages. However, the immediate-early and early phases of replication were prolonged. Infected cells entered the late replication phase only after 3 days of infection, which was associated with downmodulation of MHC class II molecules at the surface of infected cells. Strong stimulation of IE1-specific CD4+ T cells resulted from endogenous de novo antigen production and presentation by infected macrophages during the first 3 days of virus replication, despite MHC class II downmodulation in the late replication phase. Therefore, infected macrophages are assumed to contribute to the antiviral immune response in infected organs.
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Kinetics of African swine fever virus infection in Ornithodoros erraticus ticks
The kinetics of African swine fever virus (ASFV) infection in Ornithodoros erraticus ticks were investigated in specimens collected in the field at different times following an outbreak of the disease in Portugal in 1999 and in ticks infected experimentally with a virus isolated from a tick collected during this outbreak. In ticks collected from the field, initial screening for ASFV was carried out by PCR, followed by attempts to isolate the virus in macrophage cultures. Considering total numbers of ticks tested independently of developmental stages, ASFV DNA was detected in 42.3, 26.4 and 22.4 % of specimens collected at weeks 0, 32 and 63 following the outbreak, respectively. Although virus was not isolated from most of these ticks, the proportion of isolations from large nymphs and adults increased between weeks 0 and 32 from 2 to 9 % and from 5 to 11.5 %, respectively. These results, together with the higher virus titres at week 32, suggest that virus replication occurred. In contrast, virus isolations from small nymphs decreased over this period, from 5 to 1.3 %. At week 63, infection rates decreased for all stages. Experimental infections showed the occurrence of virus replication within 4 weeks post-feeding and maintenance of high titres in almost 100 % of ticks until 20 weeks post-infection. At weeks 41 and 61, a drop in virus titres and infection rates was observed. Relevant to the understanding of African swine fever epidemiology, our results show that ASFV replicates and persists in O. erraticus, but a viral clearance occurs at later times in both natural and experimental infections.
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Novel subtypes (subgenotypes) of hepatitis B virus genotypes B and C among chronic liver disease patients in the Philippines
Several hepatitis B virus (HBV) subtypes (subgenotypes), HBV/Aa (A1 : Asia/Africa), Ae (A2 : Europe), Bj (B1 : Japan) and Ba (B2 : Asia), have been reported with respect to clinical differences between patients infected with these subtypes (subgenotypes). HBV genotype distribution among patients with chronic liver diseases was investigated in the Philippines, where such studies have not been carried out previously. One hundred sera were obtained from such patients, consisting of 32 chronic hepatitis (CH), 37 cirrhosis and 31 hepatocellular carcinoma (HCC) patients. Nine complete genomes and 100 core promoter/precore genes of HBV were sequenced directly. Phylogenetic analyses revealed 51 HBV/A (Aa/A1), 22 HBV/B and 27 HBV/C strains. Interestingly, most HBV/C strains in the Philippines formed a specific cluster distinct from previous HBV/C strains (C1–4), indicating a novel subtype (subgenotype), HBV/C5. Moreover, most HBV/B strains fell within the specific cluster of the HBV/B subtype (subgenotype) B5, with viral characteristics of HBV/Ba (B2) carrying a recombination with HBV/C over the precore and core genes. Of the three genotypes, HBV/B and HBV/C were significantly more prevalent than HBV/A in cirrhosis and HCC patients (P<0.02). The prevalence of the core promoter mutations T1762/A1764 was higher in HCC patients with HBV/B and HBV/C. Multivariate analysis indicated that age [odds ratio (OR) 3.43; 95 % confidence interval (CI) 1.04–11.36; P=0.044] and the core promoter mutation (OR 14.08; 95 % CI 3.62–4.74; P<0.001) were significant factors for HCC development. In conclusion, novel HBV subtypes (subgenotypes) C5 and B5 are prevalent in the Philippines, as well as HBV/Aa (A1).
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Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ proteins through destabilization of viral core and X proteins
More LessThe hepatitis B virus core protein consists of an amino-terminal capsid-assembly domain and a carboxyl-terminal RNA-binding domain. By using the yeast two-hybrid system, two Hsp40/DnaJ chaperone-family proteins, Hdj1 and hTid1, that interact with the carboxyl-terminal region (aa 94–185) of the core protein were identified. Hdj1 is the prototype member of the family and hTid1 is the human homologue of the Drosophila tumour-suppressor protein Tid56. Binding of the viral core protein with the Hsp40 proteins was confirmed by affinity chromatography and immunoprecipitation of transiently expressed proteins. Moreover, in a sucrose gradient, the precursor form of hTid1 co-sedimented with capsid-like particles composed of the full-length core protein. Unlike the general perception of the role of the cellular chaperone proteins in assisting viral protein folding and thus enhancing virus replication, ectopic expression of Hdj1 and hTid1 suppressed replication of HBV in transfected human hepatoma cells. Conversely, RNA interference-mediated knock-down of hTid1 resulted in increased HBV replication. It was found that both Hsp40 proteins specifically accelerated degradation of the viral core and HBx proteins. Our results suggest that the cellular chaperones, through destabilization of viral proteins, exert inhibitory functions on virus replication and hence may play suppressive roles in hepatocellular carcinoma.
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Subtype I BK polyomavirus strains grow more efficiently in human renal epithelial cells than subtype IV strains
BK polyomavirus (BKPyV) is ubiquitous in human populations, infecting children without obvious symptoms and persisting in the kidney. BKPyV isolates have been classified into four subtypes (I–IV) using either serological or genotyping methods. In general, subtype I occurs most frequently, followed by subtype IV, with subtypes II and III rarely detected. As differences in growth capacity in human cells possibly determine the proportion of the four subtypes of BKPyV in human populations, here the growth properties of representative BKPyV strains classified as subtype I or IV in renal proximal tubule epithelial cells (HPTE cells) of human origin were analysed. HPTE cells were transfected with four and three full-length BKPyV DNAs belonging to subtypes I and IV, respectively, and cultivated in growth medium. Virus replication, detected using the haemagglutination assay, was observed in all HPTE cells transfected with subtype I BKPyV DNAs, whereas it was markedly delayed or not detected in those transfected with subtype IV BKPyV DNAs. It was confirmed that the transfected viral DNAs induced virus replication in HPTE cells. Furthermore, it was found that BKPyVs with archetypal transcriptional control regions replicated in HPTE cells, with only the occasional emergence of variants carrying rearranged transcriptional control regions. Essentially the same results as described above were obtained with renal epithelial cells derived from whole kidney. Thus, it was concluded that subtype I BKPyV replicates more efficiently than subtype IV BKPyV in human renal epithelial cells, supporting the hypothesis that growth capacity in human cells is related to the proportion of BKPyV subtypes in human populations.
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White spot syndrome virus VP24 interacts with VP28 and is involved in virus infection
Xixian Xie and Feng YangWhite spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp. Herein, the characterization of VP24, a major structural protein of WSSV, is described. When purified virions were subjected to Nonidet P-40 treatment to separate the envelopes from the nucleocapsids, VP24 was found to be present exclusively in the envelope fraction. Triton X-114 extraction also indicated that VP24 behaves as an envelope protein. Immunoelectron microscopy further confirmed that VP24 is located in the virion envelope. Far-Western experiments showed that VP24 interacts with VP28, another major envelope protein of the WSSV virion. To investigate the function of VP24, WSSV was neutralized with various amounts of anti-VP24 IgG and injected into crayfish. The results showed that anti-VP24 IgG could partially attenuate infection with WSSV. It is concluded that VP24 is an envelope protein and functions at an early stage in virus infection.
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Characterization of an envelope protein (VP110) of White spot syndrome virus
Li Li, Shumei Lin and Feng YangA protein of 110 kDa (termed VP110) from the envelope fraction of White spot syndrome virus (WSSV) was identified by SDS-PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (wsv035) containing an Arg–Gly–Asp (RGD) motif in the WSSV genome database. To validate the mass-spectrometry result, the C-terminal segment of the wsv035 open reading frame was expressed in Escherichia coli as a fusion protein, which was used to produce specific antibody. Analysis by Western blotting and immunoelectron microscopy demonstrated that VP110 was an envelope protein of WSSV. An interaction analysis was performed between VP110 and the host cells, using a fluorescence assay and a competitive-inhibition assay. The results showed that VP110 was capable of attaching to host cells and that adhesion could be inhibited by synthetic RGDT peptides, suggesting that the RGD motif in the VP110 sequence may play a role in WSSV infection.
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Sequence analysis of the Choristoneura occidentalis granulovirus genome
More LessThe genome of the Choristoneura occidentalis granulovirus (ChocGV) isolated from the western spruce budworm, Choristoneura occidentalis, was sequenced completely. It was 104 710 bp long, with a 67.3 % A+T content and contained 116 potential open reading frames (ORFs) covering 88.4 % of the genome. Of these, 29 ORFs were conserved in all fully sequenced baculovirus genomes, 30 were GV-specific, 53 were present in some nucleopolyhedroviruses (NPVs) and/or GVs, three were common to ChocGV and Choristoneura fumiferana GV (ChfuGV) and one was so far unique. To date, ChocGV is the only GV identified that contains a homologue of the apoptosis inhibitor protein P35/P49, present in some group I NPVs. It is also the first GV without a Xestia c-nigrum GV ORF 26 homologue. Five homologous regions (hrs)/repeat regions, lacking typical NPV hr palindromes were identified. ChocGV hrs were similar to each other but not to other GV hrs. A 1.8 kb repeat region with a high A+T content (81 %) and multiple repeats of 21–210 bp was found between choc36 and 37. This area resembled the non-homologous region origin of DNA replication (non-hr ori) identified in Cryptophlebia leucotreta GV (CrleGV) and Cydia pomonella GV (CpGV). Based on the mean amino acid identities of homologous proteins, ChocGV was closest to fully sequenced genomes CpGV (52.3 %) and CrleGV (52.1 %). The closest amino acid identity was to individual ORFs from the partially sequenced ChfuGV genome (97.2 % in 38 ORFs). Phylogenetic analysis placed ChocGV in a clade with CrleGV and CpGV.
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- Plant Viruses
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Comparison of properties of particles of Cucumber mosaic virus and Tomato aspermy virus based on the analysis of molecular surfaces of capsids
More LessThe plant RNA viruses Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) (genus Cucumovirus) have similar icosahedral particles, the crystal structures of which have been reported recently. Similarity in particle structure agrees with reports of stable capsids assembled from their capsid proteins and of viable recombinant viruses with chimeric capsid proteins derived from CMV and TAV. However, differences between the cucumoviruses have been reported for physicochemical properties. Here, structural and electrostatic features of the molecular surfaces are studied to investigate their relationship with these observations. Two coat-protein recombinants with structures modelled by taking CMV and TAV as templates were also included in the analysis. Results show that there exists an external region of negative electrostatic potential that has arisen from strictly conserved charged residues situated near the external HI loop of the subunits in the capsomers. This negative domain surrounds the fivefold and quasi-sixfold axes and locates above regions of positive potential that extend to cover, nearly homogeneously, the inner surface of capsids, where interaction with encapsidated RNA occurs. Differences between the outer electrostatic distributions in CMV and TAV explain the distinct response of both viruses to variations in physicochemical conditions required for particle stability and are essential to rationalize the biological activity of the coat-protein recombinants, in spite of their seemingly distinct electrostatic characteristics.
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Analysis of the viability of coat-protein hybrids between Cucumber mosaic virus and Tomato aspermy virus
More LessCoat-protein (CP) hybrids between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) were engineered to analyse reported CP-associated differences between these viruses. CP portions delimited by aa 1–59, 60–148 and 149–219 were exchanged in all possible combinations within TAV RNA3. The seven possible chimeras were able to replicate in tobacco protoplasts to similar levels, but only those having residues 1–59 or 60–148 from CMV were infectious to tobacco plants, a common host for CMV and TAV, and formed stable particles. When most of the movement protein (MP) of TAV was substituted for that of CMV, infectivity of CP hybrids did not vary. No hybrid was able to infect cucumber plants, a host for CMV and not for TAV. Need for MP–CP compatibility could explain these results, but shows that MP–CP compatibility conditions the use of CP chimeras to map CP-associated differences between CMV and TAV.
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Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent pepper veinal mottle virus infection of pepper
Capsicum resistance to Pepper veinal mottle virus (PVMV) results from complementation between the pvr2 and pvr6 resistance genes: recessive alleles at these two loci are necessary for resistance, whereas any dominant allele confers susceptibility. In line with previous results showing that pvr2 resistance alleles encode mutated versions of the eukaryotic translation initiation factor 4E (eIF4E), the involvement of other members of the eIF4E multigenic family in PVMV resistance was investigated. It was demonstrated that pvr6 corresponds to an eIF(iso)4E gene, predicted to encode the second cap-binding isoform identified in plants. Comparative genetic mapping in pepper and tomato indicated that eIF(iso)4E maps in the same genomic region as pvr6. Sequence analysis revealed an 82 nt deletion in eIF(iso)4E cDNAs from genotypes with the pvr6 resistance allele, leading to a truncated protein. This deletion was shown to co-segregate with pvr6 in doubled haploid and F2 progeny. Transient expression in a PVMV-resistant genotype of eIF(iso)4E derived from a genotype with the pvr6 + susceptibility allele resulted in loss of resistance to subsequent PVMV inoculation, confirming that pvr6 encodes the translation factor eIF(iso)4E. Similarly, transient expression of eIF4E from a genotype with the pvr2 + -eIF4E susceptibility allele also resulted in loss of resistance, demonstrating that wild-type eIF4E and eIF(iso)4E are susceptibility factors for PVMV and that resistance results from the combined effect of mutations in the two cap-binding isoforms. Thus, whilst most potyviruses specifically require one eIF4E isoform to perform their replication cycle, PVMV uses either eIF4E or eIF(iso)4E for infection of pepper.
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- Other Agents
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Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998–2004
Bovine spongiform encephalopathy (BSE) may have been transmitted to British sheep via contaminated feed in the 1980s. Strain-typing techniques based on immunohistochemical (IHC) detection of abnormal protein (PrPd) and the molecular analysis of proteinase-resistant protein (PrPres) by Western blotting (WB) can discriminate between natural or experimental scrapie and experimental BSE in sheep. Between 1 January 1998 and 31 October 2001, 1247 sheep, clinically suspected of scrapie, were found to be positive by statutory tests in Great Britain. Archived brain tissue from these cases was retested by using these discriminatory methods. Twelve brain samples showed PrPres WB patterns that were unlike those found in natural or experimental scrapie. Prospective screening of fresh tissue from a further 1121 scrapie cases was also carried out between 1 November 2001 and 31 May 2004. Two samples gave WB results with similarities to the results found for experimental BSE in sheep. When all 14 unusual cases were tested by IHC, no match to experimental BSE in sheep was found. There were uncertainties within the retrospective study, where some equivocal results were obtained due to poor tissue quality or the unavailability of the optimum brain region. However, for the samples where tissue condition was optimum, our results provide no evidence for the presence of BSE in sheep. Epidemiological interpretation of the 450 flocks sampled indicates that the maximum proportion of sheep transmissible spongiform encephalopathy cases that could be BSE is 0.66 %. This estimate is lower than calculated previously (5 %), when the analysis was based on the results of strain typing in mice.
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Prion protein polymorphisms in white-tailed deer influence susceptibility to chronic wasting disease
More LessThe primary sequence of the prion protein affects susceptibility to transmissible spongiform encephalopathies, or prion diseases, in mice, sheep and humans. The Prnp gene sequence of free-ranging, Wisconsin white-tailed deer was determined and the Prnp genotypes of chronic wasting disease (CWD)-positive and CWD-negative deer were compared. Six amino acid changes were identified, two of which were located in pseudogenes. Two alleles, a Q→K polymorphism at codon 226 and a single octapeptide repeat insertion into the pseudogene, have not been reported previously. The predominant alleles – wild-type (Q95, G96 and Q226) and a G96S polymorphism – comprised almost 98 % of the Prnp alleles in the Wisconsin white-tailed deer population. Comparison of the allelic frequencies in the CWD-positive and CWD-negative deer suggested that G96S and a Q95H polymorphism were linked to a reduced susceptibility to CWD. The G96S allele did not, however, provide complete resistance, as a CWD-positive G96S/G96S deer was identified. The G96S allele was also linked to slower progression of the disease in CWD-positive deer based on the deposition of PrPCWD in the obex region of the medulla oblongata. Although the reduced susceptibility of deer with at least one copy of the Q95H or G96S allele is insufficient to serve as a genetic barrier, the presence of these alleles may modulate the impact of CWD on white-tailed deer populations.
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- Jgv Direct
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Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China
Influenza A viruses are usually non-pathogenic in wild aquatic birds, their natural reservoir. However, from May to July 2005, at Qinghai Lake in China, an unprecedented outbreak of highly pathogenic H5N1 avian influenza virus caused the death of thousands of wild migratory waterbirds. Herein, H5N1 influenza virus from bar-headed geese collected during the outbreak was characterized. Genomic analysis showed that A/Bar-headed Goose/Qinghai/0510/05 (Bh H5N1 virus) is a reassortant virus. Amino acid residue (lysine) at position 627 in the PB2 gene of the Bh H5N1 virus was the same as that of the human H5N1 virus (A/HK/483/97) and different from that of H5N1 avian influenza viruses deposited in GenBank. Antigenic analysis showed that significant antigenic variation has occurred in the Bh H5N1 virus. The Bh H5N1 virus induced systemic infections and caused 100 % mortality in chickens and mice, and 80 % mortality in ducks and geese. Bh H5N1 virus titres were higher in multiple organs of chickens, ducks and geese than in mice, and caused more severe histological lesions in chickens, ducks and mice than in geese. These results support the need to pay close attention and create control programmes to prevent the transmission of highly pathogenic avian influenza virus from wild migratory waterbirds into domestic chickens, ducks, geese and mammalian hosts.
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Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern Europe support the definition of new genetic subtypes
More LessPorcine reproductive and respiratory syndrome virus (PRRSV) ORF5 and ORF7 sequences from Belarus were found to be of the European (EU) genotype, but grouped separately from all other EU genotype sequences described so far, including live-attenuated EU genotype PRRSV vaccines and Italian EU genotype sequences, some of which have been associated with reduced vaccine efficacy. Also, the Belarusian EU-PRRSV exhibited extreme ORF7 size polymorphism, ranging from 375 nt (the smallest EU genotype ORF7 yet described) to 393 nt (the largest ORF7 yet described for any arterivirus). With the Belarusian sequences, the diversity of EU genotype PRRSV now exceeds that of the North American (US) genotype PRRSV, suggesting a European origin of PRRSV. Finally, a very sharp geographical demarcation of highly diverse EU genotype PRRSV was observed along the eastern Polish border. The new Belarusian sequences have relevance for vaccine and diagnostic-antigen design and show that sequence analysis of PRRSV from more eastern parts of Europe may offer further insights into the emergence and evolution of PRRSV.
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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)