- Volume 88, Issue 12, 2007
Volume 88, Issue 12, 2007
- Animal
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- RNA viruses
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Hepatitis C virus non-structural proteins responsible for suppression of the RIG-I/Cardif-induced interferon response
Viral infections activate cellular expression of type I interferons (IFNs). These responses are partly triggered by RIG-I and mediated by Cardif, TBK1, IKKϵ and IRF-3. This study analysed the mechanisms of dsRNA-induced IFN responses in various cell lines that supported subgenomic hepatitis C virus (HCV) replication. Transfection of dsRNA into Huh7, HeLa and HEK293 cells induced an IFN expression response as shown by IRF-3 dimerization, whilst these responses were abolished in corresponding cell lines that expressed HCV replicons. Similarly, RIG-I-dependent activation of the IFN-stimulated response element (ISRE) was significantly suppressed by cells expressing the HCV replicon and restored in replicon-eliminated cells. Overexpression analyses of individual HCV non-structural proteins revealed that NS4B, as well as NS34A, significantly inhibited RIG-I-triggered ISRE activation. Taken together, HCV replication and protein expression substantially blocked the dsRNA-triggered, RIG-I-mediated IFN expression response and this blockade was partly mediated by HCV NS4B, as well as NS34A. These mechanisms may contribute to the clinical persistence of HCV infection and could constitute a novel antiviral therapeutic target.
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Multiple recombinant dengue type 1 viruses in an isolate from a dengue patient
More LessBetween 2000 and 2004, dengue virus type 1 (DENV-1) genotypes I and II from Asia were introduced into the Pacific region and co-circulated in some localities. Envelope protein gene sequences of DENV-1 from 12 patients infected on the island of New Caledonia were obtained, five of which carried genotype I viruses and six, genotype II viruses. One patient harboured a mixed infection, containing viruses assigned to both genotypes I and II, as well as a number of inter-genotypic recombinants. This is the first report of a population of dengue viruses isolated from a patient containing both parental and recombinant viruses.
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Expression of GB virus C NS5A protein from genotypes 1, 2, 3 and 5 and a 30 aa NS5A fragment inhibit human immunodeficiency virus type 1 replication in a CD4+ T-lymphocyte cell line
More LessGB virus type C (GBV-C) is a common human flavivirus that has been associated with prolonged survival in HIV-positive individuals in several, though not all, epidemiological studies. There are five distinct GBV-C genotypes that are geographically localized, and it has been speculated that GBV-C genotypic differences may explain variable outcomes observed in different clinical studies. Expression of an 85 aa fragment of the GBV-C NS5A phosphoprotein (genotype 2) in a CD4+ T cell line (Jurkat) resulted in inhibition of HIV replication, mediated in part by decreased surface expression of the HIV coreceptor CXCR4 and upregulation of SDF-1. We expressed the NS5A protein from genotypes 1, 2, 3 and 5 in Jurkat cells, and demonstrated that all genotypes inhibited HIV replication. Further deletion mapping demonstrated that expression of a 30 aa fragment resulted in decreased CXCR4 surface expression, upregulation of SDF-1 and inhibition of HIV replication.
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Norovirus recombination
More LessRNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum χ 2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.
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Complete genome sequences for nine simian enteroviruses
More LessAnalysis of the VP1 capsid-coding sequences of the simian picornaviruses has suggested that baboon enterovirus (BaEV), SV19, SV43 and SV46 belong to the species Human enterovirus A (HEV-A) and SA5 belongs to HEV-B, whereas SV4/A2 plaque virus (two isolates of a single serotype), SV6 and N125/N203 (two isolates of a single serotype) appear to represent new species in the genus. We have further characterized by complete genomic sequencing the genetic relationships among the simian enteroviruses serotypes (BaEV, N125/N203, SA5, SV4/A2 plaque virus, SV6, SV19, SV43 and SV46) and to other enteroviruses. Phylogenetic and pairwise sequence relationships for the P1 region paralleled those of VP1 alone, and confirmed that SV4/A-2 plaque virus, SV6 and N125/N203 represent unique genetic clusters that probably correspond to three new species. However, sequence relationships in the P2 and P3 regions were quite different. In 2C, SV19, SV43 and SV46 remain clustered with the human viruses of HEV-A, but BaEV, SV6 and N125/N203 cluster together; in 3CD, SA5 (HEV-B) also joined this cluster. The 3′-non-translated region (NTR) sequences are highly conserved within each of the four human enterovirus species, but the 3′-NTRs of the simian enteroviruses are distinct from those of all human enteroviruses and generally distinct from one another. These results suggest that host species may have a significant influence on the evolution of enterovirus non-capsid sequences.
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The type I interferon system protects mice from Semliki Forest virus by preventing widespread virus dissemination in extraneural tissues, but does not mediate the restricted replication of avirulent virus in central nervous system neurons
Semliki Forest virus (SFV) infection of the mouse provides a powerful model to study the pathogenesis of virus encephalitis. SFV and other alphavirus-based vector systems are increasingly used in biotechnology and medicine. This study analysed the strong susceptibility of this virus to type I interferon (IFN) responses. Following intraperitoneal infection of adult mice, SFV strain A7(74) was efficiently (100 %) neuroinvasive. In contrast, SFV4 was poorly (21 %) neuroinvasive. Upon entry into the brain, both viruses activated type I IFN responses. As determined by quantitative RT-PCR, activation of the IFN-α gene was proportional to virus RNA load. An intact type I IFN system was required for protection against both strains of SFV. IFN strongly curtailed virus spread in many cell types and in many tissues. In mice with an intact type I IFN system, infected cells were rarely observed and tissue tropism was difficult to determine. In the absence of a functional type I IFN system, the tropism and the potential for rapid and widespread infection of this virus was revealed. Virus infection was readily observed in the myocardium, endocardium, exocrine pancreas, adipose tissue, smooth muscle cells and in the brain in meningeal cells, ependymal cells and oligodendrocytes. In the brains of mice with and without type I IFN responses, virus infection of neurons remained rare and focal, indicating that the previously described restricted replication of SFV A7(74) in neurons is not mediated by type I IFN responses.
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Rescue of Akabane virus (family Bunyaviridae) entirely from cloned cDNAs by using RNA polymerase I
More LessReverse-genetic systems are often used to study different aspects of the viral life cycle. To date, three rescue systems have been developed for the family Bunyaviridae. These systems use T7 RNA polymerase, which is generally used in rescue systems for Mononegavirales. In the present study, we describe a rescue system for Akabane virus (family Bunyaviridae) that uses cDNAs and RNA polymerase I instead of T7 RNA polymerase. The utility of this system was demonstrated by the generation of a mutant with a deletion of the non-structural protein (NSs) on the S RNA segment. These results offer a new option for bunyavirus rescue.
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Enhanced lung disease and Th2 response following human metapneumovirus infection in mice immunized with the inactivated virus
More LessHuman metapneumovirus (hMPV) is a paramyxovirus that causes acute respiratory-tract infections in humans. The histopathological and immunological responses to hMPV infection in BALB/c mice immunized with inactivated hMPV were characterized. Animals were immunized intraperitoneally with PBS, supernatant from non-infected LLC-MK2 cells and from heat-inactivated influenza A- or hMPV-infected cells, all in incomplete Freund's adjuvant, or with heat-inactivated hMPV without adjuvant, and then infected intranasally with 108 TCID50 virus. Following infection, lung samples and bronchoalveolar lavages were collected for determination of viral titre and cytokine levels and for histopathological studies. On day 1, 26 % of mice immunized with inactivated hMPV and adjuvant died, compared with none in the other groups. There was more significant lung inflammation associated with eosinophilic infiltration, as well as increased levels of interleukin-4 (IL-4) and IL-5, in the bronchoalveolar lavages of mice immunized with hMPV alone or with the adjuvant. Mice from the last two groups had a 4–5 log10 decrease in their pulmonary viral titres compared with controls. Our data demonstrate the risks associated with immunization using inactivated hMPV in this animal model and that this aberrant response should be considered in the development of hMPV vaccines.
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Features of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that direct packaging and serotype restriction
More LessA series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De λ3–CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be ‘identified’ as l1 ssRNAs, the individual nucleotides in these regions that ‘mark’ each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5′ 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5′ l1. RNA secondary-structure predictions for the 5′ 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5′ ssRNA predicted a common structure.
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Genetic and epidemiological characterization of Middle Point orbivirus, a novel virus isolated from sentinel cattle in northern Australia
Middle Point orbivirus (MPOV) was isolated in 1998 from a healthy cow pastured at Beatrice Hill farm, Middle Point (formerly Coastal Plains Research Station), 50 km east of Darwin in Australia's Northern Territory. The isolate could not be identified by using conventional serological tests, and electron microscopy indicated that it belongs to the family Reoviridae, genus Orbivirus. Genetic sequencing of segments 2 and 3 revealed that this virus is related to Yunnan orbivirus, an orbivirus known only from China and not previously associated with a vertebrate host. A real-time RT-PCR test was developed to study the epidemiology of this virus in the field. Over 150 previously unidentified viruses isolated from cattle between 1994 and 2006 were positively identified as isolates of MPOV. Serology was used to demonstrate the development of antibody responses to MPOV in cattle from multiple locations across the Northern Territory.
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Genetic characterization of small ruminant lentivirus in Italian mixed flocks: evidence for a novel genotype circulating in a local goat population
In this study, characterization of the gag gene of small ruminant lentiviruses was carried out in Italian mixed flocks. The nearly complete gag gene was amplified and sequenced. Within genotype A, subtype A1 and a novel subtype, A8, were found in goats, and another novel subtype, A9, was found in both sheep and goats. Subtype B1 was found in both host species and subtype B2 was identified only in sheep. A novel, highly divergent sequence was obtained from goats in two epidemiologically related flocks and is proposed to represent a novel genotype, E. Major epitopes of matrix and capsid antigen were highly divergent, suggesting that serological identification of animals infected with genotype E may have been missed by using currently available diagnostic tests. A recombinant subunit ELISA, based on genotype E-specific epitopes, was developed and a third independent flock carrying this genotype was identified, based on serology.
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Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination
We report the isolation, purification, genome-sequencing and characterization of a picorna-like virus from dead bees in Israel. Sequence analysis indicated that IAPV (Israeli acute paralysis virus) is a distinct dicistrovirus. It is most homologous to Kashmir bee virus and acute bee paralysis virus. The virus carries a 9487 nt RNA genome in positive orientation, with two open reading frames separated by an intergenic region, and its coat comprises four major proteins, the sizes of which suggest alternate processing of the polyprotein. IAPV virions also carry shorter, defective-interfering (DI)-like RNAs. Some of these RNAs are recombinants of different segments of IAPV RNA, some are recombinants of IAPV RNA and RNA from another dicistrovirus, and yet others are recombinants of IAPV and non-viral RNAs. In several of the DI-like RNAs, a sense-oriented fragment has recombined with its complement, forming hairpins and stem–loop structures. In previous reports, we have shown that potyviral and IAPV sequences are integrated into the genome of their respective hosts. The dynamics of information exchange between virus and host and the possible resistance-engendering mechanisms are discussed.
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- DNA viruses
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The A5 gene of alcelaphine herpesvirus 1 encodes a constitutively active G-protein-coupled receptor that is non-essential for the induction of malignant catarrhal fever in rabbits
Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species. The A5 ORF of the AlHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [35S]GTPγS and reporter gene assays, we found that A5 is able to constitutively couple to α i-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AlHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AlHV-1 in MCF pathogenesis.
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Xeroderma pigmentosum C is involved in Epstein–Barr virus DNA replication
More LessCellular mismatch and base-excision repair machineries have been shown to be involved in Epstein–Barr Virus (EBV) lytic DNA replication. We report here that nucleotide-excision repair (NER) may also play an important role in EBV lytic DNA replication. Firstly, the EBV BGLF4 kinase interacts with xeroderma pigmentosum C (XPC), the critical DNA damage-recognition factor of NER, in yeast and in vitro, as demonstrated by yeast two-hybrid and glutathione S-transferase pull-down assays. Simultaneously, XPC was shown, by indirect immunofluorescence and co-immunoprecipitation assays, to interact and colocalize with BGLF4 in EBV-positive NA cells undergoing lytic viral replication. In addition, the efficiency of EBV DNA replication was reduced about 30–40 % by an XPC small interfering RNA. Expression of BGLF4 enhances cellular DNA-repair activity in p53-defective H1299/bcl2 cells in a host-cell reactivation assay. This enhancement was not observed in the XPC-mutant cell line XP4PA-SV unless complemented by ectopic XPC, suggesting that BGLF4 may stimulate DNA repair in an XPC-dependent manner. Overall, we suggest that the interaction of BGLF4 and XPC may be involved in DNA replication and repair and thereby enhance the efficiency of viral DNA replication.
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Relationship between adenovirus DNA replication proteins and nucleolar proteins B23.1 and B23.2
More LessAdenovirus infection subverts nucleolar structure and function. B23 is a nucleolar protein present in two isoforms (B23.1 and B23.2) and both isoforms have been identified as stimulatory factors for adenovirus DNA replication. Here, it is demonstrated that the two isoforms of B23, B23.1 and B23.2, interact and co-localize differently with viral DNA replication proteins pTP and DBP in adenovirus-infected cells. Thus, the mechanism by which the two proteins stimulate viral DNA replication is likely to differ. These data also demonstrate the importance of testing both isoforms of B23 for interactions with viral proteins and nucleic acids.
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Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara
Chorioallantois vaccinia virus Ankara (CVA) is the parental virus of modified vaccinia virus Ankara (MVA), which was derived from CVA by more than 570 passages in chicken embryo fibroblasts (CEF). MVA became severely host-cell-restricted to avian cells and has strongly diminished virulence in mammalian hosts, while maintaining good immunogenicity. We determined the complete coding sequence of the parental CVA and mapped the exact positions of the six major deletions that emerged in the MVA genome. All six major deletions occurred in regions of the CVA genome where one or more truncated or fragmented open reading frames (ORFs) pre-existed. The CVA genome contains 229 ORFs of which 51 are fragments of full-length orthopoxvirus (OPV) genes, including fragmented orthologues of C9L and M1L (encoding two well-conserved ankyrin-like proteins), A39R (encoding a semaphorin-like protein) and A55R (encoding a kelch-like protein). Phylogenetic analysis demonstrated that MVA was most closely related to CVA, followed by the vaccinia virus (VACV) strain DUKE, a patient-derived isolate of the Dryvax vaccine virus. Loss or mutation of genes outside the six major deletions are assumed to contribute to the restricted host range phenotype of MVA. In support of this notion, deletions, insertions and non-synonymous mutations were found in 122 of the 195 ORFs remaining in MVA when compared with their CVA counterparts. Thus, detailed knowledge of the CVA genomic sequence is a prerequisite to further dissect the genetic basis of the MVA host range phenotype as well as the particular immunological properties of MVA.
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Hepatitis B virus polymerase inhibits the interferon-inducible MyD88 promoter by blocking nuclear translocation of Stat1
More LessPrevious studies have suggested that hepatitis B virus (HBV) blocks expression of the alpha interferon (IFN-α)-inducible myeloid differential primary response protein (MyD88) gene. To study the molecular mechanism(s) of the inhibition of MyD88 expression by HBV, MyD88 promoter reporter plasmids and vectors expressing different HBV viral proteins were constructed. Co-transfection experiments showed that IFN-induced MyD88 promoter activity was inhibited by HBV polymerase expression in a dose-dependent manner and that the terminal protein (TP) domain of HBV polymerase was responsible for this antagonistic activity. Analysis of site mutants showed that the region targeted by the polymerase protein contained the signal transducer and activator of transcription (Stat) binding site. Chromatin immunoprecipitation analysis showed that the IFN-induced DNA-binding activity of Stat1 was affected. Further study demonstrated that the HBV polymerase protein inhibited the Stat1 nuclear translocation induced by IFN-α, but did not induce Stat1 degradation nor interfere with its phosphorylation. In addition, HBV polymerase could inhibit the transcriptional activity of other IFN-stimulated response element-driven promoters and the expression of interferon-stimulated genes (ISGs), such as Stat1 and ISG15. In summary, these results indicate that HBV polymerase is a general inhibitor of IFN signalling and can inhibit IFN-inducible MyD88 expression by inhibiting the activity of the MyD88 promoter through blocking the nuclear translocation of Stat1.
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Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro
More LessAPOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.
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The hepatitis B x antigen effector, URG7, blocks tumour necrosis factor α-mediated apoptosis by activation of phosphoinositol 3-kinase and β-catenin
More LessHepatitis B x antigen (HBxAg) contributes significantly to the pathogenesis of chronic infection and development of hepatocellular carcinoma. To discern some of its operative pathways, HepG2 cells were stably transduced with HBx or the bacterial chloramphenicol acetyltransferase (CAT) gene. Differential gene expression has previously revealed an upregulated gene, clone 7 (URG7), that conferred resistance to anti-Fas killing on HepG2X cells. Given that tumour necrosis factor alpha (TNFα) is also an important mediator of chronic hepatitis, and partially shares signalling with Fas, experiments were designed to test whether URG7 blocks TNFα killing of HepG2X cells. HepG2X cells expressing URG7 and HepG2 cells overexpressing URG7 in the absence of HBxAg were resistant to TNFα killing compared with HepG2CAT cells. URG7 small interfering RNA restored the sensitivity of HepG2X cells to TNFα killing. Killing was associated with the activation of caspases 3 and 8, suggesting that URG7 blocked these caspases. This resistance was also associated with activation of phosphoinositol 3-kinase/Akt. Given that Akt and HBxAg also activate β-catenin, experiments were designed to determine whether URG7 blocked apoptosis via activation of β-catenin. Both HBxAg and URG7 activated fragments of the β-catenin promoter, and also promoted expression of β-catenin target genes. Hence, URG7 inhibits TNFα-mediated killing by blocking one or more caspases in the apoptotic pathway and by activating phosphoinositol 3-kinase and β-catenin, thereby overriding the apoptotic signalling of TNFα. This suggests that URG7 helps to protect virus-infected hepatocytes during chronic hepatitis B virus infection.
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Detection of L1, infectious virions and anti-L1 antibody in domestic rabbits infected with cottontail rabbit papillomavirus
Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.
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Comparative analysis reveals frequent recombination in the parvoviruses
More LessParvoviruses are small single-stranded DNA viruses that are ubiquitous in nature. Infections with both autonomous and helper-virus dependent parvoviruses are common in both human and animal populations, and many animals are host to a number of different parvoviral species. Despite the epidemiological importance of parvoviruses, the presence and role of genome recombination within or among parvoviral species has not been well characterized. Here we show that natural recombination may be widespread in these viruses. Different genome regions of both porcine parvoviruses and Aleutian mink disease viruses have conflicting phylogenetic histories, providing evidence for recombination within each of these two species. Further, the rodent parvoviruses show complex evolutionary histories for separate genomic regions, suggesting recombination at the interspecies level.
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Baculovirus envelope fusion proteins F and GP64 exploit distinct receptors to gain entry into cultured insect cells
More LessGroup II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.
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Deletion of a Helicoverpa armigera nucleopolyhedrovirus gene encoding a virion structural protein (ORF107) increases the budded virion titre and reduces in vivo infectivity
The open reading frame Ha107 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) encodes a putative protein of 51 kDa with homologues in a few group II NPVs and a granulovirus. Ha107 was transcribed as polyadenylated transcripts in infected HzAM1 insect cells. The transcripts were initiated at two distinct locations, one upstream of Ha106 (superoxide dismutase gene, sod) and the second upstream of Ha107. By Western blot analysis, two forms of the HA107 protein were detected in infected cells, a major polypeptide of 48 kDa and a minor one of 51 kDa. Western blot and immunoelectron microscopy analyses further showed that the HA107 protein was associated with the nucleocapsids of both budded virions (BVs) and occlusion-derived virions. A Ha107 knockout virus expressing enhanced green fluorescent protein and polyhedrin was constructed using bacmid technology. A one-step virus growth curve indicated that the BV titre of the knockout virus was significantly higher than that of the parental virus and a Ha107 repair virus. Bioassays indicated that the knockout virus was able to infect third-instar H. armigera larvae; however, its median lethal dose (LD50) was significantly higher than those of the parental virus and Ha107 repair virus. These data indicate that Ha107 encodes a non-essential structural protein of HearNPV virions and that deletion of this gene increases the BV titre and LD50 of the occluded virus.
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Characterization of two genes of Cotesia vestalis polydnavirus and their expression patterns in the host Plutella xylostella
More LessCotesia vestalis is an endoparasitoid of larval stages of Plutella xylostella, the diamondback moth. For successful parasitization, this parasitoid injects a polydnavirus into its host during oviposition. Here we isolated two genes, which we named CvBV1 and CvBV2. CvBV1 was located on segment CvBV-S5 with a size of 790 bp, while CvBV2 was located on segment CvBV-S51 with a size of 459 bp. A gene copy of CvBV2 was found on segment CvBV-S48, which we name CvBV2’. Gene duplication occurred in both genes, tandem gene duplication for CvBV1 and segmental duplication for CvBV2. Gene transcripts of the two genes were detected in hosts as early as 0.5 h post-parasitization (p.p.) and continued to be detected for six days, and tissue-specific expression patterns showed that they could be detected in the haemolymph and brain at 2 h p.p., suggesting that they could participate in early protection of parasitoid eggs from host cellular encapsulation.
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- Plant
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Cauliflower mosaic virus protein P6 is a suppressor of RNA silencing
More LessWe infected a transgenic Arabidopsis line (GxA), containing an amplicon-silenced 35S : : GFP transgene, with cauliflower mosaic virus (CaMV), a plant pararetrovirus with a DNA genome. Systemically infected leaves showed strong GFP fluorescence and amplicon transcripts were detectable in Northern blots, indicating that silencing of GFP had been suppressed during CaMV-infection. Transgenic Arabidopsis lines expressing CaMV protein P6, the major genetic determinant of symptom severity, were crossed with GxA. Progeny showed strong GFP fluorescence throughout and amplicon transcripts were detectable in Northern blots, indicating that P6 was suppressing local and systemic silencing. However, levels of 21 nt siRNAs derived from the GFP transgene were not reduced. In CaMV-infected plants, the P6 transgene did not reduce levels of CaMV leader-derived 21 and 24 nt siRNAs relative to levels of CaMV 35S RNA. These results demonstrate that CaMV can efficiently suppress silencing of a GFP transgene, and that P6 acts as a silencing suppressor.
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Identification of amino acid sequences determining interaction between the cucumber mosaic virus-encoded 2a polymerase and 3a movement proteins
More LessThe cucumber mosaic virus (CMV)-encoded 3a movement protein (MP) is indispensable for CMV movement in plants. We have previously shown that MP interacts directly with the CMV-encoded 2a polymerase protein in vitro. Here, we further dissected this interaction and determined the amino acid sequences that are responsible for the MP and 2a polymerase protein interaction. Both the N-terminal 21 amino acids and the central GDD motif of the 2a polymerase protein were important for interacting with the MP. Although each of the regions alone was sufficient for the interaction with MP, quantitative yeast two-hybrid analyses showed that they acted synergistically to enhance the binding affinity. The MP N-terminal 20 amino acids were sufficient for interacting with the 2a polymerase protein, and the serine residue at position 14 played a critical role in the interaction. Multiple sequence alignment showed that the 2a protein interacting regions and the serine at position 14 in the MP are highly conserved among subgroup I and II CMV isolates.
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Successive accumulation of two size classes of viroid-specific small RNA in potato spindle tuber viroid-infected tomato plants
More LessLike many plant RNA viruses, infection by potato spindle tuber viroid (PSTVd) is known to lead to RNA silencing and a marked reduction in visible disease. To examine the relationship between RNA silencing and this recovery phenomenon in greater detail, we have carried out time-course analyses of viroid-specific small RNA accumulation using several viroid–host combinations. These analyses revealed the presence of two size classes of viroid-specific small RNAs in infected plants, and sequence analysis subsequently demonstrated the presence of a previously undescribed cluster of small RNAs derived primarily from negative-strand PSTVd RNA. Although the clustering patterns were similar, the size distribution of PSTVd small RNAs isolated from symptomatic leaf tissue became more heterogeneous with time. The process by which viroid-specific small RNAs are generated appears to be more complicated than previously believed, possibly involving multiple DICER-LIKE activities, viroid RNA substrates and subcellular compartments.
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Begomovirus ‘melting pot’ in the south-west Indian Ocean islands: molecular diversity and evolution through recombination
During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.
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- Other Agents
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Time-course studies of 14-3-3 protein isoforms in cerebrospinal fluid and brain of primates after oral or intracerebral infection with bovine spongiform encephalopathy agent
Experimental transmission of bovine spongiform encephalopathy (BSE) to cynomolgus monkeys (Macaca fascicularis) is an animal model for variant Creutzfeldt–Jakob disease (vCJD). The presence of 14-3-3 proteins in cerebrospinal fluid (CSF) samples indicates neuronal destruction and is therefore used as a clinical biomarker. However, time-course studies using 14-3-3 proteins have not been performed until now in simian vCJD. The main goals of this study were to determine isoform patterns, to examine kinetics and to correlate the clinical course with the occurrence of this biomarker in simian vCJD. In monkeys dosed intracerebrally with BSE, the earliest clinical sign of illness was a drop in body weight that was detected months before the onset of mild neurological signs. Macaques dosed orally or intracerebrally with BSE developed neurological signs 4.3 (3.7–4.6) and 4.8 (2.9–6.0) years post-infection, respectively. 14-3-3β- and -γ-positive CSF samples were found around the time of onset of mild neurological signs, but not earlier. In contrast, 14-3-3ϵ and -η isoforms were not detectable. 14-3-3 levels increased with time and were positively correlated with the degree of neurological symptoms. Post-mortem examination of brain samples revealed a positive correlation between PrPres and 14-3-3ϵ levels. Interestingly, florid plaques characteristic of human vCJD could not be detected in diseased monkeys. It was concluded that analysis of 14-3-3 proteins in CSF is a reliable tool to characterize the time course of brain degeneration in simian vCJD. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of other biomarkers.
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Prion propagation in a nerve conduit model containing segments devoid of axons
More LessPrions, the putative causative agents of transmissible spongiform encephalopathies, are neurotropic pathogens that spread to the central nervous system via synaptically linked neural conduits upon peripheral infection. Axons and their transport processes have been suggested as mediators of nerve-associated prion dissemination. However, the exact cellular components and molecular mechanisms underlying neural spread are unknown. This study used an established hamster scrapie model to pursue a novel experimental approach using nerve conduits containing segments devoid of neurites generated by incomplete nerve regeneration following Wallerian degeneration to probe the necessity of axons for the neural propagation of prions. For this purpose, animals were subjected to unilateral sciatic neurectomy 4 weeks before footpad inoculation with scrapie agent. The results showed that the regional nerve is the prime conduit for cerebral neuroinvasion and revealed, as evidenced by the accumulation of pathological prion protein PrPTSE, that prions can proceed along segments of peripheral neural projections without detectable axonal structures.
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Demographic risk factors for classical and atypical scrapie in Great Britain
Following the bovine spongiform encephalopathy (BSE) crisis, the European Union has introduced policies for eradicating transmissible spongiform encephalopathies (TSEs), including scrapie, from large ruminants. However, recent European Union surveillance has identified a novel prion disease, ‘atypical’ scrapie, substantially different from classical scrapie. It is unknown whether atypical scrapie is naturally transmissible or zoonotic, like BSE. Furthermore, cases have occurred in scrapie-resistant genotypes that are targets for selection in legislated selective breeding programmes. Here, the first epidemiological study of British cases of atypical scrapie is described, focusing on the demographics and trading patterns of farms and using databases of recorded livestock movements. Triplet comparisons found that farms with atypical scrapie stock more sheep than those of the general, non-affected population. They also move larger numbers of animals than control farms, but similar numbers to farms reporting classical scrapie. Whilst there is weak evidence of association through sheep trading of farms reporting classical scrapie, atypical scrapie shows no such evidence, being well-distributed across regions of Great Britain and through the sheep-trading network. Thus, although cases are few in number so far, our study suggests that, should natural transmission of atypical scrapie be occurring at all, it is doing so slowly.
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- ERRATUM
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- Jgv Direct
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Antigenic drift in the evolution of H1N1 influenza A viruses resulting from deletion of a single amino acid in the haemagglutinin gene
More LessTwo genetically distinct lineages of H1N1 influenza A viruses, circulated worldwide before 1994, were antigenically indistinguishable. In 1994, viruses emerged in China, including A/Beijing/262/95, with profound antigenic differences from the contemporary circulating H1N1 strains. Haemagglutinin sequence comparisons of either a predecessor virus, A/Hebei/52/94, or one representative of the cocirculating A/Bayern/7/95-like clade, A/Shenzhen/227/95, revealed a deletion of K at position 134 (H3 numbering) in the antigenic variants. The K134 deletion conferred a selective advantage to the Chinese deletion lineage, such that it eventually gave rise to currently circulating H1 viruses. Using reverse genetics to generate viruses with either an insertion or deletion of aa 134, we have confirmed that the K134 deletion, rather than a constellation of sublineage specific amino acid changes, was sufficient for the antigenic difference observed in the Chinese deletion lineage, and reinsertion of K134 revealed the requirement of a compatible neuraminidase surface glycoprotein for viral growth.
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Functional interaction of the human cytomegalovirus IE2 protein with histone deacetylase 2 in infected human fibroblasts
More LessIn human cytomegalovirus (HCMV)-infected cells, the 86 kDa immediate-early (IE) 2 protein plays a key role in transactivating downstream viral genes. Recently, IE2 has been shown to interact with histone deacetylase 1 (HDAC1) and HDAC3. HDAC1 recruited by IE2 was required for IE2-mediated autorepression of the major IE (MIE) promoter, whereas IE2–HDAC3 interaction was suggested to relieve the repressive effect of HDAC3 on viral early promoters. However, whether IE2 indeed inhibits HDAC's deacetylation activity on viral promoters and interacts with other HDACs remains unclear. Here, we provide evidence that IE2 functionally interacts with HDAC2 and negates its repressive effect on the viral polymerase promoter. IE2 interacted with HDAC2 in both virus-infected cells and in vitro, and required the conserved C-terminal half for HDAC2 binding. The subcellular localization of HDAC2 was changed in virus-infected cells, showing colocalization with IE2 in viral transcription and replication sites. The overall HDAC2 protein levels and its deacetylation activity slightly increased during the late stages of infection and the IE2-associated deacetylation activity was still sensitive to an HDAC inhibitor, trichostatin A. In transfection assays, however, histone acetylation of the viral polymerase promoter was suppressed by HDAC2, and this was relieved by IE2 binding. Therefore, our data demonstrate that IE2 functionally interacts with HDAC2 and modulates its deacetylation activity on the viral polymerase promoter. Our results also support the idea that interactions of IE2 with several HDACs to modulate the host epigenetic regulation on viral MIE and early promoters are important events in the process of productive infection.
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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 89 (2008)
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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 46 (1980)
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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 39 (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)