- Volume 75, Issue 6, 1994
Volume 75, Issue 6, 1994
- Animal
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Herpes simplex virus type 1 DNA persistence, progressive disease and transgenic immediate early gene promoter activity in chronic corneal infections in mice
More LessWe have used a mouse model system and the comeal route of inoculation to examine the issue of extraneuronal persistence of herpes simplex vims type 1 (HSV-1). HSV-1 strain F DNA and inflammatory lesions were detected in corneal tissue of mice at 5, 11, 23, 37 and 60 days post-infection (p i.). Viral DNA was localized by in situ PCR to epithelial cells and less frequently to cells in the stroma of the cornea. Viral proteins were not detected in the cornea and vims could not be isolated from tissue homogenates after 11 days p.i. even though histopathological lesions became progressively more severe at 37 and 60 days p.i. The DNA-containing cells were usually adjacent to the sites of inflammation or within these sites in the chronic stage (23, 37 and 60 days p.i.). In contrast to strain F, persistence of HSV-1 strain KOS DNA and inflammatory lesions were not detected after 11 days p.i.; this result suggests that the long-term persistence of HSV-1 DNA and the development of inflammatory lesions are vims strain-dependent. We tested for the possibility of transgenic HSV-1 immediate early gene (ICP4) promoter activity in chronically infected corneas of transgenic mice containing the ICP4 promoter fused to the bacterial β-galactosidase coding sequence. Our results indicated that the chimeric transgene was expressed in the cornea at 5, 11, 23, 37 and 41 days p.i. Possible explanations for these results and mechanisms for the generation of the chronic inflammatory lesions are discussed. The properties of chronic HSV infections in the cornea may be similar to those which have been described for persistent or defective viral infections in other systems.
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Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B
More LessPrevious studies have shown that the initial interaction of herpes simplex virus (HSV) with cells is binding to heparan sulphate and that HSV-1 glycoprotein C (gC) is principally responsible for this binding. Although gC-negative viral mutants are impaired for binding and entry, they retain significant infectivity. The purpose of the studies reported here was to explore the requirements for infectivity of gC-negative HSV-1 mutants. We found that absence or alteration of cell surface heparan sulphate significantly reduced the binding of gC-negative mutant virus and rendered cells resistant to infection, as shown previously for the wild-type virus. We isolated a recombinant double-mutated HSV strain that produces virions devoid of both of the known heparin-binding glycoproteins, gB and gC. The drastically impaired binding of these mutant virions to cells, relative to gC-negative and wild-type virions, indicates that gB mediates the binding of gC-negative virions to cells. Thus at least two HSV glycoproteins can independently mediate the binding of HSV to cell surface heparan sulphate to start the process of viral entry into cells.
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The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0
More LessND10 are nuclear domains of unknown function that become abundant in response to stress. Infection by herpes simplex virus type 1 (HSV-1) causes the apparent disappearance of these domains, an effect that requires the expression of the immediate early protein ICP0. Previously, we have shown that there are a number of cellular antigens in the ND10. In this report, we show that one of these proteins is PML, a member of the C3HC4 zinc-binding domain family which also includes ICP0. The C3HC4 domain of ICP0 is essential for the apparent release of PML from the ND10, although the interaction of ICP0 with ND10 is determined by a small region near its carboxy terminus. PML and other ND10 proteins are not lost after removal from ND10 but deposited at the nuclear envelope or nuclear envelope modifications during later parts of the replication cycle. ICP0 is required for the onset of low multiplicity infections, and has been implicated in the process of reactivation from HSV latency. Therefore, the interaction between ICP0 and the ND10 domains, specifically PML, may be important for the outcome of virus-cell interactions.
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Identification of the feline herpesvirus type 1 (FHV-1) genes encoding glycoproteins G, D, I and E: expression of FHV-1 glycoprotein D in vaccinia and raccoon poxviruses
More LessThe genome of feline herpesvirus type 1 (FHV-1), the major cause of viral upper respiratory disease in cats, contains several genes encoding homologues of herpes simplex virus type 1 (HSV-1) glycoproteins. Restriction mapping studies have indicated that the group D genome of FHV-1 contains a unique short region that is 9·0 kb long. The nucleotide sequence of a 6·2 kb portion of this region was determined. Analyses of this sequence have identified five open reading frames capable of encoding homologues to HSV-1 protein kinase and glycoproteins gG, gD, g1 and gE. Since gD of FHV-1 is most likely an immunologically important polypeptide, vaccinia and raccoon poxvirus recombinants expressing this glycoprotein were generated. In an indirect fluorescent antibody test these recombinants reacted strongly with a rabbit anti-FHV-1 serum. High titres of virus-neutralizing antibodies were also generated in rabbits inoculated with the vaccinia virus recombinant. A 53K viral polypeptide (gD) was detected with this antiserum on Western blots containing polypeptides from potassium tartrate-purified virions.
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An analysis of the in vitro and in vivo phenotypes of mutants of herpes simplex virus type 1 lacking glycoproteins gG, gE, gI or the putative gJ
More LessMutants of herpes simplex virus type 1 (HSV-1) lacking glycoproteins gG, gE, gI or the putative gJ were constructed by inserting a lacZ expression cassette within the US4, US8, US7 and US5 genes respectively. Revertant viruses were then constructed by rescue with a wild-type DNA fragment. Each of these mutant viruses, by comparison with the parental vims HSV-1 SC16, exhibited normal particle to infectivity ratios, and had no discernible phenotypic abnormalities in baby hamster kidney-21 cells following high or low multiplicity infections. Infection of mice by scarification of the ear with these mutant viruses showed the following, (i) Interruption of the US5 (gJ) gene has no effect on the ability of HSV-1 to multiply at the inoculation site or its ability to enter or multiply in the peripheral or central nervous system (CNS). This shows that the US5 gene provides a convenient site for the insertion of foreign genes for both in vitro and in vivo studies, (ii) Dismption of the US4 (gG) gene results in marginal attenuation in the mouse ear model, (iii) Dismption of the US7 (gI) or US8 (gE) genes results in pronounced attenuation; vims was rapidly cleared from the inoculation site and was barely detectable in sensory ganglia or in the CNS. The failure of gI-negative or gE-negative vimses to replicate efficiently at the inoculation site in vivo led to the investigation of vims behaviour in epithelial cells in vitro. Vimses lacking gE or gI adsorbed to and entered these cells at normal rates compared with the parental vims, but formed minute plaques. This is consistent with a failure of cell-to-cell spread by the cell contact route. This was confirmed by measurement of the rate of increase in infectious centre numbers following low multiplicity infections. The view that gE and gI influence interactions between cells at the plasma membrane was reinforced by showing that the introduction of disrupted gE or gI genes into a syncytial, but otherwise syngeneic, background resulted in a non-syncytial phenotype. We conclude that the gE-gI complex plays a part, at least in some cell types, in the interactions at the cell surface that allow transmission of the vims from infected to uninfected cells by cell contact. In syncytial strains this leads to uncontrolled membrane fusion. The observation that virions lacking gE or gI enter cells at apparently normal rates reinforces the view that cell-cell fusion is not analogous to the fusion of the virion envelope with the plasma membrane for nucleocapsid entry. It is also apparent that the phenotypes of HSV-1 mutants lacking gI or gE are similar in many respects to those reported for mutants of pseudorabies vims lacking the gE homologue.
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The analysis of polypeptides in the nuclei and cytoplasm of cells infected with murine herpesvirus 72
More LessTwenty-six polypeptides in murine herpesvirus isolate 72 (MHV-72) were identified and the synthesis and accumulation of 21 virus-specific polypeptides in Vero cells during the course of productive infection with MHV-72 were examined. Five of the infected cell polypeptides accumulated within the nuclei and nine accumulated within the cytoplasm of MHV-72-infected cells. Seven polypeptides were identified within the nuclei and cytoplasm in equivalent amounts. The major capsid protein was shown to have an M r of 161K. Thirteen virus-specific polypeptides were solubilized with Nonidet P-40 or radioimmunoprecipitation assay buffer and immunoprecipitated with rabbit and mouse immune sera. Analysis of the polypeptides of MHV-72 indicate a closer resemblance between MHV-72 and the gammaherpesviruses herpesvirus saimiri and Epstein- Barr virus than with the alphaherpesvirus herpes simplex virus type 1.
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Expression and DNA binding of budgerigar fledgling disease virus large T antigen
More LessBudgerigar fledgling disease virus (BFDV) represents the first non-mammalian member of the polyomavirus genus and possesses uncommon structural and biological properties. Recombinant baculoviruses were constructed to express BFDV small t antigen, large T antigens, as well as a large T deletion mutant Td and β- galactosidase-Td fusion proteins to high levels in infected insect cells. A recombinant virus containing a genomic copy of the BFDV early region was used for small t antigen expression, and corresponding intron- deleted cDNAs for production of large T antigen derivatives. Recombinant T as well as authentic T antigen proteins from infected chicken embryo fibroblasts were purified using both immunoaffinity and DNA affinity column chromatography. We present evidence that the large T antigen interacts specifically with DNA sequences present in the non-coding region of BFDV; by indirect DNA immunoprecipitation mapping and DNase I footprinting, four regions including 12 DNA-binding sites have been determined that cover most of the BFDV non-coding region. The T antigen binding pattern observed suggests a protein-DNA interaction system considerably different from those of simian virus 40 and other polyomaviruses.
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Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function
More LessThe polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF- 1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF- 1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.
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Proposals for a new classification of iridescent viruses
More LessThe need for comparative studies of iridoviruses to elucidate the relationships between them has been well appreciated. Sixteen iridoviruses, including type species from each of the four recognized genera of the Iridoviridae, were compared by restriction endonuclease characterization, hybridization to the major structural protein (MSP) gene of an invertebrate iridescent virus (IV) isolate at various stringencies, PCR amplification of the MSP gene region and by dot-blot hybridization studies. The results broadly supported previous serological studies. The vertebrate iridoviruses, frog virus 3 (genus Ranavirus)and flounder lymphocystivirus (genus Lymphocystivirus), appeared distinct from one another and from the invertebrate isolates. Naming and numbering invertebrate IV isolates according to history and host is no longer useful since IVs infect a number of species. A revised system, involving names based on the geographical origin of the isolate is proposed, in line with other virus families. The large IVs of invertebrates represented by Vero Beach IV (previously IV3 or mosquito IV; genus Chloriridovirus) showed little similarity to any other IVs. Members of the genus Iridovirus, the small invertebrate IVs, fell into three distinct groups of interrelated isolates. The largest group, containing the Plowden (IV1), Tia (IV2), Nelson (IV9, IV10 and IV18), Aberystwyth (IV22), Srinagar (IV24), Fort Collins (IV29) and Stoneville (IV30) iridoviruses, is named the Polyiridovirus complex. The Plowden iridovirus (IV1) is suggested as type species for this complex given the data available on its molecular biology. Based on previously published data, Timaru (IV16 and IV19) and Uitenhage (IV23) iridoviruses are also assigned to this complex. The second but smaller group is named the Oligoirido- virus complex, which includes Dazaifu (IV6) as the type species and contains Ntondwe (IV21 and IV28) on a tentative basis. Riverside IV (IV31) was distinct from both of the other groups, and is proposed as a third complex, Crustaceoiridovirus.
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Evidence for recent genetic variation in monkeypox viruses
More LessDNA from isolates of monkeypox virus, when digested with the endonuclease PstI, gave fragment-size profiles which correlated with the geographic area from which the isolate originated. Although some of the differences were located subterminally in the genome, others mapped to the central conserved region. Further differentiation of the viral genomes was sought by analysis of a short region within the central conserved part of the genome that appeared to be a partially deleted counterpart of an intact 1024 bp open reading frame (ORF) present in variola and vaccinia virus genomes. We reasoned that this region would not be conserved by functional selection and would therefore be likely to show more variation between isolates of monkeypox virus. The deletions found in monkeypox virus isolates from Liberia and from Benin were almost the same as that which we had previously found in the
Denmark strain. A much shortened ORF, potentially coding for a product of 133 amino acids, was retained in all three West African isolates, but three Zairean isolates each showed an identical series of small insertions and deletions which effectively abolish the ORF. Three deletions, present in all isolates, must pre-date the geographical separation of monkeypox virus lineages; other, presumably more recent, changes differ between the Zairean and West African isolates. In contrast, the base similarity was found to be more than 99 % when all the monkeypox virus sequences were appropriately aligned. This, in a disrupted and presumably nonfunctional gene also indicates that the changes described are recent. It is suggested that insertions and deletions occur regularly during poxvirus DNA replication, but are preserved only in sequences that are not required for continued transmission in the natural host.
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Molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type 1 and type 2 genomes
More LessMurine adenovirus (MAd) type 1 strain FL and type 2 strain K87 genomes were cloned into plasmid pAT153 as HindIII restriction fragments. The MAd-1 and MAd-2 DNA genomes, 30·10 kb and 34·71 kb in length respectively, were mapped using BglII, ClaI, EcoRI, HindIII and SphI restriction endonuclease cleavage sites. In view of the large differences found between the MAd-1 and MAd-2 genomes in terms of the number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the MAd-1 and MAd-2 physical maps. Both viruses are also genetically related to human adenovirus type 2 (HAd-2). Nucleotide sequences shared by HAd-2 and the MAds code for structural proteins, which may explain the antigenic similarities between these viruses from different origins. Our results confirm the existence of two distinct adenovirus species in the mouse.
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Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses
More LessThe sequence data presented in this paper have been submitted to the DDBJ sequence database and assigned accession numbers D26079 (CPV-Y1), D26080 (CPV-ob1) and D26081 (CPV-CP49).
Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98 % similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.
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Recombinant protein fragments from haemorrhagic septicaemia rhabdovirus stimulate trout leukocyte anamnestic responses in vitro
A. Estepa, M. Thiry and J. M. CollThis work shows that viral protein fragments are capable of stimulating fish anamnestic immunological responses in leukocytes from the rainbow trout (Oncorhynchus mykiss, W.). Recombinant protein fragments of glycoprotein and nucleoprotein from the rhabdovirus causing viral haemorrhagic septicaemia of trout (YHSY), were cloned and expressed in Escherichia coli, Yersinia ruckeri (a trout pathogen) and Saccharomyces cerevisiae. The recombinant protein fragments stimulated anamnestic responses in leukocyte cultures derived from the anterior kidney of survivors of VHSV infection but not from uninfected trout. Two types of stimulatory anamnestic responses were detected, (i) a stimulation of lymphoproliferation as measured by thymidine incorporation assays and (ii) an increase in number, spreading and size of cells as determined by fibrin-clot and/or flow cytometry techniques. The evidence presented suggests that both adherent and non-adherent trout cell populations are needed for the immunological response to VHSV in this primitive vertebrate. The possible use of in vitro lymphoproliferation assays as a preliminary screening method for candidate fish vaccines prior to their testing in vivo is discussed.
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Protective efficacy in mice of post-exposure vaccination with vaccinia virus recombinant expressing either rabies virus glycoprotein or nucleoprotein
Mice vaccinated intraperitoneally (i.p.) with 107 p.f.u. of a vaccinia virus recombinant expressing either the glycoprotein (rVac-G) or nucleoprotein (rVac-N) of rabies virus 3 weeks before challenge were protected against peripheral lethal infection. Similarly, by postexposure vaccination in which mice were first infected with rabies virus and subsequently vaccinated i.p. with the recombinant, rVac-G conferred protection when given immediately following infection and up to 24 h after infection. Prior treatment of those mice with anti-CD8 monoclonal antibodies (MAb) did not significantly affect the outcome of the infection. In contrast, rVac-N failed to confer protection even with higher doses (108 p.f.u.) of the virus or even when administered by the intradermal route. Anti-nucleoprotein antibody production by these mice was not suppressed by prior rabies virus infection and the levels and the time of antibody production were similar to those of anti-glycoprotein antibody production in mice vaccinated with rVac-G after rabies virus infection. The cytotoxic T lymphocyte response was also not down-regulated by rabies virus in the mice that were given rVac-N. Possible mechanism(s) for the ineffectiveness of rVac-N by post-exposure vaccination in contrast to pre-exposure vaccination was discussed.
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Rift valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases
More LessThe sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3′ -terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237·7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called ‘polymerase module’, we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative- stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA- dependent polymerases in the ‘polymerase module’, we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.
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Linear antigenic and immunogenic regions of the respiratory syncytial virus P protein
More LessThree linear antigenic regions on the P protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were represented by peptides that reacted with monoclonal antibodies and with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the P protein: Pro91 to Asp110, Ser161 to Lys180 and Glu221 to Phe241. The role of individual amino acids in the epitopes defined by monoclonal antibodies was determined. Two monoclonal antibodies reacting with the same antigenic site were found to detect epitopes that had different amino acid dependencies. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the P protein from RSV-infected 35S- labelled cell extracts in a radioimmune precipitation assay. These findings have implications for forthcoming structural-functional studies of RSV capsid component interactions and also for serological diagnosis of RSV infection.
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Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis
More LessHepatitis C virus (HCV) populations in vivo consist of heterogeneous mixtures of genetically different but closely related variants defined as a ‘quasispecies’. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5′-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymorphism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.
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Hepatitis delta virus replication in vitro is not affected by interferon-α or -γ despite intact cellular responses to interferon and dsRNA
More LessThe hepatitis delta vims (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like stmcture. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with antiviral activity, namely the dsRNA-dependent protein kinase (PKR) and 2′,5′ oligoadenylate (2′,5′A) synthetase. Since the vims replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the vims may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA constmct, with IFN-α or IFN-γ for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2′,5′A synthetase and class I MHC is normal and cotransfection of a constmct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase constmct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic vimses is unaffected. IFN-β is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-α or IFN-γ, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.
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Infection of human macrophages with an endogenous tumour necrosis factor-α (TNF-α)-independent human immunodeficiency virus type 1 isolate is unresponsive to the TNF-α synthesis inhibitor RP 55778
More LessMonocyte-derived macrophages (MDM) were demonstrated to be susceptible to productive infection by the monocytotropic human immunodeficiency virus type 1 (HIV-1) strain HIV-1/Ba-L and by three primary HIV-1 isolates, HIV-1/DAS, HIV-1/PAR and HIV-1/THI. Production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β was monitored between days 3 and 26 after MDM infection. TNF-α and IL-6 were detected in cell culture supernatants from days 16 to 21 following HIV-1/DAS, HIV-1/PAR and HIV-1/Ba-L infection, at the time of high viral replication. IL-1β was not found at the same time points. TNF-α mRNA expression occurred around the peak of both TNF-α levels and supernatant RT activities. In HIV-1/THI-infected macrophage cultures no endogenously produced TNF-α was observed, despite high levels of HIV-1 in MDM. This result demonstrates that a primary isolate may replicate independently of TNF-α in MDM. To investigate the relationship between TNF-α and viral replication we used a TNF-α synthesis inhibitor, RP 55778. Treatment throughout the course of cell culture resulted in a significant decrease in both TNF-α levels and viral production in HIV-1/DAS-, HIV-1/PAR- and HIV-1/Ba-L-infected MDM cultures. This phenomenon is reversed by adding recombinant human TNF-α to the RP 55778-treated cell cultures from day 14 post-infection. No effect of RP 55778 was observed in MDM cultures infected with the primary isolate HIV-1/THI, whose replication is independent of TNF-α production and therefore remained unchanged after RP 55778 treatment. We conclude that the clinical value of such a drug is directly dependent on the ability of the HIV-1 strains involved to induce TNF-α production at the time of viral replication.
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Deletion of a single N-linked glycosylation site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion
More LessThe transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus type 1 possesses four consensus sites (Asn-X-Ser/Thr) for the incorporation of N-linked sugars situated on the extracellular domain of the molecule. The purpose of this investigation was to determine the significance of each of these sites in relation to the structure and function of the viral envelope glycoprotein. Each of the four sites was removed by in vitro mutagenesis of gp160 sequence in the non-infectious viral clone pEVdl443, so that amino acids 616, 621, 642 and 679 were each changed from asparagine to serine. The effects of mutagenesis were assessed by syncytium assay after wild-type or mutant envelope clones had been transfected into CD4+ HeLa cells. Removal of the glycosylation site at position 642 resulted in the synthesis of precursor gp160 that was neither cleaved, to give gp120 and gp41, nor transported to the plasma membrane of transfected cells. A consequence of these events was that envelope mutant 642 failed to induce syncytia between neighbouring cells in which it had been expressed. The results of this study indicate that N-linked glycosylation of Asn-642 in the glycoprotein produced by the pEVdl443 expression system is necessary for the correct intracellular processing of gp160 to yield surface-expressed, fusogenic gp41.
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Antibody-dependent cellular cytotoxicity and neutralization of human immunodeficiency virus type 1 by high affinity cross-linking of gp41 to human macrophage Fc IgG receptor using bispecific antibody
Human monocytes/macrophages, which express Fc receptors for IgG are involved in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis. These receptors are known to mediate numerous immunological functions including cell-mediated killing and possibly targeting of HIV to the lysophagosome monocyte-derived macrophage (MDM) entry route for virus neutralization. To study both activities in HIV-1 infection, MDM FcγRI was specifically selected using bispecific antibody (Bs-Ab) containing whole human monoclonal antibody against gp41 and the Fab′ fragment of murine anti- FcγRI 22.2 antibody. Bs-Ab was found to mediate potent antibody-dependent cellular cytotoxicity and virus neutralization.
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Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies
More LessA quantal assay, based on syncytium formation in the human T cell leukaemia-derived C8166 cell line, was used to determine the kinetics of human immunodeficiency virus type 1 (HIV-1) strain IIIB neutralization. Three rat monoclonal antibodies (MAbs) were used, under physiological conditions of temperature and antibody concentration. MAb ICR39·3b (IgG2b) neutralized virus with no lag period while the other two MAbs, ICR39·13g (IgG2b) and ICR41· 1i (IgG2a), neutralized with lag periods of 5 min and 15 min respectively. It was calculated that the latter two MAbs mediated neutralization by about two and three molecules of IgG per virion respectively. The highest neutralization rate constant (for MAb ICR 41· li) was over 300-fold less than that of MAbs specific for the haemagglutinin of the enveloped influenza virus type A and for poliovirus type 1.
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Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication
More LessWe have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of µU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NFκB and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NFκB specificity were found in all cells. Despite this apparent deficit in NFκB activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.
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Inhibition of infectious human immunodeficiency virus type 1 particle formation by Gag protein-derived peptides
Sequential overlapping Gag protein-derived oligopeptides of human immunodeficiency virus type 1 (HIV-1) 22 to 24 amino acids long, were synthesized and tested in vitro for antiviral activity. Two synthetic peptides, one derived from the matrix protein p17 (NPGLLETSEGCRQ, amino acids 47 to 59) and one located in the capsid protein p24 (PAATLEEMMTA, amino acids 339 to 349) inhibited the production of infectious virus when added to HIV-l-infected cultures when used in the range of 20 to 200 μg/ml. As shown by thin section electron microscopy, peptide treatment resulted in the release of immature, deformed virus particles suggesting that the two peptides interfered with assembly and maturation. Other Gag protein-derived oligopeptides had little or no influence on virus production. To characterize further the functionally active regions we synthesized peptide derivatives with three consecutive amino acids substituted by alanine; they did not cause inhibition. Therefore the regions responsible for inhibition were located between amino acids 50 to 61 in pl7, and 342 to 350 in p24. These observations might lead to the development of a new antiviral strategy affecting the late stage of virus replication.
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Differential transcription, without replication, of non-structural and structural genes of human parvovirus B19 in the UT7/EPO cell line as demonstrated by in situ hybridization
More LessErythroid progenitor cells are the main target for B19 parvovirus infection. The UT7 cell line demonstrates a marked erythroid differentiation on induction by erythropoietin (EPO) (UT7/EPO cells) and therefore appears to be a potential target for B19 parvovirus. We aimed to evaluate the presence and localization of B19 nucleic acids in UT7/EPO cells by in situ hybridization.
Three digoxigenin-labelled probes were used: two recognized specifically the non-structural region of the B19 genome and one probe was structural region- specific. In our experiment UT7/EPO cells were not permissive to B19 infection. Transcription led to nonstructural and structural gene transcripts without DNA replication or capsid protein synthesis.
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Characterization of the vaccinia virus L1R myristylprotein as a component of the intracellular virion envelope
More LessIn many cases, virus-encoded acylproteins appear to localize to specific cellular and viral membranes and to be directly involved with the processes of virus morphogenesis and/or egress from the infected cell. It was therefore of interest to determine whether the major vaccinia virus (W) myristylprotein, L1R, is specifically associated with one or more of the membranes enveloping various infectious forms of W virions. To this end, single-membraned intracellular virions (INV) and extracellular enveloped virions (EEV), which are surrounded by at least two distinct membranes, were purified from W-infected cell lysates. The location of the VV L1R protein was determined by using a monospecific anti-L1R serum to detect the L1R protein by immunoblot in INV- and EEV-containing fractions, by examining the proteinase K sensitivity of the L1R protein in intact INV and EEV particles, and by immunoelectron microscopy. The data obtained clearly indicate that although the L1R protein is a constituent of both the INV and EEV particles, it is exclusively found in the inner INV-specific membrane. These results are discussed with regard to the potential role of the W LIR protein in the primary intracellular envelopment of infectious W particles.
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Genetic conservation within subtypes in the hepatitis B virus pre-S2 region
More LessThe antigenic determinants for the main hepatitis B vims (HBV) subtypes adw, adr, ayw and ayr lie in the S (surface) polypeptide. Two amino acid residues in particular, encoded by the S gene at codon positions 122 and 160, have been postulated to determine the different antigenic subtypes. In contrast, the 165 nucleotide pre- S2 gene encodes an immunodominant region common to all subtypes that can give rise to neutralizing antibodies. We have characterized the pre-S2 gene sequences of 29 HBV strains of the three main subtypes, adw, ayw and adr. Seven base positions showed variation that was entirely subtype-specific, with six of these variations leading to subtype-specific amino acid differences. This finding affords the possibility of using pre-S2 sequences for genetic subtyping. Two ayw strains from unrelated patients infected in the Middle East had identical pre-S2 sequences with a block of 12 nucleotides deleted. A geographical correlation with subtype observed from serological results was also apparent from phylogenetic analysis of DNA identities within the pre- S2 region. The results support the concept that the main HBV subtypes truly represent families of phylogen- etically different strains.
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Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates
More LesscDNA copies of the M and N genes of equine arteritis virus (EAV) isolates were synthesized by reverse transcription followed by polymerase chain reaction amplification. The cDNA was subjected to a cycle sequencing strategy using Taq polymerase, and the nucleotide and derived amino acid sequences of 10 virus isolates were compared. The M and N genes of all isolates had the same initiation and termination sites as the prototype Bucyrus strain and the encoded proteins were conserved between viruses. Comparison of nucleotide sequence homologies and phylogenetic tree analysis implied the existence of three EAV variants originating from the U.S.A. (Bucyrus), Austria (Vienna) and Switzerland (Bibuna), and suggested that RNA recombination between EAV isolates may have occurred.
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Multiple repeating motifs are found in the 3′-terminal non-translated region of Semliki Forest virus A7 variant genome
We have analysed the cDNA coding for the envelope glycoprotein (El) gene and the terminal non-translated regions (NTRs) of the avirulent Semliki Forest virus (SFV) A774 (A7) variant. The El gene exhibited 98·5 % identity to the SFV prototype strain L10 (WT) sequence at the nucleotide level. Of the 34 single base substitutions, six led to a change in the deduced amino acid sequence. The 3′ NTR of A7 consisted of a 101 nucleotide sequence, not found in WT, followed by five tandemly arranged sequence motifs, two of which were truncated forms of the others. One full-length and one truncated repeat are found at the 3′ NTR of WT. The repeats of A7 were followed by a non-repeating sequence, very similar to the equivalent region in WT. Owing to the unique sequence motif and the tandem repeats, the 3′ NTR of A7 is 334 nucleotides longer than that of WT. Each of the repeats had an internal 12 nucleotide motif complementary to a conserved sequence in the 5′ -terminal non-structural protein 1- encoding region, thought to be important in alphavirus RNA replication. In the 5′ NTR, three point mutations were found. The conserved sequence binding to the repeated 3 ′ motifs was identical in A7 and WT.
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Comparison of nucleotide and deduced amino acid sequence of the 5′ non-coding region and structural protein genes of the wild-type Japanese encephalitis virus strain SA14 and its attenuated vaccine derivatives
Nucleotide sequences of the 5′ non-coding region and the structural protein genes of the live, attenuated Japanese encephalitis vaccine virus strains SA14-2-8 and SA14-5-3 and the wild-type parental strain SA14/ USA were determined. SA14-2-8 differed from SA14/ USA by 13 nucleotides and eight amino acids whereas SA14-5-3 differed from SA14/USA by 15 nucleotides and eight amino acids. A comparison of the 5′ noncoding region and amino acid sequences of the structural proteins of these two attenuated vaccine strains and of vaccine strains SA14-14-2/PHK and SA14-14-2/PDK with three sequences of their wild-type parent SA14 virus was performed. This revealed only two common amino acid substitutions at positions 138 and 176 in the envelope (E) protein. The substitution at E138 was predicted to cause a change in the secondary structure of the E protein. These two amino acid substitutions in the E protein may contribute to attenuation of the Japanese encephalitis vaccine viruses.
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SCID mouse spleen does not support scrapie agent replication
More LessBALB/e and severe combined immunodeficiency (SCID) mice were inoculated intracerebrally or intraperitoneally with scrapie agent strain ME7 to examine the role of functional lymphocytes and follicular dendritic cells in splenic infectivity and PrPSe accumulation. Intracerebrally inoculated BALB/c and SCID mice developed the clinical signs and microscopic lesions characteristic of scrapie. Spleens from terminally affected BALB/c mice contained PrPSc which was detectable by immuno- blot analysis; SCID mouse spleens did not contain detectable PrPSc. SCID mouse spleens collected during the first 90 days after intraperitoneal infection contained neither infectivity nor PrPSc.
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Replication of the satellite RNA of pea enation mosaic virus is controlled by RNA 2-encoded functions
More LessThe helper virus mediating replication of the satellite RNA (RNA 3) of pea enation mosaic virus (PEMV) consists of two autonomously replicating, taxonomically unrelated viral RNAs with ties to the luteovirus (RNA 1) and the newly proposed umbravirus (RNA 2) genera. The following study dissects the relative contribution of each of the genomic RNAs of PEMV to the subsistence and dissemination of this satellite RNA. Infectivity assays in a pea protoplast system demonstrate that RNA 2 alone is responsible for the replication of RNA 3, an observation that is supported in part by shared regions of sequence homology at the 5′ and 3′ termini of both RNAs. In pea seedlings, infectivity assays also demonstrated that the presence of RNA 2 alone is necessary for the systemic invasion of RNA 3. In contrast, the luteovirus-like phase of PEMV (RNA 1) is solely responsible for the encapsidation and aphid transmission of both RNA 2 and the satellite RNA. In a manner comparable to several other virus-satellite systems, the satellite of PEMV also displays a differential response in its capacity to attenuate symptom expression in selected host species. Thus, the satellite RNA of PEMV exists in a trilateral arrangement with its host and two viral RNAs, comparable in many respects to the satellite-virus-host interaction occurring with groundnut rosette disease.
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Mutations in the helper component protease gene of zucchini yellow mosaic virus affect its ability to mediate aphid transmissibility
More LessThe nucleotide sequence of the helper component protease (HC-Pro) genes of three zucchini yellow mosaic virus (ZYMV) strains has been compared with that of a helper-deficient strain of ZYMV-HC. The comparisons revealed three unique deduced amino acid differences. Two of these mutations were located in regions which are conserved in other potyviruses. The role of these mutations in aphid transmissibility was examined by exchanging DNA fragments of part of the deficient HC- Pro gene with the respective section within the gene of the infectious full-length clone of the aphid-transmissible ZYMV. The first exchange included two of the three mutations, the first coding for a change from Asp to Gly (in a non-eonserved region) and the second coding for a change from Arg to lie [within the Phe-Arg-Asp-Lys (FRNK) conserved box]. This exchange resulted in a reduced transmission (20·6% for the mutated virus compared with 57·4% in the normal ZYMV when acquired from plants and 37·2 % compared with 831 %, respectively, when acquired from membranes). The second exchange incorporated a single mutation [conferring a change from Thr to Ala within the Pro-Thr-Lys (PTK) conserved box]. This single mutation resulted in almost total loss of HC activity in aphid transmission both from plants and from membranes. The Lys residue in the conserved Lys-Ile-Thr-Cys (KITC) box, which is related to loss of HC activity in potato virus Y, tobacco vein mottling virus and in the Michigan strain of ZYMV, is unchanged in the helper-deficient ZYMV. It is therefore proposed that more than one site in HC-Pro may be functionally related to aphid transmissibility. The possible reasons for the role of these mutations in helper activity in aphid transmission of ZYMV are discussed.
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Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction
More LessThe genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (HSP) 70 family. A pair of degenerate primers targeted to motifs A and E, which are highly conserved in HSP70s, was synthesized. Genomes of several definite and possible members of the closterovirus group were screened for the presence of the HSP70 gene with PCR using these degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow stunt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to represent fragments of the respective HSP70 genes. Further screening was performed with an additional degenerate primer targeted to the motif IV of the putative viral polymerase. This degenerate primer and specific primers complementary to the 5′ region of the HSP70 genes of the respective viruses were used to estimate the distance between polymerase motif IV and the start point of the HSP70 gene for BYV (approximately 1·1 kb), CTV and BYSV (around 2·0 kb) by PCR. The amplified genome regions of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned and sequenced. CTV and BYSV were found to encode the gene for an additional 3OK (BYSV) or 33K (CTV) protein between the polymerase and the small hydrophobic protein genes, which was absent in BYV. These two 30K proteins displayed very weak similarity to each other, unlike the highly conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV and BYSV. Degenerate primer- mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.
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Heterologous encapsidation of recombinant pea early browning virus
More LessThe coat protein gene of pea early browning virus (PEBV) was replaced with that of another tobravirus, tobacco rattle virus (TRV strain PPK20). The recombinant virus multiplied efficiently in the systemic host Nicotiana benthamiana and, on the local lesion host Phaseolus vulgaris, produced symptoms typical of PEBV rather than TRV showing that viral coat protein is not a determinant for lesion morphology. Both viral RNAs were encapsidated by TRV coat protein although the shorter particles (encapsidated RNA-2) did not form a discrete population. Evidence is presented to suggest involvement of nucleotide sequences upstream of the coat protein gene in virus particle assembly.
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Expression of the beet yellows closterovirus capsid protein and p24, a capsid protein homologue, in vitro and in vivo
More LessThe positive-sense RNA genome of beet yellows closterovirus (BYY) encompasses open reading frames (ORFs) for the viral capsid protein (CP, ORF 6) and for a CP homologue (p24, ORF 5). The sequences of the ORFs 5 and 6 were inserted into an Escherichia coli expression vector, pQE-9, under the control of the bacteriophage T5 promoter. The proteins were expressed in bacteria, purified, and used for antiserum production in rabbits. The recombinant BYV CP and p24 showed serological cross-reactions when probed with each antiserum on Western blots. The crossreactions of the anti-p24 serum with the CP, and of the anti-CP serum with the p24, were abolished by preadsorption with the heterologous antigens, suggesting that CP and p24 share a common epitope(s) resistant to SDS denaturation. Cross-reactivity of the soluble CP and p24 was also observed in indirect plate-trapped antigen ELISA, whereas virtually none was encountered in double-antibody sandwich ELISA. Using a polyclonal anti-p24 serum preadsorbed with the recombinant CP, the p24 was detected in BYV-infected plants. Analysis of subcellular fractions of BYV-infected Tetragonia expansa indicated that both proteins are predominantly located in the soluble fraction of the host cells. Primer extension analysis of the individual double-stranded forms of the subgenomic RNAs bearing the CP and p24 genes allowed them to be mapped and their 5′ start sites to be located at nucleotide positions 13588 and 12815, respectively, in the complete genome sequence. This corresponds to the 5′ untranslated regions of 52 and 105 nucleotides in the subgenomic RNAs for CP and p24, respectively. The data obtained indicate that the synthesis of both subgenomic RNAs is initiated on a negative RNA strand at an adenosine residue found within the conserved sequence 5′ CCAUUUPyA (shown as positive-sense), which may thus represent a core element of the subgenomic promoter. This conserved sequence also resembles the sequences at the 5′ ends of the CP subgenomic RNAs of tobamoviruses and the Bromoviridae family members, the viruses evolutionarily most closely related to BYV.
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Nucleotide sequence of apple mosaic ilarvirus RNA 4
More LessThe complete nucleotide sequence of apple mosaic ilarvirus RNA 4 was obtained from cloned cDNAs and direct sequencing of the 5′ -terminal RNA region. The sequence is 891 nucleotides long and can encode a protein of 226 amino acids (M r 25171) that, by analogy to alfalfa mosaic virus (A1MV) and tobacco streak virus (TSY), should correspond to the coat protein (CP). Database comparisons showed that no significant similarity to other proteins was apparent. Analysis of the CP sequence revealed a putative ‘zinc finger’ domain and a region rich in basic residues at the amino-terminal portion of the protein, similar to that of TSY. The secondary structure proposed for the 3′-terminal region of RNA 4 shows the presence of three hairpin structures flanked by the tetranucleotide AUGC that are highly similar to those previously described in the RNA 4 species from A1MV and TSV. These results support the idea that both features (metal-binding domain and highly conserved hairpin structures) are characteristics of ilarviruses and are probably involved in the peculiar ‘genome activation’ phenomenon described in these viruses.
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Nucleotide sequence of the original Brazilian isolate of coleus yellow viroid from Solenostemon scutellarioides and infectivity of its complementary DNA
More LessThe complete nucleotide (nt) sequence of the original coleus yellow viroid (CYYd) from Solenostemon scutellarioides, ‘Golden Bedder’, has been determined. The covalently closed single-stranded CYYd RNA molecule consists of 248 nt residues which assumes a rod-like secondary structure when folded in the model of lowest free energy. The sequence was determined by direct sequencing of RNA and from three overlapping cDNA clones. Comparison of the CYYd sequence with that of Coleus blumei viroid 1 (CbVd 1) from Germany demonstrated that they are closely related. The differences observed in the genome organization of CYVd relative to CbVd 1 were at three sites: position 25 (one U deletion), position 26 (a U was replaced by an A) and position 241 (one A insertion). The first two mutations were detected in one A-rich segment of eight nt (between positions 25 and 34). Northern blot hybridization of partially purified nucleic acids from the leaf tissue of S. scutellarioides ‘Frilled Fantasy ’ inoculated with doublestranded cDNA, demonstrated that this fragment was infectious. These data enable CYYd to be assigned to the viroid class of plant pathogens, based on its biological properties and molecular structure. This work also gives additional support to the present classification system, in which the viroids isolated from S. scutellarioides form a distinct subgroup.
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