bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of apoptosis in atherosclerosis and its therapeutic implications
Authors: STONEMAN Victoria E. A. , BENNETT Martin R.
Published: September 2004

Atherosclerotic plaques develop as a consequence of the accumulation of circulating lipid and the subsequent migration of inflammatory cells (macrophages and T-lymphocytes) and VSMCs (vascular smooth muscle cells). Advanced plaques consist of a lipid-rich core, separated from the lumen by a fibrous cap composed of VSMCs, collagen and extracellular matrix. Plaque enlargement ultimately narrows the lumen (stenosis) causing angina. However, recent studies have emphasized that acute coronary syndromes (unstable angina/myocardial infarction) are caused by lesion erosion/rupture with superimposed thrombus formation on often small non-stenotic plaques. Thus current therapies work predominantly on stabilization of plaques rather than plaque regression. Apoptosis (programmed cell death) is increasingly observed as plaques develop, although the exact mechanisms and consequences of apoptosis in the development and progression of atherosclerosis are still controversial. Increased endothelial cell apoptosis may initiate atherosclerosis, whereas apoptosis of VSMCs and macrophages localizes in ‘vulnerable’ lesions, i.e. those most likely to rupture, and at sites of rupture. This review will focus on the regulation of apoptosis of cells within the vasculature, concentrating on the relevance of apoptosis to plaque progression and clinical consequences of vascular cell apoptosis.
Tumour cell growth in culture: dependence on arginine
Authors: CASO Giuseppe , McNURLAN Margaret A. , McMILLAN Nelson D. , EREMIN Oleg , GARLICK Peter J.
Published: September 2004

The amino acid arginine has been shown to affect the growth of several tumours, although the mechanisms of its action are not clear. In the present study, using a human breast tumour cell line (MCF-7), we investigated the arginine requirements of tumour cells for optimal protein synthesis and growth, and the metabolic pathway responsible for the arginine-dependent growth. The results showed that MCF-7 cells are highly dependent on arginine for growth and that the requirement for arginine is much higher than for an indispensable amino acid, leucine, indicating that arginine is needed for pathways other than protein synthesis. In arginine-free cultures, growth could be completely restored by the urea cycle intermediate citrulline. However, arginine could not be replaced by the urea cycle intermediate and the direct precursor for polyamine synthesis, ornithine, or by the polyamine putrescine, suggesting that the high dependence on arginine is not due to a requirement for polyamine synthesis. Moreover, inhibition of NOS [NO (nitric oxide) synthase] did not affect cell protein synthesis and growth, and the arginine analogue and substrate for NOS, homoarginine, could not replace arginine, implying that the conversion of arginine into NO is not involved in the growth-promoting effects of arginine. The major determinant for the high dependence of MCF-7 cells for arginine was found to be the irreversible conversion of this amino acid into ornithine by the intracellular enzyme arginase. The conversion into ornithine caused a progressive depletion of arginine from the culture medium, which ultimately inhibited cell protein synthesis and halted growth. Intracellular arginase activity may be the major factor determining the requirement for arginine of all cells in culture.
Weight loss leads to reductions in inflammatory biomarkers after a very-low-carbohydrate diet and a low-fat diet in overweight men
Authors: SHARMAN Matthew J. , VOLEK Jeff S.
Published: September 2004

In recent years, it has become apparent that low-grade vascular inflammation plays a key role in all stages of the pathogenesis of atherosclerosis. Weight loss has been shown to improve blood inflammatory markers; however, it is unknown if weight-loss diets varying in macronutrient composition differentially affect inflammatory responses. The primary purpose of the present study was to compare a very-low-carbohydrate diet and a low-fat weight-loss diet on inflammatory biomarkers in overweight men. In a randomized cross-over design, 15 overweight men (body fat, >25%; body mass index, 34 kg/m2) consumed two experimental weight-loss diets for two consecutive 6-week periods: a very-low-carbohydrate diet (<10% energy via carbohydrate) and a low-fat diet (<30% energy via fat). Both the low-fat and the very-low-carbohydrate diets resulted in significant decreases in absolute concentrations of hsTNF-α (high-sensitivity tumour necrosis factor-α), hsIL-6 (high-sensitivity interleukin-6), hsCRP (high-sensitivity C-reactive protein) and sICAM-1 (soluble intercellular cell-adhesion molecule-1). There was no significant change in absolute sP-selectin (soluble P-selectin) concentrations after either diet. Normalized inflammatory values represented as the delta change per 1 kg reduction in body mass showed a significant difference between the two diets only for sP-selectin (P<0.05). In summary, energy-restricted low-fat and very-low-carbohydrate diets both significantly decreased several biomarkers of inflammation. These data suggest that, in the short-term, weight loss is primarily the driving force underlying the reductions in most of the inflammatory biomarkers.
Host–bacterial interactions in inflammatory bowel disease
Authors: MAHIDA Yashwant R. , ROLFE Vivien E.
Published: September 2004

Large numbers of different bacterial species are resident in the lumen of the distal gastrointestinal tract. The normal intestinal host–microbial interactions are not well understood, but the relationship is generally believed to be either mutually beneficial or beneficial to one without disadvantage to the other. Animal model and clinical studies suggest that IBD (inflammatory bowel disease) may develop in a susceptible individual when the normal host–bacterial relationship is dysregulated. In addition to rodent models, this article reviews studies that have investigated the cellular and molecular mechanisms of interactions between intestinal mucosal cells and the resident luminal bacteria in healthy individuals and patients with ulcerative colitis and Crohn's disease. Mechanisms by which the intestinal mucosa is able to avoid pro-inflammatory responses to commensal bacteria (and their products) but able to respond appropriately to luminal pathogens is currently an area of active investigation. Such studies are beginning to provide important clues regarding possible alterations in the mucosa that lead to the development of pro-inflammatory responses to resident bacteria in patients with IBD. Approaches to alter the intestinal microflora for therapeutic purposes and their potential mechanisms of action are also discussed.
Efficient reversal of Alzheimer's disease fibril formation and elimination of neurotoxicity by a small molecule
Authors: Blanchard Barbara J. , Chen Albert , Rozeboom Leslie M. , Stafford Kate A. , Weigele Peter , Ingram Vernon M.
Published: September 2004

The Aβ1-42 peptide that is overproduced in Alzheimer's disease (AD) from a large precursor protein has a normal amino acid sequence but, when liberated, misfolds at neutral pH to form “protofibrils” and fibrils that are rich in β-sheets. We find that these protofibrils or fibrils are toxic to certain neuronal cells that carry Ca-permeant α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Disrupting the structure of the Aβ1-42 fibrils and protofibrils might lead to the discovery of molecules that would be very useful in the treatment of AD. A high-throughput screen of a library of >3,000 small molecules with known “biological activity” was set up to find compounds that efficiently decrease the β-sheet content of aggregating Aβ1-42. Lead compounds were characterized by using thioflavin T (ThT) as a β-sheet assay. The most effective of six compounds found was 4,5-dianilinophthalimide (DAPH) under the following conditions: DAPH at low micromolar concentrations abolishes or greatly reduces previously existing fully formed Aβ1-42 fibrils, producing instead amorphous materials without fibrils but apparently containing some protofibrils and smaller forms. Coincubation of the Aβ1-42 peptide with DAPH produces either amorphous materials or empty fields. Coincubation of DAPH and Aβ1-42 greatly reduces the β-sheet content, as measured with ThT fluorescence, and produces a novel fluorescent complex with ThT. When the Aβ1-42 peptide was coincubated with DAPH at very low micromolar concentrations, the neuronal toxicity mentioned above (Ca
2+
influx) was eliminated. Clearly, DAPH is a promising candidate for AD therapy.
Probing conformational dynamics in single donor-acceptor synthetic molecules by means of photoinduced reversible electron transfer
Authors: Cotlet Mircea , Masuo Sadahiro , Luo Guobin , Hofkens Johan , Van der Auweraer Mark , Verhoeven Jan , Müllen Klaus , Xie Xiaoliang Sunney , De Schryver Frans
Published: September 2004

We use single-molecule fluorescence lifetimes to probe dynamics of photoinduced reversible electron transfer occurring between triphenylamine (donor) and perylenediimide (acceptor) in single molecules of a polyphenylenic rigid dendrimer embedded in polystyrene. Here, reversible electron transfer in individual donor-acceptor molecules results in delayed fluorescence that is emitted with a high photon count rate. By monitoring fluorescence decay times on a photon-by-photon basis, we find fluctuations in both forward and reverse electron transfer spanning a broad time range, from milliseconds to seconds. Fluctuations are induced by conformational changes in the dendrimer structure as well by polystyrene chain reorientation. The conformational changes are related to changes in the dihedral angle of adjacent phenyl rings located in the dendritic branch near the donor transferring the charge, a torsional motion that results in millisecond fluctuations in the “through-bond” donor-acceptor electronic coupling. Polymer chain reorientation leads to changes in the local polarity experienced by the donors and to changes in the solvation of the charge-separated state. As a result, switching between different donor moieties within the same single molecule becomes possible and induces fluctuations in decay time on a time scale of seconds.
Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification
Authors: Yanai Itai , Benjamin Hila , Shmoish Michael , Chalifa-Caspi Vered , Shklar Maxim , Ophir Ron , Bar-Even Arren , Horn-Saban Shirley , Safran Marilyn , Domany Eytan , Lancet Doron , Shmueli Orit
Published: September 2004

Motivation: Genes are often characterized dichotomously as either housekeeping or single-tissue specific. We conjectured that crucial functional information resides in genes with midrange profiles of expression.

Results: To obtain such novel information genome-wide, we have determined the mRNA expression levels for one of the largest hitherto analyzed set of 62 839 probesets in 12 representative normal human tissues. Indeed, when using a newly defined graded tissue specificity index τ, valued between 0 for housekeeping genes and 1 for tissue-specific genes, genes with midrange profiles having 0.15 < τ < 0.85 were found to constitute >50% of all expression patterns. We developed a binary classification, indicating for every gene the IB tissues in which it is overly expressed, and the 12 − IB tissues in which it shows low expression. The 85 dominant midrange patterns with IB = 2–11 were found to be bimodally distributed, and to contribute most significantly to the definition of tissue specification dendrograms. Our analyses provide a novel route to infer expression profiles for presumed ancestral nodes in the tissue dendrogram. Such definition has uncovered an unsuspected correlation, whereby de novo enhancement and diminution of gene expression go hand in hand. These findings highlight the importance of gene suppression events, with implications to the course of tissue specification in ontogeny and phylogeny.

Availability: All data and analyses are publically available at the GeneNote website, http://genecards.weizmann.ac.il/genenote/ and, GEO accession GSE803.

Contact:  doron.lancet@weizmann.ac.il

Supplementary information: Four tables available at the above site.
APART: Automated Preprocessing for NMR Assignments with Reduced Tedium
Authors: Pawley Norma H. , Gans Jason D. , Michalczyk Ryszard
Published: September 2004

Motivation: High-throughput NMR structure determination is a goal that will require progress on many fronts, one of which is rapid resonance assignment. An important rate-limiting step in the resonance assignment process is accurate identification of resonance peaks in the NMR spectra. Peak-picking schemes range from incomplete (which lose essential assignment connectivities) to noisy (which obscure true connectivities with many false ones). We introduce an automated preassignment process that removes false peaks from noisy peak lists by requiring consensus between multiple NMR experiments and exploiting a priori information about NMR spectra. This process is designed to accept multiple input formats and generate multiple output formats, in an effort to be compatible with a variety of user preferences.

Results: Automated preprocessing with APART rapidly identifies and removes false peaks from initial peak lists, reduces the burden of manual data entry, and documents and standardizes the peak filtering process. Successful preprocessing is demonstrated by the increased number of correct assignments obtained when data are submitted to an automated assignment program.

Availability: APART is available from http://sir.lanl.gov/NMR/APART.htm

Contact:  npawley@lanl.gov; rmichalczyk@lanl.gov

Supplementary information: Manual pages with installation instructions, procedures and screen shots can also be found at http://sir.lanl.gov/NMR/APART_Manual1.pdf
Differential localization of neurons susceptible to enterovirus 71 and poliovirus type 1 in the central nervous system of cynomolgus monkeys after intravenous inoculation
Authors: Nagata Noriyo , Iwasaki Takuya , Ami Yasushi , Tano Yoshio , Harashima Ayako , Suzaki Yuriko , Sato Yuko , Hasegawa Hideki , Sata Tetsutaro , Miyamura Tatsuo , Shimizu Hiroyuki
Published: September 2004

Poliovirus and enterovirus 71 (EV71) are both neurotropic enteroviruses that cause serious neurological diseases, such as poliomyelitis and encephalitis. The neurovirulence of EV71 in cynomolgus monkeys was demonstrated previously by intraspinal inoculation. In this study, an improved simian model of EV71 infection was established by using intravenous inoculation, which revealed clinical and neuropathological similarities between this model and human cases of encephalitis. Experimental EV71 infection induced direct neurological manifestations, such as tremor, ataxia and brain oedema, but not non-neurological complications, such as pulmonary oedema and cardiac failure. Using this model of EV71 infection, the neurotropic characteristics of the prototype strains of EV71 and poliovirus type 1 (PV1) were compared. Three monkeys were inoculated intravenously with 105·5 TCID50 EV71 and all developed neurological disease signs within 4–6 days of inoculation. However, after inoculation with 105·5 TCID50 PV1 strain OM1 (PV1-OM1), the major manifestation was flaccid paralysis, starting from the lower limbs 6–9 days post-inoculation. Histopathological and virological analyses of moribund monkeys revealed that disseminated EV71 infection was characterized by severe panencephalitis involving both the pyramidal and extrapyramidal systems. In contrast, the lesions induced by PV1-OM1 were mainly restricted to the pyramidal tract, particularly the spinal motor neurons, thalamus and motor cortex. In conclusion, neuropathological involvement in this model correlated well with the apparent differences in neurological disease induced by EV71 and PV1-OM1. Thus, intravenous inoculation with EV71 is an excellent model to study the neuropathology of EV71 and to evaluate candidate vaccines and potential antiviral agents.
Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed
Authors: Acutis P. L. , Sbaiz L. , Verburg F. , Riina M. V. , Ru G. , Moda G. , Caramelli M. , Bossers A.
Published: September 2004

Frequencies of polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene were studied in 1207 pure-bred and cross-bred Italian Biellese rams, a small ovine breed of about 65 000 head in Italy. Aside from the five most common alleles (VRQ, ARQ, ARR, AHQ and ARH), the rare ARK allele was also found, with the highest frequency reported so far in an ovine breed (2·5 %). ARK/--- genotypes had a total frequency of 4·9 %. The resistance-associated ARR allele was seen at a low frequency (8·3 %). Only 1·4 % of animals examined had a resistant ARR/ARR PrP genotype. Semi-resistant (ARR/ARQ, ARR/ARH and ARR/AHQ) PrP genotypes had a total frequency of 12·6 % and PrP genotypes that are associated with high scrapie susceptibility (e.g. VRQ/VRQ and ARQ/ARQ) had a total frequency of 81·1 %. Statistical analysis comparing PrP allele frequencies between pure-bred and cross-bred animals showed that the ARR allele occurred at a significantly lower frequency in pure-bred rams. Furthermore, comparison of PrP allele frequencies between pure-bred rams over 18 months of age and those below 18 months of age showed a significant decrease in the ARR allele in breeding rams over 18 months of age. Based on these results, breeding for scrapie resistance in the Biellese breed will have to take into account the low frequency of the ARR allele, which also seems to be subject to negative selection by farmers. Further investigation is required to understand whether the ARK allele is also associated with resistance to transmissible spongiform encephalopathies.
Mechanism of cell death during infectious salmon anemia virus infection is cell type-specific
Authors: Joseph Tomy , Cepica Arnost , Brown Laura , Ikede Basil O. , Kibenge Frederick S. B.
Published: September 2004

Infectious salmon anemia virus (ISAV) is a very important fish virus in the Northern hemisphere and there is continued interest in understanding the mechanisms of its pathogenesis and persistence in fish. In this study, the permissive fish cell lines SHK-1, CHSE-214 and TO were used to determine whether ISAV-induced cytopathic effect (CPE) is due to apoptosis or necrosis. Characteristic apoptotic DNA fragmentation was observed only in ISAV-infected SHK-1 and CHSE-214 cells. Apoptosis in ISAV-infected SHK-1 cells was confirmed by fragment end-labelling assay, suggesting that CPE in these cells is associated with apoptosis. ISAV-infected TO cells did not undergo apoptosis, but showed leakage of high-mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis; this suggests that CPE in these cells is associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of HMGB1 protein. Infection with two different strains of ISAV showed that induction of apoptosis was correlated with the appearance of CPE in SHK-1 cells. ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor, Z-VAD-fmk, indicating a caspase-activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 bound caspase-8 specifically in vitro, suggesting that these viral proteins may have a role in ISAV-induced apoptosis. These findings demonstrate for the first time that the mechanism of cell death during ISAV infection is dependent on the cell type, which may have implications for ISAV pathogenesis and persistence.
Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis
Authors: Bartholdy Christina , Olszewska Wieslawa , Stryhn Anette , Thomsen Allan Randrup , Openshaw Peter J. M.
Published: September 2004

A CD8+T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M282–90), linked covalently to humanβ2-microglobulin (β2m). Cutaneous gene-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+T-cell memory. After intranasal RSV challenge, accelerated CD8+T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+T-cell responses than those elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without theβ2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion of CD8+T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope andβ2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8+T-cell responses were not induced. Thus, in addition to specific CD8+T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.
Maternal transmission studies of BSE in sheep
Authors: Foster J. D. , Goldmann W. , McKenzie C. , Smith A. , Parnham D. W. , Hunter N.
Published: September 2004

If BSE (bovine spongiform encephalopathy) infected the UK sheep population concurrently with cattle, it would only now be maintained by transmission between sheep by routes which could include from mother to lamb either in utero or via perinatal close contact. In this study of experimental BSE, Cheviot ewes challenged orally with BSE cattle brain produced lambs of various PrP genotypes over the next 7 years. Of 72 surviving to >30 months of age, 29 are of the most susceptible PrP genotype (AQ/AQ) and born to mothers that were challenged with BSE. None of the progeny have shown any signs of disease. The results suggest that in these sheep, BSE could only transmit by the maternal route at a frequency of less than one in four (95 % confidence limit) from clinically affected ewes, a rate which if replicated in other breeds may not be sufficient to maintain BSE within the sheep population.
Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine
Authors: Pang Hai , Liu Yinggang , Han Xueqing , Xu Yanhui , Jiang Fuguo , Wu Donglai , Kong Xiangang , Bartlam Mark , Rao Zihe
Published: September 2004

Some of the structural proteins of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) carry major epitopes involved in virus neutralization and are essential for the induction of protective humoral responses and the development of an effective vaccine. Rabbit antisera were prepared using full-length N and M proteins and eight expressed fragments covering the S protein. Antisera to S and M proteins were found to have different neutralizing titres towards SARS-CoV infection in vivo, ranging from 1 : 35 to 1 : 128. Antiserum to the N protein did not contain neutralizing antibodies. Epitopes inducing protective humoral responses to virus infection were located mainly in the M protein and a region spanning residues 13–877 of the S protein. The neutralizing ability of antisera directed against the expressed structural proteins was greater than that of convalescent patient antisera, confirming that, as immunogens, the former induce strong, SARS-CoV-specific neutralizing antibody responses. The in vitro neutralization assay has important implications for the design of an effective, protein-based vaccine preventing SARS-CoV infection.
Association of Sf9 cell proliferating cell nuclear antigen with the DNA replication site of Autographa californica multicapsid nucleopolyhedrovirus
Authors: Iwahori Satoko , Ikeda Motoko , Kobayashi Michihiro
Published: September 2004

The accumulation of cellular proliferating cell nuclear antigen (PCNA) in the nucleus of Sf9 cells has been shown to increase upon infection withAutographa californicamulticapsid nucleopolyhedrovirus (AcMNPV). Here, analysis by DNase I treatment and chromatin immunoprecipitation revealed that cellular PCNA in the nucleus of Sf9 cells bound AcMNPV DNA. Immunocytochemical analysis showed colocalization of Sf9 cell PCNA and AcMNPV DNA replication sites. Similar colocalization was also observed in BmN-4 cells infected withBombyx moriNPV, which is inherently missing thepcnagene. The amount of cellular PCNA associated with viral DNA replication sites was greater in cells infected withpcna-defective AcMNPV mutants than in cells infected with wild-type AcMNPV. These results suggest that both cellular and viral PCNAs are involved in AcMNPV DNA replication and thatpcna-defective AcMNPV mutants are able to substitute cellular PCNA for viral PCNA.
Evidence of segment reassortment in Crimean-Congo haemorrhagic fever virus
Authors: Hewson Roger , Gmyl Anatoly , Gmyl Larissa , Smirnova Svetlana E. , Karganova Galina , Jamil Bushra , Hasan Rumina , Chamberlain John , Clegg Christopher
Published: September 2004

The complete nucleotide sequences of the small (S) and medium (M) segments of three independent strains of Crimean-Congo haemorrhagic fever (CCHF) virus isolated in Uzbekistan, Iraq and Pakistan have been determined. Partial S and M segment sequences from two additional strains and partial large segment sequences from five strains of CCHF virus have also been obtained. These data have been compiled and compared with published full-length and partial sequences of other CCHF virus strains. Analysis of virus strains for which complete and partial S and M segment sequences are available reveals that the phylogenetic grouping of some strains differ between these two segments. Data provided in this report suggest that this discrepancy is not the result of recombination, but rather the consequence of reassortment events that have occurred in some virus lineages. Although described in other genera of the Bunyaviridae family, this is the first report of segment reassortment occurring in the Nairovirus genus.
The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout
Authors: Boudinot Pierre , Bernard David , Boubekeur Samira , Thoulouze Maria-Isabel , Bremont Michel , Benmansour Abdenour
Published: September 2004

T-cell responses to viruses are still poorly investigated in lower vertebrates. In rainbow trout, a specific clonal expansion of T cells in response to infection with viral haemorrhagic septicaemia virus (VHSV) was recently identified. Expanded T-cell clones expressed a unique 8 aa Vβ4-Jβ1 junction (SSGDSYSE) in different individuals, reminiscent of a typical public response. To get further insight into the nature of this response the modifications of the T-cell repertoire following immunization with plasmid expressing the VHSV external glycoprotein (G), which is the only protein involved in protective immunity, was analysed. After G-based DNA immunization, CDR3-length spectratypes were skewed for several Vβ-Jβ combinations, including Vβ4-Jβ1. In Vβ4-Jβ1, biases consisted of 6 and 8 aa junctions that were detected from day 52, and were still present 3 months after DNA immunization. Sequence analysis of the Vβ4-Jβ1 junctions showed that the 8 aa junction (SSGDSYSE) was clearly expanded, indicating that viral G protein was probably the target of the anti-VHSV public response. Additional 6 and 8 aa Vβ4-Jβ1 junctions were also expanded in G-DNA-vaccinated fish, showing that significant clonotypic diversity was selected in response to the plasmid-delivered G protein. This higher clonotypic diversity may be related to the demonstrated higher efficiency of G-based DNA vaccines over whole virus immunization. The use of infectious hematopietic necrosis virus (IHNV) recombinant viruses, expressing the VHSV G protein, further substantiated the VHSV G-protein specificity of the 8 aa Vβ4-Jβ1 response and designated the 6 aa Vβ4-Jβ1 response as potentially directed to a T-cell epitope common to VHSV and IHNV.
The M2 gene product of murine gammaherpesvirus 68 is required for efficient colonization of splenic follicles but is not necessary for expansion of latently infected germinal centre B cells
Authors: Simas J. Pedro , Marques Sofia , Bridgeman Anne , Efstathiou Stacey , Adler Heiko
Published: September 2004

Infection of mice with murine gammaherpesvirus 68 is characterized by a marked transient expansion of latently infected splenic germinal centre (GC) B cells, which is followed by lower levels of persistent infection in GC and memory B cells. Virus transcription within GC B cells is restricted to a number of latency-associated open reading frames, including M2. This gene encodes a structurally unique protein of unknown function, which has been shown to be essential for the transient peak of virus latency during the establishment of latent infection in the spleen. This study shows that upon infection of mice with M2-defective viruses, at 14 days post-infection during the establishment of latency in the spleen, there was a reduction in the number of latently infected follicles when compared with wild-type virus. However, the mean number of latently infected cells within each follicle was equivalent between wild-type and M2-defective viruses. Late in infection, disruption of M2 resulted in sustained and abnormally high levels of virus persistence in splenic GC B cells but not memory B cells. These data indicate that during the establishment of latency in the spleen, the M2 gene product is required for efficient colonization of splenic follicles but is dispensable for the expansion of latently infected GC B cells and that M2 might be a critical modulator of B-cell function.
Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods
Authors: Tuplin A. , Evans D. J. , Simmonds P.
Published: September 2004

There is accumulating evidence from bioinformatic studies that hepatitis C virus (HCV) possesses extensive RNA secondary structure in the core and NS5B-encoding regions of the genome. Recent functional studies have defined one such stem–loop structure in the NS5B region as an essentialcis-acting replication element (CRE). A program was developed (structur_dist) that analyses multiple rna-folding patterns predicted bymfoldto determine the evolutionary conservation of predicted stem–loop structures and, by a new method, to analyse frequencies of covariant sites in predicted RNA folding between HCV genotypes. These novel bioinformatic methods have been combined with enzymic mapping of RNA transcripts from the core and NS5B regions to precisely delineate the RNA structures that are present in these genomic regions. Together, these methods predict the existence of multiple, often juxtaposed stem–loops that are found in all HCV genotypes throughout both regions, as well as several strikingly conserved single-stranded regions, one of which coincides with a region of the genome to which ribosomal access is required for translation initiation. Despite the existence of marked sequence conservation between genotypes in the HCV CRE and single-stranded regions, there was no evidence for comparable suppression of variability at either synonymous or non-synonymous sites in the other predicted stem–loop structures. The configuration and genetic variability of many of these other NS5B and core structures is perhaps more consistent with their involvement in genome-scale ordered RNA structure, a structural configuration of the genomes of many positive-stranded RNA viruses that is associated with host persistence.
Phenotype and function of monocyte derived dendritic cells in chronic hepatitis B virus infection
Authors: Tavakoli Soheila , Schwerin Wibke , Rohwer Andreas , Hoffmann Sina , Weyer Sandra , Weth Robert , Meisel Helga , Diepolder Helmut , Geissler Michael , Galle Peter R. , Löhr Hanns F. , Böcher Wulf O.
Published: September 2004

The antiviral T cell failure of patients with chronic hepatitis B virus (HBV) infection was suggested to be caused by a T cell stimulation defect of dendritic cells (DC). To address this hypothesis, monocyte derived DC (MDDC) of patients with chronic or resolved acute HBV infection and healthy controls were studied phenotypically by FACS analyses and functionally by mixed lymphocyte reaction, ELISA, ELISpot and proliferation assays of MDDC cultures or co-cultures with an allogeneic HBc-specific Th cell clone. HBV infection of MDDC was studied by quantitative PCR. MDDC from HBV patients seemed to be infected by the HBV, showed a reduced surface expression of HLA DR and CD40 and exhibited a reduced secretion of IL12p70 in response to HBcAg but not to LPS, as compared to control MDDC. However, after cytokine induced maturation, MDDC from HBV patients revealed an unimpaired phenotype. Moreover, the T cell stimulatory capacity of HBV-DC was intact, since (i) the induction of allospecific proliferative and IFN-γresponses was not affected in HBV-MDDC, and (ii) HLA DR7 restricted stimulation of an allogeneic HBc-specific Th cell clone was not impaired by HBV-MDDC compared to control MDDC. It is hypothesized that HBV infection of DC might lead to minor phenotypic and functional alterations without significantly affecting their antiviral Th cell stimulatory capacity.
Virus entry or the primary infection cycle are not the principal determinants of host specificity of Spodoptera spp. nucleopolyhedroviruses
Authors: Simón Oihane , Williams Trevor , López-Ferber Miguel , Caballero Primitivo
Published: September 2004

The multicapsid nucleopolyhedroviruses (NPVs) of Spodoptera exigua (SeMNPV), Spodoptera frugiperda (SfMNPV), and Spodoptera littoralis (SpliNPV) are genetically similar (78 % similarity) but differ in their degree of host specificity. Infection by each of the three NPVs in these three Spodoptera host species was determined by oral inoculation of larvae with occlusion bodies (OBs) or intrahaemocoelic injection with occlusion derived virions (ODVs). RT-PCR analysis of total RNA from inoculated insects, targeted at immediate early (ie-0), early (egt, DNA polymerase), late (chitinase) and very late genes (polyhedrin), indicated that each of the NPVs initiated an infection in all three host species tested. SpliMNPV produced a fatal NPV disease in both heterologous hosts, S. frugiperda and S. exigua, by oral inoculation or injection. SfMNPV was lethal to heterologous hosts, S. exigua and S. littoralis, but infected larvae did not melt and disintegrate, and progeny OBs were not observed. SeMNPV was able to replicate in heterologous hosts and all genes required for replication were present in the genome, as the virus primary infection cycle was observed. However, gene expression was significantly lower in heterologous hosts. SeMNPV pathogenesis in S. frugiperda and S. littoralis was blocked at the haemocoel transmission stage and very nearly cleared. SeMNPV mixtures with SpliMNPV or SfMNPV did not extend the host range of SeMNPV; in all cases, only the homologous virus was observed to proliferate. It is concluded that entry and the primary virus infection cycle are not the only, or the major determinants, for SeMNPV infection of heterologous Spodoptera species.
Identification of novel HLA-A*0201-restricted CD8+ T-cell epitopes on hepatitis delta virus
Authors: Huang Yi-Hsiang , Tao Mi-Hua , Hu Cheng-po , Syu Wan-Jr , Wu Jaw-Ching
Published: September 2004

Hepatitis delta virus (HDV) superinfection causes a poor prognosis in hepatitis B virus-infected patients and effective therapy is lacking. Cytotoxic T-lymphocyte (CTL) responses play an important role in the pathogenesis of chronic viral hepatitis; however, the CD8+T-cell epitopes of HDV have never been defined. Potential HLA-A*0201-restricted HDV peptides were selected from the SYFPEITHI database and screened by T2 cell-stabilization assay. HLA-A*0201 transgenic mice on a C57BL/6 background were injected intramuscularly with an HDV DNA vaccine. Splenocytes were stained directlyex vivowith HLA-A*0201–peptide tetramers after immunization. Epitope-specific CTL responses were confirmed by cytotoxic assays. HLA-A2, chronically infected HDV patients were also enrolled, to assess the existence of HDV-specific CD8+T cells, based on findings in animals. Following HDV DNA vaccination, nearly 0·9 % of the total splenic CD8+T cells were specific for peptides HDV 26–34 and HDV 43–51 in HLA-A*0201 transgenic mice, which was significantly higher than the number found in non-transgenic mice or in transgenic mice that had been immunized with control plasmid. HDV 26–34- and 43–51-specific CTL lines were able to produce CTL responses to each peptide. Interestingly, HDV 26–34- and HDV 43–51-specific CD8+T cells were also detectable in two chronically infected HDV patients in the absence of active HDV replication. In conclusion, HDV 26–34 and 43–51 are novel HLA-A*0201-restricted CTL epitopes on genotype I HDV. HDV 26–34- and 43–51-specific CTLs have been detected in chronic hepatitis delta patients without active disease. Evoking CTL responses to HDV may be an alternative approach to controlling HDV viraemia in patients with chronic hepatitis delta.
Inhibition of infectious human herpesvirus 8 production by gamma interferon and alpha interferon in BCBL-1 cells
Authors: Pozharskaya Veronika P. , Weakland Laura L. , Offermann Margaret K.
Published: September 2004

Human herpesvirus-8 (HHV-8) is aetiologically linked to Kaposi's sarcoma and primary effusion lymphoma. Although interferon-α(IFN-α) and interferon-γ(IFN-γ) are both antiviral cytokines, IFN-αblocks entry of HHV-8 into the lytic phase, whereas IFN-γinduces an increase in the percentage of cells undergoing lytic replication. Multiple events in the lytic cascade must be completed to produce infectious virus. The ability of both types of IFN to affect the production of infectious virus was explored. Both IFN-αand IFN-γinduced expression of the antiviral proteins double-stranded RNA-activated protein kinase (PKR) and 2′5′-oligoadenylate synthetase (2′5′-OAS) in HHV-8-infected BCBL-1 cells. Higher levels resulted from incubation with IFN-αthan with IFN-γ, whereas IFN-γinduced higher levels of IRF-1 than did IFN-α. IFN-γinduced a minor increase in lytic viral gene expression, which was not accompanied by a detectible increase in infectious virus. When lytic replication of HHV-8 was induced using TPA, high levels of infectious virus appeared in the conditioned medium. When IFN-γwas present during TPA stimulation, the production of infectious virus was reduced by at least a 60 %, and IFN-αfully blocked TPA-induced production of infectious virus. The greater reduction of viral production that occurred with IFN-αis consistent with the higher levels of the antiviral proteins PKR and 2′5′-OAS induced by IFN-αthan by IFN-γ. These studies indicate that the augmentation of cellular antiviral defences by IFN-γwas sufficient to prevent production of infectious virus despite IFN-γ-induced entry of some cells into the lytic phase of HHV-8 replication.
Analysis of the first complete genome sequence of an Old World monkey adenovirus reveals a lineage distinct from the six human adenovirus species
Authors: Kovács Gábor M. , Davison Andrew J. , Zakhartchouk Alexender N. , Harrach Balázs
Published: September 2004

Simian adenovirus 3 (SAdV-3) is one of several adenoviruses that were isolated decades ago from Old World monkeys. Determination of the complete DNA sequence of SAdV-3 permitted the first full genomic comparison of a monkey adenovirus with adenoviruses of humans (HAdVs) and chimpanzees, which are recognized formally as constituting six of the species (HAdV-A to HAdV-F) within the genusMastadenovirus. The SAdV-3 genome is 34 246 bp in size and has a G+C content of 55·3 mol%. It contains all the genes that are characteristic of the genusMastadenovirusand has a single VA-RNA gene and six genes in each of the E3 and E4 regions. The genetic organization is the same as that of HAdV-12, a member of the HAdV-A species. Phylogenetic analyses showed that although SAdV-3 is related marginally more closely to HAdV-A and HAdV-F than to other species, it represents a unique lineage that branched at an early stage of primate adenovirus divergence. The results imply that the genetic layout in SAdV-3 and HAdV-12 may also have characterized the common ancestor of all sequenced primate adenoviruses.
Vaccine protection from CD4+ T-cell loss caused by simian immunodeficiency virus (SIV) mac251 is afforded by sequential immunization with three unrelated vaccine vectors encoding multiple SIV antigens
Authors: Koopman Gerrit , Mortier Daniella , Hofman Sam , Niphuis Henk , Fagrouch Zahra , Norley Steve , Sutter Gerd , Liljeström Peter , Heeney Jonathan L.
Published: September 2004

Candidate human immunodeficiency virus (HIV) vaccine strategies that induce strong cellular immune responses protect rhesus macaques that are infected with recombinant simian/human immunodeficiency virus SHIV89.6p from acute CD4+T-cell loss and delay progression to AIDS. However, similar strategies have not proven as efficacious in the simian immunodeficiency virus (SIV)mac model of AIDS, an infection that causes a slow, steady loss of CD4+T-cell function and numbers in rhesus macaques similar to that caused by HIV-1, the principal cause of AIDS in humans. Efforts to increase vaccine efficacy by repeated boosting with the same vector are quickly limited by rising anti-vector immune responses. Here, the sequential use of three different vectors (DNA, Semliki Forest virus and modified vaccinia virus Ankara) encoding the same SIVmac structural and regulatory antigens was investigated and demonstrated to prevent or slow the loss of CD4+T-cells after mucosal challenge with the highly pathogenic SIVmac251 strain. Of particular interest was an inverse association between the extent of T-helper 2 cytokine responses and steady-state virus load. Although limited in the number of animals, this study provides important proof of the efficacy of the triple-vector vaccine strategy against chronic, progressive CD4+T-cell loss in the rigorous SIVmac/rhesus macaque model of AIDS.
Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase
Authors: Ting Jing-Wen , Wu Min-Feng , Tsai Chih-Tung , Lin Ching-Chun , Guo Ing-Cherng , Chang Chi-Yao
Published: September 2004

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolysis of purine (2′-deoxy)ribonucleosides to free bases and (2′-deoxy)ribose 1-phosphates. Here, a novel piscine viralPNPgene that was identified from grouper iridovirus (GIV), a causative agent of an epizootic fish disease, is reported. This putative GIVPNPgene encodes a protein of 285 aa with a predicted molecular mass of 30 332 Da and shows high similarity to the humanPNPgene. Northern and Western blot analyses of GIV-infected grouper kidney (GK) cells revealed that PNP expression increased in cells with time from 6 h post-infection. Immunocytochemistry localized GIV PNP in the cytoplasm of GIV-infected host cells. PNP–EGFP fusion protein was also observed in the cytoplasm of PNP–EGFP reporter construct-transfected GK and HeLa cells. From HPLC analysis, the recombinant GIV PNP protein was shown to catalyse the reversible phosphorolysis of purine nucleosides and could accept guanosine, inosine and adenosine as substrates. In conclusion, this is the first report of a viral PNP with enzymic activity.
ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana
Authors: Canto Tomas , MacFarlane Stuart A. , Palukaitis Peter
Published: September 2004

Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.
Cell tropism of wild-type measles virus is affected by amino acid substitutions in the P, V and M proteins, or by a truncation in the C protein
Authors: Miyajima Naoko , Takeda Makoto , Tashiro Masato , Hashimoto Koji , Yanagi Yusuke , Nagata Kyosuke , Takeuchi Kaoru
Published: September 2004

Two nucleotide differences in the P/C/V and M genes between B95a cell- and Vero cell-isolated wild-type measles viruses (MV) have previously been found from the same patient. The nucleotide difference in the P/C/V gene resulted in an amino acid difference (M175I) in the P and V proteins and a 19 aa deletion in the C protein. The nucleotide difference in the M gene resulted in an amino acid difference (P64H) in the M protein. To verify this result and to examine further whether the amino acid difference or truncation is important for MV cell tropism, recombinant MV strains containing one of the two nucleotide substitutions, or both, were generated. It was found that the P64H substitution in the M protein was important for efficient virus growth and dissemination in Vero cells and that the M175I substitution in the P and V protein or truncation of the C protein was required for optimal growth.
Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA
Authors: Rubino Luisa , Pantaleo Vitantonio , Navarro Beatriz , Russo Marcello
Published: September 2004

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.