bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Pathways in Acoustic Tumor Management
Authors: Arriaga Moisés A. , Gorum Michael , Kennedy Arthur
Published: October 2004

Clinical pathways are tools for cost containment and quality improvement in the managed care environment. This report describes our experience with developing and implementing a clinical pathway for acoustic neuroma surgery. Intensive care unit bed days and inpatient hospitalization days for cerebellopontine angle surgery significantly decreased during the months that the pathway was developed, and the incidence of complications was unchanged after adoption of the algorithm. We conclude that with joint neurootology and neurosurgery leadership clinical pathways can be a safe, cost‐effective, and clinically useful technique in acoustic neuroma surgery in a managed care setting. As this management scheme evolves, surgeons must take the leadership to ensure that decisions are based on clinically relevant parameters.
Benign Paroxysmal Positional Vertigo
Authors: Hughes C. Anthony , Proctor Leonard
Published: October 2004

Benign paroxysmal positional vertigo (BPPV) has received renewed clinical interest. At the Johns Hopkins Otological Vestibular Laboratory, 781 patients were tested on more than one occasion between September 1976 and November 1992. Of these patients, 187 (24%) were found to have positional nystagmus consistent with a diagnosis of BPPV. A retrospective analysis of these patients' records, including vestibular test and eye movement reports, audiograms, questionnaires, and hospital charts was performed. After this review, the nystagmus, initially diagnosed as due to BPPV, was believed to be a manifestation of another disease process in 36 cases. Of the remaining 151 diseases, 52 (34%) presented with no significant preceding disorder and have been termed primary BPPV. In the remaining 99 patients we found coexisting or associated disorders which included Meniere's disease, head trauma, prior ear surgery, vestibular neuronitis, migraine, and others. Forty‐five of these 151 patients (31%) had an associated diagnosis of Meniere's disease. To our knowledge a significant association between Meniere's disease and BPPV has not been reported. A review of the literature with discussion of BPPV and the associated disorders is offered.
Midline Glossectomy and Epiglottidectomy for Obstructive Sleep Apnea Syndrome
Authors: Mickelson Samuel A. , Rosenthal Leon
Published: October 2004

Obstructive sleep apnea syndrome is caused by narrowing of the pharyngeal airway and loss of dilator tone during sleep. In patients with severe apnea surgical correction often requires attention to both the oropharynx and hypopharynx. Tongue reduction surgery has been described for persistent apnea after failure of palatal surgery. We describe our experience with midline glossectomy with epiglottidectomy in 12 patients with a mean age of 48.8 ± 14.2 years and body mass index of 36.0 ± 8.8 kg/ m2. Response to treatment was defined as a postoperative respiratory disturbance index (RDI) below 20. Three patients (25%) responded to treatment. The mean apnea index decreased from 48.9 to 35.7, RDI decreased from 73.3 to 46.6, and lowest oxygen saturation increased from 65.9 to 77.9%. RDI in responders decreased from 69.7 to 10. Midline glossectomy with epiglottidectomy has variable results, yet is effective in selected patients with hypopharyngeal narrowing related to macroglossia.
Mucosal Injuries of the Upper Aerodigestive Tract After Smoking Crack or Freebase Cocaine
Authors: Meleca Robert J. , Burgio Don L. , Carr Richard M. , Lolachi Christopher M.
Published: October 2004

The introduction of “crack” and “freebase” cocaine to the United States has resulted in an increased number of patients presenting to emergency rooms with cocaine‐related burns of the upper aerodigestive tract. Because symptoms are nonspecific and histories often unreliable, the emergency room physician must keep a high index of suspicion for cocaine use when confronted with these types of patients. We present seven patients who had burns to the upper aerodigestive tract as a result of smoking crack or freebase cocaine. Two of seven patients underwent an emergency tracheotomy, probably because of delayed diagnosis and inadequate early medical intervention. This article reviews the history behind cocaine use, its different modes of administration, potential complications associated with smoking cocaine, diagnostic workup, and treatment options.
Computer‐Assisted Surgery in the Frontal and Maxillary Sinus
Authors: Gunkel Andreas Rüdiger , Freysinger Wolfgang , Thumfart Walter Franz
Published: October 2004

We report the intraoperative use of bent probes for 3‐dimensional computer‐assisted navigation in ENT surgery with the ISG Viewing Wand. Probe calibration is standardized and the new probes comply with the overall application accuracy of the navigation system. The bent probes permit an easy access to the remotest locations of the maxillary and the frontal sinus and are beneficial for application in paranasal sinus surgery. We present our clinical experiences with these probes.
Immune Responses against Streptococcus pyogenes in Human Palatine Tonsils
Authors: Hideomi Kerakawauchi , Yuichi Kurono , Goro Mogi
Published: October 2004

We investigated cellular immunity against Streptococcus pyogenes in human tonsils by measuring antigen‐specific immunoglobulin‐secreting cells and the production of cytokines from CD4+ T cells in response to M proteins. The incidence of S pyogenes in tonsils was significantly higher in patients with recurrent tonsillitis (RT) than in patients with tonsillar hypertrophy (TH). M protein‐specific immunoglobulin A (IgA) and immunoglobulin G spot‐forming cells were increased in patients with RT compared with patients with TH. In RT the number of M protein‐specific IgA spot‐forming cells was significantly greater in the S pyogenes‐negative subjects than in the S pyogenes‐positive subjects. Proliferation of CD4+ T cells and production of interferon‐gamma (IFN‐γ) and interleukins−2,− 4, −5, and −6 (IL‐2, IL‐4, IL‐5, and IL‐6) from those T cells were observed in response to M protein. The concentrations of IFN‐γ and IL‐4 were higher in RT than in TH. These findings suggest thatS pyogenes is associated with the pathogenesis of RT and that immune responses against M protein may play an important role in preventing the colonization of this bacteria in tonsils.
Contralateral External Carotid‐Middle Cerebral Artery Bypass for Carotid Artery Resection
Authors: Numata Tsutomu , Konno Akiyoshi , Takeuchi Yousuke , Katahashi Tatsuaki , Yuza Jun , Hanazawa Toyoyuki , Ono Junichi
Published: October 2004

Resection of the common carotid or internal carotid artery is occasionally unavoidable in cases of advanced head and neck carcinoma with carotid artery involvement. To prevent the consequent decrease in cerebral perfusion, we have developed a contralateral external carotid‐middle cerebral artery bypass grafting technique. From 1990 through 1995, six patients with advanced head and neck cancer involving the carotid artery underwent total tumor resection combined with vascular reconstruction using the contralateral external carotid‐ middle cerebral artery bypass grafting. No bypass‐related complications occurred in any patient postoperatively. Cerebral hemodynamics after surgery indicated that this bypass graft worked steadily over a long term and supplied sufficient volume of blood to the cerebral cortex on the opposite side in every case.
Nonseptic Lateral Sinus Thrombosis: The Otolaryngologic Perspective
Authors: Rosen Arie , Scher Nattan
Published: October 2004

Nonseptic lateral sinus thrombosis (NSLST) differs from septic lateral thrombosis (SLST) in that it is not associated with ear or sinus infection, it usually occurs in the adult population, and it has an ambiguous clinical presentation. The otolaryngologist is often consulted about diagnostic findings associated with lateral sinus thrombosis. We report three cases of NSLST that required otolaryngologic assessment. The patients were women, ages 23 to 31 years. Presenting symptoms were headaches in all three patients; one also had concomitant ear pain. Two patients had negative head computed tomography scans; only magnetic resonance imaging was diagnostic. Treatment of NSLST consists of anticoagulation and elimination of predisposing factors. Familiarity with this condition is fundamental for early diagnosis and initiation of appropriate therapy. We discuss the origins and causes of NSLST, clinical presentation, preferred diagnostic strategy, and treatment alternatives.
Predictions of microtorsional experiments by micropolar plasticity
Authors: Grammenoudis Paschalis , Tsakmakis Charalampos
Published: October 2004

Kinematic hardening rules are employed in classical plasticity to capture the so–called Bauschinger effect. They are important when describing the material response during reloading. In the framework of thermodynamically consistent gradient plasticity theories, kinematic hardening effects were first incorporated into a micropolar plasticity model by Grammenoudis and Tsakmakis. The aim of the present paper is to investigate this model by predicting size effects in torsional loading of circular cylinders. It is shown that kinematic hardening rules compared with isotropic hardening rules, as adopted in the paper, provide more possibilities for modelling size effects in the material response, even if only monotonous loading conditions are considered.
Analysis of the Drosophila gypsy endogenous retrovirus envelope glycoprotein
Authors: Misseri Yolande , Cerutti Martine , Devauchelle Gérard , Bucheton Alain , Terzian Christophe
Published: October 2004

gypsyis the only endogenous retrovirus ofDrosophilawhose infectious properties have been reported. Previous studies have shown an unexpected relationship between the gene encoding the putative envelope glycoprotein (Env) ofgypsyand genes encoding the fusion protein of several baculoviruses. The fact that fusion proteins mediate membrane fusion suggests that Env of insect retroviruses might also have fusogenic properties. The results reported here indicate thatgypsyEnv mediates cell-to-cell fusion. Cleavage of the Env precursor was also studied; it is shown that this polypeptide is cleaved at a furin-like cleavage site. This is the first report that theenv-like gene of insect retroviruses encodes a fusion protein.
Homology model of the structure of influenza B virus HA1
Authors: Tung Chang-Shung , Goodman Joshua L. , Lu Henry , Macken Catherine A.
Published: October 2004

Influenza B virus is one of two types of influenza virus that cause substantial morbidity and mortality in humans, the other being influenza A virus. The inability to provide lasting protection to humans against influenza B virus infection is due, in part, to antigenic drift of the viral surface glycoprotein, haemagglutinin (HA). Studies of the antigenicity of the HA of influenza B virus have been hampered by lack of knowledge of its structure. To address this gap, two possible models have been inferred for this structure, based on two known structures of the homologous HA of the influenza A virus (subtypes H3 and H9). Statistical, structural and functional analyses of these models suggested that they matched important details of experimental observations and did not differ from each other in any substantive way. These models were used to investigate two HA sites at which viral variants appeared to carry a selective advantage. It was found that each of these sites coevolved with nearby sites to compensate for either size or charge changes.
Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA
Authors: Laghi Luigi , Randolph Ann E. , Malesci Alberto , Boland C. Richard
Published: October 2004

Previous attempts to identify oncogenic polyomaviruses in human cancers have yielded conflicting results, even with the application of PCR technology. Here, it was considered whether the topological features of the polyomavirus genome interfere with efficient PCR amplification. Plasmid and SV40 DNAs were used as a model system for comparing the amplification efficiency of supercoiled, circular relaxed and linear templates. It was found that detection of circular templates required 10 times more molecules than detection of identical but linear templates. Supercoiling hindered the in vitro amplification of SV40 circles by a factor of 10, and erratic amplification of supercoiled SV40 occurred with subpicogram amounts of template. Accordingly, topoisomerase I treatment of DNA improved the PCR detection of supercoiled SV40, significantly decreasing the number of false-negative samples. Previously described, yet controversial, polyomavirus presence in human tissues should be reconsidered and topoisomerase I-sensitive polyomavirus amplification might help to detect polyomavirus genomes in mammalian tissues.
Propagation of a protease-resistant form of prion protein in long-term cultured human glioblastoma cell line T98G
Authors: Kikuchi Yutaka , Kakeya Tomoshi , Sakai Ayako , Takatori Kosuke , Nakamura Naoto , Matsuda Haruo , Yamazaki Takeshi , Tanamoto Ken-ichi , Sawada Jun-ichi
Published: October 2004

Human prion diseases, such as Creutzfeldt–Jakob disease (CJD), a lethal, neurodegenerative condition, occur in sporadic, genetic and transmitted forms. CJD is associated with the conversion of normal cellular prion protein (PrPC) into a protease-resistant isoform (PrPres). The mechanism of the conversion has not been studied in human cell cultures, due to the lack of a model system. In this study, such a system has been developed by culturing cell lines. Human glioblastoma cell line T98G had no coding-region mutations of the prion protein gene, which was of the 129 M/V genotype, and expressed endogenous PrPC constitutively. T98G cells produced a form of proteinase K (PK)-resistant prion protein fragment following long-term culture and high passage number; its deglycosylated form was approximately 18 kDa. The PK-treated PrPres was detected by immunoblotting with the mAb 6H4, which recognizes residues 144–152, and a polyclonal anti-C-terminal antibody, but not by the mAb 3F4, which recognizes residues 109–112, or the anti-N-terminal mAb HUC2-13. These results suggest that PrPC was converted into a proteinase-resistant form of PrPres in T98G cells.
Non-invasive testing reveals a high prevalence of simian T-lymphotropic virus type 1 antibodies in wild adult chimpanzees of the Taï National Park, Côte d'Ivoire
Authors: Leendertz Fabian H. , Boesch Christophe , Ellerbrok Heinz , Rietschel Wolfram , Couacy-Hymann Emmanuel , Pauli Georg
Published: October 2004

Little information is available on the prevalence of retrovirus infections in populations of non-human primates living in their natural habitats. To gain such information, methods were developed to detect antibodies to simian T-lymphotropic virus type 1 (STLV-1) in urine from wild chimpanzees. Samples from more than 74 chimpanzees living in three communities in the Taï National Park, Côte d'Ivoire, were analysed. The prevalence of STLV-1 antibodies in adults and adolescents was significantly higher (35/49, 71·4 %) than that in infant and juvenile chimpanzees (3/31, 9·7 %).
Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle
Authors: Vascotto Fulvia , Campagna Michela , Visintin Michela , Cattaneo Antonino , Burrone Oscar R.
Published: October 2004

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.
Identification of central nervous system genes involved in the host response to the scrapie agent during preclinical and clinical infection
Authors: Booth Stephanie , Bowman Christopher , Baumgartner Richard , Sorensen Garrett , Robertson Catherine , Coulthart Michael , Phillipson Clark , Somorjai Rajmund L.
Published: October 2004

Genes that are expressed differentially in the central nervous system of mice during infection with mouse-adapted scrapie agents were identified in this study. cDNA microarrays were used to examine gene-expression profiles at early, middle (preclinical) and late (clinical) time points after inoculation. A number of genes that showed significant changes in expression during the clinical stage of disease were identified. Of these, 138 were upregulated and 20 were downregulated. A smaller number of genes showed differential expression at the early and middle stages of the disease time course. These genes are interesting, as they may reflect biological processes that are involved in the molecular pathogenesis of the prion agent. At present, little is known about the early events in the disease process that trigger neurodegeneration. Perhaps most interestingly, one group of genes that exhibited decreased expression in all tested stages of the disease was identified in this study. This cluster included four transcripts representing haematopoietic system-related genes, which suggests that the haematopoietic system is involved in the disease process from an early stage.
Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation
Authors: Kim Chan-Lan , Karino Ayako , Ishiguro Naotaka , Shinagawa Morikazu , Sato Motoyoshi , Horiuchi Motohiro
Published: October 2004

The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrPSc), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrPScaccumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrPScformation reduced PrPScaccumulation in cells persistently infected with prions. The 50 % effective dose was as low as ∼1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrPC) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb–PrPCcomplex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrPScformation by interfering with the regular PrPCdegradation pathway.
Impact of measles virus dendritic-cell infection on Th-cell polarization in vitro
Authors: Klagge Ingo M. , Abt Marion , Fries Bianca , Schneider-Schaulies Sibylle
Published: October 2004

Interference of measles virus (MV) with dendritic-cell (DC) functions and deregulation of T-cell differentiation have been proposed to be central to the profound suppression of immune responses to secondary infections up to several weeks after the acute disease. To address the impact of MV infection on the ability of DCs to promote Th-cell differentiation, anin vitrosystem was used where uninfected, tumour necrosis factor alpha/interleukin (IL) 1β-primed DCs were co-cultured with CD45RO−T cells in the presence of conditioned media from MV-infected DCs primed under neutral or DC-polarizing conditions. It was found that supernatants of DCs infected with an MV vaccine strain strongly promoted Th1 differentation, whereas those obtained from wild-type MV-infected DCs generated a mixed Th1/Th0 response, irrespective of the conditions used for DC priming. Th-cell commitment in this system did not correlate with the production of IL12 p70, IL18 or IL23. Thus, a combination of these or other, as yet undefined, soluble factors is produced upon MV infection of DCs that strongly promotes Th1/Th0 differentiation.
Molecular identification and characterization of two proposed new enterovirus serotypes, EV74 and EV75
Authors: Oberste M. Steven , Michele Suzanne M. , Maher Kaija , Schnurr David , Cisterna Daniel , Junttila Nina , Uddin Moyez , Chomel Jean-Jacques , Lau Chi-Shan , Ridha Walid , al-Busaidy Suleiman , Norder Helene , Magnius Lars O. , Pallansch Mark A.
Published: October 2004

Sequencing of the gene that encodes the capsid protein VP1 has been used as a surrogate for antigenic typing in order to distinguish enterovirus serotypes; three new serotypes were identified recently by this method. In this study, 14 enterovirus isolates from six countries were characterized as members of two new types within the species Human enterovirus B, based on sequencing of the complete capsid-encoding (P1) region. Isolates within each of these two types differed significantly from one another and from all other known enterovirus serotypes on the basis of sequences that encode either VP1 alone or the entire P1 region. Members of each type were ⩾77·2 % identical to one another (89·5 % amino acid identity) in VP1, but members of the two different types differed from one another and from other enteroviruses by ⩾31 % in nucleotide sequence (25 % amino acid sequence difference), indicating that the two groups represent separate new candidate enterovirus types. The complete P1 sequences differed from those of all other enterovirus serotypes by ⩾31 % (26 % amino acid sequence difference), but were highly conserved within a serotype (<8 % amino acid sequence difference). Phylogenetic analyses demonstrated that isolates of the same serotype were monophyletic in both VP1 and the capsid as a whole, as shown previously for other enterovirus serotypes. This paper proposes that these 14 isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).
Bovine enterovirus 2: complete genomic sequence and molecular modelling of a reference strain and a wild-type isolate from endemically infected US cattle
Authors: Goens S. D. , Botero S. , Zemla A. , Zhou C. Ecale , Perdue M. L.
Published: October 2004

Bovine enteroviruses are members of the familyPicornaviridae, genusEnterovirus. Whilst little is known about their pathogenic potential, they are apparently endemic in some cattle and cattle environments. Only one of the two current serotypes has been sequenced completely. In this report, the entire genome sequences of bovine enterovirus 2 (BEV-2) strain PS87 and a recent isolate from an endemically infected herd in Maryland, USA (Wye3A) are presented. The recent isolate clearly segregated phylogenetically with sequences representing the BEV-2 serotype, as did other isolates from the endemic herd. The Wye3A isolate shared 82 % nucleotide sequence identity with the PS87 strain and 68 % identity with a BEV-1 strain (VG5-27). Comparison of BEV-2 and BEV-1 deduced protein sequences revealed 72–73 % identity and showed that most differences were single amino acid changes or single deletions, with the exception of the VP1 protein, where both BEV-2 sequences were 7 aa shorter than that of BEV-1. Homology modelling of the capsid proteins of BEV-2 against protein database entries for picornaviruses indicated six significant differences among bovine enteroviruses and other members of the familyPicornaviridae. Five of these were on the ‘rim’ of the proposed enterovirus receptor-binding site or ‘canyon’ (VP1) and one was near the base of the canyon (VP3). Two of these regions varied enough to distinguish BEV-2 from BEV-1 strains. This is the first report and analysis of full-length sequences for BEV-2. Continued analysis of these wild-type strains should yield useful information for genotyping enteroviruses and modelling enterovirus capsid structure.
Cucumber mosaic virus 2b protein compensates for restricted systemic spread of Potato virus Y in doubly infected tobacco
Authors: Ryang Bo-Song , Kobori Takashi , Matsumoto Tadashi , Kosaka Yoshitaka , Ohki Satoshi T.
Published: October 2004

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) inoculated with a necrotic strain of Potato virus Y (PVY, T01 isolate) developed necrotic symptoms in some systemically infected leaves, but not in younger leaves. However, PVY expressed distinct symptoms not only in the older leaves, but also in the younger leaves, of plants that had been doubly inoculated with PVY and with Cucumber mosaic virus (CMV, strain Pepo). A tissue blot immunoassay of tissues from various positions of the stem detected PVY weakly in each stem, but not in the shoot apex, of singly infected plants, whereas PVY was detected at high levels in almost all sections of doubly infected plants. CMV was also detected at high levels in sections of singly and doubly infected plants. Immunohistochemistry of stem tissues showed that in singly infected plants, PVY was confined to external phloem cells and was not detected in internal phloem cells. However, in doubly infected plants, PVY was distributed uniformly throughout whole tissues, including the external phloem, xylem parenchyma and internal phloem cells. In plants that were doubly infected with PVY and PepoΔ2b, a modified CMV that cannot translate the 2b protein, the spread of PVY was restricted as in singly infected plants. These results suggested that the plant host has a counterdefence mechanism that restricts systemic spread of PVY T01, and that the 2b protein of CMV strain Pepo negates this restriction.
Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein α-actinin 1
Authors: Mueller Sandra M. , Jung Ronny , Weiler Sigrid , Lang Sabine M.
Published: October 2004

vpxgenes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55gag. Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen,α-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101–112) of Vpx, which is important for nuclear localization, resulted in loss of interaction withα-actinin 1. These findings suggest that the interaction withα-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.
Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus
Authors: Johnstone Carolina , de León Patricia , Medina Francisco , Melero José A. , García-Barreno Blanca , Val Margarita Del
Published: October 2004

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85–93 and F92–106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85–93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249–258, which is presented by Kdas a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous Kdligand. The CD8+T-lymphocyte responses to epitopes F85–93 and F249–258 present in the F protein of RSV Long were found to be strongly skewed to F85–93 inin vitromultispecific CTL lines andin vivoduring a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8+T-lymphocyte responses to F85–93 and F249–258 epitopes was observedin vivoduring a primary response.
Abnormal prion protein in genetically resistant sheep from a scrapie-infected flock
Authors: Madec J.-Y. , Simon S. , Lezmi S. , Bencsik A. , Grassi J. , Baron T.
Published: October 2004

The central molecular event in transmissible spongiform encephalopathies, such as scrapie in sheep, is the accumulation in tissues of an abnormal isoform of the cellular prion protein. A previous investigation of 26 sheep showed that the accumulation of PrPres in brain correlated more with the prnp genotype than with the severity of the clinical disease. Here, the ability of a sandwich ELISA to detect PrPres distribution in the brain was demonstrated. Immunohistochemistry also strongly supported the hypothesis that the dorsal motor nucleus of the vagus nerve is the possible entry site in the brain for the scrapie agent. Remarkably, three asymptomatic (or possibly asymptomatic for scrapie) sheep carrying an allele known to be associated with clinical scrapie resistance (ARR), which were negative for the detection of PrPres by Western blotting and immunohistochemistry, were positive for the presence of PrPres by ELISA, raising the possibility of carriers resistant to the disease and possibly contributing to the persistence of scrapie in certain flocks.
Development of a reverse-genetics system for Avian pneumovirus demonstrates that the small hydrophobic (SH) and attachment (G) genes are not essential for virus viability
Authors: Naylor Clive J. , Brown Paul A. , Edworthy Nicole , Ling Roger , Jones Richard C. , Savage Carol E. , Easton Andrew J.
Published: October 2004

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.
Natural simian foamy virus infection in wild-caught gorillas, mandrills and drills from Cameroon and Gabon
Authors: Calattini Sara , Nerrienet Eric , Mauclère Philippe , Georges-Courbot Marie-Claude , Saïb Ali , Gessain Antoine
Published: October 2004

A survey for the presence of simian foamy retroviruses (SFVs) was performed in 44 wild-caught apes and monkeys, including 27 gorillas, 11 mandrills and six drills, originating from south Cameroon or Gabon. Combined serological and/or nested-PCR assays indicated SFV infection among fiveGorilla gorilla gorilla, sevenMandrillus sphinxand twoMandrillus leucophaeus. Sequences of a 425 bp fragment of theintegrasegene were obtained for 11 animals. Phylogenetic studies indicated that strains from gorillas, mandrills and drills each formed a highly supported phylogenetic clade with, moreover, the existence of two different gorilla SFVs. This study demonstrates for the first time that these animals are naturally infected with specific SFVs. In the context of simian-to-human interspecies transmission, the results confirm that such viruses can also infect humans, as the SFVs identified in wild-caught animals were the same as those recently reported as infecting hunters living in the same geographical areas.
Vascular endothelial and smooth muscle cells are unlikely to be major sites of latency of human cytomegalovirus in vivo
Authors: Reeves Matthew B. , Coleman Heather , Chadderton Jean , Goddard Martin , Sissons J. G. Patrick , Sinclair John H.
Published: October 2004

Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. It has also been suggested that there may be a link between HCMV and vascular disease. Both smooth muscle and endothelial cells are targets for primary infection with HCMV and have also been postulated as potential sites of HCMV latency. One of the most intensely studied sites of HCMV latency is the cells of the myeloid lineage; there is increasing evidence that the myeloid and endothelial lineages arise from a common precursor in the bone marrow, suggesting that endothelial cells could be another route of HCMV dissemination. However, using a highly sensitive PCR capable of detecting endogenous HCMV in myeloid cells, the HCMV genome in endothelial and smooth muscle cells isolated from the saphenous veins of seropositive patients was not detected. These data suggest that vascular endothelial and smooth muscle cells are unlikely to be important sites of HCMV latency in vivo.
Detection and characterization of cytoplasmic hepatitis B virus reverse transcriptase
Authors: Cao Feng , Tavis John E.
Published: October 2004

It was recently found that the Duck hepatitis B virus (DHBV) reverse transcriptase is primarily a non-encapsidated cytoplasmic molecule that is rapidly translated and has a very short half-life. Here, a non-encapsidated reverse transcriptase from the human Hepatitis B virus (HBV) was characterized. HBV polymerase accumulated in the cytoplasm in a manner similar to non-encapsidated DHBV polymerase. However, the HBV polymerase accumulated at an apparently lower concentration and had a longer half-life than the DHBV enzyme, and it displayed no evidence of the post-translational modifications observed for DHBV. Unlike the DHBV polymerase, immunofluorescence detection of the HBV polymerase in cells was suppressed by the core protein, and this suppression occurred independently of encapsidation. This implies an interaction between the polymerase and core in addition to encapsidation, but the polymerase and core did not co-immunoprecipitate, so the interaction might not be direct. These data indicate that production of cytoplasmic, non-encapsidated polymerase is conserved among the hepadnaviral genera. Furthermore, conservation of the cytoplasmic form of the polymerase suggests that it might have function(s) in virus replication or pathology beyond copying the viral genome.
Reactivation of human herpesvirus 6 during ex vivo expansion of circulating CD34+ haematopoietic stem cells
Authors: Andre-Garnier Elisabeth , Milpied Noel , Boutolleau David , Saiagh Soraya , Billaudel Sylviane , Imbert-Marcille Berthe-Marie
Published: October 2004

Human herpesvirus 6 (HHV-6) replication was evaluated during in vitro expansion of CD34-positive cells that were selected from 11 peripheral blood progenitor cell (PBPC) samples. In order to permit cellular differentiation towards the myeloid lineage, PBPCs were cultured for 14–21 days in a liquid, serum-free medium supplemented with interleukin 1 (IL1), IL3, IL6, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor and stem-cell factor. Among the 10 cultures from HHV-6-seropositive patients, the late, alternatively spliced U100 viral mRNA was detected in five of them after PBPC culture for 14 or 21 days. Recovery of infectious virus from one of the expansions, associated with an increase of HHV-6 viral load and detection of the U100 spliced messenger, confirmed the occurrence of a complete replicative cycle. These data thus demonstrate for the first time that haematopoietic differentiation can lead to HHV-6 reactivation.
RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP
Authors: Choi Chang Won , Qu Feng , Ren Tao , Ye Xiaohong , Morris T. Jack
Published: October 2004

The interaction of the coat protein (CP) of Turnip crinkle virus (TCV) with a host protein, TCV-interacting protein (TIP), from Arabidopsis thaliana has been reported previously. This interaction correlates with the ability of TCV CP to elicit the resistance response that is mediated by the resistance gene HRT in Arabidopsis ecotype Di-17. It has also been established that TCV CP is a suppressor of RNA silencing, a process by which the host plant targets viral RNA for degradation. These results have led to the speculation that TIP might be a component of the RNA-silencing pathway and that TCV CP suppresses RNA silencing through its interaction with TIP. In the current report, a number of TCV CP mutants have been investigated for their ability to suppress RNA silencing. These mutants include single amino acid substitution mutants that are known to have lost their ability to interact with TIP, as well as deletion mutants of TCV CP that are of different sizes and from different regions of the protein. Results showed that each of the single amino acid substitution mutants tested retained high levels of RNA silencing-suppressor activity. In addition, a mutant containing a 5 aa deletion in the region that is known to be critical for TIP interaction retained the ability to suppress RNA silencing significantly. Larger deletions in all regions of TCV CP abolished silencing-suppressor activity. It can be concluded from these results that the RNA silencing-suppressor activity of TCV CP cannot be attributed to its ability to interact directly with TIP.
Successful induction of ovine pulmonary adenocarcinoma in lambs of different ages and detection of viraemia during the preclinical period
Authors: Salvatori Daniela , González Lorenzo , Dewar Patricia , Cousens Christina , Heras Marcelo de las , Dalziel Robert G. , Sharp J. Mike
Published: October 2004

Ovine pulmonary adenocarcinoma (OPA) can be reproduced consistently in neonatal lambs by intratracheal injection of inocula containing jaagsiekte sheep retrovirus (JSRV). In this study, clinical disease, confirmed pathologically as OPA, was induced in a high proportion of lambs that had been inoculated intratracheally with infectious lung fluid at 1, 3 and 6 months of age. The incubation periods, however, were longer in these three age groups than in 1-week-old lambs that were used as controls. Viraemia was detected in all age groups before onset of clinical signs, but occurred later in older animals. These results suggest an age-dependent susceptibility to OPA that could be determined by the availability of JSRV target cells in the ovine lung. The feasibility of inducing OPA in older lambs and detecting JSRV viraemia in preclinical stages enables improved studies on the pathogenesis, assessment of vaccines, diagnosis and control of the disease.
Characterization and manipulation of the human adenovirus 4 genome
Authors: Jacobs Susan C. , Davison Andrew J. , Carr Sharon , Bennett Alice M. , Phillpotts Robert , Wilkinson Gavin W. G.
Published: October 2004

Human adenovirus 4 (HAdV-4), the only serotype of the species HAdV-E to be isolated from man, was first identified by its association with outbreaks of acute respiratory disease in military recruits. To combat such outbreaks, a live, oral HAdV-4 vaccine that is delivered via an enteric-coated capsule was developed. This vaccine has been used for nearly 40 years and has been shown to be safe and efficacious. In this study, the complete DNA sequence (35 994 bp) of the vaccine strain is described and its genetic content is analysed. Phylogenetic comparisons confirmed that the closest sequenced relative of HAdV-4 is another serotype of HAdV-E that infects chimpanzees (SAdV-25) and that the great majority of genes in HAdV-E are related most closely to HAdV-B genes. By using the sequence data, a system was constructed to facilitate production of replication-competent HAdV-4 recombinants.
Genetic diversity and evolution of hepatitis C virus – 15 years on
Authors: Simmonds Peter
Published: October 2004

In the 15 years since the discovery of hepatitis C virus (HCV), much has been learned about its role as a major causative agent of human liver disease and its ability to persist in the face of host-cell defences and the immune system. This review describes what is known about the diversity of HCV, the current classification of HCV genotypes within the familyFlaviviridaeand how this genetic diversity contributes to its pathogenesis. On one hand, diversification of HCV has been constrained by its intimate adaptation to its host. Despite the >30 % nucleotide sequence divergence between genotypes, HCV variants nevertheless remain remarkably similar in their transmission dynamics, persistence and disease development. Nowhere is this more evident than in the evolutionary conservation of numerous evasion methods to counteract the cell's innate antiviral defence pathways; this series of highly complex virus–host interactions may represent key components in establishing its ‘ecological niche’ in the human liver. On the other hand, the mutability and large population size of HCV enables it to respond very rapidly to new selection pressures, manifested by immune-driven changes in T- and B-cell epitopes that are encountered on transmission between individuals with different antigen-recognition repertoires. If human immunodeficiency virus type 1 is a precedent, future therapies that target virus protease or polymerase enzymes may also select very rapidly for antiviral-resistant mutants. These contrasting aspects of conservatism and adaptability provide a fascinating paradigm in which to explore the complex selection pressures that underlie the evolution of HCV and other persistent viruses.
Complementarity, sequence and structural elements within the 3′ and 5′ non-coding regions of the Bunyamwera orthobunyavirus S segment determine promoter strength
Authors: Kohl Alain , Dunn Ewan F. , Lowen Anice C. , Elliott Richard M.
Published: October 2004

The genome of Bunyamwera virus (BUN; family Bunyaviridae) consists of three segments of negative-sense, single-stranded RNA that are called L (large), M (medium) and S (small), according to their size. The genomic RNAs are encapsidated by the viral nucleocapsid protein to form ribonucleoprotein complexes (RNPs). The terminal 3′ and 5′ non-coding sequences are complementary and interact to give a panhandle-like structure to the RNP. Located within these non-coding sequences are elements that control replication and transcription. The sequences of the terminal 11 nt are conserved among the genome segments and are followed by shorter, complementary nucleotide motifs that are conserved on a segment-specific basis. Here, a detailed analysis of the 3′ and 5′ non-coding regions of the BUN S segment is presented. By using a mini-replicon system, it was shown that a functional BUN S promoter requires complementarity, as well as defined sequences, within the terminal 15 nt of either end. It was also shown that the minimal requirement for transcription is localized within the terminal 32 nt of the S segment. A comparison of known strong BUN promoters led to the prediction of a structural element outside the terminal 15 nt; introduction of this motif into the BUN S sequence resulted in increased antigenome and mRNA levels and increased expression of S segment proteins, as shown by mini-replicon assays, as well as recovery of a recombinant virus.
Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity
Authors: Kimalov Boaz , Gal-On Amit , Stav Ran , Belausov Eduard , Arazi Tzahi
Published: October 2004

Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge=0). A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CPΔ20; CP-Nt net charge=+2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious. Nevertheless, AGII-CPΔ20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge. To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants. AGII-CPKKK harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge=+3) was not systemically infectious. Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity. Addition of five negatively charged residues abolished infectivity (AGII-CPDDDDD; CP-Nt net charge=−5) even though a recombinant CPDDDDD could assemble into potyviral-like particle in bacteria. Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CPKKK and AGII-CPDDDDD. GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement. Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity.
Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product
Authors: Fenaux M. , Opriessnig T. , Halbur P. G. , Xu Y. , Potts B. , Meng X.-J.
Published: October 2004

Non-pathogenic porcine circovirus type 1 (PCV1) and pathogenic PCV2 are widespread in swine herds. In this study, the detection and characterization of PCV1 and PCV2 DNA from porcine-derived commercial pepsin are reported. The complete genomic sequences of the pepsin-derived PCV1 and PCV2 share 76 % nucleotide sequence identity with each other and 95–99 % identity with respective North American PCV1 and PCV2 isolates. However, the PCV-contaminated pepsin lacks infectivity in PK-15 cells. To further assess the infectivity of the contaminating pepsinin vivo, 16 5-week-old, specific-pathogen-free pigs were divided randomly into three groups: pigs in group 1 (n=5) were each inoculated intramuscularly and intranasally with 4 ml PBS buffer as negative controls, those in group 2 (n=6) were each inoculated with 400 mg contaminated pepsin dissolved in 4 ml PBS and those in group 3 (n=5) were each inoculated with 4×104·3TCID50PCV2 as positive controls. PCV2 viraemia, seroconversion and pathological lesions were detected in group 3 pigs, but not in group 1 or 2 pigs, confirming that the contaminating PCVs were non-infectious. Nevertheless, the detection of PCV DNA in a porcine-derived commercial product raises concern for potential human infection through xenotransplantation.