bluuebunny/crossref_metadata_2025 huggingface dataset:
Computer Aided Analysis of Bending Strength of Involute Spur Gears with Asymmetric Profile
Authors: Cavdar Kadir , Karpat Fatih , Babalik Fatih C.
Published: May 2005

This paper presents a method for the determination of bending stress minimization of involute spur gears. A computer program has been developed to investigate the variation of bending stress and contact ratio depending on the pressure angle on the drive side. Since asymmetric tooth is not standard, the tooth model, which was introduced by DIN 3990/Method C and ISO/TC 60, has been adjusted for asymmetric tooth by the authors. The determination of the tooth form and stress concentration factors for asymmetric tooth has been accomplished for each different parameter (pressure angles, tool radius, rack shift, etc.). The sample results, which were obtained by using a developed computer program, are illustrated with numerical examples.
Characterization and Coordination of Point-line Trajectories
Authors: Ting Kwun-Lon , Zhang Yi , Bunduwongse Ruj
Published: May 2005

A point-line refers to a rigid combination of a directed line and an endpoint along the line. To trace a point-line trajectory, one must control not only the trajectory of the endpoint (the directrix) but also the direction of the point-line (the indicatrix). This paper addresses three issues on point-line trajectories. First of all, by considering the relationship between the point trajectory and the corresponding point-line direction, it offers the complete characterization of point-line trajectories. It presents the coordination of the point-line axis with a free point trajectory and also with a point trajectory constrained on a free form surface. The issue of maintaining an invariant orientation relationship between the point-line axis and the point trajectory is also addressed.
A New Technique for Clearance Influence Analysis in Spatial Mechanisms
Authors: Venanzi Stefano , Parenti-Castelli Vincenzo
Published: May 2005

This paper presents a technique for assessing the influence that clearance in the kinematic pairs of a mechanism has on accuracy. The technique works for both planar and spatial, open-, and closed-chain mechanisms, but not for overconstrained mechanisms. It can be defined deterministic, because it precisely determines the actual pose of the link of interest, and does not rely on probability density functions. The most innovative aspect is that, unlike other existing techniques, knowledge of the forces acting on the mechanism is not needed; on the contrary, given a mechanism configuration, the maximum pose error of the link of interest—that is, the maximum displacement of the link of interest caused by clearance take-up—is directly determined. In this way, a problem which is intrinsically kinematic is solved by a kinematic method, rather than by a kinetostatic one as usually done in the literature. This allows for a more general approach to the clearance problem, as it removes one of the inputs (the external load) usually required for a kinetostatic analysis. The numerical effectiveness of the method is shown on a pure translational parallel manipulator.
A Decentralized Output Feedback Controller for a Class of Large-Scale Interconnected Nonlinear Systems
Authors: Pagilla Prabhakar R. , Zhu Yongliang
Published: May 2005

The decentralized output feedback control problem for a class of large-scale interconnected nonlinear systems is considered. The nonlinear interconnection function of each subsystem is assumed to satisfy a quadratic constraint on the entire state of the large-scale system. A decentralized estimated state feedback controller and a decentralized observer are designed for each subsystem. Sufficient conditions, for each subsystem, under which the proposed controller and observer can achieve exponential stabilization of the overall large-scale system are developed. Simulation results on a numerical example are given to verify the proposed design.
Development of an Experimental Correlation for a Pressure Loss at a Side Orifice
Authors: Nam Ho-Yun , Kim Jong-Man , Seo Kyung-Won , Choi Seok-Ki
Published: May 2005

An experimental study has been carried out to measure the pressure loss at the side orifice of a liquid metal reactor fuel assembly. The characteristics of the pressure loss at the side orifice are investigated using the experimental data measured from 17 different types of side orifices that have different geometric shapes, dimensions, and arrangements of nozzles, and a correlation that covers the whole flow range by one equation is developed. The error range of the correlation is within ±10%, and most of the errors occurred in a region where the Reynolds number is small. The range of Reynolds numbers based on the hydraulic diameter of the orifice is 2000–350,000. It is found that the geometric factor is the most important parameter for the pressure loss when the Reynolds number is >30,000. As the Reynolds number becomes smaller, its effect becomes larger, and when the Reynolds number is small, it is the most important parameter for the pressure loss at the side orifices. The measured data shows a trend that the pressure loss coefficient increases as the number of orifices increases, and the effect of the longitudinal arrangement is small.
Hydrodynamic Design of Rotodynamic Pump Impeller for Multiphase Pumping by Combined Approach of Inverse Design and CFD Analysis
Authors: Cao Shuliang , Peng Guoyi , Yu Zhiyi
Published: May 2005

A combined approach of inverse method and direct flow analysis is presented for the hydrodynamic design of gas-liquid two-phase flow rotodynamic pump impeller. The geometry of impeller blades is designed for a specified velocity torque distribution by treating the two-phase mixture as a homogeneous fluid under the design condition. The three-dimensional flow in the designed impeller is verified by direct turbulent flow analysis, and the design specification is further modified to optimize the flow distribution. A helical axial pump of high specific speed has been developed. To obtain a favorable pressure distribution the impeller blade was back-loaded at the hub side compared to the tip side. Experimental results demonstrate that the designed pump works in a wide flow rate range until the gas volume fraction increases to over 50% and its optimum hydraulic efficiency reaches to 44.0% when the gas volume fraction of two-phase flow is about 15.6%. The validity of design computation has been proved.
Numerical Study of Cavitation in Cryogenic Fluids
Authors: Hosangadi Ashvin , Ahuja Vineet
Published: May 2005

Numerical simulations of cavitation in liquid nitrogen and liquid hydrogen are presented; they represent a broader class of problems where the fluid is operating close to its critical temperature and thermal effects of cavitation are important. A compressible, multiphase formulation that accounts for the energy balance and variable thermodynamic properties of the fluid is described. Fundamental changes in the physical characteristics of the cavity when thermal effects become significant are identified; the cavity becomes more porous, the interface less distinct, and it shows increased spreading while getting shorter in length. The heat transfer model postulated in variants of the B-factor theory, where viscous thermal diffusion at the vapor-liquid interface governs the vaporization, is shown to be a poor approximation for cryogenic fluids. In contrast the results presented here indicate that the cavity is sustained by mass directly convecting into it and vaporization occurring as the liquid crosses the cavity interface. Parametric studies for flow over a hydrofoil are presented and compared with experimental data of Hord (1973, “Cavitation in Liquid Cryogens II—Hydrofoil,” NASA CR-2156); free-stream velocity is shown to be an independent parameter that affects the level of thermal depression.
Sniffers: Fluid-Dynamic Sampling for Olfactory Trace Detection in Nature and Homeland Security—
Authors: Settles Gary S.
Published: May 2005

Vertebrates aim their noses at regions of interest and sniff in order to acquire olfactory trace signals that carry information on food, reproduction, kinship, danger, etc. Invertebrates likewise position antennae in the surrounding fluid to acquire such signals. Some of the fluid dynamics of these natural sensing processes has been examined piecemeal, but the overall topic of sniffing is not well investigated or understood. It is, however, important for several human purposes, especially sampling schemes for sensors to detect chemical and biological traces in the environment. After establishing some background, a general appraisal is given of nature’s accomplishments in the fluid dynamics of sniffing. Opportunities are found for innovation through biomimicry. Since few artificial (“electronic”) noses can currently sniff in the natural sense, ways are considered to help them sniff effectively. Security issues such as explosive trace detection, landmine detection, chemical and biological sniffing, and people sampling are examined. Other sniffing applications including medical diagnosis and leak detection are also considered. Several research opportunities are identified in order to advance this topic of biofluid dynamics. Though written from a fluid dynamics perspective, this review is intended for a broad audience.
Computational Analysis of Pressure and Wake Characteristics of an Aerofoil in Ground Effect
Authors: Mahon Stephen , Zhang Xin
Published: May 2005

The pressure and wake of an inverted cambered aerofoil in ground effect was studied numerically by solving the Reynolds-averaged Navier-Stokes equations. Efforts were focused on the setting up of an accurate numerical model and assessing the abilities of various turbulence models in capturing major physical features associated with the flow, such as surface pressure distribution, separation, level of downforce, and wake. A number of ride heights were studied covering various force regions. Surface pressures, sectional forces, and wake characteristics were compared to experimental data. The k−ω SST and Realizable k−ε turbulence models were found to offer good overall simulations, with the k−ω SST performing better for the surface pressure and the Realizable k−ε better for the wake. The simulations at various ride heights correctly captured the trends in flow-field variations with ride height. The surface pressures, wake flow field, and region of separation on the suction surface of the aerofoil, at lower ride heights, were all modeled accurately.
Picometer-Scale Electronic Control of Molecular Dynamics Inside a Single Molecule
Authors: Lastapis M. , Martin M. , Riedel D. , Hellner L. , Comtet G. , Dujardin G.
Published: May 2005

Tunneling electrons from a low-temperature (5 kelvin) scanning tunneling microscope were used to control, through resonant electronic excitation, the molecular dynamics of an individual biphenyl molecule adsorbed on a silicon(100) surface. Different reversible molecular movements were selectively activated by tuning the electron energy and by selecting precise locations for the excitation inside the molecule. Both the spatial selectivity and energy dependence of the electronic control are supported by spectroscopic measurements with the scanning tunneling microscope. These experiments demonstrate the feasibility of controlling the molecular dynamics of a single molecule through the localization of the electronic excitation inside the molecule.
Glacial/Interglacial Changes in Subarctic North Pacific Stratification
Authors: Jaccard S. L. , Haug G. H. , Sigman D. M. , Pedersen T. F. , Thierstein H. R. , Röhl U.
Published: May 2005

Since the first evidence of low algal productivity during ice ages in the Antarctic Zone of the Southern Ocean was discovered, there has been debate as to whether it was associated with increased polar ocean stratification or with sea-ice cover, shortening the productive season. The sediment concentration of biogenic barium at Ocean Drilling Program site 882 indicates low algal productivity during ice ages in the Subarctic North Pacific as well. Site 882 is located southeast of the summer sea-ice extent even during glacial maxima, ruling out sea-ice–driven light limitation and supporting stratification as the explanation, with implications for the glacial cycles of atmospheric carbon dioxide concentration.
Induction of Protein Secretory Pathway Is Required for Systemic Acquired Resistance
Authors: Wang Dong , Weaver Natalie D. , Kesarwani Meenu , Dong Xinnian
Published: May 2005

In plants, systemic acquired resistance (SAR) is established as a result of NPR1-regulated expression of pathogenesis-related (
PR
) genes. Using gene expression profiling in
Arabidopsis
, we found that in addition to controlling the expression of
PR
genes, NPR1 also directly controls the expression of the protein secretory pathway genes. Up-regulation of these genes is essential for SAR, because mutations in some of them diminished the secretion of PR proteins (for example, PR1), resulting in reduced resistance. We provide evidence that NPR1 coordinately regulates these secretion-related genes through a previously undescribed cis-element. Activation of this cis-element is controlled by a transcription factor that is translocated into the nucleus upon SAR induction.
Single, Rapid Coastal Settlement of Asia Revealed by Analysis of Complete Mitochondrial Genomes
Authors: Macaulay Vincent , Hill Catherine , Achilli Alessandro , Rengo Chiara , Clarke Douglas , Meehan William , Blackburn James , Semino Ornella , Scozzari Rosaria , Cruciani Fulvio , Taha Adi , Shaari Norazila Kassim , Raja Joseph Maripa , Ismail Patimah , Zainuddin Zafarina , Goodwin William , Bulbeck David , Bandelt Hans-Jürgen , Oppenheimer Stephen , Torroni Antonio , Richards Martin
Published: May 2005

A recent dispersal of modern humans out of Africa is now widely accepted, but the routes taken across Eurasia are still disputed. We show that mitochondrial DNA variation in isolated “relict” populations in southeast Asia supports the view that there was only a single dispersal from Africa, most likely via a southern coastal route, through India and onward into southeast Asia and Australasia. There was an early offshoot, leading ultimately to the settlement of the Near East and Europe, but the main dispersal from India to Australia ∼65,000 years ago was rapid, most likely taking only a few thousand years.
Energetic Neutral Atom Emissions from Titan Interaction with Saturn's Magnetosphere
Authors: Mitchell D. G. , Brandt P. C. , Roelof E. C. , Dandouras J. , Krimigis S. M. , Mauk B. H.
Published: May 2005

The Cassini Magnetospheric Imaging Instrument (MIMI) observed the interaction of Saturn's largest moon, Titan, with Saturn's magnetosphere during two close flybys of Titan on 26 October and 13 December 2004. The MIMI Ion and Neutral Camera (INCA) continuously imaged the energetic neutral atoms (ENAs) generated by charge exchange reactions between the energetic, singly ionized trapped magnetospheric ions and the outer atmosphere, or exosphere, of Titan. The images reveal a halo of variable ENA emission about Titan's nearly collisionless outer atmosphere that fades at larger distances as the exospheric density decays exponentially. The altitude of the emissions varies, and they are not symmetrical about the moon, reflecting the complexity of the interactions between Titan's upper atmosphere and Saturn's space environment.
A Long and Winding Road
Authors: Duboule Denis
Published: May 2005

Endless Forms Most Beautiful
The New Science of Evo Devo and the Making of the Animal Kingdom.
By Sean B. Carroll
. Norton, New York, 2005. 371 pp. $25.95, C$38. ISBN 0-393-06016-0.

Writing for the general public, the author provides an introduction to the findings and future prospects of evolutionary developmental biology.
Ion Neutral Mass Spectrometer Results from the First Flyby of Titan
Authors: Waite J. Hunter , Niemann Hasso , Yelle Roger V. , Kasprzak Wayne T. , Cravens Thomas E. , Luhmann Janet G. , McNutt Ralph L. , Ip Wing-Huen , Gell David , De La Haye Virginie , Müller-Wordag Ingo , Magee Brian , Borggren Nathan , Ledvina Steve , Fletcher Greg , Walter Erin , Miller Ryan , Scherer Stefan , Thorpe Rob , Xu Jing , Block Bruce , Arnett Ken
Published: May 2005

The Cassini Ion Neutral Mass Spectrometer (INMS) has obtained the first in situ composition measurements of the neutral densities of molecular nitrogen, methane, molecular hydrogen, argon, and a host of stable carbon-nitrile compounds in Titan's upper atmosphere. INMS in situ mass spectrometry has also provided evidence for atmospheric waves in the upper atmosphere and the first direct measurements of isotopes of nitrogen, carbon, and argon, which reveal interesting clues about the evolution of the atmosphere. The bulk composition and thermal structure of the moon's upper atmosphere do not appear to have changed considerably since the Voyager 1 flyby.
Lichen-Like Symbiosis 600 Million Years Ago
Authors: Yuan Xunlai , Xiao Shuhai , Taylor T. N.
Published: May 2005

The fossil record of fungi and lichens is scarce. Here we report the discovery of lichen-like fossils, involving filamentous hyphae closely associated with coccoidal cyanobacteria or algae, preserved in marine phosphorite of the Doushantuo Formation (between 551 and 635 million years old) at Weng'an, South China. These fossils indicate that fungi developed symbiotic partnerships with photoautotrophs before the evolution of vascular plants.
Going On from Observation
Authors: Burbidge E. Margaret
Published: May 2005

Conflict in the Cosmos
. Fred Hoyle's Life in Science.
By Simon Mitton
. Joseph Henry, Washington, DC, 2005. 398 pp. $27.95, C$37.95. ISBN 0-309-09313-9.

Fred Hoyle
A Life in Science. Aurum, London, 2005. 320 pp. £18.99. ISBN 1-85410-961-8.

In this biography of one of the most famous and controversial astronomers of the 20th century, the author recounts the clashes between Hoyle and his critics along with the underlying science.
Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity
Authors: Buchholz Ursula J. , Biacchesi Stéphane , Pham Quynh N. , Tran Kim C. , Yang Lijuan , Luongo Cindy L. , Skiadopoulos Mario H. , Murphy Brian R. , Collins Peter L.
Published: May 2005

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [rΔM2-1, rΔM2-2, and rΔM2(1+2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The rΔM2-2 mutants exhibited a two- to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with rΔM2-1 or rΔM2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with rΔM2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV rΔM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.
Vaccinia Virus Nonstructural Protein Encoded by the A11R Gene Is Required for Formation of the Virion Membrane
Authors: Resch Wolfgang , Weisberg Andrea S. , Moss Bernard
Published: May 2005

The vaccinia virus A11R gene has orthologs in all known poxvirus genomes, and the A11 protein has been previously reported to interact with the putative DNA packaging protein A32 in a yeast two-hybrid screen. Using antisera raised against A11 peptides, we show that the A11 protein was (i) expressed at late times with an apparent mass of 40 kDa, (ii) not incorporated into virus particles, (iii) phosphorylated independently of the viral F10 kinase, (iv) coimmunoprecipitated with A32, and (v) localized to the viral factory. To determine the role of the A11 protein and test whether it is indeed involved in DNA packaging, we constructed a recombinant vaccinia virus with an inducible A11R gene. This recombinant was dependent on inducer for single-cycle growth and plaque formation. In the absence of inducer, viral late proteins were produced at normal levels, but proteolytic processing and other posttranslational modifications of some proteins were inhibited, suggesting a block in virus particle assembly. Consistent with this observation, electron microscopy of cells infected in the absence of inducer showed virus factories with abnormal electron-dense viroplasms and intermediate density regions associated with membranes and containing the D13 protein. However, no viral membrane crescents, immature virions, or mature virions were produced. The requirement for nonvirion protein A11 in order to make normal viral membranes was an unexpected and exciting finding, since neither the origin of these membranes nor their mechanism of formation in the cytoplasm of infected cells is understood.
Influenza Type A Virus Escape Mutants Emerge In Vivo in the Presence of Antibodies to the Ectodomain of Matrix Protein 2
Authors: Zharikova Darya , Mozdzanowska Krystyna , Feng Jingqi , Zhang Manxin , Gerhard Walter
Published: May 2005

The ectodomain of matrix protein 2 (M2e) of human influenza type A virus strains has remained remarkably conserved since 1918. Because M2e-specific immunity has been shown to decrease morbidity and mortality associated with influenza virus infection in several animal models and because natural infection and current vaccines do not appear to induce a good M2e-specific antibody (Ab) response, M2e has been considered as potential vaccine for inducing cross-reactive protection against influenza type A viruses. The high degree of structural conservation of M2e could in part be the consequence of a poor M2e-specific Ab response and thus the absence of pressure for change. To assess this possibility, we studied the course of infection in SCID mice in the presence or absence of passive M2e-specific monoclonal Abs (MAbs). We found that virus mutants with antigenic changes in M2e emerged in 65% of virus-infected mice treated with M2e-specific but not control MAbs. However, the diversity of escape mutants was highly restricted since only two types were isolated from 22 mice, one with a proline-to-leucine and the other with a proline-to-histidine interchange at amino acid position 10 of M2e. The implications of these findings for the use of M2e as a broadly protective vaccine are discussed.
Glycoprotein D Receptor-Dependent, Low-pH-Independent Endocytic Entry of Herpes Simplex Virus Type 1
Authors: Milne Richard S. B. , Nicola Anthony V. , Whitbeck J. Charles , Eisenberg Roselyn J. , Cohen Gary H.
Published: May 2005

Two herpes simplex virus type 1 (HSV-1) entry pathways have been described: direct fusion between the virion envelope and the plasma membrane, as seen on Vero cells, and low-pH-dependent endocytosis, as seen on CHO nectin-1 and HeLa cells. In this paper, we studied HSV entry into C10 murine melanoma cells and identified a third entry pathway for this virus. During entry into C10 cells, virion envelope glycoproteins rapidly became protected from the membrane-impermeable chemical cross-linker BS3 and from proteinase K. Protection was gD receptor dependent, and the time taken to detect protected protein was proportional to the rate of virus entry. Ultrastructural examination revealed that virions attached to the surface of C10 cells were localized to membrane invaginations, whereas those on the surface of receptor-negative B78 cells were peripherally attached. Virus entry into C10 cells was energy dependent, and intracellular enveloped virions were seen within membrane-bound vesicles consistent with endocytic entry. Entry was not inhibited by bafilomycin A1 or ammonium chloride, showing that passage of the virion through a low-pH environment was not required for infection. Resistance to similar reagents should therefore not be taken as proof of HSV entry by a nonendosomal pathway. These data define a novel gD receptor-dependent acid-independent endocytic entry pathway for HSV.
Serotype-Specific Reorganization of the Mre11 Complex by Adenoviral E4orf3 Proteins
Authors: Stracker Travis H. , Lee Darwin V. , Carson Christian T. , Araujo Felipe D. , Ornelles David A. , Weitzman Matthew D.
Published: May 2005

The early transcriptional region 4 (E4) of adenovirus type 5 (Ad5) encodes gene products that modulate splicing, apoptosis, transcription, DNA replication, and repair pathways. Viruses lacking both E4orf3 and E4orf6 have a severe replication defect, partially characterized by the formation of genome concatemers. Concatemer formation is dependent upon the cellular Mre11 complex and is prevented by both the E4orf3 and E4orf6 proteins. The Mre11/Rad50/Nbs1 proteins are targeted for proteasome-mediated degradation by the Ad5 viral E1b55K/E4orf6 complex. The expression of Ad5 E4orf3 causes a redistribution of Mre11 complex members and results in their exclusion from viral replication centers. For this study, we further analyzed the interactions of E4 proteins from different adenovirus serotypes with the Mre11 complex. Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the E1b55K/E4orf6 complex. Surprisingly, Nbs1 and Rad50 were localized to the replication centers of both Ad4 and Ad12 viruses prior to Mre11 complex degradation. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members. The E4orf3 proteins of Ad4 and Ad12 also failed to complement defects in both concatemer formation and late protein production of a virus with a deletion of E4. These results reveal surprising differences among the highly conserved E4orf3 proteins from different serotypes in the ability to disrupt the Mre11 complex.
Live Bivalent Vaccine for Parainfluenza and Influenza Virus Infections
Authors: Maeda Yasuko , Hatta Masato , Takada Ayato , Watanabe Tokiko , Goto Hideo , Neumann Gabriele , Kawaoka Yoshihiro
Published: May 2005

Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a “cocktail” vaccine with efficacy against several different infectious diseases.
Receptor Binding, Fusion Inhibition, and Induction of Cross-Reactive Neutralizing Antibodies by a Soluble G Glycoprotein ofHendra Virus
Authors: Bossart Katharine N. , Crameri Gary , Dimitrov Antony S. , Mungall Bruce A. , Feng Yan-Ru , Patch Jared R. , Choudhary Anil , Wang Lin-Fa , Eaton Bryan T. , Broder Christopher C.
Published: May 2005

Hendra virus(HeV) andNipah virus(NiV) are closely related emerging viruses comprising theHenipavirusgenus of theParamyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin κ leader sequence coupled to either an S-peptide tag (sGS-tag) or myc-epitope tag (sGmyc-tag) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.
Norovirus and Histo-Blood Group Antigens: Demonstration of a Wide Spectrum of Strain Specificities and Classification of Two Major Binding Groups among Multiple Binding Patterns
Authors: Huang Pengwei , Farkas Tibor , Zhong Weiming , Tan Ming , Thornton Scott , Morrow Ardythe L. , Jiang Xi
Published: May 2005

Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.
Identification of an Interacting Coat-External Scaffolding Protein Domain Required for both the Initiation of φX174 Procapsid Morphogenesis and the Completion of DNA Packaging
Authors: Uchiyama Asako , Fane Bentley A.
Published: May 2005

The φX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven α-helices. The protein's core, α-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all
Microviridae
(canonical members are φX174, G4, and α3) external scaffolding proteins. On the other hand, the primary sequences of the first α-helices have diverged. The results of previous studies with α3/φX174 chimeric external scaffolding proteins suggest that α-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and φX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. φX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.
Regulated and Liver-Specific Tamarin Alpha Interferon Gene Delivery by a Helper-Dependent Adenoviral Vector
Authors: Aurisicchio Luigi , De Tomassi Amedeo , La Monica Nicola , Ciliberto Gennaro , Traboni Cinzia , Palombo Fabio
Published: May 2005

Gene therapy approaches based on liver-restricted and regulated alpha interferon (IFN-α) expression, recently shown to be effective in different murine hepatitis models, appear promising alternatives to inhibit hepatitis C virus (HCV) replication in patients and minimize side effects. Tamarins (
Saguinus
species) infected by GB virus B (GBV-B) are considered a valid surrogate model for hepatitis C to study the biology of HCV infection and the development of new antiviral drugs. To test the efficacy of local delivery and expression of IFN-α in this model, we have developed HD-TET-tIFN, a helper-dependent adenovirus vector expressing tamarin IFN-α (tIFN) under the control of the tetracycline-inducible transactivator rtTA2
s
-S2. Expression of tIFN was successfully induced both in vitro and in vivo in rodents by doxycycline administration with consequent activation of IFN-responsive genes. More importantly, tIFN efficiently inhibited GBV-B replicon in a Huh-7 hepatoma cell line at low HD-TET-tIFN doses. A certain degree of transcriptional control of tIFN was achieved in tamarins injected with HD-TET-tIFN, but under the conditions used in this study, infection and replication of GBV-B were only delayed and not totally abrogated upon virus challenge. Hepatic delivery and regulated expression of IFN-α appear to be a possible approach for the cure of hepatitis, but this approach requires more studies to increase its efficacy. To our knowledge, this is the first report showing a regulated gene expression in a nonhuman primate hepatitis model.
Characterization of the Tupaia Rhabdovirus Genome Reveals a Long Open Reading Frame Overlapping with P and a Novel Gene Encoding a Small Hydrophobic Protein
Authors: Springfeld Christoph , Darai Gholamreza , Cattaneo Roberto
Published: May 2005

Rhabdoviruses are negative-stranded RNA viruses of the orderMononegaviralesand have been isolated from vertebrates, insects, and plants. Members of the genusLyssaviruscause the invariably fatal disease rabies, and a member of the genusVesiculovirus,Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to someVesiculovirusesand manyParamyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to theVesiculoviruses,Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.
Identification of a Linear Peptide Recognized by Monoclonal Antibody 2D7 Capable of Generating CCR5-Specific Antibodies with Human Immunodeficiency Virus-Neutralizing Activity
Authors: Khurana Surender , Kennedy Michael , King Lisa R. , Golding Hana
Published: May 2005

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.
Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination
Authors: Choi Vivian W. , Samulski R. Jude , McCarty Douglas M.
Published: May 2005

Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.
Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy
Authors: Barassi C. , Soprana E. , Pastori C. , Longhi R. , Buratti E. , Lillo F. , Marenzi C. , Lazzarin A. , Siccardi A. G. , Lopalco L.
Published: May 2005

The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+cells from both humans and untreated mice, (iii) inhibit Mip-1β chemotaxis of CD4+CCR5+lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.
Effect of Polypurine Tract (PPT) Mutations on Human Immunodeficiency Virus Type 1 Replication: a Virus with a Completely Randomized PPT Retains Low Infectivity
Authors: Miles Lesa R. , Agresta Beth E. , Khan Mahfuz B. , Tang Shixing , Levin Judith G. , Powell Michael D.
Published: May 2005

We introduced polypurine tract (PPT) mutations, which we had previously tested in an in vitro assay, into the viral clone NL4-3KFSΔ
nef
. Each mutant was tested for single-round infectivity and virion production. All of the PPT mutations had an effect on replication; however, mutation of the 5′ end appeared to have less of an effect on infectivity than mutation of the 3′ end of the PPT sequence. Curiously, a mutation in which the entire PPT sequence was randomized (PPTSUB) retained 12% of the infectivity of the wild type (WT) in a multinuclear activation of galactosidase indicator assay. Supernatants from these infections contained viral particles, as evidenced by the presence of p24 antigen. Two-long terminal repeat (2-LTR) circle junction analysis following PPTSUB infection revealed that the mutant could form a high percentage of normal junctions. Quantification of the 2-LTR circles using real-time PCR revealed that number of 2-LTR circles from cells infected with the PPTSUB mutant was 3.5 logs greater than 2-LTR circles from cells infected with WT virus. To determine whether the progeny virions from a PPTSUB infection could undergo further rounds of replication, we introduced the PPTSUB mutation into a replication-competent virus. Our results show that the mutant virus is able to replicate and that the infectivity of the progeny virions increases with each passage, quickly reverting to a WT PPT sequence. Together, these experiments confirm that the 3′ end of the PPT is important for plus-strand priming and that a virus that completely lacks a PPT can replicate at a low level.
Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response
Authors: Isler Jennifer A. , Skalet Alison H. , Alwine James C.
Published: May 2005

Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.
The bZIP Transcription Factor ATFx Binds Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax and Represses HTLV-1 Long Terminal Repeat-Mediated Transcription
Authors: Forgacs Eva , Gupta Saurabh K. , Kerry Julie A. , Semmes O. John
Published: May 2005

The human T-cell leukemia virus type 1 (HTLV-1) viral protein Tax is a transactivator of transcription driven by the cognate viral long terminal repeat (LTR). Tax exerts its effect through three nonidentical copies of the Tax-responsive element (TxRE), a member of the asymmetric cyclic AMP response element (CRE) family of enhancer sequences. Transactivation is mediated via interaction of Tax with members of the CREB/ATF family bound to TxRE. We have identified a cellular repressor of transcription, activating transcription factor x (ATFx), as a novel Tax-binding protein. In addition to binding directly to Tax we show by electrophoretic mobility shift assay that ATFx binds to the TxRE enhancer element via the bZIP domain. The functional impact of this bridging interaction results in repression of both basal and Tax-induced transcription from the HTLV-1 LTR. ATFx is unique among ATF family of proteins in that it is cell cycle regulated and exerts a tight repressive control over apoptotic signaling. We propose that recruitment of ATFx to the HTLV-1 LTR serves to link viral transcription with critical events in cellular homeostasis.