bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance Comparison of Internal Reforming Against External Reforming in a Solid Oxide Fuel Cell, Gas Turbine Hybrid System
Authors: Liese Eric A. , Gemmen Randall S.
Published: February 2005

Solid Oxide Fuel Cell (SOFC) developers are presently considering both internal and external reforming fuel cell designs. Generally, the endothermic reforming reaction and excess air through the cathode provide the cooling needed to remove waste heat from the fuel cell. Current information suggests that external reforming fuel cells will require a flow rate twice the amount necessary for internal reforming fuel cells. The increased airflow could negatively impact system performance. This paper compares the performance among various external reforming hybrid configurations and an internal reforming hybrid configuration. A system configuration that uses the reformer to cool a cathode recycle stream is introduced, and a system that uses interstage external reforming is proposed. Results show that the thermodynamic performance of these proposed concepts are an improvement over a base-concept external approach, and can be better than an internal reforming hybrid system, depending on the fuel cell cooling requirements.
Aerodynamics of Tip Leakage Flows Near Partial Squealer Rims in an Axial Flow Turbine Stage
Authors: Camci Cengiz , Dey Debashis , Kavurmacioglu Levent
Published: February 2005

This paper deals with an experimental investigation of aerodynamic characteristics of full and partial-length squealer rims in a turbine stage. Full and partial-length squealer rims are investigated separately on the pressure side and on the suction side in the “Axial Flow Turbine Research Facility” (AFTRF) of the Pennsylvania State University. The streamwise length of these “partial squealer tips” and their chordwise position are varied to find an optimal aerodynamic tip configuration. The optimal configuration in this cold turbine study is defined as the one that is minimizing the stage exit total pressure defect in the tip vortex dominated zone. A new “channel arrangement” diverting some of the leakage flow into the trailing edge zone is also studied. Current results indicate that the use of “partial squealer rims” in axial flow turbines can positively affect the local aerodynamic field by weakening the tip leakage vortex. Results also show that the suction side partial squealers are aerodynamically superior to the pressure side squealers and the channel arrangement. The suction side partial squealers are capable of reducing the stage exit total pressure defect associated with the tip leakage flow to a significant degree.
Computational Fluid Dynamics Study of Wake-Induced Transition on a Compressor-Like Flat Plate
Authors: Walters D. Keith , Leylek James H.
Published: February 2005

Recent experimental work has documented the importance of wake passing on the behavior of transitional boundary layers on the suction surface of axial compressor blades. This paper documents computational fluid dynamics (CFD) simulations using a commercially available general-purpose CFD solver, performed on a representative case with unsteady transitional behavior. The study implements an advanced version of a three-equation eddy-viscosity model previously developed and documented by the authors, which is capable of resolving boundary layer transition. It is applied to the test cases of steady and unsteady boundary layer transition on a two-dimensional flat plate geometry with a freestream velocity distribution representative of the suction side of a compressor airfoil. The CFD results are analyzed and compared to a similar experimental test case from the open literature. Results with the model show a dramatic improvement over more typical Reynolds-averaged Navier–Stokes (RANS)-based modeling approaches, and highlight the importance of resolving transition in both steady and unsteady compressor aerosimulations.
Effects of Pore Size Variations on Regenerative Wheel Performance
Authors: Shang Wei , Besant Robert W.
Published: February 2005

Manufacturing tolerances usually cause the air flow channel pore sizes to have a random variation in the matrices of regenerative wheels. The effects of random pore size distribution on pressure drop across a regenerative energy wheel transferring heat and moisture and effectiveness are investigated using analytical methods. Compared to an identical wheel with no pore size variation, simple algebraic expressions for pressure drop ratio, Δp/Δp0, and effectiveness ratio, ε/ε0, are developed for a Gaussian distribution of flow channel hydraulic diameters. Graphical results are presented showing that large random variations in flow channel pore size decrease the pressure drop across a wheel and the effectiveness (sensible, latent, and total) significantly for a regenerative wheel. Optical and micrometer measurements of four typical regenerative wheels showed a random variation in flow channel hydraulic diameters. These data imply significant decreases in Δp/Δp0 and ε/ε0 for each wheel.
The Impact of Oil and Sealing Air Flow, Chamber Pressure, Rotor Speed, and Axial Load on the Power Consumption in an Aeroengine Bearing Chamber
Authors: Flouros Michael
Published: February 2005

Trends in aircraft engines have dictated high speed rolling element bearings up to 3 million DN or more with the consequence of having high amounts of heat rejection in the bearing chambers and high oil scavenge temperatures. A parametric study on the bearing power consumption has been performed with a 124 mm pitch circle diameter (PCD) ball bearing in a bearing chamber that has been adapted from the RB199 turbofan engine DN∼2×106. The operating parameters such as oil flow, oil temperature, sealing air flow, bearing chamber pressure, and shaft speed have been varied in order to assess the impact on the power consumption. This work is the first part of a survey aiming to reduce power losses in bearing chambers. In the first part, the parameters affecting the power losses are identified and evaluated.
The Effect of Turbine Blade Cooling on the Cycle Efficiency of Gas Turbine Power Cycles
Authors: Wilcock R. C. , Young J. B. , Horlock J. H.
Published: February 2005

A thermodynamic cycle analysis computer code for the performance prediction of cooled gas turbines has been used to calculate the efficiency of plants with varying combustor outlet temperature, compressor pressure ratio, and turbomachinery polytropic efficiency. It is shown that the polytropic efficiency exerts a major influence on the optimum operating point of cooled gas turbines: for moderate turbomachinery efficiency the search for enhanced combustor outlet temperature is shown to be logical, but for high turbomachinery efficiency this is not necessarily so. The sensitivity of the cycle efficiency to variation in the parameters determining the cooling flow rates is also examined. While increases in allowable blade metal temperature and film cooling effectiveness are more beneficial than improvements in other parameters, neither is as important as increase in turbomachinery aerodynamic efficiency.
Laser Ignition of Methane-Air Mixtures at High Pressures and Diagnostics
Authors: Kopecek Herbert , Charareh Soren , Lackner Maximilian , Forsich Christian , Winter Franz , Klausner Johann , Herdin Gu¨nther , Weinrotter Martin , Wintner Ernst
Published: February 2005

Methane-air mixtures at high fill pressures up to 30 bar and high temperatures up to 200°C were ignited in a high-pressure chamber with automated fill control by a 5 ns pulsed Nd:YAG laser at 1064 nm wavelength. Both, the minimum input laser pulse energy for ignition and the transmitted fraction of energy through the generated plasma were measured as a function of the air/fuel-equivalence ratio (λ). The lean-side ignition limit of methane-air mixtures was found to be λ=2.2. However, only λ<2.1 seems to be practically usable. As a comparison, the limit for conventional spark plug ignition of commercial natural gas engines is λ=1.8. Only with excessive efforts λ=2.0 can be spark ignited. The transmitted pulse shape through the laser-generated plasma was determined temporally as well as its dependence on input laser energy and properties of the specific gases interacting. For a first demonstration of the practical applicability of laser ignition, one cylinder of a 1 MW natural gas engine was ignited by a similar 5 ns pulsed Nd:YAG laser at 1064 nm. The engine worked successfully at λ=1.8 for a first test period of 100 hr without any interruption due to window fouling and other disturbances. Lowest values for NOx emission were achieved at λ=2.05 NOx=0.22 g/KWh. Three parameters obtained from accompanying spectroscopic measurements, namely, water absorbance, flame emission, and the gas inhomogeneity index have proven to be powerful tools to judge laser-induced ignition of methane-air mixtures. The following effects were determined by the absorption spectroscopic technique: formation of water in the vicinity of the laser spark (semi-quantitative); characterization of ignition (ignition delay, incomplete ignition, failed ignition); homogeneity of the gas phase in the vicinity of the ignition; and the progress of combustion.
Advanced Zero Emissions Gas Turbine Power Plant
Authors: Griffin Timothy , Sundkvist Sven Gunnar , A˚sen Knut , Bruun Tor
Published: February 2005

The AZEP “advanced zero emissions power plant” project addresses the development of a novel “zero emissions,” gas turbine-based, power generation process to reduce local and global CO2 emissions in the most cost-effective way. Process calculations indicate that the AZEP concept will result only in a loss of about 4% points in efficiency including the pressurization of CO2 to 100 bar, as compared to approximately 10% loss using conventional tail-end CO2 capture methods. Additionally, the concept allows the use of air-based gas turbine equipment and, thus, eliminates the need for expensive development of new turbomachinery. The key to achieving these targets is the development of an integrated MCM-reactor in which (a) O2 is separated from air by use of a mixed-conductive membrane (MCM), (b) combustion of natural gas occurs in an N2-free environment, and (c) the heat of combustion is transferred to the oxygen-depleted air by a high temperature heat exchanger. This MCM-reactor replaces the combustion chamber in a standard gas turbine power plant. The cost of removing CO2 from the combustion exhaust gas is significantly reduced, since this contains only CO2 and water vapor. The initial project phase is focused on the research and development of the major components of the MCM-reactor (air separation membrane, combustor, and high temperature heat exchanger), the combination of these components into an integrated reactor, and subsequent scale-up for future integration in a gas turbine. Within the AZEP process combustion is carried out in a nearly stoichiometric natural gas/O2 mixture heavily diluted in CO2 and water vapor. The influence of this high exhaust gas dilution on the stability of natural gas combustion has been investigated, using lean-premix combustion technologies. Experiments have been performed both at atmospheric and high pressures (up to 15 bar), simulating the conditions found in the AZEP process. Preliminary tests have been performed on MCM modules under simulated gas turbine conditions. Additionally, preliminary reactor designs, incorporating MCM, heat exchanger, and combustor, have been made, based on the results of initial component testing. Techno-economic process calculations have been performed indicating the advantages of the AZEP process as compared to other proposed CO2-free gas turbine processes.
The Transient Liquid Crystal Technique: Influence of Surface Curvature and Finite Wall Thickness
Authors: Wagner G. , Kotulla M. , Ott P. , Weigand B. , von Wolfersdorf J.
Published: February 2005

The transient liquid crystal technique is currently widely used for measuring the heat transfer characteristics in gas turbine applications. Usually, the assumption is made that the wall of the test model can be treated as a flat and semi-infinite solid. This assumption is correct as long as the penetration depth of the heat compared to the thickness of the wall and to the radius of curvature is small. However, those two assumptions are not always respected for measurements near the leading edge of a blade. This paper presents a rigorous treatment of the curvature and finite wall thickness effects. The unsteady heat transfer for a hollow cylinder has been investigated analytically and a data-reduction method, taking into account curvature and finite wall-thickness effects has been developed. Experimental tests made on hollow cylinder models have been evaluated using the new reduction method as well as the traditional semi-infinite flat-plate approach and a third method that approximately accounts for curvature effects. It has been found that curvature and finite thickness of the wall have, in some cases, a significant influence on the obtained heat transfer coefficient. The parameters influencing the accuracy of the semi-infinite flat-plate model and the approximate curvature correction are determined and the domains of validity are represented.
Turbulent Augmentation of Internal Convection Over Pins in Staggered-Pin Fin Arrays
Authors: Ames F. E. , Dvorak L. A. , Morrow M. J.
Published: February 2005

The objective of this research was to investigate pin fin midline heat transfer in terms of our understanding of stagnation region heat transfer for cylinders in cross flow and turbine airfoils. An experimental investigation was conducted in a staggered-pin fin array at Reynolds numbers of 3000, 10,000, and 30,000 based on the maximum velocity between cylinders. Midline distributions of static pressure and heat transfer were acquired for rows 1 through 8 at the three Reynolds numbers. Turbulence measurements and velocity distributions were acquired at the inlet and in between adjacent pins in rows using hot wire anemometry. One-dimensional power spectra were calculated to determine integral and energy scales. Midline heat transfer distributions are reported as the Nusselt number divided by the square root of the Reynolds number as a function of angle. In these terms, heat transfer was found to increase through row 3 for a Reynolds number of 30,000. After row 3, heat transfer diminished slightly. The Reynolds number for each row was recast in terms of an effective approach velocity, which was found to be highest in row 3 due to the upstream blockage of row 2. Based on this effective velocity the Nusselt number divided by the square root of the Reynolds number increased through row 4. These data indicate that heat transfer is highest in row 3 pins due to the highest effective velocity, while heat transfer augmentation due to turbulence is highest in row 4 and beyond. Hot wire measurements show higher turbulence intensity and dissipation rates upstream of row 4 compared to upstream of row 3. Generally, pressure, heat transfer, and turbulence measurements were taken at all rows, providing a better understanding of turbulent transport from pin fin arrays.
Unsteady Aerodynamics and Aeroacoustics of a High-Bypass Ratio Fan Stage
Authors: Prasad Anil , Prasad Dilip
Published: February 2005

A numerical investigation of the unsteady aerodynamics of a fan stage comprised of a transonic rotor, swept fan exit guide vane (FEGV), and low-pressure compressor inlet guide vane (IGV) is described, with emphasis on acoustics. It is shown that the effects of the two downstream stator rows on the time-mean blade flow field are negligible, permitting its investigation using isolated rotor calculations. Simulations of this type are carried out along the engine operating line to quantify the acoustic sources associated with the upstream shock field and wake turbulence-stator interaction. The shock noise achieves its maximum value near the flyover acoustic certification condition, while the wake turbulence is least at this condition owing to its proximity to the design point. The behavior of these noise sources is explained physically by carrying out a detailed examination of the rotor flow field. The unsteady interaction between the rotor and stator rows at a high-power setting is investigated next. It is shown that the time-mean IGV flow is significantly affected by this interaction. Moreover, the unsteady loading on the IGV is found to be large. The behavior of the upstream-propagating acoustic field generated by rotor-IGV interaction is examined. The interaction between the rotor and FEGV is found to be linear in nature. The FEGV surface unsteady pressure and far-field acoustic field behavior are investigated.
A Study of Impeller-Diffuser-Volute Interaction in a Centrifugal Fan
Authors: Meakhail Tarek , Park Seung O.
Published: February 2005

This paper reports velocity measurement data in the interaction region between the impeller and vaned diffuser and the results of numerical flow simulation of the whole machine (impeller, vaned diffuser and volute) of a single stage centrifugal fan. Two-dimensional instantaneous velocity measurement is done using particle image velocimetry (PIV). Numerical simulation of impeller-diffuser-volute interaction is performed using CFX-Tascflow commercial code. A frozen rotor simulation model is used for the steady calculation and a rotor-stator simulation model is used for the unsteady calculation using the steady results as an initial guess. The simulation results show that the separated flow regime near the diffuser hub extends to the volute. Comparison between the unsteady computation and those of measurement indicates that the rotor/stator model employed in the simulation predicts essential characteristics of unsteady flow in the centrifugal fan. However, quantitative agreement remains rather poor.
The Effects of the Vane and Mainstream Turbulence Level on Hot Streak Attenuation
Authors: Jenkins Sean C. , Bogard David G.
Published: February 2005

This paper discusses the effects of varying pitch position on the attenuation of a simulated hot streak in a vane cascade under conditions of low and high turbulence. Measurements describe the effect of both small and large changes in pitch away from the stagnation line as evidenced by hot streak variations in the wake and at the trailing edge. Under both turbulence conditions, the vane serves to increase or decrease the attenuation rate depending on the location of the hot streak relative to the vane due to isolating of the hot streak core by the vane wall. Additional attenuation resulted due to the action of the wake on the hot streak.
Dependence of Self-Tolerance on TRAF6-Directed Development of Thymic Stroma
Authors: Akiyama Taishin , Maeda Shiori , Yamane Sayaka , Ogino Kaori , Kasai Michiyuki , Kajiura Fumiko , Matsumoto Mitsuru , Inoue Jun-ichiro
Published: February 2005

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor–associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of
TRAF6
-/-
embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.
Roles of Tumor Necrosis Factor Alpha (TNF-α) and the p55 TNF Receptor in CD1d Induction and Coxsackievirus B3-Induced Myocarditis
Authors: Huber S. A. , Sartini D.
Published: February 2005

Giving C57BL/6 mice 104PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 105or 106PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 104PFU were reduced. Tumor necrosis factor alpha (TNF-α) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-α−/−and p55 TNF receptor-negative (TNFR−/−) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR−/−mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR−/−animals showed 10-fold more inflammatory cells in the heart than p55 TNFR−/−mice, and the cell population was comprised of high concentrations of CD4+gamma interferon-positive and Vγ4+cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR−/−mice upregulate CD1d, the molecule recognized by Vγ4+cells, but infection of TNF−/−or p55 TNFR−/−endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-α, failed to elicit CD1d expression, but TNF-α treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-α treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-α signaling.
Recombinant Modified Vaccinia Virus Ankara Expressing the Spike Glycoprotein of Severe Acute Respiratory Syndrome Coronavirus Induces Protective Neutralizing Antibodies Primarily Targeting the Receptor Binding Region
Authors: Chen Zhiwei , Zhang Linqi , Qin Chuan , Ba Lei , Yi Christopher E. , Zhang Fengwen , Wei Qiang , He Tian , Yu Wenjie , Yu Jian , Gao Hong , Tu Xinming , Gettie Agegnehu , Farzan Michael , Yuen Kwok-yung , Ho David D.
Published: February 2005

Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.
Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome
Authors: Franck Nathalie , Le Seyec Jacques , Guguen-Guillouzo Christiane , Erdtmann Lars
Published: February 2005

Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.
Early Antiretroviral Therapy for Simian Immunodeficiency Virus Infection Leads to Mucosal CD4+T-Cell Restoration and Enhanced Gene Expression Regulating Mucosal Repair and Regeneration
Authors: George Michael D. , Reay Elizabeth , Sankaran Sumathi , Dandekar Satya
Published: February 2005

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infections lead to rapid depletion of CD4+T cells from gut-associated lymphoid tissue (GALT). Although the administration of antiretroviral therapy (ART) has been shown to increase CD4+T-cell levels in the peripheral blood in both SIV and HIV infections, its efficacy in restoring intestinal mucosal CD4+T cells has not been well investigated. To gain insights into the molecular mechanisms of virally induced disruptions in the mucosal immune system, we have evaluated longitudinal changes in viral burden, T-cell subsets, and mucosal gene expression profiles in SIV-infected rhesus macaques in the absence or presence of ART. Our results demonstrate a dramatic suppression of mucosal viral loads and rapid reconstitution of CD4+T cells in GALT in animals receiving ART that were not observed in untreated SIV-infected animals. DNA microarray-based gene expression profiling indicated that CD4+T-cell restoration in GALT was associated with up regulation of growth factors and genes involved in repair and regeneration of the mucosal epithelium. In contrast, untreated SIV-infected animals increased expression of lymphocyte activation and inflammatory response-associated genes and did not up regulate mucosal growth and repair associated transcription. In conclusion, these data indicate that initiating ART in primary SIV infection may lead to the restoration of the mucosal immune system through reduction of inflammation and promotion of epithelial repair in the intestinal mucosa.
Nonstructural Protein σ1s Is a Determinant of Reovirus Virulence and Influences the Kinetics and Severity of Apoptosis Induction in the Heart and Central Nervous System
Authors: Hoyt Cristen C. , Richardson-Burns Sarah M. , Goody Robin J. , Robinson Bridget A. , DeBiasi Roberta L. , Tyler Kenneth L.
Published: February 2005

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (σ1s). Although σ1s is dispensable for viral replication in vitro, we wished to investigate the expression of σ1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either σ1s
−
or σ1s
+
reovirus strains. While viral replication in target organs did not differ between σ1s
−
and σ1s
+
viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in σ1s
−
reovirus-infected animals. These results demonstrate that σ1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.
Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements
Authors: Sampaio Kerstin Laib , Cavignac Yolaine , Stierhof York-Dieter , Sinzger Christian
Published: February 2005

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.
Human Cytomegalovirus Protein US11 Provokes an Unfolded Protein Response That May Facilitate the Degradation of Class I Major Histocompatibility Complex Products
Authors: Tirosh Boaz , Iwakoshi Neal N. , Lilley Brendan N. , Lee Ann-Hwee , Glimcher Laurie H. , Ploegh Hidde L.
Published: February 2005

The human cytomegalovirus (HCMV) glycoprotein US11 diverts class I major histocompatibility complex (MHC) heavy chains (HC) from the endoplasmic reticulum (ER) to the cytosol, where HC are subjected to proteasome-mediated degradation. In mouse embryonic fibroblasts that are deficient for X-box binding protein 1 (XBP-1), a key transcription factor in the unfolded protein response (UPR) pathway, we show that degradation of endogenous mouse HC is impaired. Moreover, the rate of US11-mediated degradation of ectopically expressed HLA-A2 is reduced when XBP-1 is absent. In the human astrocytoma cell line U373, turning on expression of US11, but not US2, is sufficient to induce a UPR, as manifested by upregulation of the ER chaperone Bip and by splicing of XBP-1 mRNA. In the presence of dominant-negative versions of XBP-1 and activating transcription factor 6, the kinetics of class I MHC HC degradation were delayed when expression of US11 was turned on. The magnitude of these effects, while reproducible, was modest. Conversely, in cells that stably express high levels of US11, the degradation of HC is not affected by the presence of the dominant negative effectors of the UPR. An infection of human foreskin fibroblasts with human cytomegalovirus induced XBP-1 splicing in a manner that coincides with US11 expression. We conclude that the contribution of the UPR is more pronounced on HC degradation shortly after induction of US11 expression and that US11 is sufficient to induce such a response.
Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Authors: Kino Tomoshige , Gragerov Alexander , Valentin Antonio , Tsopanomihalou Maria , Ilyina-Gragerova Galina , Erwin-Cohen Rebecca , Chrousos George P. , Pavlakis George N.
Published: February 2005

Vpr and selected mutants were used in a
Saccharomyces cerevisiae
two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G
2
/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G
2
/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.
Detection of Antibody-Dependent Complement-Mediated Inactivation of both Autologous and Heterologous Virus in Primary Human Immunodeficiency Virus Type 1 Infection
Authors: Aasa-Chapman Marlén M. I. , Holuigue Sophie , Aubin Keith , Wong MaiLee , Jones Nicola A. , Cornforth David , Pellegrino Pierre , Newton Philippa , Williams Ian , Borrow Persephone , McKnight Áine
Published: February 2005

Specific CD8 T-cell responses to human immunodeficiency virus type 1 (HIV-1) are induced in primary infection and make an important contribution to the control of early viral replication. The importance of neutralizing antibodies in containing primary viremia is questioned because they usually arise much later. Nevertheless antienvelope antibodies develop simultaneously with, or even before, peak viremia. We determined whether such antibodies might control viremia by complement-mediated inactivation (CMI). In each of seven patients studied, antibodies capable of CMI appeared at or shortly after the peak in viremia, concomitantly with detection of virus-specific T-cell responses. The CMI was effective on both autologous and heterologous HIV-1 isolates. Activation of the classical pathway and direct viral lysis were at least partly responsible. Since immunoglobulin G (IgG)-antibodies triggered the CMI, specific memory B cells could also be induced by vaccination. Thus, consideration should be given to vaccination strategies that induce IgG antibodies capable of CMI.
Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences
Authors: Ostapchuk Philomena , Yang Jihong , Auffarth Ece , Hearing Patrick
Published: February 2005

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the
cis
-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting
trans
-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.
Positive and Negative Regulation of Chicken Anemia Virus Transcription
Authors: Miller Myrna M. , Jarosinski Keith W. , Schat Karel A.
Published: February 2005

Chicken anemia virus (CAV) is a small circular single-stranded DNA virus with a single promoter-enhancer region containing four consensus cyclic AMP response element sequences (AGCTCA), which are similar to the estrogen response element (ERE) consensus half-sites (A)GGTCA. These sequences are arranged as direct repeats, an arrangement that can be recognized by members of the nuclear receptor superfamily. Transient-transfection assays which use a short CAV promoter construct that ended at the transcription start site and drive expression of enhanced green fluorescent protein (EGFP) showed high basal activity in DF-1, LMH, LMH/2A, and primary theca and granulosa cells. The estrogen receptor-enhanced cell line, LMH/2A, had significantly greater expression than LMH cells, and this expression was significantly increased with estrogen treatment. A long promoter construct which included GGTCA-like sequences downstream of the first CAV protein translation start site was found to have significantly less EGFP expression in DF-1 cells than the short promoter, which was largely due to decreased RNA transcription. DNA-protein binding assays indicated that proteins recognizing a consensus ERE palindrome also bind GGTCA-like sequences in the CAV promoter. Estrogen receptor and other members of the nuclear receptor superfamily may provide a mechanism to regulate CAV activity in situations of low virus copy number.
Identification of Cytomegalovirus-Specific Cytotoxic T Lymphocytes In Vitro Is Greatly Enhanced by the Use of Recombinant Virus Lacking the US2 to US11 Region or Modified Vaccinia Virus Ankara Expressing Individual Viral Genes
Authors: Khan Naeem , Bruton Rachel , Taylor Graham S. , Cobbold Mark , Jones Thomas R. , Rickinson Alan B. , Moss Paul A. H.
Published: February 2005

Cytomegalovirus (CMV) elicits a potent T-cell response in humans that appears to protect the host from virus-associated disease. Despite facing strong host defense mechanisms, CMV remains as a lifelong infection that may reactivate and cause life-threatening disease in immunocompromised individuals. This persistence is probably assisted by expression of immune subversion proteins of the virus encoded by genes belonging to the US gene family. These proteins modulate major histocompatibility complex expression in infected cells and bias in vitro experiments toward the detection of only certain specificities. We have combined the use of recombinant CMV, lacking the US2 to US11 region genes, and cytoplasmic gamma interferon staining to define a more accurate assessment of CMV-specific responses in vivo. Recombinant CMV stimulation reveals a CD8 response much larger than that of parental virus in all donors tested. In some cases, this represented up to 10-fold increases in the number of cells detected. Responses were directed mainly against pp65, IE-1, and pp50 in the majority of donors. In addition, previously unreported IE-2-specific T-cell responses could be detected in a minority of cases. Furthermore, we observed a less marked increase in the response to mutant CMV by CD4 T cells in some donors. This suggests that a much broader T-cell response to CMV exists in vivo than is revealed by restimulation with wild-type virus and adds to the evidence that the efficacy of immune evasion strategies may not be as absolute as previously believed.
Establishment and Maintenance of Long-Term Murine Gammaherpesvirus 68 Latency in B Cells in the Absence of CD40
Authors: Willer David O. , Speck Samuel H.
Published: February 2005

Murine gammaherpesvirus 68 (γHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of γHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, γHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in γHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40−/−) mice. Here we report that, consistent with previous observations, γHV68 efficiently established a latent infection in B cells of CD40−/−mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40−/−mice 42 days postinfection was observed. In addition, in contrast to γHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of γHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that γHV68 may either directly infect memory B cells present in CD40−/−mice or be capable of driving differentiation of naive CD40−/−B cells. A possible explanation for the apparent discrepancy between the failure of γHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of γHV68 B-cell latency in CD40−/−mice came from examining virus replication in the lungs of infected CD40−/−mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40−/−mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.
Cellular and Humoral Immunity following Snow Mountain Virus Challenge
Authors: Lindesmith Lisa , Moe Christine , LePendu Jacques , Frelinger Jeffrey A. , Treanor John , Baric Ralph S.
Published: February 2005

Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a ≥4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-γ) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-γ and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-γ production was dependent upon CD4+cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.
Selective Down-Regulation of the NKG2D Ligand H60 by Mouse Cytomegalovirus m155 Glycoprotein
Authors: Hasan Milena , Krmpotic Astrid , Ruzsics Zsolt , Bubic Ivan , Lenac Tihana , Halenius Anne , Loewendorf Andrea , Messerle Martin , Hengel Hartmut , Jonjic Stipan , Koszinowski Ulrich H.
Published: February 2005

Both human and mouse cytomegaloviruses (CMVs) encode proteins that inhibit the activation of NK cells by down-regulating cellular ligands for the activating NK cell receptor NKG2D. Up to now, three ligands for the NKG2D receptor, named RAE-1, H60, and MULT-1, have been identified in mice. The resistance of mouse strains to murine CMV (MCMV) infection is determined by their ability to generate an effective NK cell response. The MCMV genem152, a member of them145gene family, down-regulates the expression of RAE-1 in order to avoid NK cell control in vivo. Here we report that them155gene, another member of them145gene family, encodes a protein that interferes with the expression of H60 on the surfaces of infected cells. Deletion of them155gene leads to an only partial restoration of H60 expression on the cell surface, suggesting the involvement of another, so far unknown, viral inhibitor. In spite of this, anm155deletion mutant virus shows NK cell-dependent attenuation in vivo. The acquisition of endo-β-N-acetylglucosaminidase H resistance and the preserved half-life of H60 in MCMV-infected cells indicate that the m155-mediated effect must take place in a compartment after H60 exits from the ERGIC-cis-Golgi compartment.