bluuebunny/crossref_metadata_2025 huggingface dataset:
How to Choose a Theory: Science for Nonscientists
Authors: Romer Alfred
Published: September 2005

To represent science as a reasonable activity may be a useful objective in courses for nonscientists. To do so requires teaching, among other things, the characteristics of a scientific theory and the criteria by which one theory is preferred to another. A scientific theory is based on purely speculative hypotheses from which conclusions can be deduced which are in agreement with observations. The theory is justified by these conclusions, and not by its hypotheses which may be implausible. To be acceptable, a theory must agree with observations, it should also be inflexible and comprehensive. These propositions have been derived from, and find their application in, a course on the theories of the solar system. The Copernican theory supplanted the Ptolemaic by virtue of being more comprehensive although less plausible, and these qualities were intensified in the modifications which Kepler and Newton carried out.
A Student X-Ray Diffraction Experiment
Authors: Hayn Carl H.
Published: September 2005

A diffraction experiment has been designed which takes advantage of the heterogeneous nature of x rays from tungsten targets. A rock salt crystal, whose surface was cut at a small angle relative to the cleavage plane, yielded several diffracted beams for each crystal orientation. Analysis of the data permitted a determination of the angles formed between the Bragg planes and the crystal surface.
Error Limits of Expectation Values in Quantum Mechanical Calculations
Authors: Mucci Joseph F. , Haskins Peter J.
Published: September 2005

We present a general treatment of error limits of expectation values in quantum-mechanical calculations. Important expressions and their derivations are included along with an indication of the state of research in this area.
The Absolute Electrometer Revisited
Authors: Metz Roger N.
Published: September 2005

The absolute electrometer, a device used historically to determine potential difference directly by measuring the electrostatic force between the plates of a charged capacitor, forms the basis for an experiment to determine the permittivity of free space, ε0. The inherent instability of the device at high voltage is analyzed and the optimum conditions for operation are developed. The correction to the capacitance due to a discontinuity in the guard-ring-plate surface is calculated using the method of conjugate functions. The correction differs from one calculated previously.
Classical Scattering Cross Section for a Cut-Off Coulomb Potential
Authors: MacDonald William M.
Published: September 2005

The penetration of matter by charged particles and of transport coefficients for ionized gases are often calculated by the method of impact parameters wherein the Coulomb scattering cross section is set equal to zero for orbits with impact parameters larger than the range of the force. The method does not correspond to a reasonable assumption on the interaction, however, and it is sometimes difficult for students in classical mechanics and plasma physics to accept. It is therefore interesting that the cross section for scattering by a Coulomb force which is zero beyond a certain radius of interaction can easily be calculated by the methods usually presented in junior-senior level courses in classical mechanics. The result provides a simple basis for the method of impact parameters.
Time Evolution of Predictions in Statistical Mechanics
Authors: Casper Barry M. , Freier Susan , Van Atta David
Published: September 2005

Predictions in statistical mechanics may be viewed as the result of a three-stage process. To illustrate this process we consider a classical simple harmonic oscillator whose initial position and energy are not precisely specified. It is felt that this approach can aid in illuminating similarities and points of difference between statistical mechanics and quantum mechanics.
Characterization of VP22 in Herpes Simplex Virus-Infected Cells
Authors: Mouzakitis G. , McLauchlan John , Barreca Cristina , Kueltzo Lisa , O'Hare P.
Published: September 2005

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.
Simian Immunodeficiency Virus Integration Preference Is Similar to That of Human Immunodeficiency Virus Type 1
Authors: Crise Bruce , Li Yuan , Yuan Chiuchin , Morcock David R. , Whitby Denise , Munroe David J. , Arthur Larry O. , Wu Xiaolin
Published: September 2005

Simian immunodeficiency virus (SIV) is a useful model for studying human immunodeficiency virus (HIV) pathogenesis and vaccine efficacy. As with all other retroviruses, integration is a necessary step in the replication cycle of SIV. The location of the retrovirus integration site is known to impact on viral gene expression, establishment of viral latency, and other aspects of the replication cycle of a retrovirus. In this study, 148 SIV provirus integration sites were sequenced and mapped in the human genome. Our analysis showed that SIV integration, like that of HIV type 1 (HIV-1), exhibited a strong preference for actively transcribed regions in the genome (A. R. Schroder et al., Cell
110:
521-529, 2002) and no preference for the CpG islands or transcription start sites, in contrast to observations for murine leukemia virus (X. Wu et al., Science
300:
1749-1751, 2003). The parallel integration target site preferences of SIV and HIV-1 suggest that these lentiviruses may share similar mechanisms for target site selection and that SIV serves as an accurate model of HIV-1 with respect to integration.
Mitochondrial Cell Death Suppressors Carried by Human and Murine Cytomegalovirus Confer Resistance to Proteasome Inhibitor-Induced Apoptosis
Authors: McCormick A. Louise , Meiering Christopher D. , Smith Geoffrey B. , Mocarski Edward S.
Published: September 2005

Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death.
Regulation of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Fusion by a Membrane-Interactive Domain in the gp41 Cytoplasmic Tail
Authors: Wyss Stéphanie , Dimitrov Antony S. , Baribaud Frédéric , Edwards Terri G. , Blumenthal Robert , Hoxie James A.
Published: September 2005

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this “inside-out” regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.
Covalently Closed Circular DNA Is the Predominant Form of Duck Hepatitis B Virus DNA That Persists following Transient Infection
Authors: Le Mire Marc F. , Miller Darren S. , Foster Wendy K. , Burrell Christopher J. , Jilbert Allison R.
Published: September 2005

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.
A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse
Authors: Jin Ling , Perng Guey-Chuen , Mott Kevin R. , Osorio Nelson , Naito Julia , Brick David J. , Carpenter Dale , Jones Clinton , Wechsler Steven L.
Published: September 2005

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT
−
mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT
−
mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT
−
mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse.
Kinetic Rates of Antibody Binding Correlate with Neutralization Sensitivity of Variant Simian Immunodeficiency Virus Strains
Authors: Steckbeck Jonathan D. , Orlov Irina , Chow Andrew , Grieser Heather , Miller Kenneth , Bruno JoAnne , Robinson James E. , Montelaro Ronald C. , Cole Kelly Stefano
Published: September 2005

Increasing evidence suggests that an effective AIDS vaccine will need to elicit both broadly reactive humoral and cellular immune responses. Potent and cross-reactive neutralization of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) by polyclonal and monoclonal antibodies is well documented. However, the mechanisms of antibody-mediated neutralization have not been defined. The current study was designed to determine whether the specificity and quantitative properties of antibody binding to SIV envelope proteins correlate with neutralization. Using a panel of rhesus monoclonal antibodies previously characterized for their ability to bind and neutralize variant SIVs, we compared the kinetic rates and affinity of antibody binding to soluble envelope trimers by using surface plasmon resonance. We identified significant differences in the kinetic rates but not the affinity of monoclonal antibody binding to the neutralization-sensitive SIV/17E-CL and neutralization-resistant SIVmac239 envelope proteins that correlated with the neutralization sensitivities of the corresponding virus strains. These results suggest for the first time that neutralization resistance may be related to quantitative differences in the rates but not the affinity of the antibody-envelope interaction and may provide one mechanism for the inherent resistance of SIVmac239 to neutralization in vitro. Further, we provide evidence that factors in addition to antibody binding, such as epitope specificity, contribute to the mechanisms of neutralization of SIV/17E-CL in vitro. This study will impact the method by which HIV/SIV vaccines are evaluated and will influence the design of candidate AIDS vaccines capable of eliciting effective neutralizing antibody responses.
Vectored Gag and Env but Not Tat Show Efficacy against Simian-Human Immunodeficiency Virus 89.6P Challenge in Mamu-A*01-Negative Rhesus Monkeys
Authors: Liang Xiaoping , Casimiro Danilo R. , Schleif William A. , Wang Fubao , Davies Mary-Ellen , Zhang Zhi-Qiang , Fu Tong-Ming , Finnefrock Adam C. , Handt Larry , Citron Michael P. , Heidecker Gwendolyn , Tang Aimin , Chen Minchun , Wilson Keith A. , Gabryelski Lori , McElhaugh Michael , Carella Anthony , Moyer Cheryl , Huang Lingyi , Vitelli Salvatore , Patel Deepa , Lin Jing , Emini Emilio A. , Shiver John W.
Published: September 2005

Simian-human immunodeficiency virus (SHIV) challenge studies in rhesus macaques were conducted to evaluate the efficacy of adenovirus-based vaccines in the context of different major histocompatibility complex class I genetic backgrounds and different vaccine compositions.Mamu-A*01 allele-negative rhesus monkeys were immunized with one of the following vaccine constructs: (i) replication-defective recombinant adenovirus type 5 (Ad5) expressing human immunodeficiency virus type 1 (HIV-1) Tat (Ad5/HIVTat); (ii) Ad5 vector expressing simian immunodeficiency virus (SIV) Gag (Ad5/SIVGag); (iii) Ad5 vector expressing the truncated HIV-1jrflEnv, gp140 (Ad5/gp140_jrfl); (iv) Ad5 vector expressing the SHIV-89.6P gp140 (Ad5/gp140_89.6P); or (v) the combination of Ad5/SIVGag and Ad5/gp140_jrfl. Following intravenous challenge with SHIV-89.6P, only those cohorts that received vaccines expressing Gag or Env exhibited an attenuation of the acute viremia and associated CD4-cell lymphopenia. While no prechallenge neutralizing antibody titers were detectable in either Ad5/gp140-vaccinated group, an accelerated neutralizing antibody response was observed in the Ad5/gp140_89.6P-vaccinated group upon viral challenge. The set-point viral loads in the Ad5/SIVGag- and Ad5/gp140_jrfl-vaccinated groups were associated with the overall strength of the induced cellular immune responses. To examine the contribution ofMamu-A*01 allele in vaccine efficacy against SHIV-89.6P challenge, Mamu-A*01-positive monkeys were immunized with Ad5/SIVGag. Vaccine-mediated protection was significantly more pronounced in the Mamu-A*01-positive monkeys than in Mamu-A*01-negative monkeys, suggesting the strong contributions of T-cell epitopes restricted by the Mamu-A*01 molecule. The implications of these results in the development of an HIV-1 vaccine will be discussed.
Corneal Cell Survival in Adenovirus Type 19 Infection Requires Phosphoinositide 3-Kinase/Akt Activation
Authors: Rajala Maitreyi S. , Rajala Raju V. S. , Astley Roger A. , Butt Amir L. , Chodosh James
Published: September 2005

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3β and nuclear translocation of the p65 subunit of NF-κB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.
Reciprocal Activities between Herpes Simplex Virus Type 1 Regulatory Protein ICP0, a Ubiquitin E3 Ligase, and Ubiquitin-Specific Protease USP7
Authors: Boutell Chris , Canning Mary , Orr Anne , Everett Roger D.
Published: September 2005

Herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 stimulates lytic infection and the reactivation of quiescent viral genomes. These roles of ICP0 require its RING finger E3 ubiquitin ligase domain, which induces the degradation of several cellular proteins, including components of promyelocytic leukemia nuclear bodies and centromeres. ICP0 also interacts very strongly with the cellular ubiquitin-specific protease USP7 (also known as HAUSP). We have shown previously that ICP0 induces its own ubiquitination and degradation in a RING finger-dependent manner, and that its interaction with USP7 regulates this process. In the course of these studies we found and report here that ICP0 also targets USP7 for ubiquitination and proteasome-dependent degradation. The reciprocal activities of the two proteins reveal an intriguing situation that poses the question of the balance of the two processes during productive HSV-1 infection. Based on a thorough analysis of the properties of an HSV-1 mutant virus that expresses forms of ICP0 that are unable to bind to USP7, we conclude that USP7-mediated stabilization of ICP0 is dominant over ICP0-induced degradation of USP7 during productive HSV-1 infection. We propose that the biological significance of the ICP0-USP7 interaction may be most pronounced in natural infection situations, in which limited amounts of ICP0 are expressed.
An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrP
Sc
Aggregates
Authors: Pan Tao , Chang Binggong , Wong Poki , Li Chaoyang , Li Ruliang , Kang Shin-Chung , Robinson John D. , Thompsett Andrew R. , Tein Po , Yin Shaoman , Barnard Geoff , McConnell Ian , Brown David R. , Wisniewski Thomas , Sy Man-Sun
Published: September 2005

The conversion of the normal cellular prion protein, PrP
C
, into the protease-resistant, scrapie PrP
Sc
aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP
Sc
aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP
Sc
aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.
Shared Alterations in NK Cell Frequency, Phenotype, and Function in Chronic Human Immunodeficiency Virus and Hepatitis C Virus Infections
Authors: Meier Ute-Christiane , Owen Rachel E. , Taylor Elizabeth , Worth Andrew , Naoumov Nikolai , Willberg Christian , Tang Kwok , Newton Phillipa , Pellegrino Pierre , Williams Ian , Klenerman Paul , Borrow Persephone
Published: September 2005

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3−CD56+NK subsets in HIV+and HCV+patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56dimcell fraction compared to CD56brightcells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56dimNK subset were observed in HIV+patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+and HCV+patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.
Inactivation of Retroviruses with Preservation of Structural Integrity by Targeting the Hydrophobic Domain of the Viral Envelope
Authors: Raviv Yossef , Viard Mathias , Bess Julian W , Chertova Elena , Blumenthal Robert
Published: September 2005

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.
Protection of Mice against Lethal Infection with Highly Pathogenic H7N7 Influenza A Virus by Using a Recombinant Low-Pathogenicity Vaccine Strain
Authors: de Wit Emmie , Munster Vincent J. , Spronken Monique I. J. , Bestebroer Theo M. , Baas Chantal , Beyer Walter E. P. , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Fouchier Ron A. M.
Published: September 2005

In 2003, an outbreak of highly pathogenic avian influenza occurred in the Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, ∼1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.
Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors
Authors: Mishin Vasiliy P. , Novikov Dmitri , Hayden Frederick G. , Gubareva Larisa V.
Published: September 2005

Inhibition of neuraminidase (NA) activity prevents release of progeny virions from influenza-infected cells and removal of neuraminic (sialic) acid moieties from glycans attached to hemagglutinin (HA). Neuraminic acid moieties situated near the HA receptor-binding site can reduce the efficiency of virus binding and decrease viral dependence on NA activity for replication. With the use of reverse genetics technique, we investigated the effect of glycans attached at Asn 94a, 129, and 163 on the virus susceptibility to NA inhibitors in MDCK cells and demonstrated that the glycan attached at Asn 163 plays a dominant role in compensation for the loss of NA activity.
Minimal T-Cell-Stimulatory Sequences and Spectrum of HLA Restriction of Immunodominant CD4
+
T-Cell Epitopes within Hepatitis C Virus NS3 and NS4 Proteins
Authors: Gerlach J. T. , Ulsenheimer A. , Grüner N. H. , Jung M.-C. , Schraut W. , Schirren C.-A. , Heeg M. , Scholz S. , Witter K. , Zahn R. , Vogler A. , Zachoval R. , Pape G. R. , Diepolder H. M.
Published: September 2005

The hepatitis C virus (HCV)-specific CD4
+
T-cell response against nonstructural proteins is strongly associated with successful viral clearance during acute hepatitis C. To further develop these observations into peptide-based vaccines and clinical immunomonitoring tools like HLA class II tetramers, a detailed characterization of immunodominant CD4
+
T-cell epitopes is required. We studied peripheral blood mononuclear cells from 20 patients with acute hepatitis C using 83 overlapping 20-mer peptides covering the NS3 helicase and NS4. Eight peptides were recognized by ≥40% of patients, and specific CD4
+
T-cell clones were obtained for seven of these and three additional, subdominant epitopes. Mapping of minimal stimulatory sequences defined epitopes of 8 to 13 amino acids in length, but optimal T-cell stimulation was observed with 10- to 15-mers. While some epitopes were presented by different HLA molecules, others were presented by only a single HLA class II molecule, which has implications for patient selection in clinical trials of peptide-based immunotherapies. In conclusion, using two different approaches we identified and characterized a set of CD4
+
T-cell epitopes in the HCV NS3-NS4 region which are immunodominant in patients achieving transient or persistent viral control. This information allows the construction of a valuable panel of HCV-specific HLA class II tetramers for further study of CD4
+
T-cell responses in chronic hepatitis C. The finding of immunodominant epitopes with very constrained HLA restriction has implications for patient selection in clinical trials of peptide-based immunotherapies.
Circularization of the Herpes Simplex Virus Type 1 Genome upon Lytic Infection
Authors: Strang Blair L. , Stow Nigel D.
Published: September 2005

For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral
a
sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.
Genomewide Screening Reveals High Levels of Insertional Polymorphism in the Human Endogenous Retrovirus Family HERV-K(HML2): Implications for Present-Day Activity
Authors: Belshaw Robert , Dawson Anna L. A. , Woolven-Allen John , Redding Joanna , Burt Austin , Tristem Michael
Published: September 2005

The published human genome sequence contains many thousands of endogenous retroviruses (HERVs) but all are defective, containing nonsense mutations or major deletions. Only the HERV-K(HML2) family has been active since the divergence of humans and chimpanzees; it contains many members that are human specific, as well as several that are insertionally polymorphic (an inserted element present only in some human individuals). Here we perform a genomewide survey of insertional polymorphism levels in this family by using the published human genome sequence and a diverse sample of 19 humans. We find that there are 113 human-specific HERV-K(HML2) elements in the human genome sequence, 8 of which are insertionally polymorphic (11 if we extrapolate to those within regions of the genome that were not suitable for amplification). The average rate of accumulation since the divergence with chimpanzees is thus approximately 3.8 × 10
−4
per haploid genome per generation. Furthermore, we find that the number of polymorphic elements is not significantly different from that predicted by a standard population genetic model that assumes constant activity of the family until the present. This suggests to us that the HERV-K(HML2) family may be active in present-day humans. Active (replication-competent) elements are likely to have inserted very recently and to be present at low allele frequencies, and they may be causing disease in the individuals carrying them. This view of the family from a population perspective rather than a genome perspective will inform the current debate about a possible role of HERV-K(HML2) in human disease.
The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence
Authors: Brock Sean C. , Heck Josh M. , McGraw Patricia A. , Crowe James E.
Published: September 2005

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.
Attenuated Rabies Virus Activates, while Pathogenic Rabies Virus Evades, the Host Innate Immune Responses in the Central Nervous System
Authors: Wang Zhi W. , Sarmento Luciana , Wang Yuhuan , Li Xia-qing , Dhingra Vikas , Tseggai Tesfai , Jiang Baoming , Fu Zhen F.
Published: September 2005

Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-α/β) signaling pathways and inflammatory chemokines. For the IFN-α/β signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2′-5′-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses.
Identification of Two N-Linked Glycosylation Sites within the Core of the Simian Immunodeficiency Virus Glycoprotein Whose Removal Enhances Sensitivity to Soluble CD4
Authors: Pikora Cheryl , Wittish Christine , Desrosiers Ronald C.
Published: September 2005

Using PCR mutagenesis to disrupt the NXT/S N-linked glycosylation motif of the Env protein, we created 27 mutants lacking 1 to 5 of 14 N-linked glycosylation sites within regions of gp120 lying outside of variable loops 1 to 4 within simian immunodeficiency virus strain 239 (SIV239). Of 18 mutants missing N-linked glycosylation sites predicted to lie within 10 Å of CD4 contact sites, the infectivity of 12 was sufficient to measure sensitivity to neutralization by soluble CD4 (sCD4), pooled immune sera from SIV239-infected rhesus macaques, and monoclonal antibodies known to neutralize certain derivatives of SIV239. Three of these 12 mutants (g3, lacking the 3rd glycan at position 79; g11, lacking the 11th glycan at position 212; and g3,11, lacking both the 3rd and 11th glycans) were approximately five times more sensitive to neutralization by sCD4 than wild-type (WT) SIV239. However, these same mutants were no more sensitive to neutralization than WT by pooled immune sera. The other 9 of 12 replication-competent mutants in this group were no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition.
Lys-34, Dispensable for Integrase Catalysis, Is Required for Preintegration Complex Function and Human Immunodeficiency Virus Type 1 Replication
Authors: Lu Richard , Vandegraaff Nick , Cherepanov Peter , Engelman Alan
Published: September 2005

Retroviral integrases (INs) function in the context of preintegration complexes (PICs). Two conserved Lys residues in the N-terminal domain of human immunodeficiency virus type 1 (HIV-1) IN were analyzed here for their roles in integration and virus replication. Whereas HIV-1
K46A
grew like the wild type, HIV-1
K34A
was dead. Yet recombinant IN
K34A
protein functioned in in vitro integration assays, and Vpr-IN
K34A
efficiently transcomplemented the infectivity defect of an IN active site mutant virus in cells. HIV-1
K34A
was therefore similar to a number of previously characterized mutant viruses that failed to replicate despite encoding catalytically competent IN. To directly analyze mutant PIC function, a sensitive PCR-based integration assay was developed. HIV-1
K34A
and related mutants failed to support detectable levels (<1% of wild type) of integration. We therefore concluded that mutations like K34A disrupted higher-order interactions important for PIC function/maturation compared to the innate catalytic activity of IN enzyme.
Infection of Nonhuman Primates with Recombinant Human Metapneumovirus Lacking the SH, G, or M2-2 Protein Categorizes Each as a Nonessential Accessory Protein and Identifies Vaccine Candidates
Authors: Biacchesi Stéphane , Pham Quynh N. , Skiadopoulos Mario H. , Murphy Brian R. , Collins Peter L. , Buchholz Ursula J.
Published: September 2005

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the ΔSH virus was only marginally less efficient than that of wild-type HMPV, whereas the ΔG and ΔM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The ΔG and ΔM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.
Endothelial function and inflammation in coronary artery disease
Authors: Tousoulis D , Charakida M , Stefanadis C
Published: September 2005

Evidence supports the central role of endothelium and inflammation in all phases of the atherosclerotic process. Clinical studies have shown their prognostic potential for the development of ischaemic events and for adverse outcome after acute coronary syndromes. Reduction in inflammatory levels and improving endothelial function by traditional and novel treatment strategies were associated with a proportional reduction in cardiovascular events. However, randomised controlled trials are required to explore further whether drugs targeting the inflammatory process and endothelial function will constitute a reasonable adjunctive treatment for patients with coronary artery disease.
Antithrombotic treatment in atrial fibrillation
Authors: Lip G Y H , Boos C J
Published: September 2005

Atrial fibrillation (AF) can significantly increase morbidity and mortality. It is gaining in clinical and economic importance, being the most commonly encountered tachyarrhythmia in clinical practice. Stroke is the most serious complication. Evidence from AF antithrombotic treatment trials is reviewed, risk stratification of patients with AF is discussed, and recommendations for anticoagulation are presented.
Phosphorus inactivation by aluminum in Lakes Gårdsjön and Härsvatten sediment during the industrial acidification period in Sweden
Authors: Huser Brian J , Rydin Emil
Published: September 2005

Acidification of lakes exposed to acid deposition is generally accompanied by a severe decrease in production (oligotrophication). In this study, we examined sediment from Lakes Gårdsjön and Härsvatten, Sweden, to determine whether sediment phosphorus (P) retention increased during the years corresponding to lake acidification. Sediment from both lakes had increases in aluminum (Al) in the upper 10 cm, and dating of Lake Gårdsjön sediment revealed that the Al increase occurred from 1950 to 2001 in this lake. The increase in Al input caused an increase in Al-bound P (AlP) formation and overall sediment total phosphorus retention during the same period. Lake Gårdsjön received an additional 12.9 g·m2 of Al, above preacidification background levels, that bound 1.1 g·m2 of P and removed it from the in-lake P cycle from 1950 to 2001. A substantial portion (up to 76%) of the total external P load eventually was converted to AlP and buried in the sediment over this period. The increase in sediment P burial due to increased formation of AlP in systems similar to Lake Gårdsjön may have detrimental effects on nutrient cycling, and as a result, on productivity within the lake, leading to acido-oligotrophication.
Diet and divergence of introduced smallmouth bass (Micropterus dolomieu) populations
Authors: Dunlop Erin S , Orendorff Judi A , Shuter Brian J , Rodd F Helen , Ridgway Mark S
Published: September 2005

We examine the degree and causes of divergence in growth and reproduction in two populations of smallmouth bass (Micropterus dolomieu) introduced a century ago. Despite a common source, the Provoking Lake population now has a higher population density and slower growing individuals than the Opeongo Lake population. Using this system, we test the predictions of life history theory that delayed maturation and reduced reproductive investment are expected in high density populations with slow individual growth rates. Observations on both populations run directly counter to the aforementioned expectations. Instead, Provoking males have smaller sizes and younger ages at nesting and higher gonad masses than Opeongo males; Provoking females have smaller sizes at maturity, larger egg sizes, and higher ovarian dry masses than Opeongo females. Temperature, food availability, diet ontogeny, young-of-the-year mortality, and adult mortality were examined as plausible contributors to the divergence. Results suggest that low food availability, likely caused or mediated by intraspecific competition for prey, and lack of large prey in the diet are contributing to the slow growth, increased reproductive investment, and higher mortality following reproduction in Provoking. This study provides insight into the processes that produce rapid divergence of life history in a species exhibiting parental care.