bluuebunny/crossref_metadata_2025 huggingface dataset:
E‐CRM: customer relationship marketing in the hotel industry
Authors: Luck Diana , Lancaster Geoff
Published: April 2003

Explores the degree to which UK based hotel groups had exploited the medium of electronic customer relationship marketing (E‐CRM). Research is incorporated that investigated their use of the Internet to verify whether customer relationship marketing was being implemented within online operations or whether their Internet presence merely revolved around the basic functions of “providing information” and “hotel reservations”. The findings and subsequent discussion showed that on the Internet, hotel groups used their relationship with customers to provide rather than gather information. The majority of the hotel groups had only embraced a few elements of E‐CRM and even indicated that they had no intention of being led online by the concept. Although the findings of the questionnaire indicated that hotel groups were generally aware of the potential of Web technologies and strategies, they also showed that companies were not putting this knowledge into practice when it came to implementing E‐CRM. Primary research concluded that hotel groups based in the UK were failing to take advantage of the many opportunities identified through the secondary research.
Fuzzy gradual rules in epidemiology
Authors: Ortega Neli , Barros Laécio C. , Massad Eduardo
Published: April 2003

This paper presents an application of the fuzzy gradual rules in an epidemic study of canine rabies in São Paulo city, Brazil. A linguistic epidemiological model was elaborated through fuzzy rules built by the Extension Principle. We used both the inference method of Mamdani and of Dubois et al. The results were compared with real data from São Paulo and with another MISO Model, which is entirely based on expert knowledge presented in a previous work. Questions about application of fuzzy techniques in epidemiology, different inference methods and the Dubois et al. methodology are discussed.
A biocybernetic method to learn hand grasping posture
Authors: Rezzoug N. , Gorce P.
Published: April 2003

In this paper, a biocybernetic method to learn hand grasping posture definition with few knowledge about the task is proposed. The developed model is composed of two stages. The first is dedicated to the fingers inverse kinematics learning in order to locally define a single finger posture given its desired fingertip position. This motor function is fulfilled by a modular neural network architecture that tackles the discontinuity problem of inverse kinematics function (called Fingers Configuration Neural Network (FCNN)). Following the concept of direct associative learning, a second neural model is used to search the space of hand configuration and optimize it according to an evaluative function based on the results of the FCNN. Simulation results show good learning of grasping posture determination of various object types, with different numbers of fingers involved and different contact configurations.
Fuzziology: a study of fuzziness of human knowing and being
Authors: Dimitrov Vlad
Published: April 2003

Fuzziology explores the fuzziness inherent in what we know about ourselves and our experience, about our thoughts and feelings, drives for understanding and urges to create. By studying the fuzziness – its nature, sources, causes and factors affecting its dynamics, we are able to transcend the limitations, which it constantly puts on the processes of our understanding and knowing. The basic postulate and paradox of fuzziology are formulated together with its main principles, logical tools and theorems. The link between Socrates' maieutic inquiry, the ancient Vedic wisdom and fuzziology has helped us to reveal the significance of its central maxim: “Do not reject anything, but do not remain with anything either; go beyond!”
New results on Adomian method
Authors: Himoun N. , Abbaoui K. , Cherruault Y.
Published: April 2003

In this work new results about the Adomian method are presented. Also we prove a new and general result of convergence of the Adomain method, and give two results of convergence of this method applied to ordinary differential equations. Finally, we generalize the Adomian method and prove two new results of convergence with one of them applied to the modified method.
Reduction of complexity in conic section function neural network
Authors: O¨zylmaz Lale , Yldrm Tu¨lay
Published: April 2003

This paper details how the complexity can be reduced in conic section function neural network (CSFNN) by using sensitivity analysis and the results are given for various problems. This is, particularly important for neural network hardware applications. The method used here extracts the cause and effect relationship between the inputs and outputs of the network. After training a neural network, one may want to know the effect that each of the network inputs is having on the network output. The input channels that produce low sensitivity values can be considered insignificant and can most often be removed from the network. This will reduce the size of the network, which in turn reduces the complexity and the training time.
Analytical solution of nonlinear partial differential equations of physics
Authors: García‐Olivares Antonio
Published: April 2003

A general method is proposed to approximate the analytical solution of any time‐dependent partial differential equation with boundary conditions defined on the four sides of a rectangle. To ensure that the approximant satisfies the boundary conditions problem the differential operator is modified with one additional term which takes into account the effect of boundary conditions. Then the new problem can be directly integrated in the same way as an ordinary differential equation. In this work Adomian's decomposition method with analytic extension is used to obtain the first‐order approximant to the solution of a test case. The result is an analytic approximation to the solution which is compatible with both the exact boundary conditions and the accuracy imposed in the whole domain. The solution obtained is compared with the analytic approximation obtained with a Tau‐Legendre spectral method.
Change management practices
Authors: Brown Kerry , Waterhouse Jennifer , Flynn Christine
Published: April 2003

During the last two decades the public sector has come under increasing pressure to improve performance and demonstrate greater transparency and accountability. This pressure has resulted in public sector organisations facing shifts in ways of operating. Various corporate change strategies have been adopted by different public sector agencies, many of these cloning managerial practices from the private sector. These changes in public sector organisations have enormous significance for regional economic and social development. While there is a growing body of knowledge dealing with the management of corporate change there are still significant gaps in understanding the process. While there is much written on public versus private corporate change, there is little distinguishing between change in different types of public sector organisations. This paper analyses change management processes and seeks to determine whether a hybrid model of “new public management” delivers more favourable outcomes than a model focused on cost reduction and private sector prejudice for the bottom line.
The changing nexus of power in the new public sector management of Trinidad and Tobago
Authors: Bissessar Ann Marie
Published: April 2003

Examines the extent to which the introduction of a “new” model of management in the public sector has led to a realignment in the roles, responsibilities, and relationships between the policy‐makers, the bureaucracy and civil society in Trinidad and Tobago. Suggests that the introduction of new public management in the public services of Trinidad and Tobago has led to changes in the structure, culture and functioning of the public sector. Argues that while there were tensions between the politician and the administrator during the post‐independence period these were, to a large extent, kept in check by the rules and regulations that were part and parcel of the traditional method of administration. The introduction of principles of new public management in 1991 and the stress on greater autonomy and a more “fluid” bureaucratic arrangement, however, have fundamentally altered the power relationships between the politician and the administrator so that the division between the political sphere and the administrative sphere has become increasingly blurred. With respect to the civil society, concludes that with the exception of certain non‐governmental organizations, the wider civil society continues to have a minimal input in either policy formulation or execution.
Policy advice: a practical perspective
Authors: Butterworth Robert , Horne Nicholas
Published: April 2003

Senior policy advisers face a number of basic practical issues that they must continuously negotiate if they are to successfully fulfil the policy advising function. Stemming from an Australian public sector experience, this paper offers an applied perspective on some of the more prominent of these issues: the question of political considerations in policy advising, the maintenance of good working relationships between policy advisers and ministers, the nature of timeliness, the extent of information needed before advice can be formulated, and the course to follow when policy advice is rejected. The paper seeks to arrive at some practical conclusions, not only for policy advisers already at the senior level, but also for those who are advancing their policy advising skills and find such issues material to their development.
Restructuring Ford Europe
Authors: Donnelly Tom , Morris David
Published: April 2003

Within the contexts of globalization, rationalization and modularization, this article seeks to explore why Ford Europe performed so badly in the second half of the 1990s, sustaining heavy losses and falling market share. The causes of this are deep‐rooted and are traced to poor model development and a failure to realise that the market for cars was fragmenting with the emergence of new segments such as people carriers, sports utility vehicles and premium brand cars, etc. This was made worse by high costs due to excess capacity and a crucial weakness in diesel engine technology. Moreover, the European scene of operations appeared to be marginalized compared with developments in other parts of the world in Ford 2000. Ford’s response was a reorganization of its European management structure, the development of new models, an attack on its excess capacity and costs through plant closure and redundancies, the forming of strategic alliances to improve its position in diesel engine technology and transmissions and, finally, the development of its Premier Automotive Group.
Political economy and the transition from planned to market economies
Authors: Smith Charles G. , Hills Stephen M. , Arch Gail
Published: April 2003

The revolution in the world economy, especially in the countries of Eastern Europe and the former Soviet Union, has accelerated academic and practitioner interest in the process of global change. Of special relevance to the topic are the disciplines of strategic management, human resource management, and public policy. Specifically, global changes force a rethinking of existing managerial and governmental practices with respect to economic growth and development. This rethinking is critical to the successful transformation from planned to market economies and to assist in this large systems change the West has offered a myriad suggestions in all areas of management and public policy. The paper first explains the basic tenets of institutional economics and convergence theory, second, it presents the new rights by contrasting them with past practices in former Communist countries, and it concludes with a discussion of their implications for policy makers.
Retail bank customer preferences: personal and remote interactions
Authors: Durkin Mark , McCartan‐Quinn Danielle , O’Donnell Aodheen , Howcroft Barry
Published: April 2003

The paper uses a questionnaire and a theoretical model of bank‐customer interaction preferences as the basis for examining the perceptions of retail bank customers regarding the use of remote delivery channels and the extent to which they still value traditional branch‐based face‐to‐face interactions. The empirical evidence suggests that despite the increase in remote banking, retail bank customers still place significantly greater emphasis on face‐to‐face contact. The implications of this finding are that if banks want to encourage widespread customer adoption of remote banking they must better understand customer attitudes towards alternative delivery channels and use this information to educate their customers on the tangible service benefits which emanate from remote delivery.
An empirical evaluation of US bank customer perceptions of the impact of technology on service delivery in the banking sector
Authors: Joseph Mathew , Stone George
Published: April 2003

The instalment of customer friendly technology (such as menu driven automated teller machines, telephone and Internet banking services) as a means of delivering traditional banking services has become commonplace in recent years as a way of maintaining customer loyalty and increasing market share. Traditional brick and mortar banks are using technology to meet the competitive challenge posed by online banks, as well as a method of reducing the cost of providing services that were once delivered exclusively by bank personnel. The present research investigates some of the various roles technology plays in the US banking sector and how technology in general impacts the delivery of banking service. The authors developed a grid that might prove useful to bank managers when making decisions concerning the priority of implementation of service‐oriented technology. Key strategic implications are discussed to include ways banks can improve the level of technology‐based service they provide to their customers.
Client commitment relations towards financial entities
Authors: Angeles Iniesta M. , Sanchez Manuel
Published: April 2003

Given the situation that the banking sector currently faces, this paper will focus on the importance of the presence of client commitment to retailers in general and, more specifically, to financial institutions. So, comments will be made concerning the evolution and current situation of the banking services sector in Spain. In addition, the concept of relational commitment as well as the influence that its development has on the presence of bank client loyalty will be specified. Lastly, an empirical study is developed. The results show differences in the commitment level that individuals project based on links to distinct types of financial entities.
Groupware case studies: trust, commitment and the free expression of ideas
Authors: Corbitt Gail , Martz Ben
Published: April 2003

The use of teams and groups in the workplace continues to grow and trust and commitment to decisions remain desirable characteristics for team members. Group Support Software (GSS) or groupware software has been developed to automate the basic activities of group meetings and thus, help groups. This study combines the two sets of interests; specifically, it looks at the changes in trust and commitment to decisions exhibited by five teams using groupware. The data set was collected from five real‐world (federal, state and commercial) groups as they undertook their actual (Business Process Re‐engineering (BPR) and Joint Application Development (JAD)) projects using groupware. Using a case‐based research methodology, the data set was collected over a series of 41 meetings and organized around group characteristics such as commitment, trust, openness to express ideas, etc. The analysis hints at positive changes in desirable group characteristics over time when using GSS or groupware. However, the groupware characteristic of “more open expression of ideas” does not seem to be the source of the changes. Finally, based upon these findings, the authors suggest that GSS can impact the social components of a group as well as the production components but the cause of the impact resides deeper than a simple idea of providing an environment for more open expression of ideas.
Team member acquisition of team knowledge, skills, and abilities
Authors: Hartenian Linda S.
Published: April 2003

Teams have become increasingly popular in firms of all sizes and industries. Managers believe that teams often provide better outcomes than individuals. Firms can hire such talent or develop it. This study explored whether team training, team experience, mentoring, participation in team sports, and collectivism/individualism orientations were correlated with team knowledge, skills, and abilities (KSAs) in problem solving, communication, conflict resolution, goal setting and planning. The most consistent finding was that those with mentors were more likely to be high on all team KSAs. Employees whose teams had been together longer and those in large firms were more likely to report strong team skills. Those with training in conflict resolution and team skills reported higher conflict resolution, goal setting, and planning skills. Implications of these findings for managers are provided.
Accounting for leases – the problem of rent reviews in capitalising lease liabilities
Authors: Crosby Neil
Published: April 2003

In 1996, an International Accounting Standards Committee (IASC) working party suggested that current methods of accounting for leases should be changed and in 1999 this work culminated in a position paper from the UK Accounting Standards Board (ASB) which made a number of suggestions for consultation (ASB, 1999). The paper assumes that the overall thrust of the proposed changes will be accepted and that will mean that occupying lessees will be required to capitalise the liability to pay rent for their lease and place that liability on the balance sheet. It will also require that property owners identify the value of the lease and the residual property value separately. These are by no means the only issues that are raised by the position paper but it is the implications of these two proposals for valuation methodology that is the subject of this paper. The property industry response in the UK to these two proposals is outlined and it shows that a minimalist approach is recommended, which incidentally is the preferred approach of the UK ASB. This paper argues that market valuations should already be carried out by techniques that attempt to identify the different values of the lease and the residual property value. The minimalist approach will replace one missing set of information with a misleading set, meaning that the IASC attempt to improve the ability of accounts to provide a “fair view” of companies will be thwarted. Alternative valuation models should be adopted which accurately appraise the assets and liabilities distributed by the lease and also identify the residual property value. Conventional market valuation approaches do not work in this context.
Determinants of housing prices in Spanish cities
Authors: Taltavull de La Paz Paloma
Published: April 2003

Residential price levels in Spain vary broadly among markets. Real estate theory explains that prices depend on market characteristics such as vacancy level, land availability, construction supply elasticity to respond to high or low speed to changes on the demand, as well as potential for economic growth, industrial and services activities located inside urban areas, etc. An analysis of prices in Spanish main cities shows that tensions appear to exist in some of them where economic activity shows different dynamism and price level appears to be independent of it. This paper tries to find evidence of the existing relationship between residential prices and economic and demographic factors that are demand determinants such as wages, migrations and productive structure, among others, to explain price formation in Spanish cities. It uses panel data and GLS methodology applied to 71 main Spanish province capitals and cities with more than 100,000 inhabitants. The results show evidence of determinants of housing prices and how some relationships appear to exist between price levels and families’ waged income as well as with population and productive structure in Spanish cities.
Oligopolistic bidding and pricing in real estate development
Authors: Eng Ong Seow , Jam Cheng Fook , Boon Boaz , Foo Sing Tien
Published: April 2003

Real estate developers often operate in oligopolistic environments. Pricing strategies must be made in an interactive framework that makes empirical evaluation difficult. This study appeals to economic experiments to examine how developers price their properties, especially when there is an option to market pre‐completed units. In addition, the interaction between bidding for land and pricing the end product is examined. The results indicate that competitor actions are important considerations in pricing decisions. In particular, the profit maximizing pricing strategy depends critically on being competitive, not necessarily being the most aggressive. Interestingly, pre‐completed units sell only at prices that incorporate future price expectations, and successful bids tend to precipitate more aggressive pricing. Finally, competitive bidding and pricing strategies appear to the best profit maximizing strategy.
Measuring impact and outcome of libraries
Authors: Poll Roswitha
Published: April 2003

The paper examines ways of assessing an economic value of library services, the social value imputed by users and non‐users of libraries, and the outcome on information literacy, information retrieval, and academic and professional success.
Estimating the economic value of library benefits
Authors: Sumsion John , Hawkins Margaret , Morris Anne
Published: April 2003

The theory underlying the economic value of library benefits is outlined, and research (mainly in Australia and New Zealand) is reviewed. A UK research project examined four methods of assessing benefits in economic terms with particular attention to a consensus “market value” model. In developing the “market value” model one key variable is the relationship of book reads to book prices. A prototype value added schedule gives estimates of value for different library services to compare estimated total benefits with total costs. For UK public libraries, calculations show that the economic value of library benefits exceeds costs incurred, with social and intangible benefits in addition. New performance indicators are suggested by the research. It is shown how the methodology can be extended from public libraries to a parliamentary library and also to the economic and social costs of crime.
Why do you ask? Reference statistics for library planning
Authors: Høivik Tord
Published: April 2003

Provides a study of current patterns and trends in reference services based on an analysis of data derived from Ask the Library, a national virtual reference desk that has been operated by Oslo Public Library since 1998.
LibQUAL+TM: preliminary results from 2002
Authors: Cook Colleen , Heath Fred , Thompson Bruce , Webster Duane
Published: April 2003

The LibQUAL+TM instrument derives from the Gap Theory of Service Quality, and the SERVQUAL instrument. Grounded in the constructs of discrepancy theory, the SERVQUAL protocol itself, is anchored by a singular precept: through a series of 22 questions the SERVQUAL instrument undertakes to measure the delivery of service quality across the five dimensions: reliability, assurance, empathy, responsiveness, tangibles. It has been established as defining the service quality construct.
Potential Impact of Increased Use of Biocides in Consumer Products on Prevalence of Antibiotic Resistance
Authors: Gilbert Peter , McBain Andrew J.
Published: April 2003

There has recently been much controversy surrounding the increased use of antibacterial substances in a wide range of consumer products and the possibility that, as with antibiotics, indiscriminate use of biocides might contribute to the overall pattern of susceptibility in the general environment and in the clinic. Such speculation, based on the isolation of resistant mutants from in vitro monoculture experiments, is not reflected by an emergence of biocide-resistant strains in vivo. This review provides a broad coverage of the biocide and resistance literature and evaluates the potential risks, perceived from such laboratory monoculture experiments, against evidence gathered over 50 years of field studies. An explanation for the continued effectiveness of broad-spectrum biocidal agents against the decline in efficacy of therapeutic agents is provided based on the fitness costs of resistance and the ubiquity of naturally occurring substances that possess antibacterial effect. While we conclude from this review of the literature that the incorporation of antibacterial agents into a widening sphere of personal products has had little or no impact on the patterns of microbial susceptibility observed in the environment, the associated risks remain finite. The use of such products should therefore be associated with a clear demonstration of added value either to consumer health or to the product life. Hygienic products should therefore be targeted to applications for which the risks have been established.
Parainfluenza Viruses
Authors: Henrickson Kelly J.
Published: April 2003

Human parainfluenza viruses (HPIV) were first discovered in the late 1950s. Over the last decade, considerable knowledge about their molecular structure and function has been accumulated. This has led to significant changes in both the nomenclature and taxonomic relationships of these viruses. HPIV is genetically and antigenically divided into types 1 to 4. Further major subtypes of HPIV-4 (A and B) and subgroups/genotypes of HPIV-1 and HPIV-3 have been described. HPIV-1 to HPIV-3 are major causes of lower respiratory infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. Each subtype can cause somewhat unique clinical diseases in different hosts. HPIV are enveloped and of medium size (150 to 250 nm), and their RNA genome is in the negative sense. These viruses belong to the Paramyxoviridae family, one of the largest and most rapidly growing groups of viruses causing significant human and veterinary disease. HPIV are closely related to recently discovered megamyxoviruses (Hendra and Nipah viruses) and metapneumovirus.
Pneumococcal Vaccination and Revaccination of Older Adults
Authors: Artz Andrew S. , Ershler William B. , Longo Dan L.
Published: April 2003

As individuals advance in age, the risk of infection, bacteremia, and mortality caused by Streptococcus pneumoniae rises. Retrospective data demonstrate that the licensed penumococcal polysaccharide vaccine (PPV) is effective in older persons in reducing serotype-specific invasive disease. PPV demonstrates good immunogenicity in older adults, generally comparable to that in younger subjects, although certain cohorts respond less well. The response to PPV is T cell independent, however, and does not elicit immunologic memory. The duration of the anti-capsular polysaccharide antibody response appears to wane as early as 3 years after vaccination. In older persons, revaccination induces an antibody response, although it may not be as strong as that from the initial vaccine. While revaccination of older adults has been recommended, clinical efficacy has not yet been proven. Measures of antibody function may be at least as important in determining protection as are quantitative antibody levels. Additional studies of immunogenicity, particularly regarding revaccination, will facilitate the design of an optimal pneumococcal vaccination policy. Research into conjugate- and protein-based pneumococcal vaccines, which elicit T-cell-dependent responses and induce immunologic memory, is needed in older persons. In the meantime, administering to PPV to recommended groups should be a public health priority.
Impact of Genotypic Studies on Mycobacterial Taxonomy: the New Mycobacteria of the 1990s
Authors: Tortoli Enrico
Published: April 2003

The advancement of genetic techniques has greatly boosted taxonomic studies in recent years. Within the genus Mycobacterium, 42 new species have been detected since 1990, most of which were grown from clinical samples. Along with species for which relatively large numbers of strains have been reported, some of the new species of mycobacteria have been detected rarely or even only once. From the phenotypic point of view, among the new taxa, chromogens exceed nonchromogens while the numbers of slowly and rapidly growing species are equivalent. Whereas conventional identification tests were usually inconclusive, an important role was played by lipid analyses and in particular by high-performance liquid chromatography. Genotypic investigations based on sequencing of 16S rRNA gene have certainly made the most important contribution. The investigation of genetic relatedness led to the redistribution of the species previously included in the classically known categories of slow and rapid growers into new groupings. Within slow growers, the intermediate branch related to Mycobacterium simiae and the cluster of organisms related to Mycobacterium terrae have been differentiated; among rapid growers, the group of thermotolerant mycobacteria has emerged. The majority of species are resistant to isoniazid and, to a lesser extent, to rifampin. Many of the new species of mycobacteria are potentially pathogenic, and there are numerous reports of their involvement in diseases. Apart from disseminated and localized diseases in immunocompromised patients, the most frequent infections in immunocompetent people involve the lungs, skin, and, in children, cervical lymph nodes. The awareness of such new mycobacteria, far from being a merely speculative exercise, is therefore important for clinicians and microbiologists.
Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein
Authors: Douglas Janet L. , Panis Marites L. , Ho Edmund , Lin Kuei-Ying , Krawczyk Steve H. , Grant Deborah M. , Cai Ruby , Swaminathan Swami , Cihlar Tomas
Published: April 2003

Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of ≤22°C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.
Poliovirus
cre
(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis
Authors: Morasco B. Joan , Sharma Nidhi , Parilla Jessica , Flanegan James B.
Published: April 2003

The
cre
(2C) hairpin is a
cis
-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the
cre
(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. c
re
(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the
cre
(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the
cre
(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the
cre
(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3′ poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the
cre
(2C) hairpin. The pool of excess VPgpUpU synthesized on the
cre
(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.
Expression of Inducible Nitric Oxide Synthase and Elevation of Tyrosine Nitration of a 32-Kilodalton Cellular Protein in Brain Capillary Endothelial Cells from Rats Infected with a Neuropathogenic Murine Leukemia Virus
Authors: Jinno-Oue Atsushi , Wilt Susan G. , Hanson Charlotte , Dugger Natalie V. , Hoffman Paul M. , Masuda Michiaki , Ruscetti Sandra K.
Published: April 2003

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) which causes a rapidly progressive spongiform neurodegenerative disease in rodents. The primary target of PVC-211 MuLV infection in the brain is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection. Previous studies have shown that changes in the envelope gene of PVC-211 MuLV confer BCEC tropism to the virus. However, little is known about how infection of BCECs by PVC-211 MuLV induces neurological disease. Previous results suggest that nitric oxide (NO), which has been implicated as a potential neurotoxin, is involved in PVC-211 MuLV-induced neurodegeneration. In this study, we show that expression of inducible nitric oxide synthase (iNOS), which produces NO froml-arginine, is induced in BCECs from PVC-211 MuLV-infected rats. Furthermore, elevated levels of a 32-kDa cellular protein modified by 3-nitrotyrosine, which is a hallmark of NO production, were observed in virus-infected BCECs. BCECs from rats infected with BCEC-tropic but nonneuropathogenic PVF-e5 MuLV, which is a chimeric virus between PVC-211 MuLV and F-MuLV, fail to induce either iNOS expression or elevation of tyrosine nitration of a 32-kDa protein. These results suggest that expression of iNOS and nitration of tyrosine residues of a 32-kDa protein in PVC-211 MuLV-infected BCECs may play an important role in neurological disease induction.
Functional Dissection of a Poliovirus
cis
-Acting Replication Element [PV-
cre
(2C)]: Analysis of Single- and Dual-
cre
Viral Genomes and Proteins That Bind Specifically to PV-
cre
RNA
Authors: Yin Jiang , Paul Aniko V. , Wimmer Eckard , Rieder Elizabeth
Published: April 2003

The role of the
cis
replication element (
cre
) in the 2C
ATPase
coding region of the poliovirus (PV) genome has been studied with a series of mutants derived from either a PV1 full-length genome or a replicon (P/L) containing the firefly luciferase reporter gene in place of the capsid region. Using the P/L replicon we have inserted
cre
elements at three different locations in the genome including the 5′ nontranslated region and within the open reading frame. The successful recovery of replication of a nonviable P/L (A
5
C) mutant replicon with an artificial
cre
element as “rescuer,” in addition to the results of site-directed mutagenesis and experiments with truncated forms of PV-
cre
(2C), indicated that (i) the sequence within the upper stem and loop regions contains the minimal
cre
RNA required for VPg uridylylation in vitro, (ii) the location of the
cre
RNA in the poliovirus genome is not relevant to RNA infectivity, and (iii) specific binding of 3CD
pro
to PV-
cre
(2C) occurs within the upper stem region and probably involves several contact residues. The role of a 14-nucleotide conserved “core” sequence among known
cre
structures in picornaviruses was examined by site-directed mutagenesis of individual nucleotides. In addition to a conserved AAA (4472 to 4474) triplet previously shown to be the primary RNA template for VPg uridylylation by the PV RNA polymerase 3D
pol
(E. Rieder, A. V. Paul, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol.
74:
10371-10380, 2000), we have now shown that important residues (G
4468
and A
4481
) are contained in a predicted internal bulge at the upper stem-loop of PV-
cre
(2C). We have further demonstrated that the viral proteins 3CD
pro
and 3C
pro
form stable complexes with a transcript PV-
cre
(2C) RNA that can be considered critical for VPg uridylylation.
The Papillomavirus E2 Protein Binds to and Synergizes with C/EBP Factors Involved in Keratinocyte Differentiation
Authors: Hadaschik Dirk , Hinterkeuser Korinna , Oldak Monika , Pfister Herbert J. , Smola-Hess Sigrun
Published: April 2003

The papillomavirus life cycle is closely linked to the differentiation program of the host keratinocyte. Thus, late gene expression and viral maturation are restricted to terminally differentiated keratinocytes. A variety of cellular transcription factors including those of the C/EBP family are involved in the regulation of keratinocyte differentiation. In this study we show that the papillomavirus transcription factor E2 cooperates with C/EBPα and -β in transcriptional activation. This synergism was independent of an E2 binding site. E2 and C/EBP factors synergistically transactivated a synthetic promoter construct containing classical C/EBPβ sites and the C/EBPα-responsive proximal promoter of the involucrin gene, which is naturally expressed in differentiating keratinocytes. C/EBPα or -β coprecipitated with E2 proteins derived from human papillomavirus type 8 (HPV8), HPV16, HPV18, and bovine papillomavirus type 1 in vitro and in vivo, indicating complex formation by the cellular and viral factors. The interaction domains could be mapped to the C terminus of E2 and amino acids 261 to 302 located within the bZIP motif of C/EBPβ. Our data suggest that E2, via its interaction with C/EBP factors, may contribute to enhancing keratinocyte differentiation, which is suppressed by the viral oncoproteins E6 and E7 in HPV-induced lesions.
Genome Delivery and Ion Channel Properties Are Altered in VP4 Mutants of Poliovirus
Authors: Danthi Pranav , Tosteson Magdalena , Li Qi-han , Chow Marie
Published: April 2003

During entry into host cells, poliovirus undergoes a receptor-mediated conformational transition to form 135S particles with irreversible exposure of VP4 capsid sequences and VP1 N termini. To understand the role of VP4 during virus entry, the fate of VP4 during infection by site-specific mutants at threonine-28 of VP4 (4028T) was compared with that of the parental Mahoney type 1 virus. Three virus mutants were studied: the entry-defective, nonviable mutant 4028T.G and the viable mutants 4028T.S and 4028T.V, in which residue threonine-28 was changed to glycine, serine, and valine, respectively. We show that mutant and wild-type (WT) VP4 proteins are localized to cellular membranes after the 135S conformational transition. Both WT and viable 4028T mutant particles interact with lipid bilayers to form ion channels, whereas the entry-defective 4028T.G particles do not. In addition, the electrical properties of the channels induced by the mutant viruses are different from each other and from those of WT Mahoney and Sabin type 3 viruses. Finally, uncoating and/or cytoplasmic delivery of the viral genome is altered in the 4028T mutants: the 4028T.G lethal mutant does not release its genome into the cytoplasm, and genome delivery is slower during infection by mutant 4028T.V 135S particles than by mutant 4028T.S or WT 135S particles. The distinctive electrical characteristics of the different 4028T mutant channels indicate that VP4 sequences might form part of the channel structure. The different entry phenotypes of these VP4 mutants suggest that the ion channels may be related to VP4's role during genome uncoating and/or delivery.
Specific Cleavages by RNase H Facilitate Initiation of Plus-Strand RNA Synthesis by Moloney Murine Leukemia Virus
Authors: Schultz Sharon J. , Zhang Miaohua , Champoux James J.
Published: April 2003

Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3′ terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3′ terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3′ terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3′ end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3′ terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3′ terminus.
Distinct Roles of the Adenovirus E4 ORF3 Protein in Viral DNA Replication and Inhibition of Genome Concatenation
Authors: Evans Jared D. , Hearing Patrick
Published: April 2003

Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection. Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair.
Roles for Endocytosis and Low pH in Herpes Simplex Virus Entry into HeLa and Chinese Hamster Ovary Cells
Authors: Nicola Anthony V. , McEvoy Anna M. , Straus Stephen E.
Published: April 2003

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H
+
-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.
Luciferase Imaging of a Neurotropic Viral Infection in Intact Animals
Authors: Cook Susan H. , Griffin Diane E.
Published: April 2003

The identification of viral determinants of virulence and host determinants of susceptibility to virus-induced disease is essential for understanding the pathogenesis of infection. Obtaining this information requires infecting large numbers of animals to assay amounts of virus in a variety of organs and to observe the onset and progression of disease. As an alternative approach, we have used a murine model of viral encephalitis and an in vivo imaging system that can detect light generated by luciferase to monitor over time the extent and location of virus replication in intact, living mice. Sindbis virus causes encephalomyelitis in mice, and the outcome of infection is determined both by the strain of virus used for infection and by the strain of mouse infected. The mode of entry into the nervous system is not known. Virulent and avirulent strains of Sindbis virus were engineered to express firefly luciferase, and the Xenogen IVIS system was used to monitor the location and extent of virus replication in susceptible and resistant mice. The amount of light generated directly reflected the amount of infectious virus in the brain. This system could distinguish virulent and avirulent strains of virus and susceptible and resistant strains of mice and suggested that virus entry into the nervous system could occur by retrograde axonal transport either from neurons innervating the initial site of replication or from the olfactory epithelium after viremic spread.