Search

Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.

Type I interferons (IFN-alpha/beta) rapidly confer resistance to alphavirus infection in macrophages and dendritic cells (DC) as evidenced by the dramatically increased susceptibility of these cells in mice with the IFNAR1 subunit of the IFN-alpha/beta receptor ablated (IFNAR1-/-). Normal adult mice develop only a subclinical Sindbis virus infection, whereas infected IFNAR1-/- mice rapidly succumb to a fatal disease. Here, we investigated the individual and combined contributions of the two best characterized INF-alpha/beta-mediated antiviral pathways to the control of Sindbis virus replication: (1) the coupled 2-5A synthetase/RNase L pathway and (2) the double-stranded RNA-dependent protein kinase (PKR) pathway. Surprisingly, mice deficient in PKR, RNase L, and Mx-1 (triply-deficient [TD]) developed only subclinical infection. Although the permissivity of cells in lymph nodes draining the inoculation site was increased in the absence of PKR/RNase L, systemic dissemination of the virus infection was restricted by an alternative IFN-alpha/beta receptor-dependent mechanism. In vitro, suppression of early virus protein synthesis and virion production in primary bone marrow-derived dendritic cells (BMDC) was largely dependent on the PKR pathway. However, later in infection virion production was reduced even in the absence of PKR/RNase L by an IFN-alpha/beta receptor-dependent mechanism. Priming of BMDC with IFN-alpha/beta or IFN-gamma resulted in dose-dependent restriction of virus replication, largely independent of PKR and/or RNase L expression.

Dengue is considered the most important emerging, human arboviruses, with worldwide distribution in the tropics. Unfortunately, there are no licensed dengue vaccines available or specific anti-viral drugs. The development of a dengue vaccine faces unique challenges. The four serotypes co-circulate in endemic areas, and pre-existing immunity to one serotype does not protect against infection with other serotypes, and actually may enhance severity of disease. One foremost constraint to test the efficacy of a dengue vaccine is the lack of an animal model that adequately recapitulates the clinical manifestations of a dengue infection in humans. In spite of this limitation, non-human primates (NHP) are considered the best available animal model to evaluate dengue vaccine candidates due to their genetic relatedness to humans and their ability to develop a viremia upon infection and a robust immune response similar to that in humans. Therefore, most dengue vaccines candidates are tested in primates before going into clinical trials. In this article, we present a comprehensive review of published studies on dengue vaccine evaluations using the NHP model, and discuss critical parameters affecting the usefulness of the model. In the light of recent clinical data, we assess the ability of the NHP model to predict immunological parameters of vaccine performances in humans and discuss parameters that should be further examined as potential correlates of protection. Finally, we propose some guidelines toward a more standardized use of the model to maximize its usefulness and to better compare the performance of vaccine candidates from different research groups.

Dengue viruses (DENV1-4) cause 390 million clinical infections every year, several hundred thousand of which progress to severe hemorrhagic and shock syndromes. Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated virus (LAV) vaccine candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). In a previous study, we demonstrated that a non-propagating Venezuelan equine encephalitis virus replicon expression vector (VRP), expressing the ectodomain of DENV E protein (E85), overcomes maternal interference in a BALB/c mouse model. We report here that a single immunization with a tetravalent VRP vaccine induced NAb and T-cell responses to each serotype at a level equivalent to the monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Furthermore, neonatal immunization was durable and could be boosted later in life to further increase NAb and T-cell responses. Although the neonatal immune response was lower in magnitude than responses in adult BALB/c mice, we demonstrate that VRP vaccines generated protective immunity from a lethal challenge after a single neonatal immunization. In summary, VRP vaccines expressing DENV antigens were immunogenic and protective in neonates, and hence are promising candidates for safe and effective vaccination in early life.

In this paper, we describe a Software Reconnaissance method for locating product features in code. The University of West Florida has provided a public domain Reconnaissance tool for C and C++. Characteristics of the Ada programming language present new interesting issues related to the development of a similar tool for Ada Reconnaissance, particularly its use in embedded systems, multi-tasking systems, and systems with real-time constraints. We describe our on-going efforts to develop a public domain Ada Reconnaissance tool.

Norwalk virus (NV) is a prototype strain of human caliciviruses, a group of viruses that are the major pathogens causing epidemic nonbacterial gastroenteritis (14, 49). The NV genome, a positive-sense, single-stranded RNA molecule approximately 7.7 kb in length, is predicted to contain three open reading frames (ORFs) (29). The first and third ORFs are in reading frame 2 of the cDNA, while ORF 2 is in reading frame 3. The first ORF (ORF 1) is predicted to encode the nonstructural proteins. Sequence analysis has identified similarities to the picornavirus 2C helicase, 3C protease, and 3D RNA-dependent RNA polymerase (29). The second ORF (ORF 2) encodes the capsid protein. Expression of the capsid protein in insect cells infected with baculovirus recombinants results in the self-assembly of empty recombinant virus-like particles (rVLPs) (28, 51). The third ORF (ORF 3) is located at the 3′ end of the genome and codes for a 212-amino-acid protein of unknown function. The predicted molecular weight of the NV ORF 3 protein is 22,479. The ORF 3 protein is a basic protein with a predicted isoelectric point of 10.99, which has led to speculation that it may be involved in nucleic acid binding (13). Purified 38-nm recombinant NV (rNV) VLPs have been characterized antigenically and morphologically (28). Three-dimensional reconstruction studies revealed that these particles fold into T=3 icosahedral structures formed by 180 copies of the capsid protein (40). The finding of virus capsids composed of a single structural protein is a common feature of plant viruses including tomato bushy stunt virus (22) and turnip crinkle virus (25). Caliciviruses and nodaviruses are the only animals viruses described to date with a capsid made of a single structural protein (23, 43). A notable difference between plant virus and NV capsid proteins is that the plant virus capsid proteins have an N-terminal basic domain, which is thought to interact with the RNA during assembly. The NV capsid protein lacks such a basic region, and analysis of the X-ray crystallographic structure of rNV VLPs has revealed that the inner surface of the icosahedral shell is acidic (38). For these reasons, it seems highly possible that the calicivirus ORF 3 protein may aid in RNA encapsidation. The presence of ORF 3 in the genome is conserved throughout all human and animal caliciviruses, suggesting that it plays a role in replication or assembly. ORF 3 or ORF 3-equivalent proteins were detected in feline calicivirus (FCV)-infected cells (24) and in rabbit hemorrhagic disease virus (RHDV)-infected primary hepatocytes (32) as well as RHDV virions (54). The synthesis of the ORF 3 protein in a cell-free translation system has been reported for FCV and Camberwell virus (24, 54). Considerable sequence variability of ORF 3 among members of the Norwalk-like viruses (NLVs) has been reported and is consistent with the idea that ORF 3 might be a structural protein (42, 45). The inability to grow NV and a lack of reagents to detect ORF 3 has hampered studies of the ORF 3 protein. In the present paper, we report the characterization of the NV ORF 3 protein synthesized in a cell-free translation system and expressed in insect cells, purified VLPs, and purified NV virions. These studies were facilitated by generation of ORF 3 peptide antisera which are able to recognize the original peptides by enzyme-linked immunosorbent assay (ELISA) and the ORF 3 protein by immunoprecipitation and Western blot analysis.

High levels of local immunoglobulin E (IgE) have been demonstrated in nasal polypoid tissue; however, an association between nasal polyps and allergy has not been proven. The authors have observed that polypoid edema isolated to the leading edge of the middle turbinate (MT) is highly associated with allergic rhinitis. The objective of this study was to determine if there is an association between isolated MT polyps and inhalant allergy.A single institution prospective study was performed. Twenty-five consecutive patients found to have isolated MT polyps on endoscopic exam were recruited. Nasal and allergy symptoms were documented on the intake form. Allergy testing was recommended to all patients.Of the 25 patients found to have isolated MT polypoid edema documented on endoscopic exam, 16 patients underwent skin or in vitro allergy testing. All of the patients tested positive for inhalant allergy.This small series provides strong evidence to support an association between isolated MT polypoid edema and allergy. We recommend allergy testing in all patients with isolated MT polyps or polypoid edema.

This paper describes a research program on the introduction of globally distributed teams into undergraduate software engineering courses. A pilot study, now completed, involved students at a single institution using two different virtual environments while cooperatively developing requirements artifacts in 3-person virtual teams. We describe the results of this pilot study and the plans for its extension to a three-year multi-institution, multi-culture, and multi-language setting.

Despite many years of research, a dengue vaccine is not available, and the more advanced live attenuated vaccine candidate in clinical trials requires multiple immunizations with long interdose periods and provides low protective efficacy. Here, we report important contributions to the development of a second-generation dengue vaccine. First, we demonstrate that a nonpropagating vaccine vector based on Venezuelan equine encephalitis virus replicon particles (VRP) expressing two configurations of dengue virus E antigen (subviral particles [prME] and soluble E dimers [E85]) successfully immunized and protected macaques against dengue virus, while antivector antibodies did not interfere with a booster immunization. Second, compared to prME-VRP, E85-VRP induced neutralizing antibodies faster, to higher titers, and with improved protective efficacy. Third, this study is the first to map antigenic domains and specificities targeted by vaccination versus natural infection, revealing that, unlike prME-VRP and live virus, E85-VRP induced only serotype-specific antibodies, which predominantly targeted EDIII, suggesting a protective mechanism different from that induced by live virus and possibly live attenuated vaccines. Fourth, a tetravalent E85-VRP dengue vaccine induced a simultaneous and protective response to all 4 serotypes after 2 doses given 6 weeks apart. Balanced responses and protection in macaques provided further support for exploring the immunogenicity and safety of this vaccine candidate in humans.

Interoperability is essential for modern enterprise software; one of the most promising ways of providing interoperability is though Services Oriented Architectures (SOA) usually implemented using the Web Services (WS) standards. SOA/WS has the potential to be a transformational technology but there are a number of problems that may hinder its application. One of these is the classic slowness of software evolution. This paper discusses the issues of SOA evolution and describes ongoing research experimenting with the use of search technology to speed comprehension of SOA applications. Flexible but specialized search tools may be a good match for the “open world” of a SOA system which may encounter frequent novelties in programming languages and technology during its lifetime. We describe a basic search tool adapted to SOA/WS artifacts, a knowledge-based extension to it to improve software comprehension, and ongoing work to handle additional document types and to provide ontology-based support. Development of support tools for SOA evolution could be a fruitful topic for industry-university collaboration. Such tools would be an enabler for the interoperable information systems needed to do business in the modern world.

Rule-based methods have traditionally been applied to develop knowledge-based systems that replicate expert performance on a deep but narrow problem domain. Knowledge engineers capture expert knowledge and encode it as a set of rules for automating the expert's reasoning process to solve problems in a variety of domains. We describe the development of a knowledge-based system approach to enhance program comprehension of Service Oriented Architecture (SOA) software. Our approach uses rule-based methods to automate the analysis of the set of artifacts involved in building and deploying a SOA composite application. The rules codify expert knowledge to abstract information from these artifacts to facilitate program comprehension and thus assist Software Engineers as they perform system maintenance activities. A main advantage of the knowledge-based approach is its adaptability to the heterogeneous and dynamically evolving nature of SOA environments. Rule-based methods have been very effective in supporting decision making in many complex domains. Can they also assist Software Engineers in dealing with the emerging complexities of Service Oriented Architecture (SOA) applications?

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Norwalk virus (NV) causes epidemic outbreaks of acute nonbacterial gastroenteritis in humans. The NV capsid is made up of a single protein, and expression of the capsid protein in baculovirus recombinants results in spontaneous assembly of the protein into virus-like particles (X. Jiang, M. Wang, D. Y. Graham, and M. K. Estes, J. Virol. 66:6527-6532, 1992). We have investigated whether the NV capsid protein undergoes a specific proteolytic cleavage. Recombinant NV (rNV) particles were digested with trypsin to determine if a specific cleavage occurred. A predominant band with a molecular weight of approximately 32,000 (32K protein) was observed when trypsin-treated rNV was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Determination of the N-terminal sequence of this band showed that a trypsin-specific cleavage occurred at amino acid residue 227. Early studies identified two proteins with molecular weights of 59,000 and 30,000 (59K and 30K proteins) in the stool of NV-infected volunteers that were reactive with postinfection antiserum. (H. B. Greenberg, J. R. Valdesuso, A. R. Kalica, R. G. Wyatt, V. J. McAuliffe, A. Z. Kapikian, and R. M. Chanock, J. Virol. 37:994-999, 1981). We hypothesized that the 32K rNV cleavage product might be analogous to the 30K soluble protein detected in stools of NV-infected volunteers. Immunoprecipitation of soluble protein from these stool extracts with a rabbit polyclonal antiserum made against rNV, and Western blot detection with a mouse polyclonal antiserum made against rNV, revealed a single band with an apparent molecular weight of 30,000 that migrated similarly to the trypsin cleavage product observed in vitro. The N terminus of this band was identical to that of the 32K cleavage product of rNV capsid protein. These data show that the 30K protein in stool is produced by specific cleavage of the NV capsid protein in vivo. Trypsin cleavage of isolated soluble rNV 58K capsid protein and of assembled particles showed that only soluble 58K capsid protein is susceptible to cleavage. The presence of a large amount of soluble capsid protein may influence the immune response to or pathogenicity of NV infections.

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.

Software interoperability is a pressing need to allow governments and businesses to function efficiently. The most commonly recommended technology for interoperability is Services Oriented Architecture (SOA) implemented using web services. Several authors have argued that SOA systems may be particularly challenging to maintain, largely due to difficulties in program comprehension. Program comprehension for SOA could be aided by appropriate software tools to provide information to SOA maintainers. However, there is little experience regarding the questions that SOA maintainers will need to ask. This paper describes use of a prototype SOA search tool in an informal requirements elicitation study to gather feedback from practicing programmers about what SOA maintainers will want to know. Several specific information needs were identified, including the need for a compact way of representing data types used in services, and the need for ontology support to help understand the many different elements and attributes in web services descriptions.

Services Oriented Architecture (SOA) is becoming a popular style for building complex systems-of-systems that allow businesses to work together across organizational boundaries. However concerns have been raised about the comprehensibility and maintainability of SOA composite applications. Integrating and deploying SOA applications requires artifacts in a variety of web-based languages (WSDL, XSD, BPEL, etc.) often produced by code-generation tools. It becomes difficult for a human to discover and understand the dependencies between these artifacts in an existing system. In this paper, we describe ongoing research on using search techniques to facilitate SOA maintenance by allowing users to query collections of artifacts making up a SOA composite application. The main focus in this paper is a case study using our prototype search tool SOAMiner to identify a set of abstractions that extract useful and critical information for maintainers, thereby bridging the heterogeneity of SOA artifacts while opportunistically exploiting their structure. Results of the study indicate that the highest priority abstractions for SOA are datatype summaries, service invocation (calling) relationships, and data usage relationships.

Summary Introduction There is evidence supporting an association between depression and anxiety in patients with chronic disease. Spasmodic dysphonia (SD) is a chronic, incurable, and disabling voice disorder. Reported rates of depression and anxiety in SD range from 7.1% to 72%, with a maximum number of 18 patients. The goal of this study was to define the coprevalence of depression and anxiety with SD. Materials and Methods A single-institution case-control study was performed from May to July 2010. Consecutive patients with SD and benign voice disorders were enrolled prospectively. On enrollment, patients were asked to fill out a questionnaire that reviewed the duration of the voice disorder and personal history of anxiety and depression, including current and lifetime diagnosis. Results One hundred forty-six controls with benign voice disorders and 128 patients with SD were enrolled. Patients with SD were no more likely to be diagnosed with depression or anxiety than those of the control group (odds ratio [OR] = 0.985, 95% confidence interval [CI] = 0.59–1.63; and OR = 1.314; 95% CI = 0.75–2.3, respectively). Additionally, duration of disease was a risk factor for depression in both the SD group and the control group, and the association was not significantly different between groups. Conclusion Patients with SD were no more likely to have depression or anxiety than those with other voice disorders. It is important for otolaryngologists to be aware of the increased rates of depression in patients diagnosed with chronic diseases, including voice disorders, and to refer to a psychiatrist when appropriate.

An executive software engineering program-developed to meet regional workforce needs-affords cohorts of students the opportunity to complete their Master's degree within one calendar year. This innovative program was designed with several objectives. Custom elective tracks were integrated to better meet needs within diverse application areas, such as healthcare, transportation, and the insurance industry. This program was also designed to establish a partner relationship with employers to support students through work release and opportunities for real world capstone project experiences. The program was designed as a cohort model in order to establish a strong sense of community and thus promote increased peer learning within courses because effective peer learning has been established as a best practice for online programs in higher education. Experiences regarding the development and implementation of an innovative, graduate, online program for the Master of Science in Computer Science — Software Engineering specialization are described in this paper.

Background Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses. Methodology/Principal Findings TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies. Conclusions/Significance These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.

This paper describes the use of Feature Sequence Viewer (FSV) to perform feature location in a teaching and research program suite named Open SOALab. In this context, a software feature refers to software components that provide specific functionality. The composite application encompasses a system in which hotel brokers identify rooms meeting various criteria from among several hotel chains in multiple countries, and then exchanges the necessary amount of currency, using a currency broker to get several quotes and select the best one. The currency broker in turn uses two services: an authentication service and a settlement house. The various service interfaces are exposed via WSDLs. The system, running on Apache with php and nuSOAP, uses Apache’s forensic log module and microsecond time stamps to generate data that is input into FSV which produces a browsable graphical representation of the messages in the system. FSV employs a component relevance index (pc) that is used to determine which messages are displayed within the viewer. A value of pc is computed for each message in the data set loaded into FSV. A user can raise and lower the threshold value for pc so that messages with pc values above the threshold are displayed and those below are not displayed. Three experiments of increasing complexity were performed to demonstrate the ability of this approach to extract feature messages by separating them from irrelevant messages within a SOA composite application.

Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-β and IFN-γ. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-β treatment but did inhibit Jak1 and Jak2 activation in response to IFN-γ, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.

In a world where Information Technology (IT) permeates all aspects of life and global competition is increasing, the need for trained professionals in science, technology, engineering, and mathematics (STEM) related disciplines is increasing more than ever. This paper describes some innovative strategies at the University of West Florida to encourage students to pursue STEM related disciplines and to help fill the IT workforce void. These strategies include curriculum redesign, novel University/Industry partnerships, K-12 outreach activities, and building awareness for STEM within the community. Initial feedback from the implementation of these strategies has been very positive.

A concept fundamental to viral pathogenesis is that infection induces specific changes within the host cell, within specific tissues, or within the entire animal. These changes are reflected in a cascade of altered transcription patterns evident during infection. However, elucidation of this cascade in vivo has been limited by a general inability to distinguish changes occurring in the minority of infected cells from those in surrounding uninfected cells. To circumvent this inherent limitation of traditional gene expression profiling methods, an innovative mRNP-tagging technique was implemented to isolate host mRNA specifically from infected cells in vitro as well as in vivo following Venezuelan equine encephalitis virus (VEE) infection. This technique facilitated a direct characterization of the host defense response specifically within the first cells infected with VEE, while simultaneous total RNA analysis assessed the collective response of both the infected and uninfected cells. The result was a unique, multifaceted profile of the early response to VEE infection in primary dendritic cells, as well as in the draining lymph node, the initially targeted tissue in the mouse model. A dynamic environment of complex interactions was revealed, and suggested a two-step innate response in which activation of a subset of host genes in infected cells subsequently leads to activation of the surrounding uninfected cells. Our findings suggest that the application of viral mRNP-tagging systems, as introduced here, will facilitate a much more detailed understanding of the highly coordinated host response to infectious agents., Author Summary A major element of viral pathogenesis is the induction of specific changes within the infected host, often reflected in altered gene expression patterns. However, revealing these changes in vivo has been limited by an inability to distinguish changes within the minority of infected cells from that in surrounding uninfected cells. Here we introduce a viral mRNP-tagging system, based on Venezuelan equine encephalitis virus (VEE), that enables the isolation of host mRNA specifically from infected cells in vitro and in vivo, even when they are a small minority. This system allowed us for the first time to monitor the innate response specifically within the cells initially infected in vivo. In combination with simultaneous analysis of the entire tissue response, the result was a multifaceted view of the innate response to VEE in dendritic cells, and in the draining lymph node. The results supported a two-step response in which activation of host genes within infected cells leads to activation of bystander cells, offering insight into the process by which the greater innate immune response to alphaviruses is established in vivo. This system may be employed for a wide variety of pathogens, offering broader implications to the manner in which interactions between pathogens and their hosts are studied.

A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.

Macaques are the only animal model used to test dengue virus (DENV) vaccine candidates. Nevertheless, the pathogenesis of DENV in macaques is not well understood. In this work, by using Affymetrix oligonucleotide microarrays, we studied the broad transcriptional modifications and cytokine expression profile after infecting rhesus macaques with DENV serotype 1. Five days after infection, these animals produced a potent, innate antiviral immune response by inducing the transcription of signature genes from the interferon (IFN) pathway with demonstrated antiviral activity, such as myxoprotein, 2′,5′-oligoadenylate synthetase, phospholipid scramblase 1, and viperin. Also, IFN regulatory element 7, IFN-stimulated gene 15, and protein ligases linked to the ISGylation process were up-regulated. Unexpectedly, no up-regulation of IFN-α, -β, or -γ genes was detected. Transcription of the genes of interleukin-10 (IL-10), IL-8, IL-6, and tumor necrosis factor alpha was neither up-regulated nor down-regulated. Results were confirmed by real-time PCR and by multiplex cytokine detection in serum samples.

For the last seven years students in the Masters track in Software Engineering at the University of West Florida have focused their capstone course and project work on software process and software evolution. Students initially defined a software maintenance process called GUMP which has been used in all subsequent years. Students use this process in a rolling project in which they maintain and enhance a medium-sized software tool. Simultaneously they improve the process based on their experiences, thus leaving their successors both enhanced software and an enhanced process to use in its evolution. Approximately 50 cycles of software change have been completed following this method, and two years ago a major revision to GUMP was undertaken based on an analysis of this experience. This article, by three of the instructors and two of the participating students, describes the methods used and the experience gained.

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5′ untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-α/β) activity in VEE pathogenesis. In IFN-α/βR −/− mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-α/βR −/− mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-α/β pathway. In cells derived from IFN-α/βR −/− mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-α/β plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-α/β between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-α/β in two separate in vitro assays, suggesting that the increased sensitivity to IFN-α/β plays a major role in the in vivo attenuation of V3043.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system., Author Summary Dengue virus is an escalating public health threat for over 2.5 billion people worldwide. The disease caused by dengue virus ranges from mild (dengue fever) to lethal (dengue hemorrhagic fever, dengue shock syndrome). To date, there is no cure or vaccine for dengue. One of the challenges to developing a safe and efficient dengue vaccine is that antibodies, usually induced by vaccines to protect the host from re-infection, can increase the severity of dengue disease if they are not present in sufficient amounts to neutralize the virus. An efficient vaccine is urgently needed to slow down the progression of dengue disease, but little is known about the way the immune system protects the body against dengue re-infection. Using a protective vaccine candidate for dengue, the present study evaluates in mice the relative contribution of T cells and antibodies to protection against dengue. We show that the antibody component of an immune response that is overall protective had the ability, when isolated from the other components of the immune system, to either decrease or increase viral burden, whereas T cells reduced viral burden in all situations tested. Our results suggest that vaccine development efforts should focus on approaches that induce both T cell and antibody responses against dengue virus.