HSV-1 facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in gB, gK, UL20 and other viral genes, drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, which is regulated by interactions with viral glycoproteins gD, gH/gL, gK, membrane protein UL20 and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl terminal 28 amino acids of gB, or the dominant syncytial mutation in gK (Ala-to-Val at amino acid 40), was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletions substantially affected gB, gC, gD, gE and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM, while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells slower than their parental wild-type virus. Moreover, the mouse eye model study, revealed that although the UL11-null virus replicated less efficiently in mouse corneas, it infected ganglionic neurons with greater efficiency than either the gM or gE-null viruses. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread in vivo as well as in vitro, and also that gK plays the most important role in corneal and ganglionic infection in the mouse eye model followed by gM, gE, and UL11 in a descending order of importance relative to the wild-type virus.

We designed and developed a Collaborative Annotation System for Proteins called DAS Writeback, which extends the Distributed Annotation System (DAS) to provide the functionalities of adding, editing and deleting annotations. A great deal of effort has gone into gathering information about proteins over the last few years. By June 2009, UniProtKB/Swiss-Prot, a curated database, contained over four hundred thousand sequence entries and UniProtKB/TrEMBL, a database with automated annotation, contained over eight million sequence entries. Every protein is annotated with relevant information, which needs to be eciently captured and made available to other research groups. These include annotations about the structure, the function or the biochemical residues. Several research groups have taken on the task of making this information accessible to the community, however, information flow in the opposite direction has not been extensively explored. Users are currently passive actors that behave as consumers of one or several sources of protein annotations and they have no immediate way to provide feedback to the source if, for example, a mistake is detected or they want to add information. Any change has to be done by the owner of the database. The current lack of being able to feed information back to a database is tackled in this project. The solution consists of an extension of the DAS protocol that defines the communication rules between the client and the writeback server following the Uniform Interface of the RESTful architecture. A protocol extension was proposed to the DAS community and implementations of both server and client were created in order to have a fully functional system. For the development of the server, writing functionalities were added to MyDAS, which is a widely used DAS server. The writeback client is an extended version of the web-based protein client Dasty2. The involvement of the DAS community and other potential users was a fundamental component of this project. The architecture was designed with the insight of the DAS specialized forum, a prototype was then created and subsequently presented in the DAS workshop 2009. The feedback from the forum and workshop was used to redefine the architecture and implement the system. A usability experiment was performed using potential users of the system emulating a real annotation task. It demonstrated that DAS writeback is effective, usable and will provide the appropriate environment for the creation and evolution of a protein annotation community. Although the scope of this research is limited to protein annotations, the specification was defined in a general way. It can, therefore, be used for other types of information supported by DAS, implying that the server is versatile enough to be used in other scenarios without major modifications.

Among the soil-transmitted helminths (STH), hookworms are a worldwide problem in both humans and animals. They cause non-specific gastrointestinal symptoms, and in young children and animals, they can cause stunting, malnutrition and anemia. Canine hookworms have significant zoonotic potential as a cause of cutaneous larvae migrans and eosinophilic enteritis in humans. To determine the ecological niche of human hookworm in Brazil, two risk models were developed based on the Growing Degree Day-Water Budget (GDD-WB) concept, one based on accumulation of monthly temperatures above a base temperature of 15oC and threshold WB value >0.4. The second was based on a gradient index of the product of monthly accumulated GDD and WB values. It was determined that both environmental temperature and moisture are important in the distribution of hookworm. This study supports the validity of the GDD-WB concept for mapping risk of hookworm at a national scale. A cross-sectional survey was conducted in human outpatients in Mutuípe municipality, Brazil, to determine prevalence of soil-transmitted helminths, including hookworm. Mutuípe falls within the permissive zone for the transmission of hookworm. A sucrose double centrifugation flotation technique was used for the concentration of helminth eggs in fecal samples. Hookworm infection was the most prevalent of the STH and the prevalence was highest in adults and males. PCR was then used to determine the species of hookworm present. Necator americanus was confirmed by PCR as the predominant hookworm species. A single case of Ancylostoma ceylanicum was identified. A study on the prevalence of hookworms and other gastrointestinal parasites in shelter dogs in south Louisiana and the anthelminthic protocols used in the shelters was conducted. Fecal samples examined by direct smear, flotation and sedimentation methods revealed that hookworm had the highest prevalence (53.6%) followed by Trichuris vulpis (28.7), Cystoisospora ohioensis (17.2%), Giardia duodenalis (12.0%), C. canis (7.7), Toxocara canis (6.2%), Dipylidium caninum, Alaria spp and Capillaria sp A PCR-RFLP developed to differentiate A. caninum and A. braziliense revealed A. caninum as the only species found. Evaluation of the anthelminthic protocols used in nine shelters showed current methods were inadequate for control of hookworms in shelter dogs.

Leishmaniasis is a public health problem in Brazil, and despite the control programs in place, Bahia remains one of the states with the highest incidence rates of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). This study proposed to develop and evaluate the applicability of ecological niche models (ENM) for leishmaniasis, to determine the influence of environmental and socioeconomic factors on the incidence of these diseases at three geographic scales: national, statewide and community. Water availability, garbage collection, precipitation and temperature were the most important variables in predicting areas suitable for VL and CL in the country. The extensive geography of Brazil and the coarse scale of the data used to evaluate both socioeconomic and environmental variables revealed the need of a more refined scale to define the role of these factors in risk area identification. At the statewide scale, the models were developed for Bahia state and data on vector occurrence was added to the analysis. Three environment structural indices were evaluated in addition to the environmental variables explored in the national model. Water content of vegetation was a very strong predictor of CL and VL incidence followed by NDVI. The sand fly species found in Bahia were sensitive to variations in temperature and rainfall related variables. The occurrence of Lutzomyia longipalpis, the vector of VL in the state, was most influenced by precipitation and vegetation. The district of Monte Gordo, in Bahia, was selected for development of a community level ENM using high resolution WorldView-2 imagery. CDC light traps were used to collect sand flies for a period of three months. Sand flies were tested by polymerase chain reaction (PCR) to determine host feeding preferences and natural infection by Leishmania spp. The sand flies preferably fed on chickens and humans. No natural Leishmania spp. infections were detected. NDVI was the most influencing factor in the ENM model (99.4% contribution). Implementation of a multi-scale geospatial surveillance and risk modeling capability to monitor disease incidence and their vectors, with the addition of molecular analysis, into the actions of the control program can help reduce the impact of endemic leishmaniasis in Bahia.

Severe bacterial sepsis leads to a pro-inflammatory condition that can manifest as septic shock, multiple organ failure, and death. Neutrophils are critical for the rapid elimination of bacteria, however, the role of neutrophil chemoattractant CXCL1, pattern recognition receptors (PRRs)- NLR protein 3 (NLRP3) and alcohol in bacterial clearance during sepsis remains elusive. We demonstrate that CXCL1 plays a pivotal role in mediating host defense to polymicrobial sepsis following cecal ligation and puncture (CLP) in gene-deficient mice. CXCL1 appears to be essential for restricting bacterial outgrowth and preventing multiple organ failure and death in mice. Moreover, CXCL1 is essential for neutrophil migration, expression of pro-inflammatory mediators, Recombinant interleukin 17 (IL-17) rescued impaired host defenses in cxcl1−/− mice. CXCL1 is important for IL-17A production via Th17 differentiation. CXCL1 is essential for reactive oxygen species production and neutrophil extracellular trap (NET) formation. This study reveals a novel role for CXCL1 in neutrophil recruitment via modulating T cell function and neutrophil-related bactericidal functions. These studies suggest that modulation of CXCL1 levels could reduce bacterial burden and excessive inflammatory injury in sepsis. NLRP3-/- mice or mice treated with NLRP3 inhibitor were protected in response to polymicrobial sepsis. NLRP3-/- mice showed reduced bacterial burden and production of proinflammatory cytokines. Intriguingly, neutrophils obtained from NLRP3-/- or NLRP3-inhibited mice display impaired critical functions of neutrophils, including phagocytosis, bacterial killing, NET formation, autophagy, chemotaxis, and cell death. These unique and novel findings position NLRP3 as a critical linker between neutrophil function and bacterial clearance, highlighting NLRP3 as a therapeutic target to control infection in polymicrobial sepsis. Alcoholics are more susceptible to bacterial sepsis and thus have higher mortality rate as compared to non-alcoholics. In this study, acute alcohol intoxication prior to the induction of polymicrobial sepsis show reduced NETosis. Diminished NETosis was consistent with attenuated ROS production and bacterial clearance in alcohol-challenged CLP-induced mice. Our findings demonstrate that alcohol-suppressed NETosis and NET-mediated extracellular killing of bacteria contribute to the pathogenesis of polymicrobial sepsis, and thus, furthers our understanding on alcohol-induced immune defect during bacterial infection.

The outer surface lipoprotein (osp) layer forms an interface between the internal and the external environment of the Lyme disease spirochete, Borrelia burgdorferi. The homeostatic maintenance of the osp layer effectuates adaptation of B. burgdorferi as it gets transmitted from the tick vector to a mammalian host and vice-versa. However, the regulation of the outer surface lipoproteins (osps) is still a conundrum for borrelia scientists. Part of this dissertation inquires about the homeostatic maintenance of the osp layer. We found that the deletion of the dominantly expressed tick phase osp, OspA, induces expression of two other osps. OspD, and BBJ41. Also, increased expression of OspC was seen in borrelia mutants lacking OspA, OspD, and BBJ41. These results suggest constant osp layer maintenance, irrespective of the presence or the absence of the dominant Osps, like OspA and OspC. Furthermore, our conclusive electron microscopic study demonstrates that the overall density of the osp layer remains identical in wild type and mutant B. burgdorferi, lacking either several osps or the dominantly expressed OspA. OspA is abundantly expressed on the borrelial surface as it persists in an unfed tick. A blood meal causes rapid downregulation of OspA as B.burgdorferi prepares to infect the mammalian host. The downregulation of OspA is speculated to be regulated by an unknown repressor protein. The remaining part of this dissertation pertains to the investigation of this unknown repressor protein for ospA. The borrelia oxidative stress regulator protein, BosR, has been attributed with an indirect role in OspA downregulation. However, due to its homolgy with a family of transcriptional repressors, BosR is more likely to cause direct repression of OspA. Therefore, we investigated the direct interaction of BosR and the ospA regulatory region. The DNA binding experiments demonstrated that borrelia oxidative stress regulator, BosR, binds directly to the cisI and cisII regulatory regions of ospA promoter. Thus, conclusively, BosR acts as a repressor protein which causes OspA downregulation in B. burgdorferi.

Dengue virus (DENV) is maintained in a primarily anthroponotic cycle between humans and the mosquito, Aedes aegypti. Investigations into DENV infection of the vertebrate host generally do not account for the contribution of vector saliva, an inherent part of the mosquito-borne viral inoculum. Feeding by mosquitoes on vertebrate hosts is initiated by probing, which results in physical damage to the skin and vasculature, and the simultaneous introduction of DENV and saliva into the skin. Saliva contains many individual proteins with the potential to modulate host hemostasis and immune responses, thereby facilitating blood feeding and virus transmission. As exogenous antigens, both DENV and these salivary proteins encounter the vertebrate host immune system and consequently could have an effect on the immunological environment and response of the bite site during viral establishment, as well as the ensuing viremia. My overarching hypothesis is that mosquito saliva aids in the establishment of DENV infections within the vertebrate, and that distinct immunological alterations involved in this enhancement will be attributable to individual salivary proteins. I therefore conducted investigations into the triad of vector-virus-vertebrate interactions aimed at further characterizing 1) the strain-based impact of DENV infection on salivary protein transcript expression in Ae. aegypti; 2) the probing-based modulation of vertebrate immune responses during DENV infection in the skin of a murine model of transmission; 3) the effect of individual salivary proteins on DENV production in a human hematopoietic cell line; and 4) the influence of the salivary protein aegyptin on DENV infection in the mouse; with emphasis on early, establishment-relevant time points and differences in infection kinetics with the potential to alter transmission success.

Objective: To determine the accuracy, measured by error rate, with which Clinical Research Workers (CRWs), with minimal experience in data entry, could capture data on a feature phone during an interview using two different mobile phone applications, compared to the accuracy with which they could record data on paper Case Report Forms (CRFs). Design: A comparative study was performed where 10 participating CRWs performed 90 mock interviews using either paper CRFs or one of two mobile phone applications. The phone applications were a commonly used open source application and an application custom built for this study that followed a simplified, less flexible user interface paradigm. The answers to the interview questions were randomly generated and provided to the interviewees in sealed envelopes prior to the scheduling of the mock interview. Error rates of the captured data were calculated relative to the randomly generated expected answers. Results and Conclusion: The study aimed to show that error rates of clinical research data captured using a mobile phone application would not be inferior to data recorded on paper CRFs. For the custom application, this desired result was not found unequivocally. An error in judgment when designing the custom phone application resulted in dates being captured in a manner unfamiliar to the study participants, leading to high error rates for this type of data. If this error is condoned by excluding the date type from the results for the custom application, the custom application is shown to be non-inferior, at the 95% confidence level, to standard paper forms when capturing data for clinical research. Analysis of the results for the open source application showed that using this application for data capture was inferior to paper CRFs. Secondary analysis showed that error rates for data captured on the custom mobile phone application by non-computer literate users were significantly lower at the 95% confidence level than the error rates for data recorded by the same users on paper and for data captured by computer literate users using the custom application. This result confirms that even non-computer literate users can capture data accurately using a feature phone with a simplified user interface.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous neurotropic alphaherpesvirus transmitted by contact with mucocutaneous surfaces of infected individuals. HSV-1 enters the host by fusion of the viral envelope with the host cell plasma membrane, followed by translocation of the viral capsids to the nucleus where viral DNA is injected into the host cell nucleus to initiate viral replication. To generate infectious virions, newly assembled capsids travel to the cytoplasm and undergo a process called secondary envelopment by budding into cytoplasmic vesicles derived from the trans-Golgi network. Cytoplasmic envelopment is a complex process involving interactions between a multitude of viral membrane and tegument proteins. To investigate the relative importance of a subset of viral membrane and tegument proteins in secondary envelopment, a number of recombinant viruses were constructed in the HSV-1(F) genetic background. A mutant virus unable to express gE, gM and the C-terminus of gD was characterized and compared to additional mutants unable to express both gE and gM or gE and the C-terminus of gD and to mutants lacking expression of just one of these glycoproteins, in addition to mutants lacking expression of both pUL11 and gM, and pUL20 alone. Characterization of all mutant viruses by plaque morphology, viral replication kinetics, electron microscopy and particle-to-PFU ratios revealed a hierarchy of defects in cytoplasmic envelopment and infectious virus production, with deletion of pUL20 having the greatest effect, followed by the deletion of pUL11 and gM. Characterization of additional mutants containing multiple mutations revealed that gE, gM and gD do not function in a redundant manner in cytoplasmic envelopment supporting a preeminent role for the pUL20/gK protein complex in cytoplasmic envelopment and egress. An epitope tag insertion adjacent to the pUL37 Y480 (DC480) exhibited a severe defect in cytoplasmic envelopment similar to gK and pUL20-null viruses. Importantly, this mutant virus was partially complemented when grown on cells expressing pUL20, suggesting an interaction with the pUL20/gK protein complex. This pUL37 interaction with pUL20/gK was verified by co-immunoprecipitation and proximity ligation assays suggesting that it facilitates cytoplasmic virion envelopment.

The immune response to the self-cure phenomenon seen during gastrointestinal nematode (GIN) parasitism of small ruminants was compared between sheep breeds that are resistant or susceptible to Haemonchus contortus infection. Fifty-four Gulf Coast Native (resistant) and Suffolk (susceptible) lambs were allowed to acquire a natural GIN infection on pasture and were then randomly allocated into 4 groups. After being moved to parasite free housing for 2 months, lambs were given a challenge infection of 20,000 H. contortus L3. Fecal egg counts (FEC) were monitored throughout the study and animals were necropsied at 0, 1, 3, and 7 days post infection (DPI). FEC decreased beginning at 3 DPI in both breeds, with Native lambs having a higher percent reduction in FEC at 3 and 7 DPI compared to Suffolk lambs. Both Native and Suffolk lambs were able to expel their existing adult population. However, while Native lambs also successfully cleared the larval challenge, Suffolk lambs did not. The numbers of eosinophils within the abomasal mucosa reflect the magnitude and timing of fecal egg count reductions in both breeds. Additionally, the elevated levels of eosinophils within the abomasal mucosa of Native lambs at 3 DPI are likely to be involved with the clearance of the larval burden via eosinophil mediated larval killing. Suffolk lambs displayed a delayed cellular response that resulted in larvae to entering the mucosa before sufficient eosinophilic response could be established. Elevated mast cells within the abomasal mucosa coincide with the clearance of adult GIN and along with elevated levels of IL-13 seen in both breeds suggest their involvement. The results confirm that self-cure is an immune mediated response that can occur in both resistant and susceptible breeds of sheep while differences in the magnitude and time course of immune responses may prevent susceptible sheep from fully clearing the infection.