Nearly 60 years have passed since the discovery of Yellow Fever (YF) in East Africa but the disease has remained enigmatic because of unpredictable focal periodicity, lengthy inter-epidemic periods, and a precarious potential for large epidemics. Endemic and/or enzootic transmission has been poorly documented and, as such, typically remains of little value in predicting future outbreaks. The following studies were aimed at clarifying the ecology and epidemiology of YF in East Africa and detailing a number of important interactions associated with the potential emergence and transmission of YF. Three aims were included in this investigation: (1) a study of the distribution of YF among wild monkeys in Kenya, (2) spatiotemporal descriptions of YF vectors along forest interfaces, and (3) an examination of the efficiency of domestic vectors to transmit a local YF virus genotype. Overall, the study indicates that enzootic YF activity has been limited focally and in duration within Kenya, YF vector-human interactions are relatively limited in domestic areas as compared to the forests studied, and the most important vector of epidemic YF is poorly competent. These results have important implications in regard to the emergence and epidemiological outcomes of YF. The approach taken during these studies was to highlight the dynamics of important eco-epidemiological interactions and to provide practical comparative measures. Ultimately, revealing the underlying drivers and dynamics of YF emergence, as evidenced by causal changes, will require quantifiable measure of these rare events or strongly suggestive evidence from inter-epidemic and epidemic comparisons / acase@tulane.edu

The recombinant retrovirus, MoFe2, was constructed by replacing the U3 region of Moloney murine leukemia virus (MoMuLV) with homologous sequences from the feline leukemia virus (FeLV-945) LTR. Like other gammaretroviruses, MoMuLV and FeLV induce lymphoma in the natural host through insertional activation of host oncogenes near proviral integrations sites. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. Southern blot analysis demonstrated that the MoFe2 recombinant did not integrate near the host oncogenes commonly involved in disease induction by either parent virus; thus, MoFe2 represents an opportunity to identify novel oncogenes in the induction of T-cell lymphoma. Common insertion sites (CISs) in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parental virus in wild type animals. Two of the newly identified CISs, 3-19 and Rw1, had not been previously implicated in lymphoma in any retroviral model. The exact function of Rw1 remains unknown and little information is available about the regulation of its expression. The precise location and transcriptional direction of proviral insertions in Rw1 were determined, but proviral insertion demonstrates no clear impact on the level of Rw1 expression in end-stage lymphomas. The results further indicate that Rw1 expression is highly regulated, restricted to a narrow spectrum of cell types, and may be responsive to retroviral infection. The other novel CIS, designated 3-19, encodes the regulatory subunit of phosphatidylinositol 3-kinase gamma (P13Kgamma) termed p101. P13Kgamma is known to be important in a number of cellular functions including chemotaxis, oxidative burst and regulation of T-cell proliferation. P13Kgamma activity is regulated through interactions with G protein-coupled receptors. The fact that p101 is a CIS suggests that its dysregulated expression is associated with T-cell malignancy. Tumors that contained insertions in 3-19 had elevated expression of one or more of the detected transcripts. Overexpression studies revealed that p101 has a positive effect on PI3K by activating Akt, likely by enhancing the activity of p110gamma and sensitizing it to activation. Although overexpression of p101 activates the Akt pathway and does protect cells from DNA damage-induced apoptosis it was also found to induce apoptosis. These findings indicate that substitution of FeLV-945 U3 sequences into the M-MuLV LTR significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma and supports a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. These findings demonstrate the utility of the recombinant retrovirus MoFe2-MuLV to in the identification of novel genes implicated in the induction of lymphoma / acase@tulane.edu

The Epstein-Barr virus (EBV) is a ubiquitous gamma herpes virus for which an estimated 90% of the human population is seropositive by adulthood. EBV has been implicated in a number of lymphocyte and epithelial malignancies and is considered the causative agent in anaplastic nasopharyngeal carcinoma. Evidence of EBV has been detected in the lungs of Idiopathic Pulmonary Fibrosis (IPF) patients at a higher rate than control and detection of the EBV protein Latent Membrane Protein 1 (LMP1) suggests a poorer prognosis which raises the questions whether, and how, EBV is involved in the pathogenesis of IPF. This study proposes a possible mechanism of progression of IPF through repeated alveolar epithelial insult followed by aberrant wound healing and decline in function involving synergy between LMP1 and the profibrotic cytokine TGF-beta1. LMP1 was shown to synergistically increase expression of the pro-fibrotic markers fibronectin EDA and matrix metalloproteinase 9 while additively increasing levels of PAI-1. Additionally, co-treatment with LMP1 and TGF-beta1 promote the loss of the epithelial markers E-Cadherin, zona occluding 1, surfactant protein C, and the gain of mesenchymal markers N-Cadherin, vimentin and alpha smooth muscle actin, resulting in epithelial to mesenchymal transition (EMT). Inhibition of the ERK pathway protected from LMP1/TGF-beta1 induced EMT while inhibition of the NF-kappaB pathway protected from the LMP1 contribution without affecting TGF-beta1 signaling. LMP1 reduced levels of the cytoplasmic isoform of the promyelocytic leukemia protein (PML) and disrupted the canonical TGF-beta1 signaling pathway LMP1 increased the levels of nuclear PML and intensity of the PML nuclear bodies (PML NB). Considered as part of the interferon response to viral infection, PML NB are dynamic multifunctional proteinaceous nuclear domains that affect processes as diverse as transcription, apoptosis and telomere regulation. Additionally, herpes viruses disrupt PML NB during lytic reactivation and utilize the remnants of these domains as replication compartments. Disruption of PML NB by arsenic trioxide (ATO) induced lytic reactivation of EBV in alveolar epithelial and nasopharyngeal carcinoma cells. Additionally, treatment with ATO conferred susceptibility to the antiherpesviral ganciclovir. To our knowledge, this is the first report of upregulation of PML NB by a viral protein to maintain latency / acase@tulane.edu

Our immune system protects us against viral invasion. Nevertheless, variability helps viruses to escape from immune suppression. Viruses may vary either by existing in multiple subtype forms or by mutating at a non-negligible rate. Effective design of vaccines for viral diseases requires some estimation of the escape mechanisms as well as the variability of the virus. However, prediction of viral escape is non-trivial due to the complexity of the virus system. I propose to use stochastic simulation to model viral diseases at the sequence-level. I focus on dengue virus and avian influenza. Dengue virus, having four closely related serotypes, represents a prime example of where vaccine development has been delayed because of incomplete understanding of the immune response to multiple components. Here I extend the generalized NK model theory to study dengue virus dynamics. I elucidate the mechanism for two puzzling phenomena: original antigenic sin and immunodominance. I also suggest new polytopic vaccination strategies to protect against the four serotypes of dengue virus. Previously generalized NK model was applied to B cell immunity. I extend the model to apply to T cell immunity, which is important in the control of dengue virus. Influenza is a highly mutating virus. Influenza pandemic occurs periodically. The H5N1 bird virus is regarded as one candidate pandemic-causing strain. Various countries around the world have started to create stockpiles of H5N1 avian influenza vaccines. However, since the avian influenza is mutating and multiple virus introduction is a possibility, how many and which strains should be stockpiled? I analyze the influenza database and simulate the avian influenza virus evolution to obtain optimized strains combinations. I also suggest population at risk (PaR) as a metric for infectious disease risk management. For both dengue and influenza, simulation results can not only help us to assess new vaccines and provide strategies for specific diseases, but also provide us with insights to other diseases, such as HIV and cancer.

Thesis (Ph.D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-08, Section: B, page: 4235. Adviser: Joseph M. McCune.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3783. Adviser: Cheng-Ming Chiang. Includes bibliographical references (leaves 211-253). Available on microfilm from Pro Quest Information and Learning.

HCV is a positive stranded RNA virus that infects up to 170 million people worldwide. Chronic infection is established in 80% of the cases and may lead to cirrhosis and hepatocellular carcinomas. A major debate regarding chronic HCV pathogenesis is whether liver damage is host or virus mediated. CD4+ and CD8+ immune responses are needed to control infection but these are often inefficient, resulting in chronic infection. The presence of these responses in chronic infection may contribute to liver pathogenesis. To study the relevance of immune-mediated liver damage, we created an immunization regimen which elicited humoral and cellular immune responses in B6C3F1 mice. HCV-primed donor splenocytes were stained with CFSE and transferred to a CE1E2 HCV Tg mouse model. Donor cells were recovered in various organs eight days following the transfer. HCV-primed CD4+ and CD8+ T cells selectively accumulated in the liver of the Tg mice where the CE1E2 transgene is predominantly expressed and caused liver pathology under the form of lymphocyte infiltration. The accumulation of these cells indicates a homing mechanism to this organ and a role in the development of liver pathologies. Further studies are required to unearth the mechanisms behind cellular trafficking and cell-mediated immune damage.

Noroviruses (NoV) and Hepatitis A viruses (HAV) are the most commonly implicated viruses in foodborne disease. Their transmission is mainly via the fecal-oral route and distribution of contaminated foods has lead to large outbreaks. Thus, it is crucial that contaminated foods be identified promptly. Detection by cell culture is not possible for these viruses so that researchers rely on the reverse transcription polymerase chain reaction (RT-PCR). Prior to detection, however, viruses must be isolated from foods and, as of yet, no one method has been found applicable to a large variety of food matrices. Problems with existing methods include, but are not limited to, the co-extraction of inhibitory molecules and lack of sensitivity. In addition, they are labour-intensive and time consuming. Therefore, the aims of this research project were to develop a novel methodology for the rapid and sensitive isolation and detection of NoV and HAV from select food matrices. In the first part of these studies, TRIzolRTM reagent and the RT-PCR were used to isolate and detect feline calicivirus (FCV) (a norovirus surrogate) from select artificially-inoculated foods. However, the co-extraction of PCR inhibitors resulted in false-negative results. RNA purification methods were then compared prior to detection and Oligo d (T) beads provided for the best sensitivity. However, virus capture was not sufficiently sensitive to be applied to outbreak situations. In the second part of this thesis, the Pathatrix(TM) system was applied to the isolation of HAV and NoV from a variety of foods with the use of positively-charged (cationic) magnetic beads. The use of the Pathatrix(TM) machinery has led to success in detecting a wide range of bacteria from various food matrices, and we were the first to report on its use for the concentration of viruses. When combined with the RT-PCR for detection, the Pathatrix(TM) system was able to detect HAV from inoculated foods at levels typically found in outbreak situations. Though results were not as sensitive with NoV-inoculated foods, the methodology was successful at detecting NoV in outbreak food samples. Furthermore, the cationic beads used in the Pathatrix(TM) system were able to simultaneously isolate two contaminating viruses from food samples. Therefore, the Pathatrix(TM) methodology reported herein is a very sensitive and rapid method that holds promise for the isolation and detection of HAV and NoV in outbreak situations.

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase. / Respiratory Syncytial Virus (RSV) is a RNA virus with negative polarity. RSV infections are the most common cause of hospitalization among infants. Among populations at risk, infection of RSV can be quite severe. RSV infections can cause bronchiolitis, pneumonia, while severe infections are linked to the development of asthma. Early in the infectious cycle of RSV, the cytosolic sensor RIG-I captures viral particles, and activates the immune response by engaging the transcription factors IRF-3 and NF-κB. At the heart of RSV mediated pathologies is a skewed immune response. More precisely, RSV over stimulates the release of proinflammatory chemokines and cytokines. Intriguingly, while RSV is able to stimulate the production of proinflammatory cytokines and chemokines, RSV under stimulates the antiviral response. The ability of RSV to evade the antiviral response is thought to be mediated by its non-structural proteins: NS1 and NS2. However, the mechanism by which NS1 and NS2 enable RSV to evade the antiviral response remains to be determined. In this memoir we investigated, how RSV is recognized by the innate immune response in airway epithelial cells. With this information we hope to improve our understanding of how NS1 and NS2 allow RSV to circumvent the antiviral response. We show for the first time that cytosolic sensor MDA5 plays a role in the recognition of RSV particles. Using a combination of interfering RNA directed against RIG-I, and transfection of MDA5, we show that MDA5 does not contribute to the phosphorylation of IRF-3. According to the data presented, we suggest that MDA5’s role in the immune response is to prevent the degradation of IRF-3. Contrary to previous research, we show that the inhibition of the nonstructural protein does not increase the production of the antiviral cytokine IFN-β. However, the ectopic expression of NS1 and NS2 does lead to a reduction of the promoter activity of IFN-β and the antiviral protein ISG56 when measured by luciferase assay. This research highlights the importance of MDA5 as a potential therapeutic target in the development of a cure for RSV.

La prolifération, la différenciation ainsi que les fonctions des cellules du système immunitaire sont contrôlées en partie par les cytokines. Lors de l’infection par le VIH-1, les défauts observés dans les fonctions, la maintenance, ainsi que la consistance des cellules du système immunitaire sont en large partie attribués à une production altérée des cytokines et à un manque d’efficacité au niveau de leurs effets biologiques. Durant ces études, nous nous sommes intéréssés à la régulation et aux fonctions de deux cytokines qui sont l’IL-18 et l’IL-21. Nous avons observé une corrélation inversée significative entre les concentrations sériques d’IL-18 et le nombre des cellules NK chez les patients infectés par le VIH-1. Nos expériences in vitro ont démontré que cette cytokine induit l’apoptose des cellules NK primaires et que cette mort peut être inhibée par des anticorps neutralisants spécifiques pour FasL et TNF-α. Cette mort cellulaire est due à l’expression de FasL sur les cellules NK et à la production de TNF-α par ces cellules. L’IL-18 augmente aussi la susceptibilité à la mort des cellules NK par un stimulus pro-apoptotique, en diminuant l’expression de la protéine anti-apoptotique Bcl-XL. Nous démontrons aussi que, contrairement à l’IL-18, les niveaux d’IL-18BP sont plus faibles dans les sérum de patients infectés. Ceci résulte sur une production non coordonnée de ces deux facteurs, aboutissant à des niveaux élevés d’IL-18 libre et biologiquement active chez les patients infectés. L’infection de macrophages in vitro induit la production d’IL-18 et réduit celle d’IL-18BP. De plus, l’IL-10 et le TGF-β, dont les concentrations sont élevées chez les patients infectés, réduisent la production d’IL-18BP par les macrophages in vitro. Finalement, nous démontrons que l’IL-18 augmente la réplication du VIH-1 dans les lymphocytes T CD4+ infectés. Les niveaux élevés d’IL-18 libres et biologiquement actives chez les patients infectés contribuent donc à l’immuno-pathogénèse induite par le VIH-1 en perturbant l’homéostasie des cellules NK ainsi qu’en augmentant la réplication du virus chez les patients. Ces études suggèrent donc la neutralisation des effets néfastes de l’IL-18 en utilisant son inhibiteur naturel soit de l’IL-18BP exogène. Ceci permettrait de moduler l’activité de l’IL-18 in vivo à des niveaux souhaitables. L’IL-21 joue un rôle clef dans le contrôle des infections virales chroniques. Lors de ces études, nous avons déterminé la dynamique de la production d’IL-21 lors de l’infection par le VIH-1 et sa conséquence sur la survie des cellules T CD4+ et la fréquence des cellules T CD8+ spécifiques au VIH-1. Nous avons démontré que sa production est compromise tôt au cours de l’infection et que les concentrations d’IL-21 corrèlent avec le compte de cellules T CD4+ chez les personnes infectées. Nos études ont démontré que le traitement antirétroviral restaure partiellement la production d’IL-21. De plus, l’infection par le VIH-1 de cellules T CD4+ humaines inhibe sa production en réduisant l’expression du facteur de transcription c-Maf. Nous avons aussi démontré que la fréquence des cellules T CD4+ spécifiques au VIH-1 qui produisent de l’IL-21 est réduite chez les patients virémiques. Selon nos résultats, l’IL-21 empêche l’apoptose spontanée des cellules T CD4+ de patients infectés et l’absence d’IL-21 réduit la fréquence des cellules T CD8+ spécifiques au VIH-1 chez ces patients. Nos résultats démontrent que l'IL-21R est exprimé de façon égale sur tous les sous-types de cellules NK chez les donneurs sains et chez les patients infectés. L’IL-21 active les protéines STAT-3, MAPK et Akt afin d'augmenter les fonctions effectrices des cellules NK. L'activation de STAT-3 joue un rôle clef dans ces fonctions avec ou sans un traitement avec de l'IL-21. L'IL-21 augmente l'expression des protéines anti-apoptotiques Bcl-2 et Bcl-XL, augmente la viabilité des cellules NK, mais ne possède aucun effet sur leur prolifération. Nous démontrons de plus que l'IL-21 augmente l'ADCC, les fonctions sécrétrices et cytotoxiques ainsi que la viabilité des cellules NK provenant de patients chroniquement infectés par le VIH-1. De plus, cette cytokine semble présenter ces effets sans augmenter en contrepartie la réplication du VIH-1. Elle permet donc d'inhiber la réplication virale lors de co-cultures autologues de cellules NK avec des cellules T CD4+ infectées d'une manière dépendante à l'expression de perforine et à l'utilisation de la protéine LFA-1. Les niveaux d’IL-21 pourraient donc servir de marqueurs biologiques pour accompagner les informations sur le taux de cellules T CD4+ circulantes en nous donnant des informations sur l’état de fonctionnalité de ce compartiment cellulaire. De plus, ces résultats suggèrent l’utilisation de cette cytokine en tant qu’agent immunothérapeutique pour restaurer les niveaux normaux d’IL-21 et augmenter la réponse antivirale chez les patients infectés par le VIH-1. / The proliferation, differentiation, function and maintenance of immune cells is controlled in large part by cytokines. HIV-induced dysfunctions of the antiviral immunity is in part related to defects in the cytokine network, as manifested by altered cytokine secretion and responsiveness to these cytokines. In these studies, we investigated the regulation and the functions of two cytokines, IL-18 and IL-21, during HIV-1 infection. In our studies, we observed an inverse correlation between IL-18 concentrations and absolute numbers of various subsets of NK cells in infected persons. IL-18 caused increased death of a human NK cell line, as well as of primary human NK cells in vitro. The IL-18-mediated cell death was dependent upon Fas-FasL interactions and TNF- secretion. IL-18 induced the expression of TNF-, induced the expression of FasL on NK cells, increased the transcription from the human FasL promoter, reduced the expression of Bcl-XL in NK cells, and increased their sensitivity to FasL-mediated cell death. In contrast to IL-18 levels, IL-18BP levels decreased in the serum of HIV-infected patients. This decrease resulted in enhanced levels of free IL-18 in the serum of such patients. The infection increased production of IL-18 but decreased that of IL-18BP in monocyte-derived macrophages (MDM). Furthermore, IL-10 and TGF-β, two cytokines for which concentrations are increased in HIV-infected persons, also decreased production of IL-18BP by human MDM. Finally, recombinant human IL-18 enhanced HIV-1 replication in human CD4+ T cells. The uncoordinated production of these two cytokines represents an imbalance between these two soluble factors in HIV-infected patients. Our study shows that enhanced IL-18 bioactivity in HIV-infected patients may contribute to the pathogenesis of AIDS by disrupting NK cell homoeostasis and increasing viral replication. This uncoordinated production of IL-18 and IL-18BP contribute to IL-18-induced immunopathology and pathogenesis in HIV-infected AIDS patients. Therefore, these studies suggest that the neutralization of IL-18 may represent an appropriate and useful immunotherapeutic strategy in these patients. It may delay AIDS progression and improve the immune status of infected persons. The best way to achieve this goal may be using exogenous interleukin-18 binding protein. IL-21 is a relatively newly discovered immune enhancing cytokine, which plays an essential role in controlling chronic viral infections. Therefore, we sought to determine the dynamics of the cytokine production and its potential consequences on the viability of CD4+ T cells in HIV-infected persons. We show here that the cytokine production is compromised early in the course of the infection. The serum cytokine concentrations correlated with CD4+ T cell counts in the infected persons. Among different groups of HIV-infected persons, only Elite Controllers maintain normal production of the cytokine. The HAART partially restores production of this cytokine. Interestingly, HIV-1 infection of human PBMC as well as of purified CD4+ T cells inhibits the production of the cytokine by decreasing the expression of c-Maf, a transcription factor involved in the activation of the cytokine gene, in the virus-infected cells but not in uninfected bystander cells. We also show that the frequencies of IL-21 producing HIV-specific antigen experienced CD4+ T cells are decreased in HIV-infected viremic patients. Furthermore, we show that recombinant human IL-21 acts as pro-survival factor and prevents enhanced spontaneous apoptosis of ex vivo cultured CD4+ T cells from HIV-infected patients and that increased serum levels of the cytokine are associated with higher frequencies as well as with better functions of HIV-specific CTL in HIV-infected individuals. We show that the cytokine receptors are expressed equally on all NK cell subsets. We demonstrate that the cytokine activates STAT-3, MAPK and Akt to enhance NK cell functions. IL-21 increases expression of anti-apoptotic proteins Bcl-2 and Bcl-XL, and enhances viability of NK cells, but has no effect on their proliferation. We further show that the cytokine enhances HIV-specific ADCC, secretory and cytotoxic functions as well as viability of NK cells from HIV-infected persons. Furthermore, it exerts its biological effects on NK cells with minimal enhancement of HIV-1 replication, and the cytokine-activated NK cells inhibit viral replication in co-cultured HIV-infected autologous CD4+ T cells in a perforin- and LFA-1-dependent manner. These studies suggest that serum IL-21 concentrations may serve as useful biomarker to accompany CD4+ T cell counts for monitoring HIV-1 disease progression and the fitness of the antiviral immunity. Furthermore, the cytokine may be considered for immunotherapy in HIV-infected patients in order to restore the physiological levels of the cytokine and promote their antiviral immunity.