Although avian influenza virus (AIV) infections in domestic poultry are uncommon, transmission of avian influenza from wild waterfowl reservoirs does occur. Depopulation of the infected flock is the typical response to AIV outbreaks in domestic chicken production, causing a loss in profits and accumulation of unexpected expenses. Because it is impossible to know which of many virus subtypes will cause an outbreak, it is not feasible for the U.S. to stockpile vaccines against all possible avian influenza threats. Currently, the U.S. does not routinely vaccinate chickens against influenza due to the inability to differentiate infected from vaccinated animals (DIVA), which would place limitations on its trade markets. A Sindbis virus vector expressing the PR8 influenza strain's M2e peptide was developed as a potential universal DIVA vaccine. M2e is a conserved peptide amongst influenza A viruses; M2e-specific antibodies induce antibody-dependent cytotoxicity or phagocytosis of infected cells, reducing production and shedding of AIV during infection. In this study, chickens were vaccinated at one-month-of-age with parental (E2S1) or recombinant Sindbis viruses expressing the PR8 M2e peptide (E2S1-M2e) by subcutaneous or intranasal routes at high (106 pfu) or low (103 pfu) dosages. Chickens were boosted at 2-weeks post-initial vaccination using the same virus, route, and dosage, then challenged with low pathogenic H5N3 AIV at 0.2 mL of 106/mL EID50 2-weeks post-boost. Serum samples were collected at 1-week and 2-weeks post-vaccination, 2-weeks post-boost, and 2-weeks post-challenge and screened for PR8 M2e-specific IgY antibody production by ELISA. Both high and low dose subcutaneously, as well as high dose intranasally vaccinated E2S1-M2e groups produced significantly higher levels of PR8 M2e-specific IgY antibodies as early as 1-week post-vaccination, while the uninoculated control and E2S1 groups remained negative for all pre-challenge time points. M2e-specific IgY antibodies capable of binding the challenge H5N3 M2e peptide were detected in groups with existing vaccine-induced M2e-specific antibodies pre-challenge, suggesting antibody M2e cross-reactivity. After challenge, all groups developed M2e-specific IgY antibodies and high HI titers, verifying successful AIV infection during challenge and production of hemagglutinin-specific antibodies. Viral shedding titers 4-days post-challenge were used to measure vaccine efficacy and were similar amongst all groups. Microneutralization assay results confirmed that post-boost serum samples, containing only M2e-specific antibodies, were unable to neutralize AIV in vitro. Although the E2S1-M2e vaccine was capable of producing high levels of M2e-specific IgY antibodies when inoculated subcutaneously, these antibodies were not able to reduce viral shedding and therefore did not protect chickens from AIV.

<p>Long-term plasticity, the long-lasting, activity-dependent change in synaptic efficacy, is a fundamental property of the nervous system. Presynaptic forms of long-term plasticity are widely expressed throughout the brain, having been described in regions such as the cortex, cerebellum, hippocampus, thalamus, amygdala and striatum. Presynaptic long-term potentiation (LTP) is associated with an increase in presynaptic release probability, but further evidence of the cellular basis for the change in release probability is not known. At the molecular level, presynaptic LTP is known to require protein kinase A, the synaptic vesicle protein, Rab3A, and the active zone protein, RIM1alpha. RIM1alpha, a presynaptic scaffold protein, binds to many molecules with known functions at different stages of the neurotransmitter release process and the synaptic vesicle cycle. Understanding which interactions of RIM1alpha mediate presynaptic LTP would shed light on the molecular and cellular mechanisms for presynaptic long-term plasticity.</p><p>Here I developed a novel platform to achieve robust acute genetic</p><p>manipulation of presynaptic proteins at hippocampal mossy fiber synapses, where presynaptic LTP is expressed. With this platform, I perform structure-function analysis of RIM1alpha in presynaptic LTP. I find that RIM1alpha phosphorylation by PKA at serine 413 is not required for mossy fiber LTP, nor does RIM1alpha-Rab3A interation. These findings suggest that RIM1alpha, Rab3A and PKA signaling, instead of functioning synergistically, may represent separate requirements for presynaptic long-term plasticity. I then tested whether Munc13-1, a priming protein, is an effector for RIM1alpha in presynaptic LTP and provide the first evidence for the involvement of Munc13-1 in presynaptic long-term synaptic plasticity. I further demonstrate that the interaction between RIM1alpha and Munc13-1 is required for this plasticity. These results further our understanding of the molecular mechanisms of presynaptic plasticity and suggest that modulation of vesicle priming may provide the cellular substrate for expression of LTP at mossy fiber synapses.</p> / Dissertation

Les arbovirus sont un groupe de virus transmis par des vecteurs arthropodes contaminant chaque année plusieurs centaines de millions de personnes à travers le monde. De nombreux facteurs environnementaux, comme les xénobiotiques peuvent influencer la propagation des infections virales, la susceptibilité de l'hôte et la sévérité de l'infection. Parmi eux, le cadmium, métal toxique est connu pour moduler la résistance de l'hôte lors des infections par les arbovirus. Ces travaux de thèse se sont portés sur l'étude de l'alphavirus Sindbis et du flavivirus Zika infectant des cellules épithéliales humaines. Les résultats de cette thèse ont permis de démontrer que les cellules A549 étaient permissives à la souche épidémique de 2013 du virus Zika. Le virus se réplique efficacement et induit une apoptose de type mitochondriale dans les cellules A549. La réponse immunitaire innée des cellules a été caractérisée et le rôle des interférons de type I a été mis en avant. Ces résultats peuvent contribuer à une meilleure compréhension des mécanismes de pathogénicité de ce virus chez l'homme. Par la suite, les effets d'une exposition au cadmium lors des infections par les virus Sindbis et Zika ont été évalués. L'infection par le virus Zika n'a pas été modulée par l'exposition au cadmium in vitro. Mais de façon surprenante dans le cas de l'infection des cellules HEK 293, le cadmium a protégé les cellules des effets cytopathiques induits par le virus Sindbis. En présence de cadmium, l'apoptose induite par le virus a été inhibée et la réplication du virus Sindbis a été réduite. / Arboviruses are a group of viruses transmitted by arthropod vectors infecting hundreds of millions of people each year worldwide. Many environmental factors including xenobiotics can influence the spread, the susceptibility and the outcome of viral infections. Among them, cadmium, a toxic metal is known to affect the host resistance against arboviral infection. In this thesis, our work has been focused on studying the infection capability of the Sindbis alphavirus and the Zika flavivirus in human epithelial cells. The results demonstrated that A549 cells was permissive to the 2013 epidemic strain of Zika virus. The virus replicates efficiently leading to mitochondrial apoptosis. The innate immune response was characterized and the crucial role of type I interferon was highlighted. These results may contribute to a better understanding of Zika virus pathogenesis in humans. Thereafter, the effects of cadmium exposure on Sindbis virus and Zika virus infection was evaluated. Zika virus infection was not modulated by cadmium in vitro. Interestingly, cadmium protected the HEK 293 cells from the cytopathic effect induced by Sindbis virus. Cadmium exposure inhibited the apoptosis and reduced the viral replication.

Doctor of Philosophy / Department of Division of Biology / Rollie J. Clem / Mosquitoes are carriers of a variety of harmful human pathogens, including viruses. In order to be successfully transmitted, a virus must evade mosquito immune responses. In this work, the innate immune role of apoptosis in mosquito-virus interactions was examined utilizing the disease vector Aedes aegypti and Sindbis virus. Ae. aegypti is the main vector for yellow fever and dengue virus, which result in over 100 million infections per year. Sindbis virus (Togaviridae) can be transmitted to vertebrates by Ae. aegypti in the laboratory. Sindbis is also well characterized molecularly, making it a good model system for understanding virus-vector interactions. Sindbis MRE-16 recombinant virus clones were utilized to express either an antiapoptotic or pro-apoptotic gene during virus replication. Mosquitoes were infected with recombinant virus clones during a blood meal or by intrathoracic injection. Midgut tissue and whole body samples were analyzed for virus infection and dissemination. Virus was also quantified in saliva and mosquito survival was assayed. Decreased infection in the midgut and delayed virus replication were observed in mosquitoes that were infected with virus expressing a pro-apoptotic gene. Infection with this virus clone also resulted in less virus in the saliva and reduced survival of infected mosquitoes. In addition, negative selection against pro-apoptotic gene expression during virus replication was observed. Collectively, these data suggest that apoptosis can serve as an antiviral defense in Ae. aegypti and may potentially be exploited to control virus transmission. An additional study included in this dissertation focused on zebrafish development and migration of somitic precursors from the tailbud. The tailbud consists of a population of stem cells at the posterior tip of the embryonic tail. The exit of these stem cells from the tailbud is required for the formation of tail somites. A novel double mutant was identified that lacked the t-box transcription factor spadetail and the BMP inhibitor chordin. Double mutants completely lacked somites and had an enlarged tailbud due to accumulation of stem cells that were unable to exit the tailbud. This study indicates the importance of BMP inhibition and spadetail expression in the proper exit of muscle precursors from the tailbud.

Mosquito species of the Culex genus are the enzootic vectors for several bird-associated viruses that cause disease in humans. In Europe, these viruses include Sindbis (SINV), West Nile and Usutu viruses. The morphologically similar females of Cx. torrentium and Cx. pipiens are potential vectors of these viruses, but difficulties in correctly identifying the mosquito species have caused confusion regarding their respective distribution, abundance, ecology, and consequently their importance as vectors. Species-specific knowledge from correctly identified field material is however of crucial importance since previous research shows that the relatively unknown Cx. torrentium is a far more efficient SINV vector than the widely recognized Cx. pipiens. The latter is involved in the transmission of several other viruses, but its potential importance for SINV transmission is debated. In this thesis I describe the development of a molecular method for species identification, based on reliably identified males of Cx. torrentium and Cx. pipiens. This identification method was then used in consecutive studies on the distribution and relative abundance of the two species in Sweden and 12 other European countries, SINV field infection rates in mosquitoes identified to species level, and evaluation of potential trap bias associated with common sampling techniques. The results showed that Cx. torrentium is a far more common species in Europe than previously assumed. In Sweden and Finland, it is the dominant species, accounting for 89% of the sampled Culex population. In central Europe, it is equally common to Cx. pipiens, while Cx. pipiens dominates south of the Alps Mountain range. Larvae of both species are often found together in both artificial containers (e.g. car tires) and natural sites. Also, a trapping bias against Cx. torrentium was revealed for CDC-traps. For the first time, SINV was isolated from species-identified Cx. torrentium and Cx. pipiens mosquitoes caught in the field, with Cx. torrentium being superior in infection rates (36/1,000 vs. 8.2/1,000). Future studies on SINV, as well as other mosquito-borne bird viruses in Europe, can hopefully gain from the baseline information provided here, and from principles of vector discrimination discussed in the thesis.

No description available.

Hepatitis C virus (HCV) is remarkable for its ability to establish persistent infection. Studies suggest that HCV core protein modulates immune responses to viral infection and can bind Fas receptor in vitro. To further examine the role of HCV core protein in Fas signaling, full-length (aa 1-192) and truncated (aa 1-152) HCV core proteins were expressed in Jurkat lymphocytes and cells were assayed for apoptotic response, caspase activation, and Fas activation. Jurkat expressing full-length but not truncated core protein exhibited ligand-independent apoptosis. Cytoplasmic targeting of truncated core protein recapitulated its ability to induce apoptosis. Activation of caspases 8 and 3 was necessary and sufficient for full-length core to induce apoptosis. Jurkat cells expressing full-length but not truncated core protein induced Fas receptor aggregation. HCV core activates apoptotic pathways in Jurkat via Fas and requires cytoplasmic localization of core. Infection of host lymphocytes by HCV may alter apoptotic signaling and skew host responses to acute infection.

Final publication is available at http://dx.doi.org/10.1111/ejn.12882 / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13040号 / 論医博第2115号 / 新制||医||1017(附属図書館) / 33032 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 影山 龍一郎, 教授 髙橋 良輔 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Insect Odorant-Binding Proteins (OBPs) are small, water-soluble molecules that solubilize hydrophobic odorant molecules in the sensillum lymph and transport them to their cognate receptors in the olfactory receptor neurons. With the availability of the genome sequence of the African malaria mosquito, Anopheles gambiae, there has been a profound interest in the characterization and functional analyses of Obp genes in order to understand the molecular basis of mosquito host-seeking behavior. However, no direct evidence has been found for specific functions of any mosquito OBPs. In this study, I describe the comparative genomics and expression analyses on two mosquito Obp genes (Obp1 and Obp7) as well as efforts to determine their functions. Both of these Obp genes were identified in Anopheles stephensi and only Obp7 gene was identified in Anopheles quadriannulatus by screening bacterial artificial chromosome (BAC) libraries of these species. Comparative analyses revealed several interesting features including segments of conserved non-coding sequences (CNSs) that contain potential regulatory elements relevant to olfactory tissue development and blood-feeding. The expression profiles of these genes were examined in detail in the Asian malaria mosquito An. stephensi. Obp1 and Obp7 transcripts were significantly higher in females than male mosquitoes and they were predominantly found in the antenna, which is the primary olfactory organ of mosquitoes. Twenty-four hours after a blood meal, mRNA levels of these two genes were significantly reduced in the maxillary palp and proboscis, referred to as secondary olfactory organs of mosquitoes. These findings collectively indicate that Obp1 and Obp7 genes in An. stephensi likely function in female olfactory response and may be involved in behaviors related to blood-feeding. To investigate the function of these Obp genes more directly, a Sindbis virus based expression system is established to knockdown the two Obp gene orthologs in Aedes aegypti. The effective knockdown of Obp1 and Obp7 genes (8 and 100-fold, respectively) is accomplished in female mosquito olfactory tissues. The potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique is discussed. Such analysis will provide the foundation for interfering with mosquito host-seeking behavior for the prevention of disease transmission. / Ph. D.

<p>This thesis focuses on the evolutionary interactions between birds and a parasite, the mosquito-borne Sindbis virus (Togaviridae, <i>Alphavirus</i>). In conclusion, the results show that the Sindbis virus is widespread among birds, and that the fitness of infected hosts may be reduced by the virus. Furthermore, viruclearance ability was revealed by male plumage traits, and viraemia was related to hormonal- and social status.</p><p>The distribution of Sindbis virus infections among passerine birds was examined in five areas in Sweden. Almost all species tested were infected, and three species of thrushes weridentified as the main hosts. In a series of experimental infections, greenfinches (<i>Carduelis chloris</i>) kept in aviaries were used ahosts. First, the behavioural consequences of an infection were investigated. During the infection, birds tended to reduce thespontaneous locomotion activity, and when escaping from a simulated predator attack, infected birds had reduced take-off spee Furthermore, when comparing virus clearance rate between male greenfinches, I found that males with large yellow tail ornaments hafaster virus clearance rates as compared to those with smaller ornaments. Thus, male virus clearance ability was honestly revealed by the size of an ornament. Moreover, males with experimentally elevated testosterone levels experienced a delayed, but not increased viraemia as compared to controls. When the relationship between male social ranand viraemia was examined, I found no evidence that high-ranked males suffered reduced rank during the infection. Nevertheless, viraemipatterns of males were related to their social rank, so that low-ranked birds had a delayed viraemia as compared to high-ranked birds. </p>