La faculté du virus de la stomatite vésiculeuse (VSV) à induire l'apoptose dans les cellules tumorales en fait un bon modèle d'étude. Mais contrairement au virus sauvage pour la protéine de la matrice M (TP6), certains mutants de M du VSV, comme T1026, induisent moins fortement l'apoptose. Ils peuvent également persister dans les cellules neurales humaines. Les études préalables ont montré que si la voie apoptotique intrinsèque est impliquée dans la mort cellulaire induite par le VSV, elle ne suffit pas à expliquer la totalité de la mortalité. D'autres voies apoptotiques sont donc impliquées. La calpaïne est une protéine cellulaire impliquée dans différentes voies métaboliques chez les cellules de vertébrés, dont l'apoptose. Cette cystéine protéase dépendante du calcium est particulièrement importante dans les cellules de type neural. L'infection virale d'une cellule pouvant induire une variation de la concentration en calcium cytosolique, la calpaïne peut être activée dès la phase précoce de l'infection et jouer un rôle à différents niveaux. Une fois activée, elle a la capacité de cliver le fodrine, protéine du cytosquelette, et ainsi participer aux changements morphologiques induits par le VSV. La calpaïne peut également cliver Bax, renforçant la voie apoptotique mitochondriale. Le rôle de la calpaïne a été étudié ici lors de l'infection in vitro par le VSV dans un neurogliome de type H4. L'analyse des protéines totales, extraites lors des phases précoces et tardives de l'infection par TP6 et T1026, ont pu être effectuées par immunobuvardage. De plus, des modifications dans la morphologie des cellules infectées ont été observées par microscopie optique. La présence de la calpaïne a été décelée dans la fraction cytosolique des cellules H4. Les deux mutants viraux utilisés ont montré un clivage de cette protéase tôt dans l'infection sans toutefois qu'une augmentation de la concentration en calcium cytosolique n'apparaisse. L'inhibition de la calpaïne a provoqué un ralentissement des changements morphologiques induits par TP6, mais le clivage de la fodrine n'a pu être mis en évidence étant donné l'absence de la forme classique de cette protéine dans les cellules H4. L'inhibition de la calpaïne a également permis de diminuer la mortalité cellulaire lors de la phase précoce de l'infection par TP6 (1-4h p.i). Cependant, cet effet se perd au cours de la phase tardive (16-18h p.i). Dans le cas de T1026, l'inhibition de la calpaïne n'a pas provoqué de différences, que ce soit dans les changements morphologiques ou dans la viabilité cellulaire. Entre 14h et 18h p.i. la pro-caspase-3 a été clivée, montrant ainsi l'apoptose induite par le VSV, et Bax a également été clivé en une sous-unité de 18 kDa, portion à caractère pro-apoptotique pouvant provenir de l'activité de la calpaïne. Les résultats montrent donc que cette protéase est impliquée dans l'infection par le VSV, tant dans les changements morphologiques que dans la mort cellulaire induits lors de l'infection. ______________________________________________________________________________

INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use. METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured. RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied. CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

No description available.

No description available.

No description available.

No description available.

Although induction of innate immune responses during viral infection is essential, it can cause acute inflammation and lead to devastating results. The deleterious effect of innate immune responses has been demonstrated in gene therapy where administration of a replication deficient adenoviral vector (Ad) caused fatality during a clinical trial. Despite recent advances in our understanding of the innate immunity, there is a lack of understanding on how different cell types interact to mount inflammatory responses, which may play an important role in regulating immune responses in vivo. In this study, we investigated the interaction between macrophages and epithelial cells, the two major cell types capable of sensing and responding to viral infection in the airway, in induction of inflammatory responses against replication deficient Ads. We show in Chapter 2 that Ad infection of the macrophage-epithelial cell co-culture resulted in synergistic induction of inflammatory responses. Ad infection of the co-culture compared to macrophages alone resulted in higher cytotoxicity and induction of significantly higher levels of inflammatory mediators including pro-inflammatory cytokines, chemokines, nitric oxide, and reactive oxygen species. We found that these synergistic responses require macrophages and epithelial cells to be in close proximity suggesting that a novel mechanism regulates the inflammatory responses. In Chapter 3, we studied whether ATP plays a role in regulating inflammatory responses during acute Ad infection. Using the co-culture system, we found that ATP signaling through P2X7 receptor (P2X7R) is critical as inhibition or deficiency of P2X7R resulted in reduced inflammatory responses. We demonstrate that ATP-P2X7R signaling regulates inflammasome activation and IL-1β secretion. Furthermore, intranasal administration of Ad resulted in high mortality in mice but inhibition of ATP-P2X7R signaling enhanced survival and reduced inflammatory responses. These results suggest that ATP released by the infected cells plays an important role in regulating inflammatory responses during acute viral infection.

No description available.

This thesis develops and utilizes in vitro approaches to study ciliate/fish interactions. The thesis is divided into six chapters. Chapter one reviews the literature on culturing ciliates and fish cells. Chapter two develops methods for culturing the ciliate Tetrahymena thermophila in media developed and used for mammalian and piscine cells. Chapter three explores the interactions of T. thermophila with monolayers of epithelial cells from fish and mammals. Chapter four studies the interactions of T. corlissi, T. thermophila, and T. canadensis with monolayers of epithelial and fibroblasts from a wide range of animals. The interactions of T. thermophila with the fish viruses are described for the rhabdovirus, viral hemorrhagic septicemia virus (VHSV), in chapter five and for the aquareovirus, Chum salmon reovirus (CSV) in chapter six. The summaries for these six chapters are presented in the following six paragraphs. How the ciliates of fish can be cultured and used to study ciliate/fish interactions are reviewed. The culturing of ciliates is done in media based on either freshwater, seawater, or vertebrate bodily fluids together either with bacteria, fish cells, or organic matter, which can be undefined, such as proteose peptone, or defined. Some ciliates can be pathogenic but with a variable dependency on the fish host. The most dependent and difficult to culture has been Ichthyophthirius multifiliis. Cryptocaryon irritans has been maintained successfully in co-cultures with fish cells. Pathogenic scuticociliates and tetrahymenas can be cultured axenically. Established cultures have been used to screen drugs for their potential chemotherapeutic value and to study pathogenic mechanisms. As well as being pathogens, ciliates interact with fish in other ways. Free-living forms can modulate the activities of other fish microbial pathogens and be food for fish larvae. Tetrahymena spp. have been shown in culture to phagocytose pathogenic bacteria and microsporidia spores. Large-scale cultures of both freshwater and marine ciliates have been achieved and could be a source of feed for fish larvae. In the future cell cultures should be invaluable in studying these and other possible relationships between fish and ciliates. The transfer of Tetrahymena thermophila from normosmotic solutions (~20 to 80 mOsm/kg H2O) to hyperosmotic solutions (> 290 mOsm/kg H2O) was investigated. During the first 24 h of transfer from proteose-peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H2O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L-15 (~320 mOsm/kg H2O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/mL) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L-15. In L-15 with 10 % FBS the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates. Although several species of Tetrahymena are often described as histophagous and opportunistic pathogens of fish, little is known about ciliate/fish cell interactions, but one approach for studying these is in vitro with cell lines. In this study T. thermophila, B1975 (wild type) and NP1 (temperature sensitive mutant for phagocytosis) were cultured on monolayers of three fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and of the rabbit kidney epithelial cell line, RK-13. Generally the ciliates flourished, whereas the monolayers died, being completely consumed over several days. The destruction of monolayers required that the ciliates be able to make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could concurrently be phagocytosed. The ciliates internalized into food vacuoles ZEB2J from cell monolayers as well as from cell suspensions. Phagocytosis was essential for monolayer destruction as monolayers remained intact under conditions where phagocytosis was impeded, such as 37 °C for NP1 and 4 °C for B1975. Monolayers of fish cells supported proliferation of ciliates. These results show for the first time that T. thermophila can ‘eat’ animal cells or be histophagous in vitro, with the potential to be histophagous in vivo. The activities of T. corlissi, T. thermophila, and T. canadensis were studied in co-culture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not undergo phagocytosis and did not destroy monolayers, whereas T. canadensis was able to undergo phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. Additionally the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not. Incubating the fish pathogen VHSV with the ciliate T. thermophila, inactivated the virus, depending on the incubation temperature. Without the ciliates, the VHSV titre declined significantly over 72 h at 30 °C, but remained unchanged at 22 °C and 14 °C. At 30 °C, the ciliates only slightly enhanced the heat inactivation of VHSV. At 22 °C, the ciliates inactivated a substantial proportion of the VHSV by 24 h but no inactivation had occurred by 72 h at 14 °C. The ciliates vigorously phagocytosed fluorescent beads at 22 °C but not at 14 °C. When VHSV were labeled with the nucleic acid stain SYBR Gold, internalization of the virus into food vacuoles was seen at 22 °C. Thus phagocytosis was one possible mechanism for VHSV inactivation by ciliates. However, another VHSV/ciliate interaction was revealed by immunofluorescent staining and might contribute to inactivation. After being incubated for 24 h with VHSV, washed, and stained at various times afterwards for VHSV G protein, the ciliates stained transitorily. The strongest staining was seen at approximately 30 minutes after washing and was confined largely to the cilia but after 60 minutes this staining was lost. Tetrahymena thermophila strains B1975, wild type, and NP1, a temperature sensitive mutant, activated the fish aquareovirus CSV, depending on the temperature. CSV caused fish cells to form syncytia. This cytopathic effect (CPE) was used to titre CSV in the fish cell line, CHSE-214. The CSV titre remained stable during incubations of up to 96 h in Leibovitz’s L-15 with FBS at 4, 14, 22 and 30 °C. When CSV was incubated with B1975 or NP1 at 22 °C in the same medium for between 24 and 96 h, the virus titre increased approximately 3 log. At 4 °C, the titre was unchanged by ciliates and T. thermophila was unable to phagocytose beads. At 30 °C, B1975 enhanced CSV infectivity and underwent phagocytosis, whereas NP1 did neither. When CSV were labeled with the nucleic acid stain SYBR Gold, internalization of the virus into B1975 food vacuoles was seen. Therefore the viral activation pathway likely involved phagocytosis. Tetrahymena canadensis were incubated with CSV-infected CHSE-214, washed by centrifugation through a step gradient of polysucrose, and transferred to new CHSE-214 cultures, which developed the characteristic CSV CPE. Thus as well as activating CSV, ciliates could transport CSV.