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Hepatitis C virus (HCV) infection has become a global public health concern with over 130 million people chronically infected and over 350,000 deaths every year from HCV-related liver diseases. GB virus-B (GBV-B) infection in tamarins is a surrogate model for acute HCV infection. Whilst HCV infection commonly leads to chronicity, GBV-B is naturally cleared. To better understand this natural clearance, this project aimed to study the associated humoral immune response to GBV-B. Additionally, GBV-B-specific antibodies were produced with the aim of characterising the pathology of the virus. Previously, there was no available GBV-B neutralisation assay to identify antibodies in this animal model. Therefore, a GBV-B neutralisation assay, based on a method that is known to be successful for the closely-related HCV, was developed. This method involved producing pseudotyped retroviral particles (PV) expressing the GBV-B envelope that could infect a human hepatocarcinoma cell line. GBV-B PV production was confirmed by western blotting. Future studies can now test archived tamarin sera in this assay for the presence of neutralising antibodies. Neutralising antibodies found through this model could be epitope mapped, and incorporated into HCV vaccine design strategies. To study the pathology of GBV-B infection, GBV-B-specific antibodies were also produced using two techniques in parallel- classical hybridoma technology and ribosome display. Antibodies targeting the nucleocapsid core protein of GBV-B have been previously detected in tamarins and served as the target for production of GBV-B antibodies using both aforementioned technologies. GBV-B core-specific antibodies were successfully isolated using both technologies and can now be used in downstream techniques, such as immunohistochemistry, to characterise the pathology of GBV-B infection thereby further validating the use of the animal model.

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by Winnie Yeo. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 213-256). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

In recent years, Zika virus (ZIKV) has garnered worldwide attention due to its epidemic spread throughout the Americas and due to the newly recognized link between ZIKV infection and neurological diseases, including microcephaly and Guillain-Barré syndrome. ZIKV is a mosquito-borne member of the genus Flavivirus, which includes the other prominent human pathogens Japanese encephalitis virus, West Nile virus, dengue virus, and yellow fever virus. Many questions about the biology of ZIKV and how it causes disease remain unanswered. Furthermore, there currently are no vaccines or licensed antiviral drugs available to treat ZIKV infection. The goal of this study was to create new tools to aid in ZIKV research and in the creation of new therapies for ZIKV infection. To accomplish this, we created two recombinant ZIKVs–one expressing a green fluorescent protein reporter gene and the other expressing a luciferase reporter gene. These additional genes will allow us to easily visualize infected cells and to precisely track levels of viral replication over time, thereby facilitating new experimental approaches and providing a means to gain insights about ZIKV. We believe that these two new versions of ZIKV will prove to be useful tools in the urgent task of better understanding how ZIKV causes disease and its links to other complications, as well as in the process of developing and testing new treatments to combat ZIKV infection.

L’infection par le virus de l’hépatite C (HCV) évolue dans la plupart des cas en hépatite chronique et peut conduire à une cirrhose ou un carcinome hépatocellulaire. Malgré les grandes avancées dans le traitement de l’hépatite C qui permettent d’inhiber ou même de bloquer l’évolution de cette infection vers la chronicité, l’absence de vaccin ainsi que sa répartition sur la surface du globe nous permet de classer cette pathologie en problème majeur de santé publique. La majorité des traitements actuels ciblent les protéines virales et leur fonction. Cependant un grand nombre de mécanismes du cycle viral de HCV reste à élucider.Comme pour la grande majorité des virus à ARN de polarité positive, la réplication de HCV a lieu dans des membranes cellulaires modifiées. Le remaniement de ces membranes est en lien étroit avec la voie de sécrétion précoce de la cellule. Il a été montré que GBF1, un facteur d’échange nucléotidique des protéines G de la famille Arf qui régulent la dynamique membranaire, est un facteur nécessaire à la réplication de HCV. L’inhibition de GBF1 par la bréfeldine A (BFA) inhibe la voie de sécrétion des protéines cellulaires néosynthétisées et inhibe aussi la réplication de HCV. Pour étudier le rôle de GBF1 pendant l’infection nous avons établi des lignées résistantes à la BFA. Deux de ces lignées étaient 100 fois plus résistantes que les lignées parentales à l’apoptose induite par la BFA, à l’inhibition de la sécrétion des protéines et à l’inhibition de l’infection par HCV. Ce phénotype était dû à une mutation ponctuelle dans le domaine catalytique sec7 de GBF1 de ces lignées. Un autre groupe de lignées était partiellement résistantes à l’inhibition de la sécrétion des protéines par la BFA tout en conservant un niveau d’infection proche de celui des lignées parentales dans les mêmes conditions. Ces résultats suggèrent que la fonction de GBF1 pendant l’infection HCV ne serait pas réduite à la régulation de la voie de sécrétion, évoquant ainsi un rôle additionnel de GBF1 nécessaire pour la réplication de HCV.Par ailleurs, nous avons pu montrer à l’aide des mutants de délétion de la protéine GBF1, que l’activité catalytique du domaine sec7 était nécessaire. Ceci suggère l’implication d’une protéine de la famille Arf dans l’activation de l’infection HCV via GBF1. L’implication de Arf dans l’infection HCV a été confirmée par la surexpression de dominants négatifs de la protéine Arf1 et par l’inhibition de l’activité de l’ArfGAP1 (régulateur des Arf) par l’inhibiteur spécifique QS11.Nous avons ensuite testé l’implication des différents Arf sensibles à l’inhibition par la BFA (Arf1, 3 ,4 et 5), dans l’infection HCV à l’aide de si-RNA. Il a été montré que ces protéines Arf possèdent des fonctions redondantes. Nos résultats confirment l’implication de Arf1 et indiquent que les 3 autres protéines sont aussi impliquées dans l’infection HCV. D’une manière intéressante, la déplétion combinée des Arf inhibe fortement l’infection HCV suggérant ainsi un rôle essentiel de certaines protéines Arf, probablement en activant des facteurs cellulaires nécessaires à l’étape de réplication. L’étude des facteurs cellulaires impliqués dans l’infection HCV permet de mieux comprendre l’étape de réplication et par conséquent le cycle viral de HCV. Par ailleurs, l’étude de ces facteurs pourrait permettre le développement éventuel de stratégies antivirales ciblant des facteurs de la cellule hépatique indépendamment du génotype viral, limitant ainsi le risque d’émergence de variants résistants au traitement. / The hepatitis C virus (HCV) infection progresses in most of the cases into a chronic hepatitis and can lead to cirrhosis or hepatocellular carcinoma. Despite the recent improvement of hepatitis C treatments, which inhibit or even block the progress of this infection into a chronic stage, a vaccine still not available and the worldwide distribution of the disease makes the hepatitis C a major public health problem. Most of the available treatments target viral proteins. However many mechanisms of the HCV life cycle remain unclear.As for many positive RNA viruses, HCV replication occurs in reorganized cellular membranes. These membrane rearrangements are closely linked to the early secretory pathway of the cell. It has been shown that GBF1, an exchange factor of small G proteins of the Arf family that regulates the membrane dynamics in the secretory pathway, is required for HCV replication. GBF1 inhibition by brefeldin A (BFA) inhibits the secretion of newly synthesized proteins and also inhibits HCV replication. To investigate the role of GBF1 in HCV infection, we isolated cell lines resistant to BFA. Two of these cell lines were 100 times more resistant than the parental cells to BFA-induced apoptosis, inhibition of proteins secretion and inhibition of HCV infection. This resistance was due to a point mutation in the catalytic sec7 domain of GBF1 of these cells. Another group of resistant cells was showing a partial resistance to the inhibition of proteins secretion while maintaining their sensitivity to the inhibition of HCV infection in the same conditions. These results suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication. Using GBF1 deletion mutants we showed that the catalytic activity of the sec7 domain of GBF1 is required for HCV infection. This suggests that the function of GBF1 during HCV replication is mediated by Arf activation. The involvement of Arf was confirmed with the overexpression of restricted mutants of Arf1 and by the inhibition of ArfGAP1, another regulator of Arf function. We then tested the possible involvement of different Arfs (Arf1, 3, 4 and 5) in HCV infection. It has been reported that Arfs have redundant functions. The results confirm the involvement of Arf1 and indicate that all the other BFA-sensitive Arfs (Arf3, Arf4 and Arf5) are also involved in HCV infection. The combined knockdown of Arfs strongly inhibited HCV replication, showing that the Arf proteins are working together in HCV replication probably by activating several host factors required for the virus life cycle.The study of cellular factors required for HCV infection is crucial to better understand the interaction of the virus with the host cell and thus the whole HCV life cycle. This could help to develop new therapies targeting the host cell, regardless of viral genotypes and reducing the risk of emergence of new resistant forms.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Several systems were examined for the production and delivery of recombinant vaccines for fish. C. crescentus was employed to produce a fragment of the IHNV glycoprotein. When administered by injection to 0.5 gram rainbow trout (Oncorhynchus mykiss), one of the fusion proteins (184 amino acids of the IHNV glycoprotein fused to 242 amino acids of the C-terminus of the Caulobacter crescentus) protected the fish against lethal challenge with IHNV. Attenuated strains of Yersinia ruckeri were generated using allelic exchange mutagenesis. These strains were characterized in terms of in vitro growth characteristics and invasiveness. Attenuated E. coli and Y. ruckeri were exploited to deliver plasmid DNA to fish cells in vitro; attenuated Y. ruckeri bacteria were examined in vivo as bivalent vaccine delivery vehicles, either through the expression of a fragment of the IHNV glycoprotein or by carrying a plasmid DNA vaccine encoding the complete IHNV glycoprotein. A cell wall deficient strain (11.29��dap) protected rainbow trout against lethal challenge with pathogenic Y. ruckeri. Gene transfer to fish was not detected by luciferase reporter gene assays. No clear protection from IHNV challenge was observed. / Graduation date: 2002

Oncolytic virotherapy is emerging as a new treatment option for cancer patients. At present, there are relatively few oncolytic virus clinical trials that are underway or have been conducted, however one virus that shows promise in pre-clinical models is Vesicular Stomatitis Virus (VSV). VSV is a naturally occurring oncolytic rhabdovirus that has the ability to preferentially replicate in and kill malignant versus normal cells. VSV also has a low seroprevalence, minimal associated morbidity and mortality in humans, and simple non-integrating genome that can be genetically manipulated, making it an optimal oncolytic vector. Currently, many labs are using a variety of different strategies including inserting trans genes that can modulate the innate and adaptive immune response. VSV can also be retargeted by altering its surface glycoprotein (G) or be made replication incompetent by deleting the G protein. Currently, our lab has engineered a series of new recombinant VSVs, incorporating either the murine p53 (mp53), IPS-1, or TRIF transgene. mp53, IPS-1 and TRIF were incorporated into the normal VSV-XN2 genome and mp53 was also incorporated into the mutated VSV-ΔM vector generating VSV-mp53, VSV-IPS-1, VSV-TRIF and VSV-ΔM-mp53. Our data using these new viruses indicate that these viruses preferentially replicate in and kill transformed versus non-transformed cells and efficiently express the transgene. However, despite the ability for VSV-IPS-1 and VSV-TRIF to induce a robust type 1 IFN response, VSV-ΔM-mp53 was the only construct that had reduced toxicity and elicited an increased anti-tumor response against a syngeneic metastatic mammary tumor model. VSV- ΔM-mp53 treatment lead to a reduction in IL-6 and IP-10 production, an increase in tumor specific CD8+ T cells, and immunologic memory against the tumor. Collectively these studies highlight the necessity for additional VSV construct development and the generation of new clinically relevant treatment schema.