published_or_final_version / Medicine / Master / Doctor of Medicine

Background Avian influenza A (H5N1) has been circulating around and remains to be one of the major threats to human beings since it first emerged in 1997. Besides vaccines, currently there are two major countermeasures to infection in clinical settings, which are antiviral therapies and immunomodulator treatments. Objectives To summarize evidence on the effectiveness of current treatments against H5N1 infection and to explore the potential benefits of several immunomodulatory agents. Design Systematic review of cross-sectional studies and case series. Data sources Searches of PubMed for articles using the search term “(H5N1[Title]) AND antiviral[Title/Abstract]” and also manual search on PubMed for studies that are cited in some review papers in the first automatic search. Previous search results relating to human H5N1 infection studies are also included. Study selection Included studies that were human H5N1 infection cross-sectional studies or case series in which clinical outcomes were reported, CFR and survival rate were specified or could be easily derived from original data. Results 11 articles met the selection criteria and were included in our analysis. Sample size of the included studies ranged from 8 to 308 lab confirmed cases with median age varied from 10 to 29. Leukopenia, lymphopenia, thrombocytopenia and elevated ALT and AST at admission were strongly associated with worse clinical outcomes with different significance across studies. Oseltmivir treatment was generally initiated earlier among those who survived. Survival benefit of oseltamivir was the most significant if the patient received the treatment within the first two days after symptom onset, and it is still significantly effective when treatment was given up to eight days after symptom onset according to one study we included. Corticosteroid didnot show any beneficial effect or it is associated with a higher risk of death when it is given according to the current treatment protocol and a delayed initiation time. Conclusion Oseltamivir treatment is associated with survival benefit especially when initiated within the first two days after symptom onset, while immunomodulator therapies haven’t shown such benefit so far in clinical setting but some experiments in vitro and in vivo support their use in a manner which is different from the current protocol. / published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

abstract / Microbiology / Master / Master of Philosophy

Influenza epidemics have become a major public health concern worldwide. According to the World Health Organization, the annual epidemics results in about three to five million cases of severe illness, and about 250 to 500 thousand deaths. Recurring emergence of new influenza viruses and viruses that are resistant to currently approved antiviral medications pose a critical need to explore new or alternative medications. A classical Traditional Chinese Medicine (TCM) decoction consisting of nine herbs, named Yinqiaosan (YQS, 銀翹散), has a long history for treating respiratory diseases in China. However, the efficacy of YQS has not been investigated mechanistically. In the present study, the effectiveness of YQS in treating influenza virus infection was examined. The potential mechanisms of action of two active compounds present in one of the component herbs of YQS were also investigated. Results showed that YQS increased the survival rate of the mice in an in vivo influenza virus infection model with significant reduction in lung viral titers. In order to further delineate the mechanisms of action of YQS, compounds present in a principal ingredient of YQS were examined for antiviral and immunomodulatory effects. In screening a panel of fractions extracted from YQS, forsythoside A was demonstrated to suppress the viral titers of a wide range of influenza viruses including the oseltamivir-resistant and the 2009 pandemic H1N1 viruses. Through electron microscopy, slow or abnormal viral budding events were observed upon forsythoside A treatment during influenza virus infection. Western blot analysis revealed a reduced influenza virus M1 protein expression. As previous report showed that assembly of viral components into an infectious particle required a threshold level of M1 protein, reduced M1 expression in the cells treated with forsythoside A may contribute to the virus replication suppression. On the other hand, innate immune responses provide first line protection against influenza virus infections. However, excessive responses often result in tissue damage. Cyclooxygenase (COX)-2 is an immunomodulatory factor that has been shown to play a role in the pathogenesis of influenza viruses. A previous COX-2 knock-out mice model showed that COX-2 deficiency is beneficial to the host during influenza viral infection, in which mortality was significantly reduced. Furthermore, during H5N1 infection, it has been shown that COX-2 level significantly increased and it played an essential role in coordinating the productions of inflammatory cytokines, while in another study, pharmacological inhibition of COX-2 suppressed H5N1 virus replication in primary human macrophages. In view of the roles of COX-2 during influenza virus infection, the presence of compound in YQS that reduces the influenza virus-induced COX-2 level was examined. Present results showed that jacaranone not only reduced the influenza virus-induced cyclooxygenase (COX)-2 mRNA level, it also suppressed the subsequent production of prostaglandin E2 level in primary human macrophages. At the same time, jacaranone inhibited the virus induced-activations of ERK1/2 and Akt, which are involved in the COX-2 induction. Jacaranone also suppressed, at least in part, the COX-2 mRNA level at the transcriptional level by inhibiting the nuclear translocation of NF-κB. To conclude, TCM has been recognized as an important part in complementary and alternative medicine and it is an ample source of antimicrobial drugs. The use of a mixture of herbs is the major therapeutic approach of TCM in which, the principal ingredients provide the main therapeutic actions while the others enhance the effects or diminish the side effects of the principal ones. Some components act mainly for symptomatic control. The present study not only supports the efficacy of YQS, but also gives evidences to an active antiviral compound and an immunomodulatory compound found in YQS. They may act as either principle or supporting components depending on the purpose of application. This study provides new insights on future novel drug development from the existing wisdom of TCM. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The goal of this research is to identify a compound or family of compounds that would allow effective treatment of the influenza virus without unnecessary risks and side-effects. Influenza is a substantial problem in today’s society. Each year 36,000 people die in the United States due to influenza, or influenza related causes. Influenza is caused by two types of the virus, Type-A and Type-B. There are currently four FDA approved drugs to treat influenza—two are proton channel blockers and two are neuraminidase inhibitors. The goal of my research was to design a new drug that would allow physicians to effectively treat Type-A and Type-B influenza virus without having their patients endure unnecessary risks and side-effects. Density functional theory calculations were used to optimize the geometries of the ligands. Using sophisticated resources such as the Protein Data Bank and AutoDock, a library of twenty five ligands were docked into the N4 protein. Each docking was performed five times, resulting in one overall average docked energy. The averaged energies for each of the ligands were ranked from lowest to highest. Based upon a different study, one of my ligands were shown to have antiviral activity. From the docked energy for the ligand with confirmed antiviral activity, the results of the highest ranking ligand could be determined to have promising antiviral activity. The ligand shown below is the promising ligand, which will undergo further alterations and dockings to attempt to improve the antiviral activity. / Department of Chemistry

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This study designed and computational docked a group of ligands intended to find potent inhibitors for Neuraminidase 4 which would have strong interactions with 8 conserved amino acids in the active site. Several trials of ligands were designed based on derivatives of neuraminic acid and evaluated as inhibitors of influenza neuraminidase. Optimized geometries of those ligands were determined using HF/B3LYP/6-311++G** techniques. Binding energies of the ligands bound to the N4 subtype of the neuraminidase protein were determined using AutoDock 4.0. Currently used inhibitors for influenza viruses will also be analyzed in the exactly same way. Comparing the binding information of those candidates and current ligands can provide a useful data about the potential of these species as antiviral drugs. / Department of Chemistry

I established the phage antibody library platform for the identification of specific antibodies. In the first part of my study, I tried to identify antibody against SARS-CoV. Two fragments on the spike protein, which is responsible for inducing viral entry, was chosen as target for the selection of antibody. An antibody was identified which can selectively recognize the SARS-CoV infected cells, but not non-infected cells. Although this antibody was found to retain no neutralizing ability, this specific antibody may have potential to develop for diagnostic purpose. / I utilized the phage system-based cloning method as an attractive approach to screen and identify virus-specific antibodies that can be encoded by the human genome. Once a useful phage clone is identified, unlimited amounts of human monoclonal virus-specific antibodies can be manufactured, and potentially applied clinically for prophylactic and therapeutic uses. The study focuses on two of these new infections, both of which cause severe respiratory disease: SARS and avian influenza. / Identification of specific antibodies, either for diagnostic or therapeutic use, was successfully demonstrated in the two infectious disease models. The phage antibody platform offers a fast and cost-effective method to identify phage antibodies, which can easily be converted to human viral specific monoclonal antibodies for clinical use. / In the 21st century, a number of novel infectious diseases emerged suddenly and spread rapidly, endangering the lives and well-being of people around the world. Severe acute respiratory syndrome (SARS) is a life threatening form of atypical pneumonia that ravaged Hong Kong, Taiwan, China, Canada and many cities in 2003. In the same year, novel avian influenza viruses infected human beings on two continents. Both of these diseases originated in animals and crossed over into the human population. These emerging diseases pose significant public health threats while providing a chilling reminder that another influenza pandemic could occur at any time. Thus, the development of effective therapeutics to control the disease is of paramount importance. Although several vaccines against SARS and avian influenza are available nowadays, the poor clinical performance and frequent mutation of viral strains may limit the practical use and value of the vaccines. Moreover, there are no promising antiviral drugs available for the treatment. Therefore, I aimed to develop an immunotherapy as an alternative treatment option against these diseases. / In the second part of my study, the extracellular domain of matrix protein of avian influenza virus was chosen as target for the selection of antibody. I successfully identified an antibody which can neutralize the avian influenza virus infection. This promising result indicated this antibody has potential to develop for therapeutic use and these antibodies can be easily manufactured in unlimited amounts for clinical application. / Leung, Ka Man. / Adviser: Kwok Pui Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0212. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 112-123). / 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. / Abstracts in English and Chinese.