Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 93-125). Also available in print.

Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions. In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506. In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection. MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding. The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers. / published_or_final_version / Pathology / Master / Master of Philosophy

Influenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development. The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells. A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated. In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Objectives This study focuses on the evaluation of the use of vaccine in the prevention of severe acute gastroenteritis in the community in the literature. The objectives of this project report include an in depth review of the effectiveness and cost effectiveness of rotavirus vaccination for severe acute gastroenteritis in low and middle income countries (developing countries). Methods Publications were identified using computerized bibliographic searches in PubMed (for the period from October 1994 to July 2012). The keywords “effective*”, “vaccin*”, “rotavirus” , “Randomized controlled trial” were used to search for relevant information. Also the keywords “轮状病毒”, “疫苗” , “随机对照试验” were used to search for relevant information in China Journals Full-text Database(中国期刊全文数据库). Selection criteria: SSRandomized controlled trials (RCT) in children (<5 years old) comparing rotavirus RV1/RV5/LLR vaccines for use with (1) placebo, (2) no intervention, or (3) another RV1/RV5/LLR vaccine. Once the identified articles had been screened by the inclusion and exclusion criteria, the content of each was evaluated in relation to the two research questions. International guidelines: CONSORT (for RCT) was also followed in the quality assessment process. Results In our review, there were 9 studies included and 2 of them were graded A(i.e. good quality), 5 graded B(i.e. medium quality) and 2 graded C(i.e. poor quality). For the two Grade A studies, vaccine effectiveness was estimated to be 39.3% and 48.3%. For 5 Grade B studies, vaccine effectiveness was estimated to be 19.2% to 63.9%. For 2 Grade C studies, vaccine effectiveness was estimated to be 10.6% and 74.3%. There is a smaller range in vaccine effectiveness in grade A studies. In contrast, there is a greater range in vaccine effectiveness in grade B and C studies. Many low and middle income countries may not have enough training in conducting RCT. However, the Grade A studies showed that rotavirus vaccine is effective. Our review also showed that authors from most of the low income countries suggested that rotavirus vaccine is cost effective to very cost effective, while those from middle income countries suggested that the cost of the vaccine is the key factor. Conclusion This review showed evidence of effectiveness of rotavirus vaccine and cost-effectiveness in low and middle income countries (developing countries). China has a huge population and similar situation with other developing countries, hence it is useful to conduct a study on cost-effectiveness on universal vaccination in the near future. / published_or_final_version / Public Health / Master / Master of Public Health

Objective Data on immunogenicity of hepatitis A vaccines (including inactivated and live attenuated vaccine) have been reviewed using a systematic approach in Chinese children. Our mission is to provide a comprehensive review of evidence that whether vaccine types, booster, dosage and age could affect immunogenicity. Methodology A systematic literature review was conducted including all studies reporting on immunogenicity of hepatitis A vaccine. The outcomes considered were hepatitis A Geometric Mean Concentration (GMC) and sero-conversion proportions measured by anti-HAV antibodies after immunization. Results 20 studies were identified from PubMed and Google Scholar according to searching concept. 7 manuscripts met our inclusion criteria. The Sero-conversion proportions of inactivated vaccine (Havrix and Healive)and live attenuated vaccine(H2) were close to 100% after 4-week injection, and GMC of them were 67.2mIU/ml, 71.3mIU/ml and 46.8mIU/ml respectively. Another live attenuated vaccine (LA-1) has been reported no significant differences fromH2 in terms of the Sero-conversion proportions and GMC. Booster demonstrated a stronger response both in inactivated and live attenuated vaccines. In terms of dosage, although more dosage could offer higher GMC, adequate dosage was recommended. In addition, the GMC of less dosage one was significantly lower than that of more dosage after 24 months of first injection. Conclusion Available data indicate that immunogenicity of inactivated and live attenuated Hepatitis A is extremely similar, and both could provide protection for Chinese children. Using booster of inactivated and live attenuated hepatitis A vaccine can increase the immunogenicity. / published_or_final_version / Public Health / Master / Master of Public Health

Acquired immunodeficiency syndrome (AIDS) is a major problem in our current society. There are over 35 million of the population that are currently living with human immunodeficiency virus (HIV), and the number of HIV-infected patients are still rising every year in spite of our efforts to control it. Furthermore, within the AIDS affected population, opportunistic infection is a major cause of complications and is the number one cause of death. The HIV trans-activator (Tat) protein plays a major role in the AIDS pathogenesis. HIV-1 Tat is known to cause dysregulation of cytokines such as TNF-α, IL-6, and IL-10 in AIDS patients. In this study we recognized a proto-oncogene, c-Myc, could regulate the cytokine dysregulation caused by HIV-1 Tat in primary blood monocyte derived macrophages (PBMac). By knocking down the expression of c-Myc with gene specific small-interfering RNA (siRNA), we demonstrated that c-Myc may be critical for the expression of the pro-inflammatory cytokines TNF-α and IL-6. HIV-1 Tat was subsequently found to regulate the expression of c-Myc via the activation of dsRNA-activated protein kinase (PKR), ERK1/2 and p38 mitogen-activated protein kinase (MAPK). Furthermore, c-Myc regulation of the pro-inflammatory cytokines was demonstrated to have a role in AIDS related opportunistic infections. HIV-1 Tat was shown to increase the intracellular growth of Mycobacteria avium complex (MAC) within PBMac. This increase in MAC growth was in turn found to be regulated by TNF-α expression controlled by c-Myc. HIV-1 Tat was also demonstrated to induce the expression of RIG-I, a common pattern recognition receptor of double stranded RNA viruses, in PBMac. RIG-I is known to activate the viral immune responses such as the type-I interferon (IFN) and pro-inflammatory cytokine pathways. This induction of RIG-I by HIV-1 Tat was found to be regulated by c-Myc, as well as through other signalling kinases such as p38 MAPK and PKR. Tat induction of RIG-I ultimately led to the induction of IFN-α2 and IFN-β through the expression and nuclear translocation of the interferon regulatory factor-7 (IRF-7). This alteration in type-I IFN expression regulated by HIV-1 Tat and RIG-I was also found to play a role against AIDS related opportunistic infections. HIV-1 Tat is known to increase the infectivity of Kaposi’s sarcoma-related herpesvirus (KSHV), a common opportunistic viral infection. We were able to demonstrate that this increase in KSHV infectivity was regulated by RIG-I and type-I IFN induced by HIV-1 Tat. Lastly, this study also demonstrated how HIV-1 Tat was able to manipulate the expression of IL-8 induced by KSHV in PBMac. HIV-1 Tat was able to mediate the production of IL-8 induced by KSHV by altering the phosphorylation of the p38 MAPK and the signal transducer and activator of transcription-1 (STAT-1). Taken together, the results of this study showed how c-Myc and RIG-I may be able to play critical roles in HIV-1 Tat induced cytokine dysregulation. Furthermore, the importance of these pathways is further demonstrated in their roles in regulating the immune responses against opportunistic infections in AIDS patients. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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