Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Plant resistance; Arabidopsis thaliana; turnip crinkle virus. Includes bibliographical references (p. 69-72).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Because diagnostic tests for highly pathogenic avian influenza (HPAI) viruses require the use of replication-competent viruses in a biosafety level 3 containment, numerous studies have looked at ways to develop alternative tests. Lentiviral particles pseudotyped with H5 hemagglutinin (HA), the surface glycoprotein of influenza virus, have been described as useful and safe tools for research and serological surveillance on the HPAI viruses. However, not all H5 HA give rise to efficient H5 pseudotyped lentiviral particles (H5pp) production. HA from A/Cambodia/408008/05 H5N1 (H5Cam) and HA from A/Anhui/1/05 H5N1 (H5Anh) exhibit a dramatic difference in their ability to pseudotype lentiviral particles. H5Cam gives the highest H5pp production among all HAs tested, whereas the lowest has been observed with H5Anh. The objective of this study was to investigate the molecular determinants that govern efficient H5pp production. Based on the amino acid differences between H5Cam and H5Anh, H5Anh mutants were generated by site-directed mutagenesis. Strikingly, a single amino acid change, A134V, in the 130-loop receptor-binding domain of HA, significantly increased H5pp production with H5Anh. The finding that valine 134 is crucial for H5pp production was confirmed by reciprocal H5Cam and H5Anh mutants, which displayed either a dramatic decrease or increase in H5pp production, respectively. Influenza virus and H5pp bud at the plasma membrane, therefore changes in HA cell surface expression could affect the production of H5pp. Thus, cell surface expressions of H5Cam and H5Anh were compared by flow cytometry. Intriguingly, H5Cam displayed a higher plasma membrane expression than H5Anh, suggesting that transport is important for H5pp production. Introduction of V134A mutation in H5Cam reduced its surface expression to that of H5Anh; by contrast, H5Anh mutant harboring A134V mutation largely restored its expression. Next the effect of A134V mutation on the binding of HA to sialic acid receptors was investigated. A cell-based Enzyme-linked Immunosorbent Assay was developed to measure binding of wild-type and mutated HA. Soluble recombinant proteins were produced by mammalian cells stably transfected with HA gene ectodomain and were mostly trimeric as indicated by discontinuous native gel electrophoresis. Interestingly, H5Anh proteins exhibited a stronger binding to MDCK cells than H5Cam proteins, and introduction of A134V mutation in H5Anh proteins reduced the binding. By contrast, as predicted, the reciprocal V134A mutation induced a major increase in binding to cellular receptors. It is likely that stronger binding of H5Anh to sialic acids could hinder the release of H5pp. Consistent with this notion, the ability of H5Anh to generate H5pp was significantly increased in a sialylation deficient Lec2 cell, a CHO mutant cell line. In conclusion, H5Cam allows efficient H5pp production whereas H5Anh does not. With several lines of evidence, it is likely that the behavior of H5Anh can be explained by a stronger binding to sialic acid receptors that is dependent on a single amino acid residue at position 134. Since A134V is a naturally occurring mutation observed occasionally in human host, these results may have implications for the understanding of human host adaptations of H5N1 viruses. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Hepatitis C Virus “HCV” is the major factor to develop the chronic liver disease. Accurate genotyping is important to decide the choice and duration of therapy. The most common method for the determination of the genotype in HCV is the direct sequencing of the 5’UTR region, CORE, CORE/E1 and NS5B region. The current study is to compare different subgenomic regions for the molecular characterization of genotype 6 in Hong Kong and study the molecular epidemiology by phylogenetic analysis. Ninety-four patients were included in the study from 2006 to 2009. There was no discordant result between different subgenomic regions. The percentage of the patients sequenced from CORE, CORE/E1 and NS5B were 95%, 93% and 78% respectively. All the HCV strains were genotype 6a except two patients were other subtypes of genotype 6. The phylogenetic analysis in the neighbor-joining tree in CORE/E1 and NS5B region can clearly discriminate between the subtypes of genotype 6, however it cannot show in the NJ tree of the CORE region sequences. It is to conclude that CORE/E1 is the best choice both for the characterization of genotype 6 and used for phylogenetic analysis in the study. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Our systematic influenza surveillance in southern China revealed that two lineages of H9N2 influenza viruses, represented by Chicken/Beijing/1/94 and Quail/Hong Kong/G1/97, became endemic in the poultry in southern China since 1990’s. These established H9N2 lineages continually evolved to generate many different reassortants (or genotypes) and caused sporadic human infection cases. As co-circulating with H5N1 influenza viruses, the increasing genetic diversity and the capability to cause sporadic human infection make the H9N2 viruses become one of the major candidates with pandemic potential. Even though highly pathogenic H5N1 influenza viruses were seldom detected at the live-poultry markets of Hong Kong since 2002, H9N2 viruses were still commonly isolated in our surveillance program. The accumulated H9N2 isolates provided an opportunity to get insights into the continual evolution of this subtype virus in the region. In present study, we have systematically analyzed the H9N2 influenza viruses isolated from 2005 to 2010. Antigenic and phylogenetic analyses of 60 representative H9N2 viruses showed that the Ck/Bei-like H9N2 virus lineage continued endemic in the terrestrial poultry during the survey period in southern China. Genotyping analyses revealed four prevalent genotypes or reassortant variants in the field. Fifty-three of the viruses analyzed belonged to genotype B14 and B15, which were also the major reassortant variants prevailing in southern China from 2000 to 2005. The remaining seven viruses belonged to novel genotypes that have not been identified before. Our findings suggested that the Ck/Bei-like lineage continually maintained high genetic diversity in this region. The epidemiological findings showed that the isolation rate of H9N2 virus at the marketing poultry in Hong Kong was dramatically dropped down since 2009, which was different from what have observed in other provinces in southern China, but was closely correlated with the hygiene measures implemented in live-poultry markets in Hong Kong, e.g. not keeping live chicken overnight. These findings suggest the proper market policy would directly impact the prevalence of influenza virus in the field. / published_or_final_version / Microbiology / 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