Thesis (M.S. in Management)--Naval Postgraduate School, December 2009. / Thesis Advisor(s): Apte, Aruna. Second Reader: Ferrer, Geraldo. "December 2009." Description based on title screen as viewed on January 27, 2010. Author(s) subject terms: Pandemic Influenza, Vaccine Distribution, Vaccine Distribution with DoD Assets, DoD Pandemic Influenza Response Plan. Includes bibliographical references (p. 63-64). Also available in print.

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

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

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

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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

Background Influenza causes annual, worldwide epidemics of respiratory disease that affects all segments of the population. Mass vaccination of healthy children, who are playing an important role in the transmission of influenza, is promoted to be a complementary approach in prevention and control of influenza. However, lack of published systemic review evidencing the indirect protection of vaccinating healthy children makes the implementation under uncertainty. Method A systemic review was conducted by computerized bibliographic searches in PubMed and the Cochrane Library identifying the published studies on the effectiveness and cost-effectiveness of vaccinating healthy children to control influenza epidemics by reducing transmission in the community. Any study design with vaccinating healthy children as the intervention versus control group with no influenza vaccine was included. Only outcomes measured on the contacts of children, either the community or household members were considered. Result Twenty-two articles were selected to be reviewed in this project, in which 17 of them covered the public health benefit of vaccinating healthy children to protect others in the community against influenza, and five of them were economic studies. Overall the result suggested that vaccinating health children produces a public health benefit in protecting others in the community against influenza and that it is a cost-effective measure. Discussion Targeting vaccines to healthy children should be promoted for optimal vaccine allocation, maximizing the vaccination effectiveness. Community planning on vaccine delivery infrastructure as well as educational and communicational strategies is necessary to improve influenza vaccine coverage. Further well-designed studies such as RCT with larger sample sizes, as well as studies in Hong Kong or other sub-tropical regions should be carried out and included. Moreover, large and population-based studies should be conducted to examine the overall impact of universal childhood influenza immunization. / published_or_final_version / Public Health / Master / Master of Public Health

Influenza epidemics have always been a constant public health threat to human populations. Recent societal developments and demographics changes have put us at increased risk of a widespread and potentially deadly influenza epidemic. Antiviral prophylaxis may provide an important epidemic intervention measure especially against influenza epidemic caused by novel influenza viruses where there will be no effective epidemic-specific vaccines available at the initial phase of the epidemic. Antiviral prophylaxis is listed as a fundamental component of the Hong Kong Preparedness Plan for Influenza Pandemics, however the rationale of such plan is not supported by public presentation of scientific evidence and no details of the actual antiviral prophylaxis plan are provided. With a majority of its stockpile set to expire in the very near future, we would like to know if antiviral prophylaxis is an effective intervention strategy against influenza epidemic and should antiviral stockpiling be continued. We identified relevant studies and reviewed the effectiveness of antiviral prophylaxis in preventing influenza infections at the individual, household and population level. We found that prophylactic treatment with Oseltamivir or Zanamivir are both effective in preventing influenza infections at the individual and household level. Both antivirals are well tolerated and neither is associated with major adverse events. Antiviral prophylaxis is effective in mitigating the public health impact of an influenza epidemic such as number of clinical influenza infections, hospitalization and deaths. However, antiviral prophylaxis alone may not be sufficient to contain or avert an influenza epidemic or delay the epidemic progress long enough for public health resources such as epidemic-specific vaccines to be acquired. In addition, the emergence of antiviral resistance and various logistics constraints will hamper the effectiveness of antiviral prophylaxis in containing influenza epidemics. We suggest the use of antiviral prophylaxis as the key intervention to mitigate influenza epidemics, however, with special considerations taken into hedging antiviral resistance and fulfilling the logistics requirements in order to make antiviral prophylaxis effective. In addition, we recommend public health authorities to take a multi-factorial approach in tackling highly transmissible influenza epidemics by incorporating other non-pharmaceutical interventions such as quarantine, school closures and boarder restrictions into the their antiviral prophylaxis-based containment plan. / published_or_final_version / Public Health / Master / Master of Public Health