Enveloped viruses, such as influenza, infect cells by fusing their viral envelope with the cell membrane. The fusion pore is a macromolecular structure that links two membranes that are fusing. This paper will focus on the fusion pore initiated by the hemagglutinin (HA) protein of influenza virus upon infection of a host cell. Mutations in the HA protein can alter the time-course and structure of the developing fusion pore. While there is a clear relationship between HA's structure and the dynamic opening of the pore, the initial 3D structure of the fusion pore as it first begins to form remains unknown. We have attempted to address this unanswered question by measuring fusion pore conductance - a one dimensional electrophysiological measurement - at millisecond time resolution for both wild type and mutant HA proteins, using an automated patch clamp apparatus. Correlating the entire life history of the fusion pore with the snapshots we get from 3D imaging (cryo-electron tomography) would allow us to capture the initial pore opening, as well as better understand the effect that mutating the structure of HA has on influenza viral infection. At this time, we have not yet been able to observe the fusion event; however, we do believe that future experimentation using fusion pore conductance to investigate the effects of HA's structure on influenza viral infection are both promising and necessary.

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

Thesis (M.S.)--Baylor University, 2007. / Includes bibliographical references (p. 53-64).

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1988. / Includes bibliographical references (leaves 169-208).

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Influenza infection occurs in as much as 5–15% of the world population, resulting in 3–5 million cases of severe illness and up to 500,000 deaths annually. According to the CDC, on average 24% of all influenza positive respiratory samples during 2001 to 2011 tested positive for Influenza B. Influenza has two main surface glycoproteins, neuraminidase (NA) and hemagglutinin (HA), HA being responsible for the binding of the virus to the host cell. Currently, seasonal influenza vaccines are produced using two strains of Influenza A and one or two strains of Influenza B viruses recommended by the World Health Organization (WHO). These vaccines are mainly targeting the head domain of the HA protein, which mutates constantly, hence the need for annual vaccine updates. The goal of this research is to develop an experimental universal vaccine against influenza B and increase our knowledge to help pave the way for finding a one-time vaccination alternative, reducing the need for a yearly flu shot. To achieve the above, protection and toxicity studies were conducted in DBA/2 mice immunized with a designed HA2 adenoviral-vectored vaccine targeting the HA stem region of influenza B. Results showed that this designed vaccine was able to confer 100% survival protection, this was supported by lower viral titer in trachea and lung tissues. Additionally, we studied the influence of CD40L as a targeting adjuvant, by analyzing its effect on the humoral and cellular immune response, where results showed that it has a significant effect by inducing a higher TH1-bias response. This research is the first report that leads us to a better understanding of the potential use of a conserved consensus HA2 sequence to induce protection against influenza B virus.

Thesis advisor: Kathleen Dunn / Nearly a century ago, a deadly pandemic swept the globe, taking with it over 25 million lives. This pandemic was caused by the elusive Spanish influenza of 1918. Although many decades have passed since this pandemic, research has yet to uncover the exact origin of the Spanish influenza and the cause of its increased virulence. By examining the current research on the Spanish influenza, some of the secrets of this virus can be uncovered. Most of today's research supports the theory that the hemagglutinin receptor of the Spanish influenza was the most likely source of its potency and that it was an amalgamation of swine and human strains created from a common avian strain that created this virus. Based upon the information that has been uncovered, there is a considerable chance that the Spanish influenza or a similar strain could return in the future. The processes of recombination and reassortment create an endless amount of genetic variants of the virus and any one of them has the potential to be lethal. Although a natural emergence of lethal influenza is a potential threat, the artificial reconstruction of the Spanish influenza or another lethal strain for the purposes of bioterrorism may be an even bigger threat. Thus, it is necessary for researchers to press on with their search for the secrets of the Spanish influenza so that a future outbreak can be avoided. As researchers continue to do their job, the government must also take action and develop the most efficient approach to protecting the public from deadly strains of influenza. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Thesis (M.S.)--Northern Michigan University, 2009. / Includes bibliographical references (leaves 65-73).