Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Enhancing gene delivery to the mammalian nucleus for applications in viral reverse genetics and human artificial chromosome development.(2017) Brown, David; Dinman, Jonathan D; Glass, John I; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Delivery of transgenic DNA into mammalian cells is critical to realizing the potential of synthetic biology in advancing gene therapy, construction of entire chromosomes and production of new vaccines and therapeutics in cultured mammalian cells. New synthetic biology techniques such as rapid, inexpensive DNA synthesis have opened the door to engineering biology. However, now the delivery of these synthetic DNA constructs to the nucleus of a living cell is the limiting step in the development of these applications. Living cells possess numerous cellular barriers that a synthetic DNA construct needs to cross to be successfully expressed. In this dissertation, I explore two methods to enhance DNA delivery across the nuclear membrane barrier. First, a plasmid delivery system was developed involving a papillomavirus scaffolding protein that when expressed by the transfected cell line, more consistently delivered plasmids bearing a specific DNA binding site to the nucleus of mammalian cells. This technique enabled us to produce infectious influenza virus more effectively when transfecting mammalian cells with DNA copies of influenza virus genes. These improvements accelerate production of vaccine against influenza virus. Second, we improved an existing method of transferring large DNA molecules cloned in Saccharomyces cerevisiae into cultured cells through polyethylene glycol mediated fusion of the yeast and cultured cells. Creating a reporter yeast strain allowed us to track the percentage of fused cells and the percentage that achieved YCp delivery allowing us to easily optimize the process. By synchronizing recipient cells in mitosis when the nuclear envelope is broken down we increased the delivery efficiency of large YCps ten-fold. This was accomplished by fusing yeast spheroplasts harboring large YCps (up to 1.1 Mb) with cultured cell lines. A statistical design of experiments approach was employed to further boost the vector delivery rate 300-fold to achieve a YCp delivery rate of 1/840 cells. This method was adapted to deliver a 152-kb herpes simplex virus genome cloned in yeast into mammalian cells to produce infectious virus. Finally, we discuss future applications for this technology including the development of human artificial chromosomes and applications in viral reverse genetics for vaccine development.Item Zoonotic Transmission of Influenza H9 subtype through Reassortment(2013) Kimble, James Brian; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A virus causes disease across a broad host range including avian and mammalian species. Most influenza viruses are found in wild aquatic birds, are of low consequence and refrain from zoonotic transmission. However, some strains occasionally cross the species barrier, into domestic birds and a plethora of mammalian species, most notably swine and humans. Many of these infections are dead ends and quickly disappear from the species, but occasionally, a stable lineage is established and becomes endemic in an animal population. Avian Influenza virus (AIV) H9N2 was predominantly found in wild ducks and shore birds across the globe with occasional infections in turkeys until the late 1980's, at which point the virus became established in Eurasian poultry populations. In the late 1990's the virus again jumped hosts, first into swine, and then into humans. Across many regions, these viruses appear to be gaining human-like virus characteristics. Here, the influenza receptor distribution in a range of poultry species has been characterized and shown that many of the birds were able to bind human-like binding viruses. While no large-scale H9N2 human infections have occurred, the threat is there. The most likely route for this to occur is through reassortment with human viruses. The 2009 human pandemic H1N1 (pH1N1) is a likely candidate as it is found in multiple species and seems to readily reassort. The two viruses were shown to be compatible for reassortment and H9:pH1N1 viruses would readily infect and transmit in both ferrets (a human model animal) and swine. Finally, a novel method of modeling reassortment in vivo was developed, which simultaneously tests the breadth of possible reassortant and utilizes natural host selective pressure to select the most-fit progeny. Furthermore, the characterization of these viruses in ferrets showed they readily infect, efficiently transmit, and exhibit mild to moderate pathological consequences. Taken together, these findings broaden our understanding of natural observations, characterize the potential for zoonosis, highlight the dangers H9 viruses may pose to humans, and give scientists a new tool to deepen our understanding of reassortment.Item A Difference in Heterosubtypic Immunity Induced by a Modified Live Attenuated Avian Influenza Backbone in Mice and Ferrets(2011) Hickman, Danielle; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The unprecedented emergence of multiple avian influenza virus (AIV) subtypes with a broad host range poses a major challenge in the design of vaccination strategies that are effective against multiple subtypes of influenza. The present study focused on the protective effects of a modified AIV as a backbone for epidemic and pandemic influenza. In addition, the ability of this backbone to induce heterosubtypic immunity (Het-I) was also analyzed. Het-I is the ability of one influenza subtype to protect against a different influenza subtype. Previously, a live attenuated AIV with the internal backbone of A/guinea fowl/Hong Kong/WF10/99 (H9N2) (WF10), called WF10att, protected chickens against a lethal influenza challenge. To characterize the WF10att backbone as a master donor strain and determine its ability to induce Het-I, we evaluated its protective efficacy in mice and ferrets. Vaccinated mice were protected against homologous challenge with A/WSN/1933 (H1N1) (WSN), mouse-adapted A/California/04/2009 (pH1N1) and A/Vietnam/1203/2004 (H5N1) (HPAI H5N1) viruses, and ferrets survived homologous challenge with HPAI H5N1. H7N2att vaccinated mice were protected against both H1N1 and HPAI H5N1 challenge; however, Het-I was observed in H9N2att vaccinated ferrets challenged with HPAI H5N1. We found that both B and T cells are involved in the Het-I induced by our WF10att backbone. Cross-reactive non-neutralizing antibodies to viral proteins were detected. JhD-/- mice, which lack mature B-lymphocytes, were vaccinated with the recombinant vaccines and challenged with HPAI H5N1. None of the vaccinated mice survived challenge further suggesting a role for Het-I. In addition, cells isolated from the lungs of H7N2att vaccinated mice had cross-reactive antibody-secreting cells targeted to HPAI H5N1. Together, these results suggest a role for B cells in Het-I. Although B cells are important, T cells may also play a role in Het-I. Both IFN-γ and Granzyme B secreting cells were detected in lung and spleen cells isolated from H7N2att vaccinated mice and stimulated with HPAI H5N1 suggesting a role for T cells in Het-I. The ability of our WF10att backbone to induce Het-I depends on the surface glycoproteins expressed and the challenge virus subtype. In addition, WF10att uses both B and T cells to induce Het-I.Item Phylogenetic analysis of swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan(2009) Padmanabhan, Rangarajan; Perez, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continuous surveillance of influenza becomes important considering the economic, epidemic and pandemic implications of influenza infections. This study details phylogenetic & molecular analysis of the genes of four swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan. Phylogenetic analysis placed the eight segments of the four viruses in the classical H1N1 swine clade, along with the isolate A/sw/Jamesburg/1942, except for the HA of A/Alma-Ata/32/98, which was placed in the human H1N1 lineage, along with the isolate A/WS/1933. On amino acid analysis, the viruses displayed mutations on HA and ribonucleoproteins which putatively disrupt antigenic recognition of the virus by the host immune system. The presence of these viruses relatively unchanged for 6 decades after their initial isolation could be speculated to be a combination of laboratory leaks in southern USSR in 1980s, low divergence of classical H1N1 viruses in pigs, and the low population density of Kazakhstan.