Cell Biology & Molecular Genetics
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Item REASSORTMENT AND GENE SELECTION OF INFLUENZA VIRUSES IN THE FERRET MODEL AND POTENTIAL PLATFORMS FOR IN VIVO REVERSE GENETICS(2014) Angel, Matthew Gray; Perez, Daniel R; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Influenza A virus is a highly infectious agent that cause seasonal epidemics affecting 5-15% of the world population with mild to severe illness and possibly death. While this pathogen represents a significant disease burden to the human population, it can also infect a wide range of animals including swine and land-based poultry, which are thought to serve as intermediate hosts between the human and natural wild aquatic bird reservoir. Here, two viruses, a swine-origin pandemic H1N1 and a seasonal human H3N2 are examined for segment fitness during co-infection of in vivo animal models. In three independent co-infections, reassortment between seasonal and pandemic viruses resulted in the selection of an H1N2 virus with a seasonal PB1 with an otherwise pandemic internal gene constellation. Selection for the seasonal PB1 and NA as well as the pandemic M segment was observed to occur rapidly during segment resolution. As pandemic M gene reassortant strains are being consistently identified in the field, studies were performed to identify the genetic determinants in pandemic M gene selection. Research here shows that both the M1 capsid protein and M2 ion channel from the pandemic virus are sufficient to drive the selection of the entire M segment. As swine represent an important intermediate host for the adaptation of potentially pandemic viruses, including pandemic M gene reassortant strains, alternative DNA and recombinant baculovirus-based platforms are investigated for their ability to generate influenza viruses from porcine polymerase I promoters and serve as potential vaccine candidates. Research here shows that influenza A virus can be rescued de novo using the porcine polymerase I promoter in an eight plasmid system. Furthermore, a single bacmid can be constructed that rescues influenza virus or baculovirus encoding the influenza reverse genetic system in mammalian tissue culture or Sf9 cells, respectively. These represent a new generation of species-tailored vaccine platforms.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.