Electronic Theses and Dissertations

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Browsing Electronic Theses and Dissertations by Subject "17D"

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    Characterization of the molecular and biological determinants of viral dissemination from the mosquito midgut: Yellow feber virus in Aedes aegypti
    (2006-08-15) Kate Lewin McElroy; Stephen Higgs; Walter J. Tabachnick; Stanley J. Watowich; Peter W. Mason; Alan D. T. Barrett
    In order for a mosquito-borne virus to be transmitted from an infected mosquito to a vertebrate host, virus must disseminate from the mosquito midgut- the initial site of infection following uptake of an infectious bloodmeal- to infect the salivary glands and enter the saliva. The genetic determinants of viral dissemination from the midgut were characterized using the flavivirus yellow fever virus (YFV) in the Aedes aegypti mosquito as a model system. Fifteen chimeric viruses were generated between infectious clones of genotypically and phenotypically distinct YFV strains: the wild-type, disseminating Asibi strain, and the attenuated, non-disseminating 17D vaccine strain. These viruses were characterized in vitro in Vero green monkey kidney cells and C6/36 Ae. albopictus mosquito cells and in vivo for virus production, infection and dissemination in orally infected Ae. aegypti. Data from these studies map the YFV genetic determinants of dissemination from the midgut to position 36 in the membrane (M) structural protein gene, domain III of the envelope (E) protein gene, non-structural protein gene 2A (NS2A), and position 95 in non-structural protein gene 4B (NS4B). \r\nThe virus distribution and tissue tropisms Asibi, 17D, and a chimera 17D/Asibi M-E were evaluated in whole-sectioned Ae. aegypti by immunohistochemistry to characterize the temporal and spatial aspects of YFV dissemination. Data from these studies suggest the following sequence of events takes place after infection of the posterior and anterior midgut: virus travels through the basal lamina underlying the midgut epithelium to the hemocoel and infects the abdominal and thoracic fat body tissues. Virus then may infect the salivary glands following amplification in the fat body tissues or infect and amplify in the intussuscepted foregut, cardia, and thoracic ganglia and return to the hemocoel to infect the salivary glands. Successful completion of these events is dependent on efficient receptor binding, replication, and virus packaging, maturation and release from infected cells, events which are mediated by the M, E, NS2A, and NS4B protein genes. This information contributes to our fundamental understanding of mosquito-virus interactions and may be used in the rational design of live attenuated flavivirus vaccines which are not transmitted by mosquitoes.\r\n
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    Population Structure of Yellow Fever Virus: Influence of Viral Diversity on Vaccine Attenuation
    Beck, Andrew Stephen; Barrett, Alan DT; Pyles, Richard B; Wang, Tian; Freiberg, Alexander; Holbrook, Michael
    Yellow fever virus (YFV) is the prototype of all arthropod-borne viruses, the study of which encompasses some of the landmark paradigm shifts in modern vaccinology. Recently, studies on the lifecycle of RNA viruses have indicated that population structure of these viruses is in many cases complex, which in itself may confer fitness within hosts or host ecologies. Thus, these dissertation studies were performed under a set of hypotheses that the YFV population structure is in itself complex, and that features of these populations define a signature that differentiates wild-type from serially passaged and attenuated vaccine strains. Using deep sequencing techniques, a comparison was made between the prototype West African YFV strain Asibi, and a commercial aliquot of the YFV 17D-204 vaccine. In silico methods revealed the vaccine to be of lower diversity than the parental strain, leading to subsequent hypotheses that the level of complexity in the vaccine populations would be related to phenotypic stabilities. A deep sequencing study was performed comparing a set of rare 17D vaccine seeds, which represent all main lineage (substrain) relationships in modern production of the vaccine, and millions of doses administered. The population structures of the vaccine seeds were found to be stable, showing both low diversity and repeatability of subpopulation content. A deep sequencing study was performed on a set of institutional collection strains representing the discontinued YFV French neurotropic vaccine (FNV), which was developed concurrently to 17D. FNV strains with high neurovirulence contained homogeneity in their population structures, showing that the neurotropic adverse events caused by FNV were the result of mouse brain adaptation to attenuate. A deep sequencing study of an adapted strain of Asibi which causes viscerotropy in hamsters was shown to be of low complexity, suggesting that YFV disease of the liver is rare due to low content of viscerotropic alleles. Finally as confirmation, the Japanese encephalitis vaccine strain SA14-14-2 was compared to its wild-type parental strain SA14 by deep sequencing, revealing that loss of diversity for the vaccine was localized to regions of the genome previously thought to influence pathogenicity. The combined results from both JEV and YFV suggest that population studies are useful in the control of empiric vaccine stability and monitoring of safety.