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Characterization of the Nipah Virus P gene Products And the Innate Immune Responses of Nipah Virus Infected Endothelial and Neuronal Cells

Lo, Michael K. (2009)
Dissertation (152 pages)
Committee Chair / Thesis Adviser: Rota, Paul A
Committee Members: Katz, Jacqueline M ; Perng, Guey Chuen 'Oscar' ; Steinhauer, David ; Compans, Richard W
Research Fields: Biology, Virology; Health Sciences, Immunology
Keywords: Nipah virus; Interferon beta; chemokines; mRNA editing; P gene; NiV W protein; endothelial cells; neuronal cells
Program: Laney Graduate School, Biological and Biomedical Sciences
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Characterization of the Nipah Virus P gene Products And the Innate Immune Responses of Nipah Virus Infected Endothelial and Neuronal Cells By Michael K. Lo Nipah virus (NiV) is a highly pathogenic paramyxovirus which frequently causes fatal encephalitis in humans, and the molecular mechanisms of NiV pathogenesis are unclear. Endothelial cells and neurons are important cellular targets in the pathogenesis of this disease. The goals of this dissertation are to characterize the expression of NiV phosphoprotein (P)-derived gene products in the context of infection, and to characterize the endothelial and neuronal cell innate immune responses against NiV infection. In this study, our sequence analysis of multiple cloned mRNAs from infected cells showed that henipavirus P gene mRNA editing frequencies are higher than those reported for most other paramyxoviruses. Antisera generated against synthetic peptides from the P, V, W, and C proteins of NiV were able to detect all four proteins in NiV infected cells and in purified virions. In infected Vero cells, the W protein was detected in the nucleus while P, V, and C were found in the cytoplasm. The W protein co-immunoprecipitated with karyopherin alpha3. Although plasmid expression studies indicated the ability of individual NiV P gene products to antagonize the innate antiviral response in several cell lines, it is not known whether live NiV infection of physiologically relevant cellular targets reflect those results. Little has been done in regards to the molecular mechanisms of vasculitis seen in human cases. In this study, we characterized the growth kinetics and the innate immune responses of primary endothelial cells and a neuronal cell line. NiV infected endothelial cells produced a functional IFN-β response, which correlated with a differential localization of the NiV W protein when compared with infected neuronal cells, which lacked any detectable antiviral response. We demonstrated that NiV infection of endothelial cells induced a significant increase of inflammatory chemokines secreted into the cellular supernatant, which induced a corresponding increase in monocyte and T-lymphocyte chemotaxis. This study is the first in vitro characterization of the innate immune response against NiV infection in physiologically relevant cell types.

Table of Contents

Table of Contents -- Page -- Chapter 1. Literature Review 1 -- Introduction 2 -- Epidemiology 3 -- Virus Reservoir 5 -- Clinical presentation & pathological manifestations 6 -- Classification and Morphology 10 -- The Genome Organization of NiV 12 -- General Overview of the NiV Replication Cycle 13 -- NiV Membrane Proteins 16 -- The Ribonucleoprotein Complex 24 -- The Innate Cellular Antiviral Response 31 -- Virus-Host interactions of the NiV P gene products 33 -- References 37 -- Chapter 2. Determination of Henipavirus P gene mRNA Editing55 -- Frequencies and Detection of the C, V, and W Proteins -- of Nipah Virus in Virus-Infected Cells -- Abstract 56 -- Introduction 57 -- Results 60 -- Discussion 65 -- Materials and Methods 70 -- References 79 -- Chapter 3. Characterization of the growth kinetics and the93 -- innate immune response against Nipah virus -- in endothelial cells and neurons -- Abstract 94 -- Introduction 95 -- Results 98 -- Discussion 103 -- Materials and Methods 108 -- References 115 -- Chapter 4. Discussion and Conclusions 128 -- References 139


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