Date on Master's Thesis/Doctoral Dissertation

5-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Lukashevich, Igor

Committee Co-Chair (if applicable)

Sokoloski, Kevin

Committee Member

Beier, Juliane

Committee Member

Chung, Donghoon

Committee Member

Kosiewicz, Michele

Committee Member

Mitchell, Thomas

Author's Keywords

mammalian arenaviruses; arenaviruses; lassa virus; LCMV; epithelial cells; intestinal epithelia

Abstract

Mammalian Arenaviruses are a geographically and genetically diverse family of viruses, which is separated into two sub-groups; the Old World (OW) and New World (NW) groups. Of the OW viruses, Lassa virus (LASV), found endemically in Western Africa, is an important human pathogen, causing hundreds of thousands of infections, and several thousand deaths annually. Interestingly, some villages in endemic regions, up to 45% of the population show seropositivity for the virus. It is hypothesized that seropositivity is a result of natural infection through inhalation or ingestion of infectious particles. However, the exact mechanism is still unknown. LASV’s natural reservoir is Mastomys natalensis, a common rat found in sub-Saharan Africa. Epidemiological studies have identified the inhalation, and/or ingestion of infectious rodent excreta as the primary route of transmission from rodent reservoir to human hosts. Additionally, controlled experiments investigating intragastric (i.g.) versus intravenous (i.v.) routes of inoculation of non-human primates (NHPs) have continued transmission through these routes. viii These studies utilized Lymphocytic Choriomeningitis Virus (LCMV)-WE, a strain of LCMV that results in Lassa Fever (LF)-like disease in NHPs, and LCMVArmstrong (ARM), a strain of LCMV that mimics subclinical infection. When administered i.v., LCMV-WE-infected NHPs became systemically infected, showing clinical signs much like that of LF, and died. However, when orally infected with this virus through i.g. inoculation, some of these animals recovered, and later, were protected from lethal doses of i.v. WE challenge. Due to the nature of natural transmission from rodent to humans, epithelial cells are amongst the first cells to come in contact with the virus. However, the role(s) of the epithelial barrier during these infections have yet to be investigated. In order to investigate the role of these cells during arenaviral infection, here, a cell culture model was developed to investigate the interaction of OW mammalian arenaviruses at the site of intragastric inoculation. An important finding of this works is that the patterns of entry and release are viral dependent, and attachment to epithelial surfaces may play a role in these phenomena. Furthermore, regardless of their pathogenic potential in NHPs, both strains of LCMV, as well as LASV’s close relative, MOPV, showed similar patterns of entry and release when exposed to the apical and basolateral surfaces of polarized intestinal epithelia. Additionally, the replication patters of vaccine candidate ML- 29; a reassortant virus that contains the L segment of MOPV, and S segment of LASV, providing the exact same GP1 of LASV, were characterized. Interestingly, ML-29 virus entered and released in a different pattern than was observed with LCMV and MOPV. ix To determine if patterns of viral entry and release were driven by attachment differences, LCMV, MOPV, and ML-29 viral attachment to the surface of polarized epithelia was analyzed. LCMV an MOPV attaches to the apical and basolateral surfaces of the cells with similar efficiency. However, ML-29 showed decreased attachment to the basolateral surface of these cells as compared to the apical surface. Due to differences in pathogenicity seen in NHPs infected i.v. with LCMVWE and ARM, we hypothesized that these viruses would show differences in entry and release patterns in the polarized Caco-2 cells. However, these viruses replicated in much the same way. From these observations, we sought to further investigate differences in viral replication that may explain pathogenic differences between these closely related viruses. To do so, we investigated intracellular trafficking under the hypothesis that it may be responsible for these differences. Through the use of chemical inhibitors and immunofluorescence with confocal microscopy, a number of differences through the intracellular trafficking of LCMV-ARM and WE. The data indicates that LCMV-WE bypasses the TLR-2 receptor interaction in early endosome, and does not produce an IL-6 response in infected macrophages, opposed to LCMV-ARM-infected cells. Additionally, co-staining with LCMV and late endosome marker RAB7, showed more colocalization with LCMV-ARM than that of LCMV-WE. Furthermore, when blocking acidification of late endosome/lysosome with bafilomycin treatments, LCMV-ARM was more sensitive to pH change in the late endosome, indicating x that fusion occurs at less acidic conditions. These less acidic conditions promote earlier release at viral RNA in the case of LCMV-ARM versus that of WE. Together, these results signify differences in viral replication are tissue and viral specific. Furthermore, this research provides a platform to continue investigating key differences in viral replication between viruses of close genetic relatedness.

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