Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Serotype I feline infectious peritonitis virus (FIPV) is an alphacoronavirus of high veterinary importance due to the 99% mortality rate observed in cats. Since the 1980s, robust experimentation on these viruses has been limited by availability of culturable pathogenic strains, few permissive cell lines, and a lack of standardized methods to study their basic virology. These complications have resulted in variable and conflicting literature reports, have slowed study of clinical strains and hindered effective vaccine design. The goal of this dissertation was to develop a laboratory "toolbox" containing standardized methods, permissive cell lines and genetic techniques to alleviate some of the technical barriers to investigating type I feline coronavirus (FCoV). To this end, our group characterized the replication kinetics of serotype I FIPV Black in three feline cell lines. From this work, colleagues and I described a new macrophage-like cell line, termed Fcwf-4 Cornell University (CU), which rapidly grew FCoV to high titer. Using these cells, we established a standardized plaque assay to accurately determine type I FCoV titer. To facilitate study of live-attenuated and clinical isolate FCoVs, I used Crispr/Cas to disrupt IFNαR, the major antiviral signaling component, in two feline cell lines: AK-D and Fcwf-4 CU. Further, I isolated a clone, termed Fcwf-4 CU IRN, and demonstrated that expression of a feline protease enhanced FIPV infection in these cells. Finally, using a deep-sequenced laboratory strain of FIPV Black, we constructed an infectious clone (ic) reverse genetics system and successfully produced icFIPV.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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