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Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines

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2019, Doctor of Philosophy, Ohio State University, Comparative and Veterinary Medicine.
Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, weight loss, dehydration, and up to 100% mortality in neonates. Vaccination of sows with a live attenuated vaccine (LAV) stimulates protective immunity in sows that can provide passive protection to piglets against PEDV via colostrum and milk. The goal of this dissertation is to determine attenuation mechanisms of deletions in two viral genes and to develop an effective and safe PEDV LAV candidates using reverse genetics technology. Our first objective was to study the pathogenesis of a PEDV variant TC-PC177 which contains a 197-aa deletion in the spike protein. In 4-day-old conventional piglets, TC-PC177 caused mild diarrhea, lower titers of fecal viral RNA shedding and no mortality, compared with a virulent strain PC21A. To investigate the role of this deletion in the attenuation of TC-PC177, we generated a mutant (icPC22A-S1Δ197) bearing this deletion from an infectious cDNA clone of the highly virulent PEDV PC22A strain (icPC22A). In 4-day-old gnotobiotic pigs, we found that the icPC22A-S1Δ197 virus caused mild to moderate diarrhea, lower titers of viral shedding and no mortality, compared with the virulent icPC22A. Our second objective was to elucidate the functions of motifs YxxΦ; and KVHVQ at the cytoplasmic tail of the S protein in intracellular sorting and in viral pathogenesis. By transiently expressing PEDV S proteins with mutations in these motifs, we confirmed that the motif KVHVQ is involved in the retention of the S protein in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and the YxxΦ; motif triggers endocytosis of the S protein. Next, we generated mutants by introducing deletions or a mutation into the two motifs of the icPC22A. Infection of Vero cells with icΔ10aa (ΔYxxΦEKVHVQ) resulted in larger syncytia and reduced numbers of S protein projections on the surface compared with icPC22A. In 5-day-old gnotobiotic piglets, mutant icΔ10aa caused less severe diarrhea and significantly moderated intestinal pathology compared with icPC22A. The third objective was to engineer and evaluate a PEDV LAV platform by inactivating the 2'-O methyltransferase (2'-O MTase). Using icPC22A as the backbone, we generated two PEDV mutants: KDKE4A, harboring an inactivated 2'-O MTase, and KDKE4A-SYA, containing the inactivated 2'-O MTase and an ablated endocytosis signal of the spike protein. Compared with icPC22A, KDKE4A and KDKE4A-SYA replicated less efficiently but induced stronger type I and type III interferon responses in vitro. In 4-day-old gnotobiotic piglets, the virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with the icPC22A. At 21 days post-inoculation, all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge. Furthermore, we have serially passaged the KDKE4A-SYA in gnotobiotic pigs three times and did not find any reversion of the introduced mutations. In summary, a reverse genetics platform for icPC22A has been established that allows us to investigate virulence determinants and to design effective and safe LAVs for PEDV. The knowledge obtained from this dissertation may also aid in the development of LAVs against other emerging coronaviruses using reverse genetics technology.
Qiuhong Wang (Advisor)
Linda Saif (Advisor)
Anastasia Vlasova (Committee Member)
Chang-Won Lee (Committee Member)
Scott Kenney (Committee Member)
354 p.

Recommended Citations

Citations

  • Hou, Y. (2019). Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1562349484215276

    APA Style (7th edition)

  • Hou, Yixuan. Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1562349484215276.

    MLA Style (8th edition)

  • Hou, Yixuan. "Porcine Epidemic Diarrhea Virus: Molecular Mechanisms of Attenuation and Rational Design of Live Attenuated Vaccines." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1562349484215276

    Chicago Manual of Style (17th edition)