Title Page
Contents
ABSTRACT 10
Chapter Ⅰ. General Introduction 14
1.1. Porcine epidemic diarrhea virus 14
1.2. Antivirals as a control measure for infectious disease 17
1.3. Rottlerin 19
1.4. Liposome, one of the drug delivery systems 20
Chapter Ⅱ. Comparative Genome Analysis and Molecular Epidemiology of Reemerging Porcine Epidemic Diarrhea Virus in Korea from 2014 to 2023: A Time-Scaled Study 22
2.1. Abstract 22
2.2. Introduction 24
2.3. Materials and methods 26
2.3.1. Sample processing and complete genome sequencing of reemerging PEDV in 2014 26
2.3.2. Comparative genome analysis of the reemerging strain in 2014 28
2.3.3. Genetic sequence analysis of the PEDV virus that reinfected the same farm in 2023 29
2.4. Results 31
2.4.1. Korean PEDV strains isolated in 2014 are distinct from previous Korean field strains 31
2.4.2. re-emerging Korean PEDV strains are identical to US PEDV isolated in 2013 33
2.4.3. Time-Scaled Phylogeographic Analysis using the Sequence of PEDV Reinfected in the Same Farm in 2023 35
2.5. Discussion 36
Chapter Ⅲ. Rottlerin inhibits replication of PED virus through internalization associated pathway. 43
3.1. Abstract 43
3.2. Introduction 44
3.3. Materials and methods 48
3.3.1. Cells, Virus, and Antibodies 48
3.3.2. Preparation of rottlerin 49
3.3.3. Cell Viability Assay 50
3.3.4. Dose-Dependent Inhibition Assay 51
3.3.5. RNA Extraction and RT-qPCR Assay 52
3.3.6. Time of Drug Addition Assay 53
3.3.7. Determining the Target of Rottlerin During the Early Stage of PEDV Infection 54
3.3.8. Localization of PEDV during Internalization using Laser Scanning Microscope 55
3.3.9. Observation of PEDV-induced Syncytium Using Immunofluorescence Assay 57
3.3.10. Statistical Analysis 58
3.4. Results 59
3.4.1. Rottlerin inhibits PEDV replication 59
3.4.2. Rottlerin Suppresses PEDV's Viral RNA Replication 61
3.4.3. Rottlerin Influences at Least Two Stages of Virus Replication 63
3.4.4. Rottlerin Disrupts the Internalization of PEDV Rather Than Binding 65
3.4.5. Rottlerin Inhibits PEDV endosomal exit 67
3.4.6. Rottlerin reduces the size of syncytium induced by PEDV 68
3.5. Discussion 69
Chapter Ⅳ. Control of PEDV-Infected herd with Liposome encapsulated Rottlerin 80
4.1. Abstract 80
4.2. Introduction 82
4.3. Materials and methods 85
4.3.1. Preparing Rottlerin-Liposome and Verification of Its Antiviral Effect 85
4.3.2. Animal experiments #1 87
4.3.3. Animal experiments #2 88
4.3.4. Sequence Analysis and Comparative Genome Analysis of S gene 89
4.4. Results 90
4.4.1. Characteristics of Rottlerin-Liposome and its antiviral effect 90
4.4.2. The rottlerin-liposome administered to suckling piglets controlled the PEDV infection 92
4.4.3. Administering rottlerin-liposome to both sows and suckling piglets increases the effectiveness of infection control 93
4.4.4. Genetic sequence analysis reveals that the outbreak is due to the G2b HP strain 95
4.5. Discussion 96
Chapter Ⅴ. Conclusion 105
References 108
Abstract (in Korean) 129
〈Table 2-1〉 Primers used for the amplification of full-length genomes of the reemerging Korean PEDV strains. 39
〈Table 4-1〉 Summary of piglets in each compartment during the first PED outbreak episode at Farm OO. Upon immediate detection of symptoms... 103
〈Table 4-2〉 Summary of piglet fluctuations in each compartment during the second PED outbreak episode at Farm OO. Clinical symptoms suspected of PED were observed at Farm OO (day 0) and the outbreak was confirmed through... 104
〈Figure 2-1〉 Complete Genome Nucleotide Sequence Alignment and Phylogenetic Tree Analysis of PEDV Strains (J.-C. Choi et al. 2014). (A) The... 40
〈Figure 2-2〉 Phylogenetic Tree Depicting the Relationship Among Reemerging Korean Strains, Previous Endemic Korean Strains, and Overseas... 41
〈Figure 2-3〉 Maximum clade credibility tree inferred using the complete coding sequences of spike gene of Porcine epidemic diarrhea virus with... 42
〈Figure 3-1〉 Rottlerin inhibits the replication of PED virus in a dose-dependent manner. (A) Confluent Vero cells were incubated for 72 hours in... 74
〈Figure 3-2〉 Rottlerin not only inhibits the production of infectious viruses but also suppresses RNA replication. (A) Vero cells treated with 2.5µM... 75
〈Figure 3-3〉 Rottlerin inhibits PED virus replication at least in two distinct stages. (A) Confluent Vero cells were infected with PED virus for 1 hour,... 76
〈Figure 3-4〉 Rottlerin inhibits internalization through a mechanism more associated with cathepsin L rather than cathepsin B. (A) Confluent Vero cells... 77
〈Figure 3-5〉 Rottlerin inhibits the internalization of PED virus. (A) Confluent Vero cells were treated with rottlerin and then infected with PED... 78
〈Figure 3-6〉 Rottlerin reduces the size of syncytium induced by PED virus. (A) Vero cells treated with rottlerin were infected with PED virus at an MOI... 79
〈Figure 4-1〉 Rottlerin-liposomes exhibit a spherical structure and consistent size (J.-C. Choi et al. 2023). (A) The shape of the rottlerin-... 101
〈Figure 4-2〉 Liposome-encapsulated rottlerin demonstrates reduced cellular toxicity and enhanced efficacy. (A) Confluent Vero cells were... 102