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Title Page

Contents

Abstract 10

Ⅰ. Introduction 11

Ⅱ. Materials and Methods 21

1. Ethics 21

2. Sample collection 21

3. RNA extraction 21

4. Nested reverse transcription-polymerase chain reaction 22

5. Real-time reverse transcription quantitative polymerase chain reaction 25

6. Multiplex polymerase chain reaction-based next generation sequencing 27

7. Phylogenetic analysis 34

8. Genetic reassortment analysis 34

9. Mitochondrial DNA cytochrome b gene analysis 34

Ⅲ. Results 36

Part 1. A Southern Genotype of Hantaan Virus as an Etiologic Agent of Hemorrhagic Fever with Renal Syndrome, ROK 36

1. Clinical characteristics and epidemiologic study of patients with hemorrhagic fever with renal syndrome 36

2. Apodemus agrarius is the most common animal in Jeollabuk and Jeollanam Provinces 38

3. Southern Hantaan orthohantavirus sequencing shows higher coverage under the cycle threshold value of 32 40

4. Near whole genome sequences of southern Hantaan orthohantavirus were obtained from six patients with hemorrhagic fever with renal syndrome and seven Apodemus agrarius 42

5. Southern Hantaan orthohantavirus generates a distinct lineage from northern Hantaan orthohantavirus 44

6. Genetic reassortment event was observed in a single clinical sample 48

Part 2. Improving the Diagnosis of Severe Fever with Thrombocytopenia Syndrome in Suspected Patients Using Amplicon-Based MinION Sequencing 51

1. Clinical characteristics and epidemiologic study of patients suspected of having severe fever with thrombocytopenia syndrome 51

2. Comparison of laboratory diagnostics of patients suspected of having severe fever with thrombocytopenia syndrome 54

3. Amplicon-based MinION sequencing complements the laboratory diagnostics 56

4. Severe fever with thrombocytopenia syndrome virus obtained from Chuncheon patients belongs to genotype A and B 59

5. Genetic reassortment was observed in patient N50 62

Ⅳ. Discussion 65

Ⅴ. References 69

ABSTRACT 79

List of Tables

Table 1. Primer sequences used for reverse transcription-polymerase chain reaction 24

Table 2. Primer sequences used for real-time reverse transcription quantitative polymerase chain reaction 26

Table 3. Primer sequences used for southern Hantaan orthohantavirus multiplex polymerase chain reaction 30

Table 4. Primer sequences used for severe fever with thrombocytopenia syndrome virus multiplex polymerase chain reaction 32

Table 5. Clinical characteristics and epidemiological analysis of patients with hemorrhagic fever with renal syndrome 37

Table 6. Trapping results of small mammals collected at sites in the southern part of the Republic of Korea from 2017 to 2019 39

Table 7. The correlation between cycle threshold value and amplicon MinION sequencing coverage rate 41

Table 8. Summary of total reads and read mapping to the Hantaan orthohantavirus strain Ac20-5 reference genome by multiplex polymerase chain reaction-based next-generation sequencing 43

Table 9. Clinical characteristics of 17 patients with suspected severe fever with thrombocytopenia syndrome enrolled at Chuncheon Sacred Heart Hospital from... 52

Table 10. Epidemiology of 17 patients with suspected severe fever with thrombocytopenia syndrome 53

Table 11. Comparison of real-time reverse transcription quantitative polymerase chain reaction results of 17 patients with suspected severe fever with thrombocytopenia syndrome 55

Table 12. Summary of total reads and read mapping to severe fever with thrombocytopenia syndrome virus strain SPL114A reference genome using multiplex polymerase chain reaction-based next-generation sequencing 57

Table 13. Consensus results of amplicon-based MinION next-generation sequencing and both real-time reverse transcription quantitative polymerase chain reaction assays 58

List of Figures

Figure 1. An illustrative structure of Hantaan virus, one of the species in the Bunyavirales. 12

Figure 2. An incident map of hemorrhagic fever with renal syndrome in the Republic of Korea in 2021. 14

Figure 3. A schematic timeline of discoveries of rodent- and shrew-borne Orthohantaviruses from 1976 to 2021. 15

Figure 4. The discovery of the southern genotype of Hantaan orthohantavirus in Jeju Island, the Republic of Korea (ROK) in 2021. 16

Figure 5. The global distribution map of Haemaphysalis longicornis. 18

Figure 6. Illustration of active targeted surveillance to identify sites of infection in emerging Orthohantavirus outbreaks. 20

Figure 7. A map for small mammal trapping in the southern part of the Republic of Korea. 23

Figure 8. Experimental scheme for amplicon-based MinION sequencing. 29

Figure 9. Phylogenetic analysis of the Hantaan orthohantavirus L segment. 45

Figure 10. Phylogenetic analysis of the Hantaan orthohantavirus M segment. 46

Figure 11. Phylogenetic analysis of the Hantaan orthohantavirus S segment. 47

Figure 12. Tanglegrams comparing the phylogenies of Hantaan orthohantavirus tripartite genomes in the southern part of Republic of Korea. 50

Figure 13. Phylogenetic analyses of the severe fever with thrombocytopenia syndrome virus. 61

Figure 14. Tanglegrams comparing the phylogenies between each segment of severe fever with thrombocytopenia syndrome virus in the Republic of Korea. 64

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