Title Page
Contents
ABSTRACT 11
General Introduction 15
1. Etiology of avian influenza virus 15
2. Epidemiology of avian influenza virus 16
4. Current situation of South Korea 17
5. Host factors influencing influenza virus infection 18
Chapter 1. The genetics and pathobiological characteristics of H5Nx high pathogenicity avian influenza viruses in South Korea, 2003-2023 20
1.1. Abstract 20
1.2. Introduction 21
1.3. Results 23
1.3.1. 2003-2004: clade 2.5 H5N1 23
1.3.2. 2006-2007: clade 2.2 H5N1 24
1.3.3. 2008: clade 2.3.2 H5N1 24
1.3.4. 2010-2011: clade 2.3.2.1 H5N1 25
1.3.5. 2014-2016: clade 2.3.4.4 H5N8 26
1.3.6. 2016-2017: clade 2.3.4.4 H5N6/H5N8 29
1.3.7. 2017-2018: clade 2.3.4.4b H5N6 30
1.3.8. 2020-2021: clade 2.3.4.4b H5N8 31
1.3.9. 2021-2022, 2022-2023: clade 2.3.4.4b H5N1 32
1.4. Conclusion 33
Chapter 2. Wild birds play a major role in the maintenance and spread of H5N8 high pathogenicity avian influenza viruses in South Korea during the winter of 2020-2021 38
2.1. Abstract 38
2.2. Introduction 39
2.3. Materials and Methods 42
2.3.1. Sample collection and virus isolation 42
2.3.3. Full genome sequencing 42
2.3.4. Phylogenetic tree construction 43
2.3.5. Construction of the K148 clone containing HA gene of ES2 HPAIV 43
2.3.6. Experimental design (Figure 2-1) 44
2.3.7. Virus detection and quantification 45
2.3.8. Serology 45
2.3.9. Histopathology and immunohistochemistry 45
2.4. Results 46
2.4.1. Isolation and genetic characterization of the Korean isolates 46
2.4.2. ML phylogenetic tree analysis for the Korean isolates 46
2.4.3. MCC phylogenetic tree analysis for the Korean isolates 46
2.4.4. Pathogenicity and transmissibility of K20-551-4/2020 (H5N8) in wild birds 47
2.4.5. Histopathologic findings 48
2.5. Discussion 50
Chapter 3. Prolonged Survival Time and Onset of Disease in Chickens Naturally Infected or Vaccinated with H9N2 Avian Influenza Virus against High Pathogenicity Avian Influenza Virus clade 2.3.4.4b isolated in 2022, South Korea 75
3.1. Abstract 75
3.2. Introduction 77
3.3. Materials and Methods 80
3.3.1. Ethics statement 80
3.3.2. Virus 80
3.3.3. Preparations of antigens and vaccines 81
3.3.4. Experimental designs and sampling (Figure 3-2) 81
3.3.5. Serology 82
3.3.6. Histopathology and immunohistochemistry 82
3.3.7. Statistical analysis 83
3.3.8. Viral quantification 82
3.4. Results 84
3.5. Discussion 88
Conclusion 109
References 112
Abstract (in Korean) 136
Table 1-1. Case counts of all HPAI virus reports in South Korea retrieved from the epidemiological investigation report 34
Table 1-2. Experimental infections of various species of animals with HPAI viruses isolated in South Korea from 2003 to 2022. 35
Table 2-1. Information of HPAI isolates used in this study. 53
Table 2-2. The statistical support value, including Bayes factor, posterior probability, and actual migration rate, for comparisons between host groups. 54
Table 2-3. Summary of clinical signs, viral shedding, and serology in Mandarin ducks, Mallards and Eurasian wigeon infected with clade 2.3.4.4b H5N8 HPAI virus. 55
Table 3-1. Mortality, mean death time (MDT), BID 50 and BLD 50 of clade 2.3.4.4b H5N1 HPAI virus in chickens with different treatments. 91
Figure 2-1. Study design including infection, and sampling time points. 56
Figure 2-2. Maximum-likelihood phylogenetic tree of 8 segments of HPAI virus isolated in wild birds in South Korea, 2020-2021. 57
Figure 2-3. Bayesian phylogenetic Maximum clade credibility (MCC) tree of H5 HPAI virus isolated during 2019-2021. 66
Figure 2-4. Viral transitions between host groups. The populations consist of H5N8 HPAIVs isolated from 2019 to 2021. 68
Figure 2-5. Viral shedding of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus isolated in 2020, South Korea. 69
Figure 2-6. Histopathology and immunohistochemistry of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus... 70
Figure 2-7. Histopathology and immunohistochemistry of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus... 71
Figure 2-8. Histopathology and immunohistochemistry of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus... 72
Figure 2-9. Histopathology and immunohistochemistry of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus... 73
Figure 2-10. Histopathology and immunohistochemistry of Mandarin ducks, Eurasian wigeons, and Mallards infected with H5N8 clade 2.3.4.4b HPAI virus... 74
Figure 3-1. Maximum-likelihood phylogenetic trees of HA genes of the Korean clade 2.3.4.4b H5N1 HPAI virus and the Korean H9N2 LPAI viruses used in this study. 92
Figure 3-2. Study design including vaccination, infection, and sampling time points. 95
Figure 3-3. Immunogenicity of the vaccine and cross-reactivity against homologous and heterologous viruses used in this study. 96
Figure 3-4. Survival curves of chickens challenged with clade 2.3.4.4b H5N1 HPAI virus with different treatments. 97
Figure 3-5. Survival curve analysis across three dose levels in groups with varied pre-immune status. 98
Figure 3-6. Comparative survival curve analysis between two groups with varied pre-immune status and dose levels. 99
Figure 3-7. Oropharyngeal (OP) and cloacal (CL) virus shedding in chickens with different treatments after challenging the clade 2.3.4.4b H5N1 HPAI virus. 100
Figure 3-8. Gross lesions of HPAI H5N1-infected chickens dead at 3dpc. 101
Figure 3-9. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc (Trachea and lung). 102
Figure 3-10. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc (Muscle and heart). 103
Figure 3-11. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc... 104
Figure 3-12. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc (Liver and pancrease). 105
Figure 3-13. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc (Thymus and spleen). 106
Figure 3-14. Histopathology and immunohistochemical staining for AIV antigen in tissues of chickens infected with clade 2.3.4.4b HPAI virus at 3 dpc (Brain and kidney). 107
Figure 3-15. Viral titers in tissues of three groups of chickens infected with clade 2.3.4.4b H5N1 virus. 108