Title Page
Contents
ABSTRACT 9
General Introduction 11
1. Recurrency of Newcastle Disease outbreaks in Egypt 11
2. Limitations of current commercial vaccines 11
3. Epidemiology of recent virulent NDV strains in Egypt 12
4. Prevalent genotypes of NDV in Egypt 14
5. Urgent need for homologous NDV vaccine in Egypt 14
1. Introduction 15
2. Materials and Methods 25
2.1. RNA Preparation 25
2.2. Full-length Fusion gene Sequence analysis 25
2.3. Phylogenetic analysis 26
2.4. Fusion gene cleavage site mutation 26
2.5. Full-Length cDNA Clone of K148 Virus 27
2.6. Substitution of Fusion gene in pK148/08 27
2.7. Viruses and Cells 28
2.8. Transfection 28
2.9. Virus Rescue and Propagation 29
2.10. Recombinant virus titration and EID₅₀ 29
2.11. Viral Growth Properties 29
2.12. Genetic stability of cleavage site 30
2.13. Thermostability 30
2.14. Pathogenic properties of the Rescued Virus 30
2.15. Animal experiments 31
2.15.1. Chicken lethal dose study 31
2.15.2. Protective dose, immunization, and challenge experiments 31
2.15.3. Serological Analysis 32
2.15.4. Detection of Viral Shedding post-Challenge 33
2.15.5. EID₅₀ /mL-Ct values equivalent 33
2.15.6. K148/GVII-F vaccine replication and tropism 33
2.15.7. Ethical approval 34
2.15.8. Statistical Analyses 34
3. Results 35
3.1. Virulent NDV 35
3.2. Sequence alignment and phylogenetic analysis 35
3.3. Fusion gene cleavage site mutation 35
3.4. Fusion gene substitution 35
3.5. Virus Rescue and Propagation 36
3.6. EID₅₀ and Growth kinetics 36
3.7. Genetic stability of attenuated cleavage site 36
3.8. Thermostability 36
3.9. Pathogenic properties of the Rescued Virus 37
3.10. Animal experiments 37
3.10.1. Chicken lethal dose study 37
3.10.2. Assessment of Optimal Vaccine Dosage for rK148/GVII-F 38
3.10.3. Survival Analysis 38
3.10.4. Protective Efficacy against mortality and clinical signs 38
3.10.5. Immune response 39
3.10.6. Viral Shedding Assessment Post-challenge 39
3.10.7. Replication and Tropism 40
4. Discussion 41
Conclusion 50
References 72
Abstract (in Korean) 90
Table 1. Oligonucleotide primers that were used for the current study. 51
Table 2. Illustration of chick's groups used in vaccine safety and efficacy experiment. 54
Table 3. Amino acid sequence mutations in the multi-basic cleavage site (MBCS) of Egy-8_NDV_VII_Sohag_2018 Fusion gene. 55
Table 4. Pathogenic indices of K148/08, rK148/GVII-F and Egy-8_NDV_VII_Sohag_2018. 55
Figure 1. (a) Phylogenetic analysis of complete Fusion gene of Egyptian strain Egy-8_NDV_VII_Sohag_2018 was performed based on gene open reading... 57
Figure 1. (b) Phylogenetic analysis of complete Fusion gene of Egyptian strain Egy-8_NDV_VII_Sohag_2018 was performed based on gene open reading... 59
Figure 2. Diagrammatic illustration of the construction of recombinant pK148/GVII-F. The cDNA of the GVII-F was amplified, cloned to RBC-TA... 60
Figure 3. Growth Kinetics of K148/08 and rK148/GVII-F in SPF 10 day old embryonated chicken eggs. 61
Figure 4. HA activity and infectivity titer of K148/GVII-F and K148/08 strains thermostability test. 62
Figure 5. Survival rates in mock and vaccinated SPF-chickens (13 chicken per group) post challenge with (Egy-8_NDV_VII_Sohag_2018) (10⁶ ELD₅₀). 64
Figure 6. The HI antibody titers (log₂) were scored using genotype VII NDV antigen at weekly intervals in the 3 groups immunized with 10⁷ EID₅₀ of... 66
Figure 7. Shedding of challenge virus in birds vaccinated at 1 week-old and challenged at 3-week post-vaccination. Oropharyngeal and cloacal swabs were... 69
Figure 8. Viral replication and tropism for rK148/GVII-F, K148/08 and LaSota detection in 5 days old SPF chicks' trachea, lung and spleen (a, b, c)... 71