Characterization of the genome of a hepatopancreatic parvovirus (HPV) Korean strain and development of molecular diagnostic tools
- Abstract
- Viral pathogens, alongside other pathogens, have major effects on crustacean aquaculture. Hepatopancreatic parvovirus (HPV) is an emerging virus in the shrimp industry and has been detected in shrimp farms worldwide. The HPV genome has greater diversity than other shrimp viruses due to its wide host range and geographical distribution. Therefore, developing diagnostic tools is essential to detect even small copy numbers from the target region of native HPV isolates. We have developed two easy to use quantitative real-time PCR kits, called Green Star and Dual Star, which contain all of the necessary components for real-time PCR, including HPV primers, using the primers obtained from the sequences of HPV isolates from Korea and analyzed their specificity, efficiency, and reproducibility. These two kits could detect from 1 to 1 ≠ 109 copies of cloned HPV DNA. The minimum detection limits obtained from HPV-infected shrimp were 7.74 ≠ 101 and 9.06 ≠ 101 copies in the Green Star and Dual Star assay kits, respectively. These kits can be used for rapid, sensitive, and efficient screening for HPV isolates from Korea before the introduction of postlarval stages into culture ponds, thereby decreasing the incidence of early development of the disease.
- Issued Date
- 2012
- Awarded Date
- 2012. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 미생물학과
- Advisor
- 최태진
- Table Of Contents
- Abstract .....i
Contents ..... v
List of Tables ..... viii
List of Figures ..... ix
List of Abbreviations ..... x
Chapter 1 ..... 1
General Introduction ..... 1
1.1. Marine Viruses ..... 1
1.2. Shrimp aquaculture ..... 1
1.3. Viral disease of shrimp..... 3
1.3.1. RNA Viruses of shrimp ..... 3
1.3.2. DNA Viruses of shrimp..... 7
1.4. Parvoviruses ..... 8
1.5. Shrimp parvoviruses ..... 9
1.6. General characteristics of HPV ..... 9
1.7. Hosts and geographical distribution of HPV ..... 10
1.8. HPV genotypes...... 11
1.9. Diagnostic methods for shrimp diseases ...... 12
1.9.1. Microscopy ...... 12
1.9.2. Hematology and clinical chemistry ..... 13
1.9.3. Serological methods ..... 13
1.10. Molecular diagnostic methods ...... 15
1.11. Factors responsible for shrimp viral diseases..... 17
1.12. Control of shrimp disease ..... 18
1.12.1. Probiotics ...... 18
1.12.2. Phage therapy ..... 19
1.12.3. Shrimp？Cvirus interactions ..... 19
1.12.4. Shrimp viral vaccines..... 20
1.12.5. RNA interference (RNAi)...... 21
1.13. Objective of the research ...... 22
1.14. References ...... 23
Chapter 2 ...... 29
Complete nucleotide sequence analysis of a Korean strain of hepatopancreatic parvovirus (HPV) from Fenneropenaeus chinensis ..... 29
2.1. Abstract ..... 29
2.2. Introduction ..... 30
2.3. Materials and methods ..... 33
2.3.1. Isolation of viral DNA and screening of HPV infection..... 33
2.3.2. PCR amplification and cloning of the genome ..... 36
2.3.3. Cloning of the 5’- and 3’-termini ..... 36
2.3.4. Nucleotide sequence analysis ..... 37
2.4. Results and discussion ..... 39
2.4.1. Genome characterization of the HPV Korean strain ..... 39
2.4.2. Open reading frames and encoded proteins ...... 44
2.4.3. Comparative analysis of the encoded proteins...... 47
2.4.4. Structure of the terminal sequences...... 51
2.5. References ...... 58
Chapter 3 ..... 62
Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection ...... 62
3.1. Abstract..... 62
3.2. Introduction ...... 64
3.3. Materials and methods ...... 68
3.3.1. Sample collection ....... 68
3.3.2. Nucleic acid extraction...... 68
3.3.3. Primer design and shrimp infecting virus screen ..... 68
3.3.4. Cloning of WSSV and HPV DNA ..... 69
3.3.5. Multiplex PCR for WSSV and HPV detection ..... 69
3.4. Results and discussion ..... 70
3.4.1. Primer design ...... 70
3.4.2. Optimization and sensitivity test ..... 74
3.4.3. Field testing of WSSV and HPV coinfection...... 80
3.5. References ..... 86
Chapter 4 ...... 93
Development of two quantitative real-time PCR diagnostic kits for HPV Korean isolates ...... 93
4.1. Abstract ..... 93
4.2. Introduction ..... 94
4.3. Materials and Methods ..... 98
4.3.1. DNA Isolation ...... 98
4.3.2. Preparation of the standard plasmids ........ 98
4.3.3. Primers for real-time PCR ..... 99
4.3.4. Test of primers by SYBR green real ？C time PCR ..... 101
4.3.5. Sequence analysis of the selected target ...... 101
4.3.6. Development and test of Green Star quantitative real-time PCR diagnostic kit... 101
4.3.7. Development and test of Dual Star quantitative real-time PCR diagnostic kit ..... 102
4.3.8. Determination of detection limits ..... 103
4.4. Results and Discussion ..... 104
4.4.1. Selection of best primer combination ..... 104
4.4.2. Sequence analysis ..... 104
4.4.3. Analysis of the Green Star real-time PCR diagnostic kit ...... 107
4.4.4. Analysis of the Dual Star real-time PCR diagnostic kit ..... 110
4.4.5. Comparisons of the two kits ..... 113
4.5. Reference ..... 121
Korean Summary ...... 126
Acknowledgements ..... 130
- Degree
- Doctor
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