표제지
목차
요약 7
1. 서론 13
2. 재료 및 방법 16
1. Koi herpes virus 감염시료 16
2. PCR분석 18
2.1. PCR 18
2.2. Cloning 19
2.3. Real-time PCR 20
3. 유전형 분석 23
3.1. 계통발생학 특성 23
3.2. 유전 아형 분석 24
4. Dexamethasone과 polyinosinic-polycytidylic acid 처리에 따른 CCB세포에서의 KHV 감염 특성 25
4.1. Koi Herpesvirus 배양 25
4.2. Dexamethasone과 polyinosinic-polycytidylic acid 처리 농도 결정 27
4.3. Dexamethasone과 polyinosinic-polycytidylic acid 처리에 따른 KHV 역가 분석 29
5. Dexamethason과 polyinosinic-polycytidylic acid 처리 후 Koi Herpesvirus감염에 따른 어류의 영향 30
5.1. 초기면역인자 발현 및 조직 내 virus genome copy분석 30
3. 결과 33
1. Koi Herpesvirus의 identification 33
1.1. PCR amplification 33
2. 유전형 분석 36
3. CCB(Common Carp Brain) 세포에서 Dexamethasone과 polyinosinic-polycytidylic acid 처리에 따른 KHV 배양 특성 40
3.1. KHV 배양 40
3.2. Dexamethasone과 polyinosinic-polycytidylic acid 처리 농도 설정 42
3.3. Dexamethasone과 polyinosinic-polycytidylic acid 전 처리에 따른 Koi Herpesvirus 감염역가 비교 44
3.4. Dexamethasone과 polyinosinic-polycytidylic acid 처리 후 KHV접종에 따른 CCB cell의 초기 면역인자 발현 비교 47
4. 잉어에서 Dexamethasone과 polyinosinic-polycytidylic acid 처리에 따른 virus 감수성 비교 50
4.1. 초기면역인자 발현 비교 50
4.2. 시간별 koi herpesvirus 역가 변화 비교 53
4. 고찰 55
V. 참고문헌 62
Table 1. Primers and conditions used in this study 21
Table 2. SYBR-Green based real-time PCR primers used in this study. 28
Table 3. Sub-genotyping of KHV by analysis variant regions of ORF136 gene, Enlarged 9/5 gene, Enlarged TK gene 37
Figure 1. Clinical signs of domestic koi carp; (A) Gill necrosis, (B) Excess mucus. 17
Figure 2. Overview of carp injection study design. 1st injection means immunomodulation (DEX or Poly I:C) exposure and 2nd injection is KHV.... 32
Figure 3. The quantification of KHV genome copies in the kidney of koi carp with clinical symptoms. The low-level group is determined as having ≤1.5 x... 34
Figure 4. Cytopathic effect of KHV in CCB cells (A) 80% confluency of control cells, (B) 14 days post KHV inoculation. 35
Figure 5. Phylogenetic analysis of the Enlarged TK gene of koi herpesvirus identified from koi carp. The phylogenetic tree was constructed using the Maximum-likelihood algorithm with 1,000 bootstrap replicates using MEGA software (ver.11.0.10). Specific genogroups... 38
Figure 6. Sequence analysis of the Enlarged TK gene and ORF136 gene. (A) The Asian type variants were determined based on the number of serial "T"... 39
Figure 7. Correlation between TCID₅₀/mL results from titration on CCB cells with the Reed and Muench method and genomic copies of KHV. 41
Figure 8. Expression of NF-κB in CCB cells treated with various concentrations of immunomodulators (DEX, Poly I:C). The cDNA was quantified using real-... 43
Figure 9. Virus genomic titer (means ± SD) after pre-treated with DEX or Poly I:C. Then KHV genomic copy was quantified in (A) Intracellularly, (B) 200μL... 46
Figure 10. CCB cells inoculated KHV after being pre-treated with DEX or Poly I:C. The expression of innate immune factors was then measured after 6, 12, 24, and 48 hours from virus infection ((A) IFN, (B) IRF7, (C) NF-κB, (D) TNF-α). 49
Figure 11. Expression of innate immune factors in only treatment DEX or Poly I:C groups, KHV injection with pre-treated each immunomodulator. Another group is injected with only KHV. 52
Figure 12. Quantification of KHV genome copies in groups (n=3) injected with KHV after pre-treated with DEX or Poly I:C, and injected with only KHV group. 54