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Characteristics and Anti-betanodavirus Mechanism of One Intestinal Bacterium Derived from Grouper
Nervous Necrosis Virus,probiotics,antiviral substance,small molecular purification,LC/MS,FPLC,HPLC,
|Publication Year :||2008|
|Abstract:||病毒性神經壞死症是造成多種海水及淡水魚類的魚苗或幼魚高死亡率之重要病毒性疾病。防疫方法除了洗卵與施打疫苗外，益生菌的使用等生態防治方法也逐漸倍受重視。從石斑魚腸道中所篩選到具抗石斑魚神經壞死病毒 (GNNV) 能力之益生菌，代號0409，經16S rDNA鑑定後與Shewanella decolorationis有99% 的相似度。利用物理化學方法分析0409菌株所分泌之抗病毒物質，發現具有熱穩定性、耐酸鹼值的變化、不受DNase I、proteinase K及trypsin影響、帶陰離子、分子量小於3 kDa，並且具有高極性。以快速蛋白液相色譜、陰離子交換樹脂及高效能液相層析技術進行純化，發現抗病毒物質具有芳香基團及含氮鹼基的特性，推測可能為小分子的核酸類似物。利用石斑魚鰭細胞GF-1研究抗病毒物質對GNNV的作用機制，發現抗病毒物質在病毒感染六小時即可抑制病毒RNA聚合酶 (RNA dependent RNA polymerase) 轉錄RNA2，並抑制RdRp及外鞘蛋白的表現，進而達到抑制病毒的增殖。以菌液處理金目鱸腦細胞cBB，可誘發干擾素反應下游基因Mx的表現，並可降低RNA2的產量。以混合菌株0409之飼料餵食石斑魚苗兩個月後，攻毒GNNV，可降低石斑苗的發病率及死亡率，顯示菌株 0409 有潛力成為防治神經壞死症病毒的生物之ㄧ。|
Betanodavirus is the pathogen of viral nervous necrosis disease (VNN), causing mass mortality of many species of marine and freshwater fish at larval and juvenile stages. Besides ovum sterilization and vaccination, probiotics also play an important role in the ecological control of viral infection. In this study, a bacterial strain 0409, derived from grouper intestinal microflora, exhibited antiviral activity against grouper nervous necrosis virus (GNNV). The similarity of 16S rDNA sequences between strain 0409 and Shewanella decolorationis was 99%. Anti-NNV components in the culture supernatant of strain 0409 were demonstrated to be less than 3 kDa, heat-stable, wide-pH tolerance, DNase, proteinase K and trypsin-resistant, high polar and having anion molecules. By FPLC, anion exchange chromatography and HPLC analysis, the anti-NNV components were suggested to be a small chemical or nucleotide analogue with aromatic group or nitrogenous bases. According to the results of real-time PCR and Western blot analysis, the anti-NNV components could inhibit the transcription of RNA-dependent-RNA polymerase (RdRp) since 6 hpi. , and decreased the production of NNV RdRp and capsid protein. Moreover, the anti-NNV componens could induce the expression of Mx protein in cBB cell after the treatment of 0409 culture supernatant. Grouper larvae were challenged by NNV two months post feeding with strain 0409-mixed artificial food, and the morbidity and mortality of the fish fed with strain 0409 were much lower than that without feeding strain 0409, indicating that strain 0409 has potential to be one of the biological control organisms against NNV infection.
|Appears in Collections:||動物學研究所|
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