- 學位論文
石斑魚腸道希瓦氏菌株0409抗神經壞死症病毒的機制
The Anti-betanodavirus Mechanism of Shewanella strain 0409 Isolated from Grouper Intestine
摘要
神經壞死症病毒(Nervous Necrosis Virus, NNV)是造成世界各地海水養殖魚苗高死亡率的重要病原體。該病毒無封套膜，基因體由兩段單股正意RNA組成，RNA1負責轉譯RNA聚合酶(RNA-dependent RNA polymerase，RdRp)，RNA2負責轉譯外鞘蛋白。自石斑魚腸道分離到一株希瓦氏菌株0409 (Shewanella strain 0409)，在石斑苗活體餵食實驗中證明能降低NNV攻毒後的罹病率及死亡率(Cheng, 2008)，本研究則利用細胞株進一步探討此菌抗NNV的機制。以即時定量PCR及免疫螢光染色，發現當0409 胞外產物(extracellular product，ECP)與NNV 同時加入細胞, NNV的RNA1及RdRp的表現量會在病毒感染細胞後24小時內受到抑制；若0409 ECP在病毒吸附細胞1小時完畢後才加入細胞，對NNV RNA1複製的影響就變得非常低，顯示0409 ECP對NNV的干擾是發生在病毒感染細胞的早期。0409 ECP若在NNV感染後12小時才加入細胞，則完全不會影響NNV RNA1的複製與RdRp的表現量，證明0409 ECP 並不會干擾RdRp的活性。在電子顯微鏡觀察下，NNV顆粒經酸性(pH 6, pH 5)溶液處理後，負染劑會滲入病毒顆粒中，但中性(pH 7)或鹼性(pH 10)溶液處理後的NNV，外鞘蛋白仍保持完整，沒有負染劑滲入病毒顆粒內,說明酸性環境能改變病毒外鞘蛋白的完整性。在0409 ECP中可測到高濃度銨根離子(80 mM NH4+)且可抑制似內囊胞的酸化。以開蓋加熱方式將0409 ECP中的銨根離子氣化為氨氣（NH3），再將降低銨根離子後的0409 ECP和NNV一起加入細胞，則發現0409 ECP失去抗NNV的能力，因此證明，0409 ECP主要抗病毒活性的成份是足夠濃度的銨根離子，其抗病毒機制是經由銨根離子來抑制內囊胞(endosomes)等胞器的酸化，進而阻止NNV外殼構型的改變，並可能干擾到後續病毒脫殼或核酸釋放等步驟，而達到降低病毒複製的效果。
並列摘要
Nervous necrosis virus (NNV), a world-wide pathogen of marine fish, has caused mass mortality of fish at the larval stage. It is a non-enveloped virus with two single-stranded, positive-sense RNA genomes. RNA1 encodes RNA-dependent RNA polymerase, and RNA2 encodes capsid protein. A strain 0409 belonging to Shewanella was isolated from grouper gut, and has been demonstrated to decrease morbidity and mortality of grouper larvae in NNV challenge test after feeding for a period of time (Cheng, 2008). In this study, the anti-NNV mechanism of 0409 was examined in vitro. By real-time PCR and immunofluorescence staining, 0409 extracellular product (ECP) was found to inhibit viral RNA1 or RdRp synthesis within 24 h post infection (hpi), when it was added into cells at the same time of NNV addition. However, the anti-NNV activity significantly decreased if 0409 ECP was added into cells after viral one-hour adsorption, indicating that the interference of 0409 ECP acted during the early stage of NNV infection. When 0409 ECP was added into cells at 12 hpi, the synthesis of RNA1 and the following RdRp was similar to the control, revealing that 0409 ECP did not inhibit the activity of NNV RdRp. Under TEM observation, negative stain diffused into NNV particles if the purified virions were treated under pH 5 or 6, but instead, no diffusion was observed inside the virions if the treatment was under pH 7 or 10, suggesting that acid environment could change the integrity of NNV capsid protein. High concentration of ammonium ions (80 mM NH4+) was detected in 0409 ECP, and was able to inhibit the acidification of endosome-like organelles. The concentration of ammonium ions in 0409 ECP was down-regulated by heating in an open-lid tube, and its anti-NNV activity was then lost. Therefore, the main component of 0409 ECP against NNV replication was proved to be the sufficient concentration of ammonium ions, which could inhibit the acidification of endosome-like organelles, and possibly interfered with the following uncoating or releasing viral genome into the cytoplasm of infected cells, and resulted in the inhibition of viral replication.