仙鹤草水提物对石斑鱼虹彩病毒作用途径与机制的研究

Study on the role and mechanism of Agrimonia pilosa Ledeb water extract in the infection of grouper iridovirus

关键词:  仙鹤草水提物，石斑鱼虹彩病毒，病毒感染，抗病毒作用，抗病毒机制

Key words:  Agrimonia pilosa Ledeb water extract  Singapore grouper iridovirus  virus infection  antiviral effects  antiviral mechanisms

摘要:

为了探究仙鹤草(Agrimonia Pilosa)在新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus, SGIV)感染中的作用及作用机制，以仙鹤草水提物和SGIV为研究对象，首先通过光学显微镜观察及细胞活力检测实验确定仙鹤草水提物在细胞中的安全工作浓度。其次利用光学显微镜观察、实时荧光定量PCR (quantitative real-time PCR, qRT-PCR)检测技术检测病毒基因的表达以及流式细胞仪分析技术评估了仙鹤草水提物在SGIV感染中的作用。最后使用qRT-PCR检测技术、流式细胞仪分析技术或病毒滴度的测定深入探究了仙鹤草水提物在SGIV感染过程中的作用机制。研究结果发现，仙鹤草的细胞安全工作浓度为156.3 μg/mL，该浓度的仙鹤草水提物处理细胞未影响细胞的细胞活力及形态结构。浓度为156.3、78.2和39.1 μg/mL的仙鹤草水提物均能抑制SGIV在细胞中的感染，且仙鹤草水提物的抗病毒活性随着浓度的增加而增强。安全浓度的仙鹤草水提物与SGIV共孵育后处理细胞发现SGIV的感染能力减弱。同时，在SGIV感染细胞的吸附、侵入与复制阶段用仙鹤草水提物处理细胞，观察到SGIV在细胞中的含量显著低于未用仙鹤草水提物处理的细胞，且仙鹤草水提物的抗病毒作用在SGIV吸附细胞的阶段最为显著。综上可知，仙鹤草水提物可能通过破坏SGIV病毒粒子结构、干扰SGIV感染细胞过程中的吸附、侵入与复制阶段发挥抗病毒作用，由此表明，仙鹤草是一种具有明确抗病毒机制的抗SGIV特效药用植物，具有研发成为高效抗SGIV绿色药物的潜力。本研究首次探讨仙鹤草在SGIV感染中的作用及作用机制，研究结果为研发SGIV特效药物提供理论依据。

Abstract:

To identify the role of Agrimonia pilosa Ledeb on Singapore grouper iridovirus (SGIV) infection, this study firstly determined the safe working concentration of Agrimonia pilosa Ledeb water extract (AWE) in cells using microscopy observation and cell viability detection experiments. Secondly, the role of the AWE in SGIV infection was evaluated by microscopy observation, quantitative real-time PCR (qRT-PCR) detection technology, and flow cytometry analysis. Finally, qRT-PCR detection, flow cytometry analysis, or virus titer measurement was used to investigate the mechanism of action of the AWE in SGIV infection. The results showed that the safe working concentration of AWE on cells is 156.3 μg/mL, AWE at this concentration did not affect the cell viability and cell’s morphological structure. AWE at concentrations of 156.3, 78.2, and 39.1 μg/mL could inhibit SGIV infection in cells, and the antiviral activity of AWE increased with increasing concentration. The infectivity of SGIV was weakened in cells when the virus treated with AWE. Meanwhile, it was observed that the content of SGIV in cells was significantly lower than that of cells did not treat with AWE during the adsorption, invasion, and replication stages of SGIV infection, and the antiviral effect of AWE was most significant during the SGIV adsorption stage in cells. In summary, AWE could exert antiviral effects by disrupting the structure of SGIV virus particles, interfering with the adsorption, invasion, and replication stages of SGIV infection, indicating that Agrimonia pilosa Ledeb is a medicinal plant with a clear antiviral mechanism that has the potential to develop into an efficient green drug against SGIV. This study is the first time to explore the role and mechanism of Agrimonia pilosa Ledeb in SGIV infection, providing a theoretical basis for the development of SGIV specific drugs.