Ph.D. Program of Aquatic Science and Technology in Industry, College of Hydrosphere Science, National Kaohsiung University of Science and Technology, Kaohsiung City 80778, Taiwan.
Department and Graduate Institute of Aquaculture, National Kaohsiung University of Science and Technology, Kaohsiung City 80778, Taiwan.
J Microbiol Biotechnol. 2021 Aug 28;31(8):1088-1097. doi: 10.4014/jmb.2103.03036.
Grouper nervous necrosis virus (GNNV) infection causes mass grouper mortality, leading to substantial economic loss in Taiwan. Traditional methods of controlling GNNV infections involve the challenge of controlling disinfectant doses; low doses are ineffective, whereas high doses may cause environmental damage. Identifying potential methods to safely control GNNV infection to prevent viral outbreaks is essential. We engineered a virus-binding bacterium expressing a myxovirus resistance (Mx) protein on its surface for GNNV removal from phosphate-buffered saline (PBS), thus increasing the survival of grouper fin (GF-1) cells. We fused the grouper Mx protein (which recognizes and binds to the coat protein of GNNV) to the C-terminus of outer membrane lipoprotein A (lpp-Mx) and to the N-terminus of a bacterial autotransporter adhesin (Mx-AIDA); these constructs were expressed on the surfaces of BL21 (BL21/lpp-Mx and BL21/Mx-AIDA). We examined bacterial surface expression capacity and GNNV binding activity through enzyme-linked immunosorbent assay; we also evaluated the GNNV removal efficacy of the bacteria and viral cytotoxicity after bacterial adsorption treatment. Although both constructs were successfully expressed, only BL21/lpp-Mx exhibited GNNV binding activity; BL21/lpp-Mx cells removed GNNV and protected GF-1 cells from GNNV infection more efficiently. Moreover, salinity affected the GNNV removal efficacy of BL21/lpp-Mx. Thus, our GNNV-binding bacterium is an efficient microparticle for removing GNNV from 10‰ brackish water and for preventing GNNV infection in groupers.
神经坏死病毒（GNNV）感染会导致石斑鱼大量死亡，给台湾带来巨大的经济损失。传统的控制 GNNV 感染的方法涉及到控制消毒剂剂量的挑战；低剂量无效，而高剂量可能会对环境造成破坏。寻找安全控制 GNNV 感染的潜在方法以防止病毒爆发是至关重要的。我们构建了一种表达病毒结合蛋白的细菌，该蛋白在其表面表达一种抗粘液病毒（Mx）蛋白，用于从磷酸盐缓冲液（PBS）中去除 GNNV，从而提高石斑鱼鳍细胞（GF-1）的存活率。我们将石斑鱼 Mx 蛋白（识别并结合 GNNV 的外壳蛋白）融合到外膜脂蛋白 A（lpp-Mx）的 C 末端和细菌自转运黏附素（Mx-AIDA）的 N 末端；这些构建体在 BL21 表面表达（BL21/lpp-Mx 和 BL21/Mx-AIDA）。我们通过酶联免疫吸附试验检测细菌表面表达能力和 GNNV 结合活性；我们还评估了细菌吸附处理后细菌对 GNNV 的去除效果和病毒细胞毒性。虽然两种构建体都成功表达，但只有 BL21/lpp-Mx 表现出 GNNV 结合活性；BL21/lpp-Mx 细胞能更有效地去除 GNNV 并保护 GF-1 细胞免受 GNNV 感染。此外，盐度会影响 BL21/lpp-Mx 去除 GNNV 的效果。因此，我们的 GNNV 结合细菌是一种从 10‰咸水中去除 GNNV 和预防石斑鱼感染 GNNV 的有效微粒。
