Alenton Rod Russel R, Mai Hung N, Dhar Arun K
Aquaculture Pathology Laboratory, School of Animal and Comparative Biomedical Sciences, The University of Arizona, Tucson, AZ 85721, USA.
PNAS Nexus. 2023 Aug 23;2(9):pgad278. doi: 10.1093/pnasnexus/pgad278. eCollection 2023 Sep.
Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV carrying the GFP RNA cargo resulting in MrNV-GFP. Upon incorporating MrNV-GFP in the diet of the marine Pacific white shrimp (), MrNV particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within 8 days postadministration of the viral vector, which indicated a lack of MrNV-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.
病毒性疾病大流行是全球甲壳类养殖经济损失的主要原因。虽然基于RNA干扰（RNAi）的疗法在实验室规模上已显示出前景，但如果没有有效的口服给药平台，基于RNA的疗法就无法充分发挥其控制甲壳类病毒性疾病的潜力。利用反向工程的对虾RNA病毒——诺达病毒（MrNV），我们开发了一种用于递送工程化RNA货物的对虾病毒载体。通过用编码绿色荧光蛋白（GFP）的货物RNA替换MrNV的RNA依赖性RNA聚合酶（RdRp）蛋白编码区作为概念验证，我们产生了携带GFP RNA货物的无复制能力的突变体MrNV，即MrNV-GFP。将MrNV-GFP掺入海洋太平洋白对虾的饲料中后，在血细胞中观察到MrNV颗粒，证明载体成功内化。血细胞的荧光成像显示了GFP蛋白、MrNV衣壳RNA（RNA2）以及掺入的GFP RNA货物的表达。在肝胰腺和腹足中检测到货物RNA，表明病毒载体发生了全身扩散。在施用病毒载体后8天内，MrNV RNA2和GFP RNA的定量负荷逐渐减少，这表明MrNV-GFP在对虾中没有复制。此外，在处理过的对虾的靶组织中，使用组织病理学未检测到野生型MrNV感染的病理特征。这些数据明确证明了成功构建了一种用于RNA递送的无复制能力的病毒载体，为在养殖甲壳类动物中口服递送抗病毒疗法铺平了道路。
