Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, 629 502, Tamilnadu, India; Institute of Marine and Environmental Technology (IMET), University of Maryland Baltimore Country (UMBC), Baltimore, MD, 21202, USA.
Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, 629 502, Tamilnadu, India.
Fish Shellfish Immunol. 2019 Mar;86:1123-1129. doi: 10.1016/j.fsi.2018.12.010. Epub 2018 Dec 27.
White Tail Disease (WTD) is one of the important viral diseases of fresh water giant prawn Macrobrachium rosenbergii, which is caused by Macrobrachium rosenbergii nodavirus (MrNV). In the present study, the capsid protein gene of MrNV containing a His-tag was cloned into a baculovirus vector pVL1393 and expressed the recombinant MrNV protein in insect cells, using a baculovirus expression system. A band corresponding to the MrNV protein of 43 kDa was characterized after fractionating the proteins of baculovirus-infected cell lysates by SDS-polyacrylamide gel, and immunostaining with His-tag monoclonal antibody. Furthermore, purified MrNV capsid protein assembled into virus-like particles (VLPs) of ∼30 nm in diameter, when examined by transmission electron microscopy (TEM). To vaccinate the larvae by oral route, the recombinant MrNV (r-MrNV) protein was coated with artificial prawn feed and fed to M. rosenbergii larvae (90 ± 10 mg) for 60 days. After 30 and 60 days of vaccine treatment, group of prawns were challenged with virulent MrNV orally. Samples were collected at different time intervals to evaluate the survival of larvae and to analyze the presence of MrNV by double-step PCR and expression of immune/ toll-like receptor (TLR) genes. Non-vaccinated group of M. rosenbergii larvae succumbed to death and had 90% mortality, whereas the r-MrNV protein treated groups exhibited 65 and 80% survival (P ≤ 0.001) for 30 and 60 days post-vaccination (dpv), respectively. Double-step PCR diagnosis revealed that there was 100% positive signals observed in non-vaccinated prawn group, whereas the infection was reduced significantly (P < 0.001) to 32 and 17% respectively in 30 and 60 dpv. Among the four different immune/ TLR genes such as antimicrobial peptide (Mramp), lysozyme (MrLY), proPhenol Oxidase (MrPPO) and Toll-Like Receptor (MrToll) expression screening, Mramp was successfully expressed in the MrNV subunit protein vaccinated prawns, whereas the non-vaccinated prawn had no immune/TLR gene expression. Taken together, our results demonstrate that oral vaccination of M. rosenbergii larvae with baculovirus-expressed MrNV capsid protein confer up to 78% protection against MrNV infection.
白斑综合征（WTD）是淡水罗氏沼虾（Macrobrachium rosenbergii）的重要病毒性疾病之一，由罗氏沼虾诺达病毒（MrNV）引起。在本研究中，MrNV 的衣壳蛋白基因被克隆到杆状病毒载体 pVL1393 中，并在昆虫细胞中表达重组 MrNV 蛋白，使用杆状病毒表达系统。通过 SDS-聚丙烯酰胺凝胶分离杆状病毒感染细胞裂解物中的蛋白质后，用 His 标签单克隆抗体进行免疫染色，可鉴定出约 43 kDa 的 MrNV 蛋白条带。透射电子显微镜（TEM）检查显示，纯化的 MrNV 衣壳蛋白组装成直径约 30nm 的病毒样颗粒（VLPs）。为了通过口服途径给幼虫接种疫苗，将重组 MrNV（r-MrNV）蛋白包被在人工对虾饲料上，并给 90±10mg 的罗氏沼虾幼虫投喂 60 天。在疫苗处理 30 和 60 天后，通过口服用毒力 MrNV 对虾进行攻毒。在不同时间间隔采集样本，以评估幼虫的存活率，并通过两步 PCR 和免疫/ toll 样受体（TLR）基因的表达来分析 MrNV 的存在。未接种疫苗的罗氏沼虾幼虫全部死亡，死亡率为 90%，而 r-MrNV 蛋白处理组在接种后 30 和 60 天分别表现出 65%和 80%的存活率（P≤0.001）。两步 PCR 诊断显示，未接种疫苗的虾组有 100%的阳性信号，而在 30 和 60 天攻毒后，感染显著减少（P<0.001），分别为 32%和 17%。在抗菌肽（Mramp）、溶菌酶（MrLY）、原酚氧化酶（MrPPO）和 Toll 样受体（MrToll）等四种不同的免疫/TLR 基因表达筛选中，Mramp 在接种 MrNV 亚单位蛋白的罗氏沼虾中成功表达，而未接种疫苗的罗氏沼虾则没有免疫/TLR 基因表达。总之，我们的结果表明，用杆状病毒表达的 MrNV 衣壳蛋白对罗氏沼虾幼虫进行口服免疫接种可提供高达 78%的保护，防止 MrNV 感染。
