罗氏沼虾苗种肌肉白浊病诺达病毒的分离和特性研究

Isolation and characterization of Nodavirus caused whitish muscle diseases in Macrobrachium rosenbergii larvae

本实验在以往罗氏沼虾(Macrobrachiumrosenbergii)苗种肌肉白浊病病毒致病性的基础上对病毒特性进行深入研究。病虾除菌过滤液以1:50和1:250浸泡感染可复制出典型的症状,病毒对氯仿抵抗;用PEG法浓缩病毒后,35000r/min超速离心部分纯化病毒,在电镜下可观察到大量直径24nm的球形病毒颗粒,无囊膜,此外还观察到大小为14nm的病毒粒子;采用Trizol试剂对病毒进行了核酸提取,电泳结果发现有4条核酸条带,分别为3.0kb、1.3kb、0.8kb、0.75kb,对RNA核酸酶、S1核酸酶敏感,对DNA酶抵抗,为单链RNA病毒。用罗氏沼虾诺达病毒核酸探针进行分子杂交,探针可与提纯核酸及病虾样品匀浆液反应,Southern杂交表明3.0kb、1.3kb条带分别属于诺达病毒的RNA1和RNA2。对不同的发病样品进行了核酸电泳,4个典型症状的样品均存在诺达病毒条带,2个样品还存在小病毒核酸。上述结果表明,诺达病毒是引起罗氏沼虾苗种肌肉白浊病的主要病原。14nm小病毒与罗氏沼虾肌肉白浊病中的关系有待进一步的研究。

The virus, which has caused whitish muscle diseases of M. rosenbergii, and led to a new epidemic spread out since 1996 in larvae and postlarvae of giant freshwater prawn in South China , was isolated and characterized. The typically diseased M. rosenbergii larvae were collected from some culture farms in Zhejiang province and Jiangsu province. The healthy individuals were from the experimental farm of Zhejiang Institute of Freshwater Fisheries. The virus can cause natural symptoms when healthy postlarvae was used for immersion challenging by using bacteriafree tissue virus homogenate at concentration of 1:50 and 1:250, and is resistant to 10% chloroform treatment. The virus was partly purified with PEG precipitation followed by centrifugal at 35 000 r/min, and observed under electronic microscope after 2% PTA negative staining. Plenty of spherical virus could be found at the size of 24 nm in diameter and the same smaller particles sized 14 nm could be found also. The nucleic acid of virus was extracted by using the Trizol reagents and analyzed with 1% agarose electrophorosis. It shows the nucleic acid has four bands sized 3.0, 1.3, 0.8 and 0.75 kb respectively, and is sensitive to RNase and S1 nuclerase and resistant to DNase, indicating these bands as singlestrand RNA. The molecular hybridization test was carried out with Nodavirus probe in M. rosenbergii, showing strong signal to the extracted RNA and diseased prawn homogenate. Two larger bands were confirmed as Nodavirus RNA1 and RNA2 by Southern blot. The bands of Nodavirus were found in all samples with typical symptoms collected from 2000 to 2002, while the 0.8 and 0.75 kb bands were only found in two of four samples. These results indicated that Nodavirus was the main pathogen of whitish muscle diseases in M. rosenbergii larvae in China in recent years. The virus was named as Macrobrachium rosenbergii Nodavirus (MrNV). The classification and function of the smaller viral particles of 14 nm in diameter remained unclear.