Universidade Estadual do Ceará (UECE) - Campus Itaperi - Bloco D - Laboratório de Bioquímica Humana., Av. Parajana, 1700, Cep. 60.000-007, Fortaleza, Ceará, Brazil.
J Virol Methods. 2011 Oct;177(1):10-4. doi: 10.1016/j.jviromet.2011.05.032. Epub 2011 Jun 30.
In Brazil, shrimp farming has been developed most intensely in the Northeast Region. Recently, however, exporters have become concerned over the appearance of Infectious Myonecrosis (IMN), the etiological agent of which is a virus called Infectious Myonecrosis Virus (IMNV). Although IMNV has been characterized extensively, purification methods are complicated to reproduce and very expensive. The objective of this study was to purify the IMNV virus using an easy reproductive method and to produce anti-IMNV antibodies to be used in diagnostic methods. Shrimp samples showing symptoms of IMN obtained from two aquaculture farms in Ceará were used for this purpose. IMNV-positive shrimps were macerated in phosphate buffer, pH 7.5, enriched with antioxidants, clarified with chloroform and the supernatant was submitted to differential centrifugation, precipitated using PEG and NaCl and finally loaded on a discontinuous gradient of sucrose. Purified IMNV was submitted to RT-PCR and electrophoresis either in agarose gel or SDS-PAGE, which revealed RNA and protein bands, characteristic of IMNV. IMNV induced humoral immune response in Swiss mice when administered subcutaneously. Anti-IMNV antibodies were identified by ELISA (enzyme-linked immunosorbent assay) and Western blotting methods and produced a response against purified IMNV and the crude extract obtained from the infected shrimp. However, antibodies specific to the crude extract obtained from uninfected shrimp were not detected. This is the first report of IMNV having been purified in Brazil and the first time that specific antibodies against IMNV proteins have been produced. These results suggest that easy methods can be developed to produce specific antiserum for viral diagnosis on a large scale.
在巴西，虾养殖业在东北地区发展最为活跃。然而，最近出口商开始对传染性肌肉坏死症（IMN）的出现感到担忧，其病原体是一种名为传染性肌肉坏死病毒（IMNV）的病毒。尽管已经对 IMNV 进行了广泛的特征描述，但纯化方法复杂，难以复制且非常昂贵。本研究的目的是使用简单的繁殖方法来纯化 IMNV 病毒，并生产用于诊断方法的抗 IMNV 抗体。为此，使用了来自塞阿拉州两个养殖场的出现 IMN 症状的虾样本。用 pH 值为 7.5 的磷酸盐缓冲液将 IMNV 阳性虾捣碎，用抗氧化剂富集，用氯仿澄清，然后将上清液进行差速离心，用 PEG 和 NaCl 沉淀，最后加载到蔗糖不连续梯度上。纯化的 IMNV 进行了 RT-PCR 和电泳，无论是在琼脂糖凝胶还是 SDS-PAGE 中，都显示出 RNA 和蛋白质条带，这是 IMNV 的特征。皮下注射 IMNV 可诱导瑞士小鼠产生体液免疫反应。通过 ELISA（酶联免疫吸附试验）和 Western blot 方法鉴定抗 IMNV 抗体，并对纯化的 IMNV 和从感染虾中获得的粗提物产生反应。然而，未检测到针对未感染虾粗提物的特异性抗体。这是巴西首次报道纯化的 IMNV，也是首次生产出针对 IMNV 蛋白的特异性抗体。这些结果表明，可以开发出简单的方法来大规模生产用于病毒诊断的特异性抗血清。
