国内首次发现的新型虫媒病毒——南定病毒致C6/36细胞病变情况研究

Study on CPE of the first identified NDiV in China on cultured C6/36 cells

目的探讨国内首次分离的南定病毒致白纹伊蚊细胞病变情况。方法用长成单层的白纹伊蚊细胞感染本课题组在国内首次发现的南定病毒,观察细胞病变情况和进行分子生物学检测。结果南定病毒接种白纹伊蚊细胞后,7d开始出现病变,细胞固缩,单层细胞稀疏,细胞之间空隙增大,直到细胞数量减少,细胞间空隙增大,单层消失,空白对照生长正常,TCID50为1.41×10-6PFU/ml。应用TaqMan-MGB Probe Real Time PCR技术,检测细胞病变孔的培养液,荧光PCR结果均出现"S"扩增曲线,显示均存在NDiV核酸。用TaqMan-MGB Probe Real Time PCR制作相对定量标准曲线,6个稀释度的Ct值标准曲线R2≈1.0,呈线性关系。结论南定病毒能够引起白纹伊蚊细胞出现明显细胞病变效应。

Objective To explore the cytopathic effect （CPE） of the first isolated Nam Dinh Virus （NDiV） in China on cultured C6/36 cells. Methods NDiV was inoculated in monolayer of C6/36 cells to observe the CPE and molecular biological detection. Results NDiV had caused CPE in C6/36 cells, since 7 days after infected the cells. Cells became shrinkage,increasing the gap between cells, until the cell number decreased, and disappearing of cell monolayer. Void increased between cells,TCIDs0 was 1.41 × 10^-6 PFU/ml. The application of TaqMan-MGB Probe Real Time PCR technology to detect the NDiV in the culture fluid of lesion cell. The NDiV proliferation changing with time were showed that Ct value of isolates gradient reduced as the time went on. These indicated the viral nucleic acid increased with increasing of the incubation time. Ct value standard curve and six dilution showed linear relation, interrelated,R2 = 1.0. Conclusion NDiV can cause obvious cytopathic effect in C6/36 Cells.