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草鱼呼肠孤病毒Ⅱ型(GCRVⅡ)是当前导致草鱼出血病流行和暴发的主要基因型,本研究拟通过分析GCRVⅡ代表株HZ08在不同鱼类细胞中的增殖情况来筛选GCRVⅡ的敏感细胞系。首先以不同浓度的HZ08株接种草鱼吻端成纤维细胞(PSF)和草鱼鳔细胞(GSB),以确定病毒接种的最佳浓度;然后以最佳接种浓度同时感染PSF、GSB、草鱼肝细胞(L8824)、草鱼卵巢细胞(CO)等10种鱼类细胞,接毒后每天观察细胞状态,用荧光定量PCR(q PCR)方法定量分析HZ08在各种细胞系中的增殖量,并在感染5 d后用间接免疫荧光定性分析病毒在各种细胞中的增殖情况。结果显示,HZ08感染细胞的最佳接种浓度为1.0×104拷贝/μL,该毒株接种的10种鱼类细胞均无明显的细胞病变效应(CPE);q PCR分析病毒感染5~8 d后的结果显示,HZ08在GSB、L8824、PSF、草鱼鳍条细胞(CF)、CO、草鱼脑细胞(CIB)、锦鲤吻端细胞(KS)7种细胞中均能增殖,其中在GSB、L8824和PSF细胞中的增殖量较大,最大分别为1.14×107、5.90×106和6.30×104拷贝/μL。而在鲤上皮细胞(EPC)、锦鲤脑细胞(KB)、鲫脑细胞(Cc B)中不增殖,免疫荧光定性检测结果与荧光定量检测结果相吻合,在GSB、L8824和PSF中的荧光信号较多、较强,其他细胞中则较弱或没有荧光信号。研究表明,GSB、L8824和PSF是GCRVⅡ较为敏感的细胞系,该研究结果对今后Ⅱ型GCRV的研究和防控产品开发具有重要意义。
Grass carp reovirus type Ⅱ (GCRVⅡ) is the major genotype that causes the epidemic and outbreak of grass carp hemorrhage. In this study, GCRVⅡ sensitive cell lines were screened by analyzing the proliferation of GCRVⅡ representative strain HZ08 in different fish cells. Firstly, grass carp fibroblasts (SCF) and grass carp (SCB) were inoculated with different concentrations of HZ08 to determine the best concentration of virus inoculation. Then the PSF, GSB and grass carp hepatocytes L8824) and grass carp ovary cells (CO). The proliferation of HZ08 cells in various cell lines was quantified by quantitative PCR (qPCR) d by indirect immunofluorescence qualitative analysis of virus proliferation in a variety of cells. The results showed that the optimal inoculation concentration of HZ08 infected cells was 1.0 × 104 copies / μL, and no significant cytopathic effect (CPE) was observed in 10 kinds of fish cells inoculated with this strain. After 5 to 8 days of virus infection by q PCR, The results showed that HZ08 could proliferate in seven kinds of cells including GSB, L8824, PSF, CF, CO, CIB and KS, among which, GSK, L8824 And PSF cells proliferated at a maximum of 1.14 × 107, 5.90 × 106 and 6.30 × 104 copies / μL, respectively. But not in EPCs, KB cells, and Cc B cells. The results of immunofluorescence assay coincided with the results of fluorescence quantitative detection, and the fluorescence in GSB, L8824 and PSF More signals, stronger, the other cells are weak or no fluorescence signal. Studies have shown that GSB, L8824 and PSF are more sensitive GCRV Ⅱ cell lines, the results of this study for the future Ⅱ GCRV research and prevention and control product development is of great significance.