5种虫媒病毒的细胞培养及空斑试验

Cell culture and plaque forming test of five arboviruses

目的 :选择一株对 5种虫媒病毒 :马雅罗病毒 (MAY)、西门利克病毒 (SFV)、墨累谷脑炎病毒 (MVE)、伊利乌斯病毒 (ILH)和布尼安姆韦拉病毒 (BUN)敏感的细胞用于这 5种病毒生物学性状的研究。方法 :将不同稀释度的乳鼠脑病毒悬液接种到BHK2 1 ,LLC_MK2 ,A549和C6 36传代细胞上 ,进行这 5种病毒的敏感性试验 ,选出一株对 5种病毒较敏感的细胞株 ;对敏感细胞株进行三因素三水平正交试验 ,探索细胞的最佳培养条件 ;进行 5种病毒的空斑试验。结果 :BHK2 1 细胞对 5种病毒细胞病变特征明显 ,出现时间较早 ,滴度较高 ,适用于 5种病毒的培养。在合适条件下 ,BHK2 1 细胞单层可以维持 7d以上 ,5种虫媒病毒在BHK2 1 细胞上出现了大小、形态都不相同的空斑。结论 :BHK2 1 细胞在 6 0 %DMEM培养液、30 %的 0 .5 %水解乳蛋白、10 %小牛血清、常量青、链霉素和 5 0 μg ml卡那霉素 ,保持pH 7.2条件下生长最好 ,能够满足 5种虫媒病毒生物学性状研究的需要。

Objective:To select a sensitive cell and to establish the best cell culture conditions used in studying biological characters of five arboviruses.Methods:Suckling mouse brain suspensions at different dilutions were inoculated to monolayer of BHK 21 , LLC_MK 2, A 549 and C6/36 cells to test their sensitivity to the viruses.Cytopathic effect(CPE) appearing time, virus titers and character of CPE were also observed. Then, the L 9(3 3) orthogonal design test of BHK 21 cell culture and plaque forming test were conducted.Results:All the five arboviruses could be cultured in the four cell strains and all caused earlier appearance of CPE in BHK 21 cells which showed distinct CPE character and higher titer than in the other three cell strains.The result of orthogonal test of five arboviruses in culturing cells showed plaques in BHK 21 cell monolayer. Conclusions:Under the condition of 60%DMEM, 30% hydrolyzed lactalbumin(HL)(0.5%), 10% serum, 100*!U/ml penicillin and streptomycin and 50*!μg/ml kanamycin, pH 7.2, BHK 21 cell could grow well enough to fulfil the requirement of studying biological characters of five arboviruses.