人肺腺癌细胞A549对蜱媒脑炎病毒的易感性分析

Susceptibility of human lung adenocarcinoma cell line A549 to tick-borne encephalitis virus

目的研究蜱媒脑炎病毒(TBEV)在易感细胞中的培养特性,并鉴定病毒RNA的复制、蛋白质表达和感染性。方法利用非洲绿猴肾细胞Vero培养TBEV,采用qPCR检测TBEV RNA的复制水平,采用蛋白质印迹法检测黄病毒包膜蛋白的表达,采用空斑实验检测培养基中TBEV的滴度。用TBEV感染人肺腺癌细胞A549,于显微镜下观察TBEV感染A549细胞的细胞病变效应。通过免疫荧光法检测TBEV感染的A549、Vero细胞中黄病毒包膜蛋白的表达。结果与孵育48 h的细胞相比,孵育72 h的Vero细胞内TBEV RNA水平更高,并检测到有黄病毒包膜蛋白表达。空斑实验结果显示,在孵育72 h Vero细胞的培养基中,TBEV病毒滴度为(2.0±1.4)×10^(6)空斑形成单位(PFU)/mL。TBEV感染可引起A549细胞的细胞病变效应。在1∶1000稀释TBEV感染的细胞中,A549细胞中黄病毒包膜蛋白表达的阳性率(61.0%)高于Vero细胞(9.3%)。TBEV感染A549细胞48 h培养基中的病毒滴度高于Vero细胞培养基中的病毒滴度[(2.0±0.4)×10^(7)PFU/mL vs(8.5±2.1)×10^(3)PFU/mL,P<0.05]。结论Vero细胞可用于培养TBEV,A549细胞对TBEV更易感。

Objective To study the culture characteristics of tick-borne encephalitis virus(TBEV)in susceptible cells and identify the RNA replication,protein expression and infectivity.Methods The TBEV was cultured using African green monkey kidney cells(Vero cells).The RNA replication of TBEV was detected by quantitative polymerase chain reaction,the expression of flavivirus envelope protein was detected by Western blotting,and the titer of TBEV was detected by plaque assay.The human lung adenocarcinoma cells(A549 cells)was infected with TBEV,and the cytopathic effect of A549 cells infected with TBEV was observed under microscope.The expression of flavivirus envelope protein in A549 and Vero cells infected with TBEV was detected by immunofluorescence staining.Results The levels of TBEV RNA were increased in Vero cells at 72 h incubation compared with those at 48 h incubation,and the expression of flavivirus envelope protein was detectable.The results of plaque assay showed that the TBEV titer in the supernatant of Vero cells was(2.0±1.4)×10^(6) plaqueforming unit(PFU)/mL after culturing for 72 h.There was obvious cytopathic effect in A549 cells with TBEV infection.The positive rate of flavivirus envelope protein expression in A549 cells(61.0%)was higher than that in Vero cells(9.3%)after infection with 1∶1000-diluted TBEV.Compared with Vero cells,the titer in the supernatant of A549 cells infected with TBEV for 48 h was significantly higher([2.0±0.4]×10^(7)PFU/mL vs[8.5±2.1]×10^(3)PFU/mL,P<0.05).Conclusion Vero cells can be used to culture TBEV,and A549 cells are more susceptible to TBEV.