稳定表达胰蛋白酶原的重组MDCK细胞分离流感病毒效果初步分析

Preliminary analysis of isolation effect of influenza virus by recombinant MDCK cells stably expressing trypsinogen

目的 初步考察稳定表达胰蛋白酶原的重组MDCK细胞(即MTY6细胞)分离流感病毒的效果。方法 按照世界卫生组织(World Health Organization,WHO)全球流感监测网络和国家流感中心(National Influenza Centers,NICs)推荐的病毒分离方法,使用MDCK和MTY6细胞同步分离包含H1N1、H3N2和B型流感病毒核酸阳性的咽拭子样本共20份,对分离培养液分别使用豚鼠红细胞和鸡血红细胞进行凝集试验,统计比较不同类型红细胞凝集效果,以及不同细胞分离病毒阳性率和血凝素滴度。结果 相同病毒分离物对两种类型红细胞的凝集效果不同,豚鼠红细胞完全凝集时间约为鸡血红细胞2倍,沉积形状呈环状,血凝滴度平均为鸡红细胞的23.6±1.2倍。相同条件下,有3份样本两种细胞均分离阴性,11份样本两种细胞均分离阳性,其余6份样本MDCK细胞分离阴性,而MTY6细胞分离阳性,MTY6细胞分离阳性率较MDCK细胞高30%;分离阳性样本包括H1N1亚型8份,MTY6细胞分离病毒血凝素滴度显著高于MDCK细胞,平均为13.0(1.7,23.0)倍;H3N2亚型和B型各2份,MTY6细胞分离病毒血凝素滴度均高于MDCK细胞,平均为11.3和32.0倍。结论 综合考虑,对于细胞分离流感病毒检测,豚鼠红细胞优于鸡血红细胞。相同条件下,MTY6细胞较MDCK细胞分离流感病毒的灵敏度更高,具有作为流感病毒分离优质细胞基质的潜力。

Objective To investigate the isolation effect of influenza virus by recombinant MDCK cells(MTY6 cells)stably expressing trypsinogen.MethodsAccording to the virus isolation method recommended by the World Health Organization(WHO)Global Influenza Surveillance Network(GISN)and the National Influenza Centers(NICs),a total of 20 throat swab specimens containing positive nucleic acid for H1N1,H3N2 and B influenza virus were isolated simultaneously using MDCK and MTY6 cells.Guinea pig red blood cells and chicken red blood cells were used for agglutination test respectively and the agglutination effects of different types of red blood cells,the positive rate of virus and the titer of hemagglutinin isolated from different cells were statistically compared.ResultsThe agglutination effect of the same virus isolate on the two types of red blood cells was different.The complete agglutination time of guinea pig red blood cells was about 2 times that of chicken red blood cells,and the deposition shape showed a ring shape.The average hemag-glutinin titer was 23.6 ± 1.2times that of chicken red blood cells.Under the same conditions,3 samples were negative for both types of cells,11 samples were positive for both types of cells,and the other 6 samples were negative for MDCK cells while positive for MTY6 cells.The positive rate of MTY6 cells was 30% higher than that of MDCK cells.The isolated positive samples included 8 cases of H1N1 subtype and the hemagglutinin titer of virus isolated by MTY6 cells was significantly higher than that by MDCK cells[13.0(1.7,23.0)times on average].2 cases of H3N2 and 2 cases of B were isolated,the hemagglutinin titer of each virus isolated by MTY6 cells was 11.3 and 32.0 times higher than that by MDCK cells on average respectively.ConclusionIn conclusion,guinea pig red blood cells were superior to chicken red blood cells for influenza virus detection by cell isolation.Under the same conditions,MTY6 cells were more sensitive than MDCK cells for influenza virus isolation,and had the potential to be used as a high-quality cell matrix for influenza virus isolation.