四种病毒灭活方法用于登革病毒灭活效果的比较

Assessment of the inactivation efficacy of Dengue Virus by four different methods

目的评价常用病毒灭活方法对血液制品中登革病毒(DENV)的灭活效果。方法将单采新鲜冰冻血浆(FFP),人凝血因子Ⅷ(FⅧ)和静脉注射免疫球蛋白(IVIG)3种血浆及其制品中加入高滴度(8.00-9.25)的登革病毒液,分别采用亚甲蓝(MB)光化学法灭活FFP、有机溶剂/去污剂(S/D)法灭活FⅧ、低pH常温孵化法和巴氏消毒法灭活IVIG;以1×10~6/mL A549细胞接种于T25的培养瓶中作为病毒传代及滴度滴定指示细胞,将灭活前后的血浆及制品接种于A549细胞并检测其DENV滴度,并通过qRT-PCR对DENV RNA做定量检测;评估不同的灭活方法对DENV的灭活效果。结果 DENV滴度下降:MB光化学法灭活FFP下降滴度≥5.92 log,S/D法灭活FⅧ下降滴度≥5.17 log、巴氏法和低pH法灭活IVIG均下降滴度≥5.92 log,其中MB光化学法灭活FFP、SD灭活FⅧ及巴氏法灭活IVIG后DENV RNA(cp/mL)降低1.25 log-2.25 log,低pH法灭活IVIG后DENV RNA(cp/mL)降低0.17 log。所有血浆和血液制品样品经灭活后,在DENV宿主细胞上3代盲传后均未检测到DENV RNA。结论 4种常用病毒灭活方法均能有效灭活血浆及血液制品中的DENV。

Objective To access the efficacy of four commonly-used inactivation methods on Dengue virus(DENV) spiked in plasma and derivatives. Methods High titers(8.00-9.25 log) of DENV were spiked into three blood products as fresh frozen plasma(FFP), coagulation factor Ⅷ(FⅧ) and intravenous immunoglobulin(IVIG). Methylene blue(MB) photochemical method, solvent/detergent(S/D) method, and pasteurization/low pH at room temperature were performed to inactivate DENV in FFP, FⅧ and IVIG, respectively. A549 cells(1×106/mL) were inoculated in T25 culture flask as indicator cells for virus passage and titer titration. DENV infectious titers and RNA loads were measured by cell culture-based assays and qRT-PCR before and after different inactivation treatment, as well as three passages. Results The mean reduction of DENV titers after treatment of MB, S/D, and pasteurization/low pH was 5.92, 5.17 and 5.92 logTCID50/0.1 mL, respectively. DENV RNA load decreased markedly via S/D, pasteurization and MB treatment. The reduction of RNA loads(log copies/mL) after treatment of MB, S/D and pasteurization ranged from 1.25 to 2.25;low pH treatment affected DENV RNA mildly, as only marginal decrease of RNA load(0.17 log copies/mL) was observed. No infectious virus was detected after inactivation treatment and three serial passages. Conclusion These four commonly used methods for virus inactivation can effectively inactivate high titers of DENV in spiked blood products.