摘要目的 针对中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV),建立安全、操作简易的空斑试验检测方法.方法 将MERS-CoV(EMC/2012)感染Vero或Huh7.5细胞,通过研究MERS-CoV感染后镜下细胞病变与染色后蚀斑,比较染毒时间(2 d和3 d)以及细胞培养覆盖物[琼脂、微晶纤维素-羧甲基纤维素钠复合物(Avicel)]对空斑试验的影响,确定MERS-CoV空斑试验适用的细胞类型、染毒时间以及细胞培养覆盖物,并将该方法用于MERS-CoV中和抗体的检测.结果 MERS-CoV感染Vero细胞后能形成清晰的病毒空斑;染毒后3 d后能稳定清晰地形成MERS-CoV特异性空斑;微晶纤维素-羧甲基纤维素钠复合物覆盖细胞所产生的病毒空斑较琼脂覆盖物更为清晰,操作简易,更易于在BSL-3实验室进行操作;该方法可用于MERS-CoV中和抗体的检测.结论建立了基于Avicel-结晶紫染色技术的适用于BSL-3实验室操作的MERS-CoV空斑试验方法,为MERS-CoV的滴定、中和抗体检测等提供了实验资料.

abstractsObjective To establish a plaque assay for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Methods Vero and Huh7. 5 cells were infected with MERS-CoV (EMC/2012) strains for different days (two or three days) and covered with different overlays (agarose or Avicel). The optimal host cells, time of infection and overlay were screened out by analyzing plague formation to establish the plaque assay for detection of MERS-CoV infection. Neutralizing antibodies against MERS-CoV were detected by the established assay. Results Clear plaques were observed on Vero cells infected with MERS-CoV at 3 days post-infection ( p. i. ) , while only tip-size plaques were observed at 2 days p. i. . Clearer and bigger sizes of plaques were observed under the overlay of Avicel than those under the agarose. The Avicel-based plaque assay was suitable for detecting neutralizing antibodies against MERS-CoV. Conclusion We develop a modified plaque assay for detecting MERS-CoV based on Avicel-crystal violet staining. It is easier to operate the modified plaque assay in biological safety level 3 ( BSL-3 ) laboratories. This study paves a way for quantification of MERS-CoV and detection of neutralizing antibodies against MERS-CoV.