狂犬病病毒PM株病毒滴度检测改良蚀斑法的建立

Development of an improved plaque assay for determination of titer of rabies virus PM strain

目的建立检测狂犬病病毒PM株病毒滴度的改良蚀斑法,并验证其精密度。方法于细胞接种病毒后第6天,分别加入不同浓度(0、50、75、100、200、400 mmol/L)HEPES溶液,以及分别于细胞接种病毒后第2、4、6、8天,加入最适浓度的HEPES溶液,确定HEPES最适加入浓度及蚀斑最适判定时间,建立改良的蚀斑法,检测48批狂犬病病毒毒力,并与小鼠脑内滴定法进行相关性比较;重复检测3次10批狂犬病病毒收获液毒力,计算变异系数(CV)。结果接种病毒后第6天后加入200 mmol/L HEPES溶液,形成的蚀斑清晰,易于计数。建立的蚀斑法与小鼠脑内滴定法具有高度相关性,3次检测结果的CV在0. 4%～3. 38%之间,重复性良好。结论加入HEPES溶液改良后的蚀斑法,操作便捷,是较为理想的滴定狂犬病病毒PM株毒力的方法,可替代小鼠脑内滴定法。

Objective To develop an improved plaque assay for determination of titer of rabies virus PM strain and verify its precision. Methods BHK21 cells 6 d after inoculation with PM strain were treated with HEPES at concentrations of 0,50,75,100,200 and 400 mmol/L respectively to optimize the HEPES concentration. The cells on days 2,4,6 and 8 after inoculation were treated with HEPES at the optimal concentration to optimize the time for judgment of the result.On the basis of this,an improved plaque assay was developed and used for determination of titers of 48 batches of rabies virus,of which the results were compared with those of intracerebral titration in mice. The titers of 10 batches of rabies virus were determined for 3 times,and the CVs of results were calculated. Results The plaques formed in the cells treated with 200 mmol/L HEPES on day 6 after virus inoculation were clear and easy to count. The test results by the developed plaque assay was highly related to that by intracerebral titration in mice. The CVs of determination results of 10 batches of virus for 3 times were 0. 4%~ 3. 38%,indicating a high reproducibility. Conclusion The improved plaque assay by addition of HEPES was simple to handle,which might be a satisfactory method for titration of rabies virus PM strain instead of intracerebral titration in mice.