以腺病毒为模式分析影响病原微生物2种常用灭活方法可靠性的因素

目的以表达绿色荧光蛋白的重组腺病毒（rAdvGFP）为模式病毒，模拟实验室内利用次氯酸钠灭活含病毒废液和利用紫外线对生物安全柜台面进行消毒的实验室操作，并评价其可靠性及影响因素。 方法终浓度为0（阳性对照）、0．5％、0．2％、0．1％、0．05％的次氯酸钠溶液（0．5％、0．2％、0．1％、0．05％次氯酸钠组）分别与rAdvGFP溶液（1×10^6 TCID50）混合后，将混合液原液与混合液的1：10～1：10^5倍比稀释液分别接种人胚肾293细胞，在荧光显微镜下观察GFP蛋白的表达。将rAdvGFP溶液（1×10^3 TCID50）置于紫外灯下分别照射0（阳性对照）、15、30、60、120min（15、30、60、120min照射组），各组再按垂直照射高度的不同分为5、10、20、30、67cm亚组，照射后收集各组病毒液接种293细胞，在荧光显微镜下观察GFP蛋白的表达。实验均重复3次。 结果各组次氯酸钠病毒混合液原液均造成细胞不同程度的损伤，其中0．5％次氯酸钠组混合液原液对细胞的毒性作用较强；稀释10倍后，除0．05％次氯酸钠组外，其余各浓度次氯酸钠组稀释液均导致细胞不同程度的死亡；稀释100倍后，仅0．5％次氯酸钠组稀释液仍对细胞具有毒性作用。荧光显微镜下观察显示，仅0．1％次氯酸钠组的1：10、1：10^2倍稀释液和0．05％次氯酸钠组的1：10、1：10^2、1：10^3倍稀释液接种细胞可见绿色荧光。紫外线照射后在荧光显微镜下观察显示，15min照射组的各亚组细胞均可见明显绿色荧光；30min照射组的20、30、67cm亚组与60、120min照射组的67cm亚组细胞仍可见绿色荧光。 结论有效氯浓度须在0．2％以上、安全柜内1．12mW／cm^2强度紫外线照射30min以上方能有效灭活重组腺病毒，建立实验室操作的可靠性评价技术体系并据此制定标准操作规程很有必要性。

Analysis of the factors interfering the reliability of two microorganism inactivation methods by using adenovirus as a model

Objective To explore the necessity and possibility of developing laboratory biosafety assessment technology by mimic two most frequently used laboratory procedures, i.e. inactivating virus with disinfectant sodium hypochlorite or with ultraviolet (UV) radiations, by using a recombinant adenovirus (rAdv) expressing green fluorescent protein (GFP) as a model and analyzing the factors those interfere the inactivating effects. Methods The model adenovirus rAdvGFP (1 × 10⁶ TCID50) was treated with sodium hypochlorite at different final concentration of 0 (positive control), 0.5%, 0.2%, 0.1% and 0.05% (0.5%, 0.2%, 0.1%, and 0.05% sodium hypochlorite groups), respectively. The mixture of sodium hypochlorite and virus was 10 - 10⁵-fold diluted. The effects of sodium hypochlorite treatment at different concentration were evaluated by the isolation and culture of original and serial dilution of sodium hypochlorite treated rAdvGFP on monolayer HEK 293 cells and the observation of expression of GFP with fluorescent microscopy. The model virus rAdvGFP (1 × 10⁶ TCID50) was treated with UV irradiations for 0 (positive control), 15, 30, 60, and 120 min (15, 30, 60, and 120 min UV irradiation groups), respectively, and each group was divided into subgroups of 5, 10, 20, 30 and 67 cm according to the vertical height from the UV lamp. The effects of UV irradiation were evaluated by the isolation and culture of rAdvGFP on monolayer HEK 293 cells after treatment. All experiments were repeated for three times. Results The original solution of different concentration of sodium hypochlorite treated virus mixture affected the viability of HEK 293 cells and the 0.5% sodium hypochlorite group had the most apparent effect. HEK 293 cells were damaged even after 10-fold dilution in the sodium hypochlorite treating groups exception the 0.05% sodium hypochlorite group. HEK 293 cells were damaged only in the 0.5% sodium hypochlorite group after 10²-fold dilution. GFP expression was observed only at 10 - 10²-fold dilution of 0.1% sodium hypochlorite group and at 10 - 10³-fold dilution of 0.05% sodium hypochlorite group, respectively. After the UV irradiation, GFP fluorescence was apparently observed by using fluorescent microscopy in all the UV-irradiated subgroups of 15 min UV irradiation group, in subgroups of 20, 30, and 67 cm after 30 min treatment, and in subgroups of 67 cm after 60 and 120 min treatment. Conclusions The 1 × 10⁶ TCID50 rAdvGFP could only be inactivated by using sodium hypochlorite at more than 0.2% concentration, while at more than 1.12 mW/cm2 after 30 min by UV irradiation. Our preliminary data raises the necessity to develop related methodologies to evaluate the reliability of laboratory biosafety measures and to make the standard operating procedures based on such evaluations.