H5N1亚型禽流感病毒实验全过程实验室微环境集群监测和评价
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摘要
在实验室研究流感病毒的过程中很多操作都会产生气溶胶，除了产生一些沉降于物体表面的粒径较大的气溶胶颗粒以外，更多的操作会产生粒径在1~5μm的气溶胶，比如离心，混匀等操作。这种微小粒径的气溶胶可长时间悬浮于空气中，并保持其感染性，一旦吸入进呼吸道最终将沉降在肺泡，未发病前，很难被诊断和治疗。实验人员在未知状态下吸入气溶胶引发病毒的感染，是实验室内感染的主要原因。
由于病毒气溶胶收集困难，在空气中相对浓度低，检测的方法灵敏度不高，导致无法对实验室内微环境进行检测和评价。因此本课题组采用了一种新的气溶胶收集系统，该系统是由一个控制终端和六台便携式生物气溶胶收集器通过无线网络连接，可以同时、多点的收集微生物气溶胶。通过已知ELD50的H5N1亚型禽流感病毒的滴度递减稀释，比较细胞培养吸附法、RT-PCR法、膜吸附洗脱-PCR法、RT-LAMP法几种方法的灵敏度，建立了一种针对H5N1亚型禽流感病毒气溶胶的高敏感度检测方法，即固相病毒吸附-增殖PCR法。
为了确保实验室的安全，研究实验操作禽流感病毒产生气溶胶的可能性。我们分组模拟了实验室内正常操作流感病毒的过程，分别为动物解剖组、研磨组、离心组、移液组、磁力搅拌组、鸡胚接种组和实验动物感染组，以及对照组。此外，还模拟了与正常操作相关的常见事故，包括打碎装有病毒液的玻璃容器，注射器喷射病毒液和离心管破裂。在各组操作过程中利用设计的集群式空气微生物采集系统分别进行空气样本采集。对采集到的样本进行病毒吸附-增殖的检测，即利用流感病毒凝集红细胞的特点，浓缩后接种鸡胚进行病毒增殖，再用RT-PCR的方法对增殖后得病毒液进行检测。
选取2种能产生H5N1亚型禽流感病毒气溶胶的实验室操作，评价紫外照射消毒、过氧乙酸喷雾消毒、过氧化氢喷雾消毒和二氧化氯喷雾消毒的效果，并撰写操作标准和应急预案，为实验室安全评价和规范化处理提供依据。
研究结果表明，细胞培养吸附法和膜吸附洗脱-PCR法灵敏度最低，无法检测到10倍稀释后的病毒液；RT-PCR法仅可以检测到10倍稀释的病毒液；RT-LAMP法灵敏度略高，可以检测到100倍稀释的病毒液，以上方法均不能用于H5N1亚型禽流感病毒气溶胶的检测。新建立的固相病毒吸附-增殖法结合RT-PCR，可以检测到1013倍稀释的病毒液，具有高灵敏度和特异性。
利用这种方法，对实验室各种正常操作及相关的失误操作进行病毒气溶胶检测，判断实验室可能产生气溶胶的操作程序。结果显示，离心组及对照组为阴性，其他组均为阳性。
选择磁力搅拌病毒液和打碎装有病毒液的玻璃容器2种模拟操作，对2种操作过程产生的病毒气溶胶进行消毒显示，紫外线照射消毒无法消杀H5N1亚型禽流感病毒气溶胶，而过氧化氢喷雾、过氧乙酸喷雾和二氧化氯喷雾都有很好的消毒效果，由于二氧化氯对人体无伤害，因此在H5N1亚型禽流感病毒气溶胶的应急处理中，首选二氧化氯喷雾式消毒。
实验的结果证明了实验室很多种H5N1亚型禽流感的操作都会产生病毒气溶胶，只是由于病毒气溶胶在空气中的浓度低，不易收集和检测，本实验利用新建立的高敏感度的方法，对各模拟实验操作产生的气溶胶进行检测，推荐了最佳的气溶胶消毒方法，以确保实验室的环境健康。
实验人员应该了解实验操作过程暴露的风险，积极做好安全防护工作，才能更好的防止实验室内病毒气溶胶感染以及实验室泄露风险。
Many of the routine procedures used to process influenza virus for laboratory research,such as centrifugation or mixing, have a high potential of producing aerosols, and theparticle load of each has been estimated to be up to1–5μm. In addition, it is expected thatlarger particles will tend to fall out of the air and contaminate surfaces. Fundamentally,aerosols are suspensions in the air of solid or liquid particles small enough that they willremain transmissible and airborne for a prolonged period of time. Particles of5μm or lessincrease the risk of establishing an infection upon airborne transmission, as they areremarkably capable of penetrating the physical cellular barrier of the respiratory tract andtraveling all the way to the alveolar region. As with naturally-acquired infections, mostindividuals are not diagnosed before onset of symptoms, impeding the time to initiation oftreatment.
Avian H5N1influenza viruses present a challenge in the laboratory environment, asthey are difficult to collect from the air due to their small size and relatively lowconcentration. The prototype sampling device consisted of a controller and six pumps of awireless networking technology. This instrument can collect several samples of aerosolssimultaneously, or can be set to have one of the six pumps operate independently. Thesensitivity to detect AIV was evaluated by using a10-fold diluted virus series (ELD50).Establish a sensitivity method of virus adsorb-proliferation to detect H5N1AIV aerosolsthough compared to infect a host cell, membrane adsorption elution, RT-PCR andRT-LAMP methods.
Normal laboratory procedures used to process the influenza virus were carried outindependently and the amount of virus polluting the on-site atmosphere was measured. Inparticular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation,and experimental zoogenetic infection were performed. In addition, common accidentsassociated with each process were simulated, including breaking glass containers, syringeinjection of influenza virus solution, and rupturing of centrifuge tubes. A micro-clustersampling ambient air pollution collection device was used to collect air samples. Thecollected viruses were tested for activity by measuring their ability to inducehemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription PCR.
Evaluation of ultraviolet irradiation sterilization, peracetic acid disinfection, hydrogenperoxide disinfection and chlorine dioxide disinfectant affect though Simulation twolaboratory procedures. According to the results, we proposed operating standards andcontingency plans, and provide the basis for laboratory safety evaluation and standardizationof treatment.
Traditional methods of determining the presence of virus in an aerosol include directlyapplying the concentrated aerosol to infect a host cell, or membrane adsorption elutionmethods wasn’t detectable10^dilution of the virus solution. RT-PCR to detect virus-specificgenes was detectable10^dilution of the virus solution. The sensitivity of the RT-LAMPprocedure to detect AIV was detectable100-fold dilution of the virus solution. AIV wasdetectable by virus adsorb-proliferation method up to10^13dilution of the virus solutionbecause of its remarkable sensitivity and specificity.
The results proofed that the air samples from the normal centrifugal group and thenegative-control group were negative, while all other groups were positive for H5N1.
Spray hydrogen peroxide, peracetic acid and chlorine dioxide was effective to H5N1AIV aerosols disinfection. And we recommend using the atomization chlorine dioxidebecause it is harmless.
The results showed that many of the routine procedures used to process influenza virusfor laboratory research have a high potential of producing aerosols Measuring infectiousvirus from air samples is logistically difficult. Few researchers have detected the infectiouscapacity of influenza viruses sampled from air as they are low concentration and difficult tocollect. Laboratory procedures were carried out under controlled conditions monitoring theamount and character of aerosol produced by virus adsorb-proliferation method. Besides,disinfection of virus aerosol aimed to create a range of environmentally friendly cleaning.
Our findings suggest that there are numerous sources of aerosols in laboratoryoperations involving H5N1. Thus, laboratory personnel should be aware of the exposure riskthat accompanies routine procedures involved in H5N1processing and take proactivemeasures to prevent accidental infection and decrease the risk of virus aerosol leakagebeyond the laboratory.
由于病毒气溶胶收集困难，在空气中相对浓度低，检测的方法灵敏度不高，导致无法对实验室内微环境进行检测和评价。因此本课题组采用了一种新的气溶胶收集系统，该系统是由一个控制终端和六台便携式生物气溶胶收集器通过无线网络连接，可以同时、多点的收集微生物气溶胶。通过已知ELD50的H5N1亚型禽流感病毒的滴度递减稀释，比较细胞培养吸附法、RT-PCR法、膜吸附洗脱-PCR法、RT-LAMP法几种方法的灵敏度，建立了一种针对H5N1亚型禽流感病毒气溶胶的高敏感度检测方法，即固相病毒吸附-增殖PCR法。
为了确保实验室的安全，研究实验操作禽流感病毒产生气溶胶的可能性。我们分组模拟了实验室内正常操作流感病毒的过程，分别为动物解剖组、研磨组、离心组、移液组、磁力搅拌组、鸡胚接种组和实验动物感染组，以及对照组。此外，还模拟了与正常操作相关的常见事故，包括打碎装有病毒液的玻璃容器，注射器喷射病毒液和离心管破裂。在各组操作过程中利用设计的集群式空气微生物采集系统分别进行空气样本采集。对采集到的样本进行病毒吸附-增殖的检测，即利用流感病毒凝集红细胞的特点，浓缩后接种鸡胚进行病毒增殖，再用RT-PCR的方法对增殖后得病毒液进行检测。
选取2种能产生H5N1亚型禽流感病毒气溶胶的实验室操作，评价紫外照射消毒、过氧乙酸喷雾消毒、过氧化氢喷雾消毒和二氧化氯喷雾消毒的效果，并撰写操作标准和应急预案，为实验室安全评价和规范化处理提供依据。
研究结果表明，细胞培养吸附法和膜吸附洗脱-PCR法灵敏度最低，无法检测到10倍稀释后的病毒液；RT-PCR法仅可以检测到10倍稀释的病毒液；RT-LAMP法灵敏度略高，可以检测到100倍稀释的病毒液，以上方法均不能用于H5N1亚型禽流感病毒气溶胶的检测。新建立的固相病毒吸附-增殖法结合RT-PCR，可以检测到1013倍稀释的病毒液，具有高灵敏度和特异性。
利用这种方法，对实验室各种正常操作及相关的失误操作进行病毒气溶胶检测，判断实验室可能产生气溶胶的操作程序。结果显示，离心组及对照组为阴性，其他组均为阳性。
选择磁力搅拌病毒液和打碎装有病毒液的玻璃容器2种模拟操作，对2种操作过程产生的病毒气溶胶进行消毒显示，紫外线照射消毒无法消杀H5N1亚型禽流感病毒气溶胶，而过氧化氢喷雾、过氧乙酸喷雾和二氧化氯喷雾都有很好的消毒效果，由于二氧化氯对人体无伤害，因此在H5N1亚型禽流感病毒气溶胶的应急处理中，首选二氧化氯喷雾式消毒。
实验的结果证明了实验室很多种H5N1亚型禽流感的操作都会产生病毒气溶胶，只是由于病毒气溶胶在空气中的浓度低，不易收集和检测，本实验利用新建立的高敏感度的方法，对各模拟实验操作产生的气溶胶进行检测，推荐了最佳的气溶胶消毒方法，以确保实验室的环境健康。
实验人员应该了解实验操作过程暴露的风险，积极做好安全防护工作，才能更好的防止实验室内病毒气溶胶感染以及实验室泄露风险。
Many of the routine procedures used to process influenza virus for laboratory research,such as centrifugation or mixing, have a high potential of producing aerosols, and theparticle load of each has been estimated to be up to1–5μm. In addition, it is expected thatlarger particles will tend to fall out of the air and contaminate surfaces. Fundamentally,aerosols are suspensions in the air of solid or liquid particles small enough that they willremain transmissible and airborne for a prolonged period of time. Particles of5μm or lessincrease the risk of establishing an infection upon airborne transmission, as they areremarkably capable of penetrating the physical cellular barrier of the respiratory tract andtraveling all the way to the alveolar region. As with naturally-acquired infections, mostindividuals are not diagnosed before onset of symptoms, impeding the time to initiation oftreatment.
Avian H5N1influenza viruses present a challenge in the laboratory environment, asthey are difficult to collect from the air due to their small size and relatively lowconcentration. The prototype sampling device consisted of a controller and six pumps of awireless networking technology. This instrument can collect several samples of aerosolssimultaneously, or can be set to have one of the six pumps operate independently. Thesensitivity to detect AIV was evaluated by using a10-fold diluted virus series (ELD50).Establish a sensitivity method of virus adsorb-proliferation to detect H5N1AIV aerosolsthough compared to infect a host cell, membrane adsorption elution, RT-PCR andRT-LAMP methods.
Normal laboratory procedures used to process the influenza virus were carried outindependently and the amount of virus polluting the on-site atmosphere was measured. Inparticular, zootomy, grinding, centrifugation, pipetting, magnetic stirring, egg inoculation,and experimental zoogenetic infection were performed. In addition, common accidentsassociated with each process were simulated, including breaking glass containers, syringeinjection of influenza virus solution, and rupturing of centrifuge tubes. A micro-clustersampling ambient air pollution collection device was used to collect air samples. Thecollected viruses were tested for activity by measuring their ability to inducehemagglutination with chicken red blood cells and to propagate in chicken embryos after direct inoculation, the latter being detected by reverse-transcription PCR.
Evaluation of ultraviolet irradiation sterilization, peracetic acid disinfection, hydrogenperoxide disinfection and chlorine dioxide disinfectant affect though Simulation twolaboratory procedures. According to the results, we proposed operating standards andcontingency plans, and provide the basis for laboratory safety evaluation and standardizationof treatment.
Traditional methods of determining the presence of virus in an aerosol include directlyapplying the concentrated aerosol to infect a host cell, or membrane adsorption elutionmethods wasn’t detectable10^dilution of the virus solution. RT-PCR to detect virus-specificgenes was detectable10^dilution of the virus solution. The sensitivity of the RT-LAMPprocedure to detect AIV was detectable100-fold dilution of the virus solution. AIV wasdetectable by virus adsorb-proliferation method up to10^13dilution of the virus solutionbecause of its remarkable sensitivity and specificity.
The results proofed that the air samples from the normal centrifugal group and thenegative-control group were negative, while all other groups were positive for H5N1.
Spray hydrogen peroxide, peracetic acid and chlorine dioxide was effective to H5N1AIV aerosols disinfection. And we recommend using the atomization chlorine dioxidebecause it is harmless.
The results showed that many of the routine procedures used to process influenza virusfor laboratory research have a high potential of producing aerosols Measuring infectiousvirus from air samples is logistically difficult. Few researchers have detected the infectiouscapacity of influenza viruses sampled from air as they are low concentration and difficult tocollect. Laboratory procedures were carried out under controlled conditions monitoring theamount and character of aerosol produced by virus adsorb-proliferation method. Besides,disinfection of virus aerosol aimed to create a range of environmentally friendly cleaning.
Our findings suggest that there are numerous sources of aerosols in laboratoryoperations involving H5N1. Thus, laboratory personnel should be aware of the exposure riskthat accompanies routine procedures involved in H5N1processing and take proactivemeasures to prevent accidental infection and decrease the risk of virus aerosol leakagebeyond the laboratory.
引文
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