【作者】 于龙；

【导师】 李劲松；

【作者基本信息】 中国人民解放军军事医学科学院 ， 军事预防医学， 2010， 博士

【摘要】 自20世纪70年代以来,在世界范围内发现和确认的新发传染病已多达40余种,在这些传染病中约50%可通过空气传播,而且几乎每年至少有一种新的传染病被发现,它们对人类健康、社会经济发展造成了严重的威胁。如2003年爆发的SARS,仅9个月,全世界30个国家有8439人感染,812人死亡;2004年起发生的高致病性禽流感;以及去年爆发的甲型H1N1流感,均造成了人员和财产的重大损失。防护装备和设施在隔离传染源、阻断微生物气溶胶的传播途径和保护易感人群三方面均发挥着重要作用,在面对大范围未知生物恐怖袭击事件、自然突发事件时,是阻止疫病扩散最快最有效的方式之一,可起到立竿见影的效果。尽管如此,有些防护装备和设施仍然存在生物学防护效果评价方法或标准不完善甚至缺乏的情况。本研究旨在建立和完善生物防护装备和设施对呼吸道病毒替代病毒气溶胶防护效果评价技术与平台,提升对生物防护装备和设施进行模拟病毒气溶胶防护效果评价的检测能力;开展对生物防护口罩、生物防护服、正压医用防护头罩、负压隔离转运舱、负压救护车排风净化装置、Ⅱ级生物安全柜和高等级生物安全实验室排风系统等生物防护装备和设施的安全性能评价。内容:1.从污水中分离大肠杆菌和粘质沙雷氏菌噬菌体并对其生物学特点进行系统研究;2.研究模式病毒耐受发生和采样压力特点;3.建立和完善生物防护装备和设施评价技术与平台并对多种防护装备和设施进行病毒气溶胶防护效果测试评价;方法:1.采用四步法从污水中分离大肠杆菌和粘质沙雷氏菌噬菌体,通过单层平板噬菌斑和双层平板噬菌斑实验筛选烈性噬菌体,挑取单个典型噬菌斑进行噬菌体的培养增殖及纯化,电镜观察噬菌体的形态,对噬菌体最佳感染复数、一步生长曲线特点、紫外线灭活特点、噬菌体宽噬性进行了研究,手工提取噬菌体核酸并进行电泳分析,对噬菌体结构蛋白进行了聚丙烯酰胺凝胶电泳分析;2.在密闭柜内使用玻璃发生器Devilbiss 40发生模式病毒气溶胶,发生液分别选用肉汤、SM液和PBS,TSI-3321气溶胶粒子分析仪测量空气中模式病毒气溶胶粒子谱,分别于发生前后取少量发生液,双层琼脂平板法测定噬菌体滴度,使用统计软件对结果进行分析;3.使用全玻璃液体冲击式采样器(AGI-10)对模式病毒进行耐冲击实验,采样液选用蒸馏水(DW)、磷酸盐缓冲液(PBS)和SM液,每种采样液又分为加橄榄油组和不加橄榄油组,在以7L/min的气流冲击30min后测定采样液中噬菌体滴度和终末采样液体积,采用校正存活率评价噬菌体耐冲击性,使用统计软件对结果进行分析;4.发生模式病毒气溶胶,空气微生物采样器采集防护装备和设施气溶胶暴露区和被保护区空气中模式病毒粒子,采用防护效率评价防护装备和设施对病毒气溶胶的防护效果。结果:1.成功分离出粘质沙雷氏菌烈性噬菌体2株(SM701、SM702),噬菌体SM701和SM702在双层平板上分别培养6h和8h后可出噬菌斑,前者噬菌斑形态为圆形,直径1mm左右(培养12h),噬菌斑透亮度较高;后者噬菌斑圆形,直径2mm～3mm左右(培养12h),透亮度较前者低。两株噬菌体形态极为相似,均有一个正多面体立体对称的头部,头径约64nm,无囊膜,有一长尾,无收缩尾鞘,尾长约143nm。两者最佳感染复数均为10。SM701感染宿主菌的潜伏期约为30min,爆发时间约为100min,裂解量约为63;SM702感染宿主菌的潜伏期约为40min,爆发时间约为90min,裂解量约为5。SM701和SM702在紫外线(光强221μW/cm2)下分别暴露14min和16min全部失活。噬菌体SM701和SM702核酸类型为dsDNA,衣壳和尾至少分别由14和16个不同大小蛋白构成,两者之间相似大小条带8条。成功分离出大肠杆菌(285)烈性噬菌体2株(EcP1、EcP2), EcP1噬菌斑直径3mm～5mm(培养12h),逆光观察噬菌斑呈全透明状,该噬菌体有一个长多面体立体对称的头部,头长径(L)约47nm,头横径(W)约35nm,L/W=1.34,无囊膜,有一短尾,尾长约20nm;EcP2噬菌斑直径约1mm(培养12h),逆光观察噬菌斑呈全透明状,该噬菌体有一个长多面体立体对称的头部,头长径(L)约89nm,头横径(W)约54nm,L/W=1.65,无囊膜,有一长尾,有尾鞘,尾长约81nm。EcP2可噬大肠杆菌(8099)形成直径约1mm的圆形噬菌斑。EcP1和EcP2最佳感染复数分别为10和0.1,在紫外灯(光强221μW/cm2)下暴露8min和4min可全部失活。EcP1和EcP2核酸类型为dsDNA,衣壳和尾至少分别由12和16个不同大小蛋白构成。2.在19.5min内,发生器Devilbiss 40对悬液中噬菌体活性影响小,影响随模式病毒和发生液的不同而不同;不同发生液之间发生前噬菌斑数差别无统计学意义(P>0.05),发生后19.5min三种不同发生液之间噬菌斑数差异具有统计学意义(P<0.01),均值结果为肉汤>SM液>PBS,说明相同滴度的噬菌体,使用三种不同的发生液,发生一定时间后,噬菌体活性之间差别显著,使用肉汤作为发生液对噬菌体的保护作用最好,不同模式病毒气溶胶粒子数量中值直径在0.7μm到0.8μm之间。3.同一种噬菌体在不同采样液中,耐冲击性不同,以SM液作为采样液时,噬菌体存活率较高,SM液中是否加入橄榄油对噬菌体的耐冲击性没有影响。噬菌体SM701、SM702、PhiX174、EcP1和F2在SM液中经7L/min的气流冲击60min后,校正存活率分别为79%、77%、86%、50%和71%左右。在短时间(10min)内,噬菌体SM701、SM702、PhiX174和f2存活率无差别。4.国内市售医用防护口罩质量良莠不齐,随检测产品的不同病毒学检测达标率为0、20%、60%和100%;正压医用防护头罩对病毒气溶胶的防护效率大于99.98%;从医用防护服测试结果来看,我国目前没有能够达到国际标准ISO16604的市售防护服;固定式和充气式负压隔离转运舱病毒气溶胶隔离效率均大于99.999%;负压救护车排风负压净化装置病毒气溶胶过滤效率大于99.99%;三级生物安全实验室中9块被检测HEPA中,有1块HEPA存在模式病毒气溶胶漏;生物安全柜检测结果表明,不同指示微生物在人员、产品和交叉污染保护实验中结果无明显差异,但高效空气粒子过滤器生物学检漏结果随指示微生物是细菌还是病毒而不同。结论:1.四步法是一种有效的噬菌体污水分离方法;2.噬菌体SM701和SM702属于长尾噬菌体科噬菌体,噬菌体EcP1和EcP2分别属于短尾噬菌体科噬菌体和肌尾噬菌体科噬菌体;3.成功分离的噬菌体SM702易于培养计数且对发生和采样冲击压力耐受,是理想的空气微生物学研究示踪微生物;营养肉汤是理想的气溶胶发生液;SM液是理想的液体冲击式采样器采样液;4.噬菌体SM702可模拟病毒气溶胶对多种生物防护装备和设施病毒气溶胶防护效果进行评价;5.成功建立和完善了生物防护装备和设施病毒气溶胶防护效果评价技术方法。更多还原

【Abstract】 There have been more than 40 kinds of emerging infectious diseases found and validated all over the world since 1970s. More than 50% of these infectious diseases are airborne and at least one is found each year. They have seriously threatened the human health and social economy. For example, SARS which broke out in 2003 caused 8439 infections and 812 deaths in 30 countries within 9 months, highly pathogenic avian influenza in 2004 and type A H1N1 influenza last year also have caused heavy loss of lives and assets.Protective equipments and facilities play an important role in three aspects: isolating the infectious source, cutting off the transmission route of microbiological aerosols and protecting the susceptible population. It is one of the fastest and most effective measures facing the unknown vast bio-terroristic attacks and natural outbreaks. However, some of the protective equipments and facilities are deficient in biological evaluation methods and standards or even lack of them.The purpose of this study is to establish and imporve the evaluation technology and flat of bioprotective equipments and facilities agaist model virus aerosol for respiratory viruses, to advance the detection ability of bioprotective equipments and facilities and to carry out the safety evaluation for bio-protective respirator, bio-protective suit, positive pressure powered air-filter protective hood, negative pressure powered air-filter litter, exhaust HEPA of negative pressure ambulance, classⅡbiological safety cabinet and exhaust HEPA of high biosafety level laboratory.Contents:1. To isolate the bacteriophages of Eshcherichia coli and Serratia marcescens and to study their biological characteristics.2. To figure out the bacteriophage resistance to the viral aerosol generation and sampling pressure.3. To set up and improve the the evaluation technology and flat of bioprotective equipments and facilities and to evaluate their protective effect agaist model virus aerosol through testing. Methods:1. The bacterium Escherichia coli(285) and Serratia marcescens (8039) were used as the hosts to isolate phages from the raw sewage treatment center of hospital. Lytic phages were screened out using single or double layer agar plate method. Phages from single plaque were multiplicated and purified. Morphological properties of phages were examined by electron microscopy. Optimal multiplicity of infection (MOI),characteristics of one-step growth curve,phage inactivation characteristics by ultraviolet, host range of lytic phages were examined. The nucleic acid of phages were extracted and analyzed by agarose gel electrophoresis and the structure proteins of phages were analyzed by SDS-PAGE.2. Model virus aerosol was generated by using glass nebulizer Devilbiss 40 in an exposure tank. Nutrient broth, suspension medium and phosphate buffered saline was used to dilute phage solution as nebulizing solution respectively.TSI-3321 aerodynamic particle sizer was utilized to measure the spectrum of model virus aerosol. The phage concentration in nebulizing solution was titered by double layer agar plate method before and after nebulization. The outcome was analyzed by the statistical software.3. AGI-10(All Glass Impinger) was used as the representative for all the impingers that would bubble during operation (for example,AGI-4,AGI-30,and other newly developed biosamplers) to fulfill the bubbling experiment. Three different sampling solutions distilled water(DW),phosphate buffered saline(PBS),and suspension medium(SM) were used, and they were divided into two groups by adding olive oil(50μL) or not(0μL).The impingers were operated 30 min at a flow rate of 7.0 liters/min. The titers of bacteriophages and the volume of final sampling solutions were determined and then the corrected survival probability was used to evaluate the stress resistance of several different bacteriophages. The outcome was analyzed by the statistical software.4. Model virus aerosol was nebulized by Devibiss 40 and virus aerosol in exposure room and protected room was sampled by microbial air sampler. The protective effect of protective equipments and facilities was evaluated by the protective efficiency.Results:1. Two phage strains of Serratia marcescens 8039 (named SM701,SM702) were isolated successfully from the raw sewage, and their lysis properties were constant. All of them belonged to tailed phage. They could form plaques after 6 h and 8 h incubation. Although SM701 and SM702 both had an isometric polyhedral head (about 64nm in diameter) and a long noncontractile tail (about 143nm in length), their plaques were different in shape and size. The former were transparent less than 1mm in diameter and the latter were semi-transparent about 2mm-3mm in diameter after 12 h incubation. The optimal multiplicity of infection(MOI) of phage SM701 and SM702 equaled 10. The latent periods in bacterial host of SM701 and SM702 were about 30min and 40min, respectively. The burst times for SM701 and SM702 were about 100min and 90min and the burst sizes were about 63 and 5, respectively. And they lost their abilities of forming plaques on double layer agar plate after 14min and 16min exposure in ultraviolet (221μW/cm2) respectively. The SDS-PAGE illustrated that SM701 and SM702 at least consists of 14 and 16 structure proteins respectively and they have 8 similar size proteins.Two phage strains of Escherichia coli (285)(named EcP1、EcP2) were isolated successfully from the raw sewage, and their lysis properties were constant. Plaques of EcP1 and EcP2 were both transparent about 3~5mm and nearly 1mm in diameter after 12 h incubation, respectively. And EcP2 can also lyse Escherichia coli 8099 except Escherichia coli (285) forming clear plaques about 1mm in diameter. All of them belonged to tailed phage. Phage EcP1 had an elongated head (length about 47nm and width about 35nm) and a short tail (about 20nm in length).Phage EcP2 had an elongated head (length about 89nm and width about 54nm) and a contractile tail (about 81nm in length).The optimal multiplicity of infection(MOI) of phage EcP1 and EcP2 equaled 10 and 0.1 respectively. And they lost their abilities of forming plaques on double layer agar plate after 8min and 4min exposure in ultraviolet respectively. The SDS-PAGE illustrated that EcP1 and EcP2 at least consists of 12 and 16 structure proteins respectively.2. The phage viability could be influenced by aerosol generator Devilbiss 40 insignificantly in a certain operation time, which varied with different model viruses and even solution types. The plaque forming unit counts between different solutions before nebulization were not significantly different(P>0.05),but there were significant differences between them after 19.5min nebulization(P<0.01).The mean value of plaque forming unit count was in the order of nutrient broth>suspension medium>phosphate buffered saline, indicating that the viable phage count differed significantly between different nebulizing solutions after a certain time of nebulization. Nutrient broth as the nebulizing solution could provide phages with the optimal protection. The count median diameter of model virus aerosol ranged from 0.7-0.8μm.3. It was found that the survival probability of the same bacteriophage bubbling with different sampling solutions was different except that there was no significant difference observed for the bacteriophage f2.The use of SM as the collection fluid was relative to a high survival probability which was not different between 50μL olive oil and 0μL.The corrected survival probability were 79%,77%,86%,50% and 71% for phage SM701,SM702,PhiX174,EcP1 and f2 respectively after 60 min impingement at a flow rate of 7.0 liters/min. There were no significant differences in survival probability for phage SM701,SM702,PhiX174 and f2 within 10min.4. The quality of medical protective masks saled on domestic market was uneven. The virological testing attainment rate of masks were 0,20%,60% and 100% along with different mask brands. The protective efficiency of the positive pressure powered air-filter protective hood against virus aerosol surpassed 99.98%. From the testing results of medical protective suit, none of the tested medical protective suits satisfied with the international standard ISO16604. The protective efficiencies of both fixed type and inflatable type negative pressure powered air-filter litter were more than 99.999%. The filtration efficiencies of exhaust HEPA of negative pressure ambulance surpassed 99.99%. One of nine tested HEPAs in biosafety level 3 laboratory existed model viral aerosol leakage. The tested classⅡbiological safety cabinets met the requirements of personnel, product, and cross-contamination protection test no matter which agent was used to challenge the system. However, the HEPA filter leak testing results indicated that the penetration ability of viral aerosol through HEPA filter was superior to bacterial.Conclusions:1. Four step procedures is an effective phage isolation method from sewage water;2. Phage SM701 and SM702 belong to tailed family: siphoviridae. Phage EcP1 and EcP2 belong to tailed families: podoviridae, myoviridae respectively;3. One of the isolation strains is easy to cultivation and count plaque and it is resistant to nebulization and sampling stress. It is an ideal aero-microbiological tracer and model virus. Nutrient broth is an ideal nebulization solution. Suspension medium(SM) is an ideal sampling solution for liquid impinger;4. The isolation could use as model virus for evaluation of bio-protective equipments and facilities against virus aerosol.5. The evaluation technology and method of bio-protective equipments and facilities against virus aerosol is set up and improved.更多还原