模式病毒（噬菌体）分离、特性及在防护装备和设施评价中的应用研究
|
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。
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.
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。
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.
引文
[1]Tseng Chun-Chieh,Chih-Shan Li.Collection efficiencies of aerosol samplers for virus-containing aerosols[J].J Aerosol Sci,2004,36:593-607.
[2]Balazy Anna,Toivola Mika,Adhikari Atin,et al.Do N95 respirators provide 95% protection level against airborne viruses,and how adequate are surgical masks[J]?Am J Infect Control,2006,34(2):51-57.
[3]帖金凤,张文福.几种噬菌体与脊髓灰质炎病毒对三氯异氰尿酸抗力的比较[J].中国消毒学杂志.2007,24(3),208-211.
[4]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志.2004,21(3):189-192.
[5]于龙,王洁,赵建军,温占波,杨文慧,李娜,鹿建春,李劲松.两株大肠杆菌烈性噬菌体的分离与生物学特征研究[J].军事医学科学院院刊, 2008,32(5):432-435.
[6]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.分子克隆实验指南[M].金冬雁,黎孟枫,等译.第2版.北京:科学出版社,1992.
[7]Lu Z,Breidt F Jr,Fleming HP,et al.Isolation and characterization of a Lactobacillus plantarum bacteriophage,ΦJL-1,from a cucumber fermentation[J].Int J Food Microbiol,2003,84(2):225-235.
[8]司稚东,何晓青.噬菌体学[M].北京:科学出版社,1996.30.
[9]Ackermann H–W.5500 Phages examined in the electron microscope[J].Archives of virology,2007,152:227-243.
[10]Bradley DE.The morphology of some bacteriophages specific to Serratia marcescens [J].J Appl Bacteriol,1965,28:271-277.
[11]陆德源主编.医学微生物学[M].第5版.北京:人民卫生出版社,2002.51.
[12]徐焰,熊鸿燕,苏明权,等.f2噬菌体及其宽噬株的生物学特性比较[J].第四军医大学学报,2004,25(7):653-655.
[13]Hamilton RL,Brown WJ.Bacteriophage typing of clinically isolated Serratia marcescens [J].Applied Microbiology,1972,24(6):899-906.
[14]Traub WH.Antibiotic susceptibility of clinical isolates of Serratia marcescens compared with sensitivity to group A (phage tail) bacteriocins[J].Chemotherapy,1978,24:301-313.
[15]Frederick GL,Lloyd BJ.Evaluation of Serratia marcescens bacteriophage as a tracer and a model for virus removal in waste stabilisation ponds[J].Water Science and Technology,1995,31:291-302.
[16]Frederick GL,Lloyd BJ.An evaluation of retention time and short-circuiting in wastestabilisation ponds using Serratia marcescens bacteriophage as a tracer[J].Water Science and Technology,1996, 33:49-56.
[17]Drury DF, Wheeler DC.Applications of a Serratia marcescens bacteriophage as a new microbial tracer of aqueous environments[J].J Appl Bacteriol,1982,53:137-142.
[18]Carstens EMJ,Coetzee OJ, Malherbe HH ,et al.Bacteriophage of Serratia marcescens as an index of human virus survival during sewage purification[J].Water Pollution Abstracts,1968,41:129.
[19]Ashelford KE, Day MJ, Fry JC.Elevated abundance of bacteriophage infecting bacteria in soil[J].Applied and Environmental Microbiology,2003,69:285-289.
[20]Betts WB,Mould A,Clough G.A note describing a bacteriophage aerosol model of droplet infection to assess the viral barrier properties of filters[J].Microbios Letters,1990,45:57-60.
[21]Harstad JB.Sampling submicron T1 bacteriophage aerosols[J].Applied Microbiology, 1965,13(6):899–908.
[22]Grinshpun SA,Willeke K,Ulevicius V,et al.Collection of airborne microorganisms: advantages and disadvantages of different methods[J].J Aerosol Sci,1996,27(2):247-248.
[23]Alexandersen S and Donaldson AI.Further studies to quantify the dose of natural aerosols of foot and mouth disease virus for pigs[J].Epidemiol Infect,2002,128(2):313–323.
[24]Donaldson AI,Gibson CF,Oliver R,et al.Infection of cattle by airborne foot-and-mouth disease virus:minimal doses with O1 and SAT 2 strains[J].Res Vet Sci,1987,43(3):339–346.
[25]Willeke K,Grinshpun SA,Ulevicius V,et al.Microbial stress,bounce,and reaerosolization in bioaerosol samplers[J].J Aerosol Sci,1995,26:s883–s884.
[26]于龙,温占波,杨文慧,等.一株粘质沙雷氏菌烈性噬菌体污水分离及特性[J].微生物学报,2008,48(4):498-502.
[27]Adams MH.Surface inactivation of bacterial viruses and of proteins[J].Journal of general physiology,1948,31(5):417-431.
[28]Agranovski IE,Safatov AS,Borodulin AI,et al.Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring[J].Appl Environ Microbiol,2004, 70(12):6963–6967.
[29]Lin X,Willeke K,Ulevicius V,et al.Effect of sampling time on the collection efficiency of all-glass impingers[J].AM Ind Hyg Assoc J,1997,58(7):480-488.
[30]Ackermann HW.Bacteriophages.In:Lederberg JP ed.Encylopedia of Microbiology,2nd ed.New York:Academic Press,1999.398-411.
[31]Harstad JB,Decker HM and Buchanan LM, et al.Air filtration of submicron virusaerosols[J].Amercian journal of public health,1967,57(12):2186-2193.
[32]Morawska L.Droplet fate in indoor environments,or can we prevent the spread of infection[J]?Indoor Air,2006,16:335-347.
[33]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志,2004,21(3):189-192.
[34]GB/T6223-1997,自吸式过滤式防微粒口罩[S].中华人民共和国国家标准.
[35]GB19083-2003,医用防护口罩技术要求[S].中华人民共和国国家标准.
[36]YY 0469-2004,医用外科口罩技术要求[S].中华人民共和国医药行业标准.
[37]赵建军,王洁,李娜,等.医用外科口罩生物防护效果测试平台的建立和应用[J].军事医学科学院院刊,2007,31(5):460-462.
[38]温占波,陈洁君,赵建军,等.医用外科口罩细菌气溶胶过滤效果评价[J].中国消毒学杂志,2007,24(4):306-309.
[39]McCullough NV,Brosseau LM,Vesley D.Collection of three bacterial aerosols by respirator and surgical mask filters under carrying conditions of flow and relative humidity[J].Ann Occup Hyg,1997,41:677-690.
[40]Qian Y,Willeke K,Grinshpun SA,et al.Performance of N95 respirators:filtration efficiency for airborne microbial and inert particles[J].Am Ind Hyg Assoc J,1998,59:128-132.
[41]刘蔼成,易小红,张佳愉.预防呼吸道传染病口罩的研究[J].实用预防医学,2003,10(6):933-934.
[42]沈恒根,杨磊,刘刚,等.新型医用防护口罩过滤环境气溶胶的性能[J].第二军医大学学报,2003,24(6):629-631.
[43]Robert M. Eninger,Takeshi Honda,Atin Adhikari,et al.Filter Performance of N99 and N95 Facepiece Respirators Against Viruses and Ultrafine Particles[J].Ann Occup Hyg, 2008,52(5):385-396.
[44]王政,田丰,杨荆泉,等.正压医用防护头罩过滤效率和微环境研究及其临床试验[J].生物防护防疫装备,2005,26(2):26.
[45]胡良平.检验医学科研设计与统计分析[M].北京:人民军医出版社,2004:238.
[46]王政,陈世谦,石梅生,等.生物污染物理防护技术研究-过滤器及正/负压生成系统设计[J].生物防护防疫装备,2005,26(8):31.
[47]GB/T 13554-1992,高效空气过滤器[S].中华人民共和国国家标准.
[48]Testing HEPA and ULPA Filter Media[S].IEST RP021.2.USA:Institute of Environmental Sciences and Technology,2005.
[49]High Efficiency Air Filters (HEPA and ULPA)-part 3:Testing Flat Sheet FilterMedia[S].EN 1822-3.Brussels:European Committee for Standard,2007.
[50]车凤翔,于玺华,鹿建春,等.空气微生物采样理论及其技术应用[M].北京:中国大百科全书出版社,1998:1～216.
[51]GB 190822003,医用一次性防护服技术要求[S].中华人民共和国国家标准.
[52]WSB5822003,生物防护服通用规范[S].中国人民解放军总后勤部卫生部标准.
[53]张建春,郝新敏,周国泰,等.医用防护服研究现状及SARS防护服的性能要求[J].西安工程科技学院学报,2003,17(3):194-199.
[54]ISO 16604.Clothing for protection against contact with blood and body fluids -Determination of resistance of protective clothing materials to penetration by blood borne pathogens-Test method using Phi-X174 bacteriophage[S].International Organization for Standardization.2004.
[55]姜慧霞.医用防护服材料的性能评价研究[D].天津工业大学硕士学位论文,2008.1-61.
[56]张文福,蒋莉,王太星,等.新型生物防护服对病毒防护效果的研究[J].中国消毒学杂志,2004,21(2):83-85.
[57]沈伟,何静芳,苏怡,王一鸣.医用防护服与防护口罩阻隔性能研究[J].中国消毒学杂志,2005,22(4):386-390.
[58]ASTM F 1671.Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using PhiX 174 Bacteriophage Penetration as a Test System [S].2003.
[59]http://www.cleanairtechnology.com/medical.php
[60]http://www.bjcleanair.com/product.php?id=58
[61]http://detail.china.alibaba.com/buyer/offerdetail/565417149.html
[62]http://b2b.ewsos.com/2/14131.shtml
[63]杜新安,曹务春,主编.生物恐怖的应对与处置[M].北京,人民军医出版社,2005.
[64]马静,李劲松,杜新安,等.外军生物武器医学防护装备现状与发展[J].医疗卫生装备,2003,24(2):28-31.
[65]Lisowski A,Jankowska E,Thorpe A,et al.Performance of textile fibre filter material measured with monodisperse and standard aerosols[J].Powder Technol,2001,118(l): 149-159.
[66]刘亚军,孙景工,谭树林,等.传染病员救护车病员室模拟气溶胶流动仿真与实验研究[J].医疗卫生装备,2005,26(5):27-29.
[67]徐新喜,刘亚军,崔向东,等.防生物污染伤病员急救车的车厢环境质量研究[J].医疗卫生装备.2009,30(7):2-6.
[68]徐新喜,刘亚军,赵秀国.负压防护急救车车厢内生物污染物运动扩散的数值模拟与试验验证[J].中国安全科学学报,2009,19(8):28-33.
[69]徐新喜,刘亚军,王太勇,等.具有超压/负压防护功能的急救车防生物污染的安全性试验研究[J].中国安全科学学报,2008,18(7):105-110.
[70]刘亚军,徐新喜,王政,等.具有过滤净化超压和负压防护功能的车用生物防护系统研究[J].军事医学科学院院刊,2008,32(6):550-554.
[71]温占波,鹿建春,赵建军,等.几种高效过滤器材对微生物气溶胶防护效果的评价[J].中国消毒学杂志,2007,24(2):112-114.
[72]温占波,赵建军,王洁,等.生物安全柜的使用选择和生物防护性能的检测与评价[J].医疗卫生装备,2008,29(4):14-24.
[73]温占波,赵建军,李劲松,等.过滤罐微生物气溶胶过滤效率及其评价方法的研究[J].中华预防医学杂志,2009,43(8):686-689.
[74]National Sanitation Foundation.NSF49-2002《ClassⅡ(laminar flow) biosafety cabinetry》[S].Michigan:NSF international,2002.
[75]GB19489-2008《实验室生物安全通用要求》[S].中华人民共和国国家标准.
[76]祁建城,李艳菊,吕京,等.关于ABSL-3实验室分类、设计要求的分析、探讨[J].洁净与空调技术,2007,4:15-21.
[77]David LS.Laboratory-associated infections and biosafety[J].Clinical microbiology reviews,1995,8(3):389-405.
[78]NSF/ANSI49—2002 Class(laminar flow)biosafety cabinetry.
[79]EN12469:2000 Biotechnology-Performance criteria for microbiological safety cabinets.
[80]YY0569—2005.生物安全柜[S].中华人民共和国医药行业标准.
[81]Woods JBLC, MC, USAF. USAMRIID's Medical Management of Biological Casualties Handbook. 6th ed. United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland; 2005. p. 61-74.
[82]Decker HM, Buchanan LM, Lawrence BH, and Goddard KR. Air Filtration of Microbial Particles[J].Am J Public Health Nations Health,1963,53:1982-1988.
[83]Rapp ML, Thiel T, and Arrowsmith RJ. Model system using coliphage Phix174 for testing virus removal by air filters[J].Appl Environ Microbiol,1992,58:900-904.
[84]Burnett LAC,Lunn G,and Coico R.Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr Protoc Microbiol,2009,5: chapter 1:unit 1A.1.
[1]Tseng Chun-Chieh,Chih-Shan Li.Collection efficiencies of aerosol samplers for virus-containing aerosols[J].J Aerosol Sci,2004,36:593-607.
[2]Balazy Anna,Toivola Mika,Adhikari Atin,et al.Do N95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks?[J]Am J Infect Control,2006, 34(2):51-57.
[3]Fauquet CM,Mayo MA,Maniloff J,et al.Virus Taxonomy, Eighth Report of the International Committee Taxonomy Viruses [M].Amsterdam: Elsevier Academic Press,2005.
[4]Ackermann H–W.5500 Phages examined in the electron microscope[J].Archives ofvirology,2007,152:227-243.
[5]Davis JE,Strauss JH,Sinsheimer RL.Bacteirophage MS2:Another RNA phage[J]. Science,1961,134:1427.
[6]Stockley PG,Stonehouse NJ and Valegard K.Molecular mechanism of RNA phage morphogenesis[J].Int J Biochem,1994,26:1249–1260.
[7]Peabody,DS,and Al-Bitar L.Isolation of viral coat protein mutantswith altered assembly and aggregation properties[J].Nucleic Acids Res,2001,29,E113.
[8]Jolis D,Hirano R and Pitt P.Tertiary treatment using microfiltration and UV disinfection for water reclamation[J].Water Environ Res,1999,71:224–231.
[9]Lykins BW, Koffskey WE and Patterson KS.Alternative disinfectants for drinking water treatment[J].J Environ Eng,1994,120:745–758.
[10]Heal KG,Hill HR,Stockely PG,Hollongdale MR and Taylorrobinson AW.Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA-free MS2 bacteriophage capsids[J].Vaccine,2000,18:251–258.
[11]Stockley PG and Mastico RA.Use of fusions to viral coat proteins as antigenic carriers for vaccine development[J].Methods Enzymol,2000,326:551–56.
[12]ISO10705—1 Water Qulality—Detection and Enumeration of Bacteriophages.Part1:Enumeration of F—specific RNA Bacteriophgaes[S].International organizaiton for Standardization.1995:15.
[13]Hsu YC,Nomura S and Kruse CW.Some bactericidal and virucidal properties of iodine not affecting infectious RNA and DNA[J]. Am J Epidemiol,1966,82:317-328.
[14]Cramer WN,Kawata K and Kruse CW.Chlorination and iodination of poliovirus and f2[J].J Am Water Works Assoc,1976,68:61-76.
[15]Olivieri VP,Kruse CW,Hsu YC,Griffiths AC and Kawata K.The comparative mode of action of chlorine,bromine,and iodine on f2 bacterial virus.1975,p.145-162. In J.D. Johnson(ed.),Disinfection-water and wastewater. Ann Arbor Science Publishers, Ann Arbor, Mich.
[16]何静芳,马伟,陈泰尧,等.f2噬菌体对常用消毒处理的抗力测定[J].上海预防医学杂志,2005,17(9):448-449.
[17]王思熊,熊鸿燕,林辉,等.吖啶橙与长波紫外线灭活血浆中f2噬菌体的形态变化[J].第三军医大学学报,2004,26(3):220-224.
[18]田丰,王政,杨荆泉,等.正压送风过滤式防护头罩防护性能研究[J].生物医学工程学杂志,2006,23(4):766-769.
[19]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志,2004,21(3):189-192.
[20]Sanger F,Air GM,Barrell BG,Brown NL,Coulson AR,Fiddes CA,Hutchison CA, Slocombe PM,and Smith M.Nucleotide sequence of bacteriophage phiX174 DNA[J]. Nature,1977,265(5596):687-695.
[21]Fiers W and Sinsheimer RL.The structure of the DNA of bacteriophage PhiX 174. III. Ultracentrifuge evidence for a ring structure[J].J Mol Biol,1962,5:424-434.
[22]Demik G. and Degroot I.Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene[J].J Hyg,1977,78(2):199-211.
[23]Tseng Chun-Chieh and Li Chih-Shan.Ozone for inactivation of aerosolized bacteriophages[J].Aerosol science and technology,2006,40(9):683-689.
[24]ISO 16604.Clothing for protection against contact with blood and body fluids-Determination of resistance of protective clothing materials to penetration by blood-borne-pathogens-Test method using Phi-X174 bacteriophage[S].International Organization for Standardization.2004.
[25]Delbr?ck M.The burst size distribution in the growth of bacterial viruses[J].J Bacteriol,1945,50:131–135.
[26]Trouwborst T,De jong JC and Winkler KC.Mechanism of inactivation in aerosols of bacteriophage T1[J].J gen Virol,1972,15:235-242.
[27]Trouwborst T and Winkler KC. Protection against aerosol-inactivation of bacteriophage T1 by peptides and Amino Acids[J].J gen Virol,1972,17:1-11.
[28]Harstad JB.Sampling submicron T1 bacteriophage aerosols[J].Applied Microbiology, 1965,13(6):899–908.
[29]Ehrlich Richard,Miller sol,and Idoine LS.Effects of environmental factors on the survival of airborne T-3 coliphage[J]. Appl Microbiol,1964,12(6):479-482.
[30]Trouwborst T. and Kuyper Sjoukje.Inactivation of bacteriophage T3 in aerosols:effect of prehumidification on survival after spraying from solutions of salt,peptone,and saliva[J]. Appl Microbiol,1974,27(5):834-837.
[31]Warren J.C.and Hatch M.T.Survival of T3 coliphage in varied extracellular environments I. Viability of the coliphage during storage and in aerosols [J].Appl Microbiol,1969,17(2):256-261.
[32]Dahlgren CM,Decker HM,and Harstad JB.A slit sampler for collecting T-3 bacteriophage and Venezuelan equine encephalomyelitis virus.I.Studies with T-3 bacteriophage[J].Appl Microbiol,1961,9(2):103-105.
[33]Coriell LL and Mcgarrity G.J.Biohazard hood to prevent infection duringmicrobiological procedures[J]. Appl Microbiol,1968,16(12):1895-1900.
[34]Hatch MT and Warren JC.Enhanced recovery of airborne T3 coliphage and Pasteurella pestis bacteriophage by means of a presampling humidification technique[J].Appl Microbiol,1969,17(5):685-689.
[35]Experimental studies on environmental contamination with infected blood during haemodialysis[J].J Hyg,1975,74(1):133-148.
[36]Sullivan JF and Songer JR. Role of differential air pressure zones in the control of aerosols in a large animal isolation facility[J]. Appl Microbiol,1966,14(4):674-678.
[37]Merrill JT.Evaluation of selected aerosol-control measures on flow sorters[J]. Cytometry,1981,1:342–345.
[38]Schmid Ingrid,Hultin Lance E.,and Ferbas John.Testing the efficiency of aerosol containment during cell sorting[J].Curr Protoc Cytom,2001,Chapter3:3.3.1-3.3.15.
[39]Lennartz K,Lu M,Flasshove M,Moritz T,and Kirstein U.Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet[J]. Cytometry,2005, 66(2):119-127.
[40]Ferbas J,Chadwick KR,Logar A,Patterson AE,Gilpin RW,Margolick JB.Assessment of aerosol containment on the ELITE flow cytometer.Cytometry(Communications in Clinical Cytometry)1995,22:45–47.
[41]Eninger RM,Honda takeshi,Adhikari A,et al.Filter performance of N99 and N95 facepiece respirators against viruses and ultrafine particles[J].Ann Occup Hyg, 2008,52(5): 385-396.
[1]Ali M,Lemoine NR,Ring CJ.The use of DNA viruses as vectors for gene therapy[J].Gene Ther,1994,l(6):367-384.
[2]Jolly D.Viral vector systems for gene therapy[J].Cancer Gene Ther,1994,1(1):51-64.
[3]Levine AJ.The molecular biology of adenovirus.In:Bercoff RP ed.The molecular basis ofviral replication[M].Plenum Press,1987,483-498.
[4]Harvey B,Maroni J,O’Donoghue KA,et al.Safety of local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid condition [J].Hum Gene Ther,2002,(13):15-63.
[5]龚非力主编.医学免疫学[M].第1版,北京:科学出版社,2003,34-35.
[6]Mestecky J.The common mucosal immune system and current strategies of induction of immune responses in external secretions [J].Clin mmunol,1987,7(4):265–276.
[7]McGhee JR,Mestecky J,Dertzbaugh MT,et al.The mucosal immune system: from fundamental concepts to vaccine development[J].Vaccine,1992,10(2):75–88.
[8]Nugent J,Po A L,Scott EM.Design and delivery of non-parenteral[J].Clin Pharm Ther,1998,23(4):257-285.
[9]Walker RI.New strategies for using mucosal vaccination to achieve more effective immunization[J].Vaccine,1994,12(5):387–400.
[10]Frey A,Neutra MR.Targeting of mucosal vaccines to Peyer’s patch M cells[J].Behring Inst Mitt,1997,98:376–389.
[11]Mestecky J,Michalek SM,Moldoveanu Z,et al.Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans[J].Behring Inst Mitt,1997,98:33–43.
[12]Mestecky J,McGhee J R.Prospects for human mucosal vaccines[J].Adv Exp Med Biol,1992,327:13–23.
[13]Zhang Y,Chirmule N,Gao GP,et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages[J].Mol Ther,2001,3:697–707.
[14]Crystal RG, Harvey B,Wisnivesky JP,et al.Analysis of risk factors for local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid condition[J].Hum Gene Ther,2002,13:65-100.
[15]Morrow CD,Novak MJ,Ansardi DC,et al.Recombinant viruses as vectors for mucosal immunity[J].Curr Top Microbiol Immunol,1999,236:255–273.
[16]Medzhitov R and Janeway C Jr.Innate immune recognition: mechanisms and pathways [J].Immunol Rev,2000,173:89–97.
[17]Schnell MA,Zhang Y,Tazelaar J,et al.Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors[J].Mol Ther,2001, 3:708-722.
[18]Morelli AE,Larregina AT,Ganster RW,et al.Recombinant adenovirus inducesmaturation of dendritic cells via an NF kappaB dependent pathway[J].J Virol,2000,74: 9617–9628.
[19]Molinier FV,Prevost BA,Hong SS,et al.The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain[J].J Biol Chem,2003,278(39):37175–37182.
[20]Philpott NJ,Nociari M,Elkon KB,et al.Adenovirus induced maturation of dendritic cells through a PI3 kinase-mediated TNF-alpha induction pathway[J].Proc Natl Acad Sci USA,2004,101:6200–6205.
[21]Hong SS,Habib NA,Franqueville L,et al.Identification of adenovirus (Ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative Ad (Addl1520) for treatment of liver tumors [J].J Virol,2003,77:10366–10375.
[22]Olive M,Eisenlohr L,Flomenberg N,et al.The adenovirus capsid protein hexon contains a highly conserved human CD4+ T-cell epitope[J].Hum Gene Ther,2002,13(10):1167-1178.
[23]Fitzgerald JC,Gao GP,Reyes-Sandoval A,et al.A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag[J].J Immunol,2003,170(3):1416–1422.
[24]Xiang ZQ, Yang Y,Wilson JM,et al.A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier[J].Virology,1996,219:220–227.
[25]He Z,Wlazlo AP,Kowalczyk DW,et al.Viral recombinant vaccines to the E6 and E7 antigens of HPV-16[J].Virology,2000,270(1):146–161.
[26]Yang Y,Li Q,Ertl HC,et al.Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses[J].J Virol,1995,69:2004–2015.
[27]Yang Y,Ertl HC and Wilson JM.MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses[J].Immunity,1994,1:433–442.
[28]Prevec L,Campbell JB,Christie BS,et al.A recombinant human adenovirus vaccine against rabies[J].J Infect Dis,1990,161:27–30.
[29]Casimiro DR,Chen L,Fu TM,et al.Comparative immunogenicity in rhesus monkeys of DNA plasmid,recombinant vaccinia virus,and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene[J].J Virol,2003,77: 6305–6313.
[30]Wang Y,Xiang Z,Pasquini S,et al.The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus[J].J Virol,1997,71:3677–3683.
[31]Xiang ZQ,Gao GP,Reyes-Sandoval A,et al.Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier[J].J Virol, 2003,77: 10780–10789.
[32]Sharpe S,Fooks A,Lee J,et al.Single oral immunization with replication deficient recombinant adenovirus elicits long-lived transgene-specific cellular and humoral immune responses[J].Virology,2002,293:210–216.
[33]Pinto AR,Fitzgerald JC,Gao GP,et al.Induction of CD8+ T cells to an HIV-1 antigen upon oral immunization of mice with a simian E1-deleted adenoviral vector[J].Vaccine, 2004,22:697–703.
[34]李焱,岳盈盈,张凤丽,等.腺病毒载体疫苗免疫途径的研究[J].中华医学研究杂志2004,4(5):392–393.
[35]Natuk RJ,Davis AR,Chanda PK,et al.Adenovirus vectored vaccines[J].Dev Biol Stand, 1994,82:71–77.
[36]何金生,王健伟,姜秀丽,等.复制缺陷型重组腺病毒rv Ad G1VP7免疫学效果的研究[J].安徽医科大学学报,2002,37(2):86–88.
[37]Lemiale F,Kong WP,Akyurek LM,et al.Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system[J].J Virol,2003,77:10078–10087.
[38]Smith JG,Raper SE,Wheeldon EB,et al.Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self-limiting inflammatory Response[J].Hum Gene Ther,1997,8(8):943–954.
[39]Charlton KM,Artois M,Prevec L,et al.Oral rabies vaccination of skunks and foxes with a recombinant human adenovirus vaccine[J].Arch Virol,1992,23(1-2):169-179.
[40]Meszaros J,Szemeredi M,Tamasi G.Immunization of day-old chickens against newcastle disease[J].Acta Vet Hung,1992,40:121-127.
[41]Van Eck JHH.Vaccination of fowl with inactivated New-Castle disease vaccine by the respiratory route[J].Avian pathol,1990,19:313-330.
[42]Sharma JM.Introduction to poultry vaccines and immunity[J].Adv Vet Med,1999, 41:481-494.
[43]Al-tarcha B,Kojnok J,Varro C.Immunization of day-old chicks having maternally derived antibodies against infectious bronchitis: degree of protection as monitored by ciliary activity after intratracheal challenge [J].Acta Vet Hung,1991,39:83-93.
[44]Sandhu TS.Immunogenecity and safety of a live pasteulla anatipestifer vaccine in white pekin ducklings: laboratory and field trials[J].Avian Pathol,1991,20:423-432.
[45]Deuter A,Southee DJ,Mockett APA.Fowlpox virus:pathogenicity and vaccination of day-old chicks via the aerosol route[J].Res Vet Sci,1991,50:362-364.
[46]Murphy D,Van Alstine WG,Clark LK,et al.Aerosol vaccination of pigs against mycoplasma hyopneumoniue infection[J].Am J Vet Res,1993,54:1874-1880.
[47]Gruber WC,Hinso HP,Holland KL,et al.Comparative trial of large-particle aerosol and nase drop administration of live attenuated influenza vaccines[J].J Infect Dis,1993, 168(5):1282-1285.
[48]徐琳,李劲松,周育森.炭疽杆菌保护性抗原重组腺病毒疫苗的构建及气溶胶免疫探索性研究[D].中国人民解放军军事医学科学院硕士学位论文,2006,25-34.
[49]Bellon G,Michel CL,Thouvenot D,et al.Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial[J].Hum Gene Ther,1997,8(1):15-25.
[50]Bellon G,Calmard L,Thouvenot D,et al.Aerosol administration of a replication defective recombinant adenovirus expressing normal human cDNA-CFTR in the respiratory tractus in patients with cystic fibrosis[J].C R Seances Soc Biol Fil,1996, 190(1):109-142.
[51]Lerondel S,Vecellio N L,Faure L,et al.Gene therapy for cystic fibrosis with aerosolized adenovirus-CFTR:characterization of the aerosol and scintigraphic determination of lung deposition in baboons[J].J Aerosol Med,2001,14(1):95-105.
[2]Balazy Anna,Toivola Mika,Adhikari Atin,et al.Do N95 respirators provide 95% protection level against airborne viruses,and how adequate are surgical masks[J]?Am J Infect Control,2006,34(2):51-57.
[3]帖金凤,张文福.几种噬菌体与脊髓灰质炎病毒对三氯异氰尿酸抗力的比较[J].中国消毒学杂志.2007,24(3),208-211.
[4]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志.2004,21(3):189-192.
[5]于龙,王洁,赵建军,温占波,杨文慧,李娜,鹿建春,李劲松.两株大肠杆菌烈性噬菌体的分离与生物学特征研究[J].军事医学科学院院刊, 2008,32(5):432-435.
[6]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.分子克隆实验指南[M].金冬雁,黎孟枫,等译.第2版.北京:科学出版社,1992.
[7]Lu Z,Breidt F Jr,Fleming HP,et al.Isolation and characterization of a Lactobacillus plantarum bacteriophage,ΦJL-1,from a cucumber fermentation[J].Int J Food Microbiol,2003,84(2):225-235.
[8]司稚东,何晓青.噬菌体学[M].北京:科学出版社,1996.30.
[9]Ackermann H–W.5500 Phages examined in the electron microscope[J].Archives of virology,2007,152:227-243.
[10]Bradley DE.The morphology of some bacteriophages specific to Serratia marcescens [J].J Appl Bacteriol,1965,28:271-277.
[11]陆德源主编.医学微生物学[M].第5版.北京:人民卫生出版社,2002.51.
[12]徐焰,熊鸿燕,苏明权,等.f2噬菌体及其宽噬株的生物学特性比较[J].第四军医大学学报,2004,25(7):653-655.
[13]Hamilton RL,Brown WJ.Bacteriophage typing of clinically isolated Serratia marcescens [J].Applied Microbiology,1972,24(6):899-906.
[14]Traub WH.Antibiotic susceptibility of clinical isolates of Serratia marcescens compared with sensitivity to group A (phage tail) bacteriocins[J].Chemotherapy,1978,24:301-313.
[15]Frederick GL,Lloyd BJ.Evaluation of Serratia marcescens bacteriophage as a tracer and a model for virus removal in waste stabilisation ponds[J].Water Science and Technology,1995,31:291-302.
[16]Frederick GL,Lloyd BJ.An evaluation of retention time and short-circuiting in wastestabilisation ponds using Serratia marcescens bacteriophage as a tracer[J].Water Science and Technology,1996, 33:49-56.
[17]Drury DF, Wheeler DC.Applications of a Serratia marcescens bacteriophage as a new microbial tracer of aqueous environments[J].J Appl Bacteriol,1982,53:137-142.
[18]Carstens EMJ,Coetzee OJ, Malherbe HH ,et al.Bacteriophage of Serratia marcescens as an index of human virus survival during sewage purification[J].Water Pollution Abstracts,1968,41:129.
[19]Ashelford KE, Day MJ, Fry JC.Elevated abundance of bacteriophage infecting bacteria in soil[J].Applied and Environmental Microbiology,2003,69:285-289.
[20]Betts WB,Mould A,Clough G.A note describing a bacteriophage aerosol model of droplet infection to assess the viral barrier properties of filters[J].Microbios Letters,1990,45:57-60.
[21]Harstad JB.Sampling submicron T1 bacteriophage aerosols[J].Applied Microbiology, 1965,13(6):899–908.
[22]Grinshpun SA,Willeke K,Ulevicius V,et al.Collection of airborne microorganisms: advantages and disadvantages of different methods[J].J Aerosol Sci,1996,27(2):247-248.
[23]Alexandersen S and Donaldson AI.Further studies to quantify the dose of natural aerosols of foot and mouth disease virus for pigs[J].Epidemiol Infect,2002,128(2):313–323.
[24]Donaldson AI,Gibson CF,Oliver R,et al.Infection of cattle by airborne foot-and-mouth disease virus:minimal doses with O1 and SAT 2 strains[J].Res Vet Sci,1987,43(3):339–346.
[25]Willeke K,Grinshpun SA,Ulevicius V,et al.Microbial stress,bounce,and reaerosolization in bioaerosol samplers[J].J Aerosol Sci,1995,26:s883–s884.
[26]于龙,温占波,杨文慧,等.一株粘质沙雷氏菌烈性噬菌体污水分离及特性[J].微生物学报,2008,48(4):498-502.
[27]Adams MH.Surface inactivation of bacterial viruses and of proteins[J].Journal of general physiology,1948,31(5):417-431.
[28]Agranovski IE,Safatov AS,Borodulin AI,et al.Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring[J].Appl Environ Microbiol,2004, 70(12):6963–6967.
[29]Lin X,Willeke K,Ulevicius V,et al.Effect of sampling time on the collection efficiency of all-glass impingers[J].AM Ind Hyg Assoc J,1997,58(7):480-488.
[30]Ackermann HW.Bacteriophages.In:Lederberg JP ed.Encylopedia of Microbiology,2nd ed.New York:Academic Press,1999.398-411.
[31]Harstad JB,Decker HM and Buchanan LM, et al.Air filtration of submicron virusaerosols[J].Amercian journal of public health,1967,57(12):2186-2193.
[32]Morawska L.Droplet fate in indoor environments,or can we prevent the spread of infection[J]?Indoor Air,2006,16:335-347.
[33]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志,2004,21(3):189-192.
[34]GB/T6223-1997,自吸式过滤式防微粒口罩[S].中华人民共和国国家标准.
[35]GB19083-2003,医用防护口罩技术要求[S].中华人民共和国国家标准.
[36]YY 0469-2004,医用外科口罩技术要求[S].中华人民共和国医药行业标准.
[37]赵建军,王洁,李娜,等.医用外科口罩生物防护效果测试平台的建立和应用[J].军事医学科学院院刊,2007,31(5):460-462.
[38]温占波,陈洁君,赵建军,等.医用外科口罩细菌气溶胶过滤效果评价[J].中国消毒学杂志,2007,24(4):306-309.
[39]McCullough NV,Brosseau LM,Vesley D.Collection of three bacterial aerosols by respirator and surgical mask filters under carrying conditions of flow and relative humidity[J].Ann Occup Hyg,1997,41:677-690.
[40]Qian Y,Willeke K,Grinshpun SA,et al.Performance of N95 respirators:filtration efficiency for airborne microbial and inert particles[J].Am Ind Hyg Assoc J,1998,59:128-132.
[41]刘蔼成,易小红,张佳愉.预防呼吸道传染病口罩的研究[J].实用预防医学,2003,10(6):933-934.
[42]沈恒根,杨磊,刘刚,等.新型医用防护口罩过滤环境气溶胶的性能[J].第二军医大学学报,2003,24(6):629-631.
[43]Robert M. Eninger,Takeshi Honda,Atin Adhikari,et al.Filter Performance of N99 and N95 Facepiece Respirators Against Viruses and Ultrafine Particles[J].Ann Occup Hyg, 2008,52(5):385-396.
[44]王政,田丰,杨荆泉,等.正压医用防护头罩过滤效率和微环境研究及其临床试验[J].生物防护防疫装备,2005,26(2):26.
[45]胡良平.检验医学科研设计与统计分析[M].北京:人民军医出版社,2004:238.
[46]王政,陈世谦,石梅生,等.生物污染物理防护技术研究-过滤器及正/负压生成系统设计[J].生物防护防疫装备,2005,26(8):31.
[47]GB/T 13554-1992,高效空气过滤器[S].中华人民共和国国家标准.
[48]Testing HEPA and ULPA Filter Media[S].IEST RP021.2.USA:Institute of Environmental Sciences and Technology,2005.
[49]High Efficiency Air Filters (HEPA and ULPA)-part 3:Testing Flat Sheet FilterMedia[S].EN 1822-3.Brussels:European Committee for Standard,2007.
[50]车凤翔,于玺华,鹿建春,等.空气微生物采样理论及其技术应用[M].北京:中国大百科全书出版社,1998:1～216.
[51]GB 190822003,医用一次性防护服技术要求[S].中华人民共和国国家标准.
[52]WSB5822003,生物防护服通用规范[S].中国人民解放军总后勤部卫生部标准.
[53]张建春,郝新敏,周国泰,等.医用防护服研究现状及SARS防护服的性能要求[J].西安工程科技学院学报,2003,17(3):194-199.
[54]ISO 16604.Clothing for protection against contact with blood and body fluids -Determination of resistance of protective clothing materials to penetration by blood borne pathogens-Test method using Phi-X174 bacteriophage[S].International Organization for Standardization.2004.
[55]姜慧霞.医用防护服材料的性能评价研究[D].天津工业大学硕士学位论文,2008.1-61.
[56]张文福,蒋莉,王太星,等.新型生物防护服对病毒防护效果的研究[J].中国消毒学杂志,2004,21(2):83-85.
[57]沈伟,何静芳,苏怡,王一鸣.医用防护服与防护口罩阻隔性能研究[J].中国消毒学杂志,2005,22(4):386-390.
[58]ASTM F 1671.Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Blood-Borne Pathogens Using PhiX 174 Bacteriophage Penetration as a Test System [S].2003.
[59]http://www.cleanairtechnology.com/medical.php
[60]http://www.bjcleanair.com/product.php?id=58
[61]http://detail.china.alibaba.com/buyer/offerdetail/565417149.html
[62]http://b2b.ewsos.com/2/14131.shtml
[63]杜新安,曹务春,主编.生物恐怖的应对与处置[M].北京,人民军医出版社,2005.
[64]马静,李劲松,杜新安,等.外军生物武器医学防护装备现状与发展[J].医疗卫生装备,2003,24(2):28-31.
[65]Lisowski A,Jankowska E,Thorpe A,et al.Performance of textile fibre filter material measured with monodisperse and standard aerosols[J].Powder Technol,2001,118(l): 149-159.
[66]刘亚军,孙景工,谭树林,等.传染病员救护车病员室模拟气溶胶流动仿真与实验研究[J].医疗卫生装备,2005,26(5):27-29.
[67]徐新喜,刘亚军,崔向东,等.防生物污染伤病员急救车的车厢环境质量研究[J].医疗卫生装备.2009,30(7):2-6.
[68]徐新喜,刘亚军,赵秀国.负压防护急救车车厢内生物污染物运动扩散的数值模拟与试验验证[J].中国安全科学学报,2009,19(8):28-33.
[69]徐新喜,刘亚军,王太勇,等.具有超压/负压防护功能的急救车防生物污染的安全性试验研究[J].中国安全科学学报,2008,18(7):105-110.
[70]刘亚军,徐新喜,王政,等.具有过滤净化超压和负压防护功能的车用生物防护系统研究[J].军事医学科学院院刊,2008,32(6):550-554.
[71]温占波,鹿建春,赵建军,等.几种高效过滤器材对微生物气溶胶防护效果的评价[J].中国消毒学杂志,2007,24(2):112-114.
[72]温占波,赵建军,王洁,等.生物安全柜的使用选择和生物防护性能的检测与评价[J].医疗卫生装备,2008,29(4):14-24.
[73]温占波,赵建军,李劲松,等.过滤罐微生物气溶胶过滤效率及其评价方法的研究[J].中华预防医学杂志,2009,43(8):686-689.
[74]National Sanitation Foundation.NSF49-2002《ClassⅡ(laminar flow) biosafety cabinetry》[S].Michigan:NSF international,2002.
[75]GB19489-2008《实验室生物安全通用要求》[S].中华人民共和国国家标准.
[76]祁建城,李艳菊,吕京,等.关于ABSL-3实验室分类、设计要求的分析、探讨[J].洁净与空调技术,2007,4:15-21.
[77]David LS.Laboratory-associated infections and biosafety[J].Clinical microbiology reviews,1995,8(3):389-405.
[78]NSF/ANSI49—2002 Class(laminar flow)biosafety cabinetry.
[79]EN12469:2000 Biotechnology-Performance criteria for microbiological safety cabinets.
[80]YY0569—2005.生物安全柜[S].中华人民共和国医药行业标准.
[81]Woods JBLC, MC, USAF. USAMRIID's Medical Management of Biological Casualties Handbook. 6th ed. United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland; 2005. p. 61-74.
[82]Decker HM, Buchanan LM, Lawrence BH, and Goddard KR. Air Filtration of Microbial Particles[J].Am J Public Health Nations Health,1963,53:1982-1988.
[83]Rapp ML, Thiel T, and Arrowsmith RJ. Model system using coliphage Phix174 for testing virus removal by air filters[J].Appl Environ Microbiol,1992,58:900-904.
[84]Burnett LAC,Lunn G,and Coico R.Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr Protoc Microbiol,2009,5: chapter 1:unit 1A.1.
[1]Tseng Chun-Chieh,Chih-Shan Li.Collection efficiencies of aerosol samplers for virus-containing aerosols[J].J Aerosol Sci,2004,36:593-607.
[2]Balazy Anna,Toivola Mika,Adhikari Atin,et al.Do N95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks?[J]Am J Infect Control,2006, 34(2):51-57.
[3]Fauquet CM,Mayo MA,Maniloff J,et al.Virus Taxonomy, Eighth Report of the International Committee Taxonomy Viruses [M].Amsterdam: Elsevier Academic Press,2005.
[4]Ackermann H–W.5500 Phages examined in the electron microscope[J].Archives ofvirology,2007,152:227-243.
[5]Davis JE,Strauss JH,Sinsheimer RL.Bacteirophage MS2:Another RNA phage[J]. Science,1961,134:1427.
[6]Stockley PG,Stonehouse NJ and Valegard K.Molecular mechanism of RNA phage morphogenesis[J].Int J Biochem,1994,26:1249–1260.
[7]Peabody,DS,and Al-Bitar L.Isolation of viral coat protein mutantswith altered assembly and aggregation properties[J].Nucleic Acids Res,2001,29,E113.
[8]Jolis D,Hirano R and Pitt P.Tertiary treatment using microfiltration and UV disinfection for water reclamation[J].Water Environ Res,1999,71:224–231.
[9]Lykins BW, Koffskey WE and Patterson KS.Alternative disinfectants for drinking water treatment[J].J Environ Eng,1994,120:745–758.
[10]Heal KG,Hill HR,Stockely PG,Hollongdale MR and Taylorrobinson AW.Expression and immunogenicity of a liver stage malaria epitope presented as a foreign peptide on the surface of RNA-free MS2 bacteriophage capsids[J].Vaccine,2000,18:251–258.
[11]Stockley PG and Mastico RA.Use of fusions to viral coat proteins as antigenic carriers for vaccine development[J].Methods Enzymol,2000,326:551–56.
[12]ISO10705—1 Water Qulality—Detection and Enumeration of Bacteriophages.Part1:Enumeration of F—specific RNA Bacteriophgaes[S].International organizaiton for Standardization.1995:15.
[13]Hsu YC,Nomura S and Kruse CW.Some bactericidal and virucidal properties of iodine not affecting infectious RNA and DNA[J]. Am J Epidemiol,1966,82:317-328.
[14]Cramer WN,Kawata K and Kruse CW.Chlorination and iodination of poliovirus and f2[J].J Am Water Works Assoc,1976,68:61-76.
[15]Olivieri VP,Kruse CW,Hsu YC,Griffiths AC and Kawata K.The comparative mode of action of chlorine,bromine,and iodine on f2 bacterial virus.1975,p.145-162. In J.D. Johnson(ed.),Disinfection-water and wastewater. Ann Arbor Science Publishers, Ann Arbor, Mich.
[16]何静芳,马伟,陈泰尧,等.f2噬菌体对常用消毒处理的抗力测定[J].上海预防医学杂志,2005,17(9):448-449.
[17]王思熊,熊鸿燕,林辉,等.吖啶橙与长波紫外线灭活血浆中f2噬菌体的形态变化[J].第三军医大学学报,2004,26(3):220-224.
[18]田丰,王政,杨荆泉,等.正压送风过滤式防护头罩防护性能研究[J].生物医学工程学杂志,2006,23(4):766-769.
[19]鹿建春,孙振海,李劲松,等.生物防护口罩的研制及其防护效果评价[J].中国消毒学杂志,2004,21(3):189-192.
[20]Sanger F,Air GM,Barrell BG,Brown NL,Coulson AR,Fiddes CA,Hutchison CA, Slocombe PM,and Smith M.Nucleotide sequence of bacteriophage phiX174 DNA[J]. Nature,1977,265(5596):687-695.
[21]Fiers W and Sinsheimer RL.The structure of the DNA of bacteriophage PhiX 174. III. Ultracentrifuge evidence for a ring structure[J].J Mol Biol,1962,5:424-434.
[22]Demik G. and Degroot I.Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene[J].J Hyg,1977,78(2):199-211.
[23]Tseng Chun-Chieh and Li Chih-Shan.Ozone for inactivation of aerosolized bacteriophages[J].Aerosol science and technology,2006,40(9):683-689.
[24]ISO 16604.Clothing for protection against contact with blood and body fluids-Determination of resistance of protective clothing materials to penetration by blood-borne-pathogens-Test method using Phi-X174 bacteriophage[S].International Organization for Standardization.2004.
[25]Delbr?ck M.The burst size distribution in the growth of bacterial viruses[J].J Bacteriol,1945,50:131–135.
[26]Trouwborst T,De jong JC and Winkler KC.Mechanism of inactivation in aerosols of bacteriophage T1[J].J gen Virol,1972,15:235-242.
[27]Trouwborst T and Winkler KC. Protection against aerosol-inactivation of bacteriophage T1 by peptides and Amino Acids[J].J gen Virol,1972,17:1-11.
[28]Harstad JB.Sampling submicron T1 bacteriophage aerosols[J].Applied Microbiology, 1965,13(6):899–908.
[29]Ehrlich Richard,Miller sol,and Idoine LS.Effects of environmental factors on the survival of airborne T-3 coliphage[J]. Appl Microbiol,1964,12(6):479-482.
[30]Trouwborst T. and Kuyper Sjoukje.Inactivation of bacteriophage T3 in aerosols:effect of prehumidification on survival after spraying from solutions of salt,peptone,and saliva[J]. Appl Microbiol,1974,27(5):834-837.
[31]Warren J.C.and Hatch M.T.Survival of T3 coliphage in varied extracellular environments I. Viability of the coliphage during storage and in aerosols [J].Appl Microbiol,1969,17(2):256-261.
[32]Dahlgren CM,Decker HM,and Harstad JB.A slit sampler for collecting T-3 bacteriophage and Venezuelan equine encephalomyelitis virus.I.Studies with T-3 bacteriophage[J].Appl Microbiol,1961,9(2):103-105.
[33]Coriell LL and Mcgarrity G.J.Biohazard hood to prevent infection duringmicrobiological procedures[J]. Appl Microbiol,1968,16(12):1895-1900.
[34]Hatch MT and Warren JC.Enhanced recovery of airborne T3 coliphage and Pasteurella pestis bacteriophage by means of a presampling humidification technique[J].Appl Microbiol,1969,17(5):685-689.
[35]Experimental studies on environmental contamination with infected blood during haemodialysis[J].J Hyg,1975,74(1):133-148.
[36]Sullivan JF and Songer JR. Role of differential air pressure zones in the control of aerosols in a large animal isolation facility[J]. Appl Microbiol,1966,14(4):674-678.
[37]Merrill JT.Evaluation of selected aerosol-control measures on flow sorters[J]. Cytometry,1981,1:342–345.
[38]Schmid Ingrid,Hultin Lance E.,and Ferbas John.Testing the efficiency of aerosol containment during cell sorting[J].Curr Protoc Cytom,2001,Chapter3:3.3.1-3.3.15.
[39]Lennartz K,Lu M,Flasshove M,Moritz T,and Kirstein U.Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet[J]. Cytometry,2005, 66(2):119-127.
[40]Ferbas J,Chadwick KR,Logar A,Patterson AE,Gilpin RW,Margolick JB.Assessment of aerosol containment on the ELITE flow cytometer.Cytometry(Communications in Clinical Cytometry)1995,22:45–47.
[41]Eninger RM,Honda takeshi,Adhikari A,et al.Filter performance of N99 and N95 facepiece respirators against viruses and ultrafine particles[J].Ann Occup Hyg, 2008,52(5): 385-396.
[1]Ali M,Lemoine NR,Ring CJ.The use of DNA viruses as vectors for gene therapy[J].Gene Ther,1994,l(6):367-384.
[2]Jolly D.Viral vector systems for gene therapy[J].Cancer Gene Ther,1994,1(1):51-64.
[3]Levine AJ.The molecular biology of adenovirus.In:Bercoff RP ed.The molecular basis ofviral replication[M].Plenum Press,1987,483-498.
[4]Harvey B,Maroni J,O’Donoghue KA,et al.Safety of local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid condition [J].Hum Gene Ther,2002,(13):15-63.
[5]龚非力主编.医学免疫学[M].第1版,北京:科学出版社,2003,34-35.
[6]Mestecky J.The common mucosal immune system and current strategies of induction of immune responses in external secretions [J].Clin mmunol,1987,7(4):265–276.
[7]McGhee JR,Mestecky J,Dertzbaugh MT,et al.The mucosal immune system: from fundamental concepts to vaccine development[J].Vaccine,1992,10(2):75–88.
[8]Nugent J,Po A L,Scott EM.Design and delivery of non-parenteral[J].Clin Pharm Ther,1998,23(4):257-285.
[9]Walker RI.New strategies for using mucosal vaccination to achieve more effective immunization[J].Vaccine,1994,12(5):387–400.
[10]Frey A,Neutra MR.Targeting of mucosal vaccines to Peyer’s patch M cells[J].Behring Inst Mitt,1997,98:376–389.
[11]Mestecky J,Michalek SM,Moldoveanu Z,et al.Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans[J].Behring Inst Mitt,1997,98:33–43.
[12]Mestecky J,McGhee J R.Prospects for human mucosal vaccines[J].Adv Exp Med Biol,1992,327:13–23.
[13]Zhang Y,Chirmule N,Gao GP,et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages[J].Mol Ther,2001,3:697–707.
[14]Crystal RG, Harvey B,Wisnivesky JP,et al.Analysis of risk factors for local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid condition[J].Hum Gene Ther,2002,13:65-100.
[15]Morrow CD,Novak MJ,Ansardi DC,et al.Recombinant viruses as vectors for mucosal immunity[J].Curr Top Microbiol Immunol,1999,236:255–273.
[16]Medzhitov R and Janeway C Jr.Innate immune recognition: mechanisms and pathways [J].Immunol Rev,2000,173:89–97.
[17]Schnell MA,Zhang Y,Tazelaar J,et al.Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors[J].Mol Ther,2001, 3:708-722.
[18]Morelli AE,Larregina AT,Ganster RW,et al.Recombinant adenovirus inducesmaturation of dendritic cells via an NF kappaB dependent pathway[J].J Virol,2000,74: 9617–9628.
[19]Molinier FV,Prevost BA,Hong SS,et al.The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain[J].J Biol Chem,2003,278(39):37175–37182.
[20]Philpott NJ,Nociari M,Elkon KB,et al.Adenovirus induced maturation of dendritic cells through a PI3 kinase-mediated TNF-alpha induction pathway[J].Proc Natl Acad Sci USA,2004,101:6200–6205.
[21]Hong SS,Habib NA,Franqueville L,et al.Identification of adenovirus (Ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative Ad (Addl1520) for treatment of liver tumors [J].J Virol,2003,77:10366–10375.
[22]Olive M,Eisenlohr L,Flomenberg N,et al.The adenovirus capsid protein hexon contains a highly conserved human CD4+ T-cell epitope[J].Hum Gene Ther,2002,13(10):1167-1178.
[23]Fitzgerald JC,Gao GP,Reyes-Sandoval A,et al.A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag[J].J Immunol,2003,170(3):1416–1422.
[24]Xiang ZQ, Yang Y,Wilson JM,et al.A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier[J].Virology,1996,219:220–227.
[25]He Z,Wlazlo AP,Kowalczyk DW,et al.Viral recombinant vaccines to the E6 and E7 antigens of HPV-16[J].Virology,2000,270(1):146–161.
[26]Yang Y,Li Q,Ertl HC,et al.Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses[J].J Virol,1995,69:2004–2015.
[27]Yang Y,Ertl HC and Wilson JM.MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses[J].Immunity,1994,1:433–442.
[28]Prevec L,Campbell JB,Christie BS,et al.A recombinant human adenovirus vaccine against rabies[J].J Infect Dis,1990,161:27–30.
[29]Casimiro DR,Chen L,Fu TM,et al.Comparative immunogenicity in rhesus monkeys of DNA plasmid,recombinant vaccinia virus,and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene[J].J Virol,2003,77: 6305–6313.
[30]Wang Y,Xiang Z,Pasquini S,et al.The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus[J].J Virol,1997,71:3677–3683.
[31]Xiang ZQ,Gao GP,Reyes-Sandoval A,et al.Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier[J].J Virol, 2003,77: 10780–10789.
[32]Sharpe S,Fooks A,Lee J,et al.Single oral immunization with replication deficient recombinant adenovirus elicits long-lived transgene-specific cellular and humoral immune responses[J].Virology,2002,293:210–216.
[33]Pinto AR,Fitzgerald JC,Gao GP,et al.Induction of CD8+ T cells to an HIV-1 antigen upon oral immunization of mice with a simian E1-deleted adenoviral vector[J].Vaccine, 2004,22:697–703.
[34]李焱,岳盈盈,张凤丽,等.腺病毒载体疫苗免疫途径的研究[J].中华医学研究杂志2004,4(5):392–393.
[35]Natuk RJ,Davis AR,Chanda PK,et al.Adenovirus vectored vaccines[J].Dev Biol Stand, 1994,82:71–77.
[36]何金生,王健伟,姜秀丽,等.复制缺陷型重组腺病毒rv Ad G1VP7免疫学效果的研究[J].安徽医科大学学报,2002,37(2):86–88.
[37]Lemiale F,Kong WP,Akyurek LM,et al.Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system[J].J Virol,2003,77:10078–10087.
[38]Smith JG,Raper SE,Wheeldon EB,et al.Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self-limiting inflammatory Response[J].Hum Gene Ther,1997,8(8):943–954.
[39]Charlton KM,Artois M,Prevec L,et al.Oral rabies vaccination of skunks and foxes with a recombinant human adenovirus vaccine[J].Arch Virol,1992,23(1-2):169-179.
[40]Meszaros J,Szemeredi M,Tamasi G.Immunization of day-old chickens against newcastle disease[J].Acta Vet Hung,1992,40:121-127.
[41]Van Eck JHH.Vaccination of fowl with inactivated New-Castle disease vaccine by the respiratory route[J].Avian pathol,1990,19:313-330.
[42]Sharma JM.Introduction to poultry vaccines and immunity[J].Adv Vet Med,1999, 41:481-494.
[43]Al-tarcha B,Kojnok J,Varro C.Immunization of day-old chicks having maternally derived antibodies against infectious bronchitis: degree of protection as monitored by ciliary activity after intratracheal challenge [J].Acta Vet Hung,1991,39:83-93.
[44]Sandhu TS.Immunogenecity and safety of a live pasteulla anatipestifer vaccine in white pekin ducklings: laboratory and field trials[J].Avian Pathol,1991,20:423-432.
[45]Deuter A,Southee DJ,Mockett APA.Fowlpox virus:pathogenicity and vaccination of day-old chicks via the aerosol route[J].Res Vet Sci,1991,50:362-364.
[46]Murphy D,Van Alstine WG,Clark LK,et al.Aerosol vaccination of pigs against mycoplasma hyopneumoniue infection[J].Am J Vet Res,1993,54:1874-1880.
[47]Gruber WC,Hinso HP,Holland KL,et al.Comparative trial of large-particle aerosol and nase drop administration of live attenuated influenza vaccines[J].J Infect Dis,1993, 168(5):1282-1285.
[48]徐琳,李劲松,周育森.炭疽杆菌保护性抗原重组腺病毒疫苗的构建及气溶胶免疫探索性研究[D].中国人民解放军军事医学科学院硕士学位论文,2006,25-34.
[49]Bellon G,Michel CL,Thouvenot D,et al.Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial[J].Hum Gene Ther,1997,8(1):15-25.
[50]Bellon G,Calmard L,Thouvenot D,et al.Aerosol administration of a replication defective recombinant adenovirus expressing normal human cDNA-CFTR in the respiratory tractus in patients with cystic fibrosis[J].C R Seances Soc Biol Fil,1996, 190(1):109-142.
[51]Lerondel S,Vecellio N L,Faure L,et al.Gene therapy for cystic fibrosis with aerosolized adenovirus-CFTR:characterization of the aerosol and scintigraphic determination of lung deposition in baboons[J].J Aerosol Med,2001,14(1):95-105.