Isolation And Characterization Of Model Viruse And Its Application In Bio-protective Equipments And Facilities

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.