Advances in Applied Research. Dr. Steven Theriault Applied Biosafety Research Program Public Health Agency of Canada

Transcription

1 Advances in Applied Research Dr. Steven Theriault Applied Biosafety Research Program Public Health Agency of Canada

2 Applied Biosafety Research Program ABRP Our focus at ABRP is to promote the Science of Biosafety, Bio-containment, Applied Research, and Biosecurity Develop stronger national/international based knowledge on Applied Biosafety Research and develop Training Programs to assist in biosafety issues

3 Mandate Develop, Design, and Validate Engineering practices related to containment laboratories Develop standards for disinfectant practices in containment laboratories and field outbreaks Inactivation protocols for inactivation of CL3/4 samples (DNA,RNA, and Protein) Efficacy studies of commonly used disinfectants against infectious disease Infectious Clone Systems, used as a genetically marked standard

4 Program Research Areas ABRP Decontamination Studies -CL3/4 disinfectants - Inactivation of infectious agents -Develop standards for decontamination -Efficacy studies of disinfectants Infectious Clone Systems -Generate traceable/ quantifiable standards -Develop genetic backbones vaccine -Pathogenic Studies -Develop new diagnostic tools Training -Containment Training -Biosafety Training -Emergency Egress -Applied Research techniques -National/International

5 Infectious Clone Systems

6 Infectious Clone Systems Generation of florescent emitting organisms to be used as standard detection tools during validation experiments Genetic Backbones vaccine development Diagnostics Pathogenesis site directed mutagenesis

7 Ebola Reverse Genetics NP T7 pol Ebola FL cdna VP 30 L VP 35 Ebola RNA N

8 Ebola Virus Rescue 30 NP 35 T7 pol L Ebo-Rib? VeroE6/293T Fresh VeroE6 Percentage Rescue Anti VP40 0 All -L -NP -VP30 -VP35 20/20 Priming Plasmids

9 ZEBOV-GFP Construction A NP NP STOP NON-CODING REGION ATT AAG AAA AAA CTA AT GATG AAG ATT AA VP35 Poly A Tail VP35 Start NP NP STOP NON-CODING REGION BssHII site ATT AAG AAA AAA GCGCGC GATG AAG ATT AA VP35 B Poly A Tail VP35 Start NP BssHI I SacI BssHII GCGCGC GAGGAAGATTAA GAGCTC ATTAAGAAAAAA GCGCGC VP35 Transcriptional Start Transcriptional Stop SacI GAGCTC GFP SacI GAGCTC

10 GFP-ZEBOV Virus Rescue 30 NP T7 pol L Ebo-Rib GFP 35? VeroE6/293T Ultraviolet Fresh VeroE6 GFP-ZEBOV

11 GFP Passage Stability STABILITY Stability ASSAY Assay GFP 105 GP/GFP PERCENT Percent CO-EXPRESSION Expression P1 P2 P3 P4 P5 P6 P7 P8 P Microscope MICROSCOPE Point POINT

12 ZEBOV-GFP Stability Assay Texas Red GFP/Texas Red Anti-GP GFP

13 In-vitro studies GFP emitting ZEBOV viruses grew similar to wildtype ZEBOV GFPs growth curve 7 6 Titer 10x( ) Wild-Type GFP-NP GFP Days PI

14 Other Genetic Clones Systems ssrna Cell free Cloning - Generate traceable standards - Develop vaccine backbones - Pathogenic Studies - Develop new diagnostic tools Avian Influenza-GFP ds.cdna Green Fluorescent Fusion protein Decontamination studies Aerosolisation studies Kyasanur Forest Disease Virus (KFDV) Flaviviridae infectious RNA Decontamination studies

15 Decontamination

16 Decontamination Studies (Overview) Determine the optimal concentration range of disinfectants commonly used in containment laboratories Validation of CL3/4 extraction/inactivation protocols Define the efficacies of gaseous decontamination methods

17 Where has all the data gone? Limited efficacy data for all viral families People assume data can be crossreferences Closely related viruses may differ in susceptibility Research conducted under laboratory conditions vary from laboratory to laboratory Test conditions vary with virus, tissue culture type, and experimental design Very few research groups carrying out this type of research

18 Decontamination Studies Experimental Goals Determine the optimal concentration range of disinfectants commonly used in containment laboratories (CL4) Test disinfectants include: Microchem Plus Percept Ethanol - Virkon - Bleach Test organisms will include: Filoviruses Zaire Ebola virus 76 (Wild-type) Zaire Ebola virus GFP Recombinant virus

19 Experimental Procedures Quantitative Carrier Tests QCT-2 Inoculate carrier disk with soil load + virus Add dilution series of disinfectant Soil Load: Mixture of BSA, Tryptone, and Mucin Defined dilutions: 10%- 5%- 1%- 0.1%- 0.01% Carrier disk: Brushed stainless steel disk Defined contact time 1 min 5 min 10 min Terminate test by adding neutralizer Determine log reduction of virus TCID 50 or FFU

20 Disinfectant Range Disinfectant (Min) Contact (Min) Concentration Log Reduction MicroChem 10min 5% 7 logs Virkon 10min 1% 7 logs Bleach 5min 1% 7 logs Percept 5min 5% 7 logs Ethanol 10min 70% 7 logs Ethanol 10min 95% 4 logs

21 Utilizing a green fluorescent Ebola virus to test the efficacy of common used disinfectants in CL4 containment laboratories Jay Krishnan, Laura Douglas, Everett Milotte, Heinz Feldmann, and Steven Theriault (Peer Review Stage) Key Points of Publication Within this paper we defined the range for contact time as well as the specific concentration of disinfectant to inactivate Zaire Ebola virus. We utilized both wild-type ZEBOV and a ZEBOV-GFP to evaluate the use of this fluorescent construct as a standard Both viruses were comparable in growth and inactivation patterns Allowing us to propose ZEBOV-GFP as a standard read out system which is quantifiable when testing filoviral inactivation

22 Determination of the Virucidal Efficacy of Liquid Disinfectants Against a Surrogate virus for Viral Hemorrhagic Fever Viruses Jay Krishnan, Diane Gordon, Stefan Wagner, and Steven Theriault (Peer Review Stage) Key Points of Publication: Using the same five disinfectants we evaluate the relationship between Vesicular Stomatitis Virus (VSV) and ZEBOV during inactivation The similarities between the two viruses has allowed us to suggest that VSV a CL2 virus could be used as a surrogate for viral hemorrhagic fever viruses Determined the efficacy of commonly used CL2 disinfectants against VSV

23 Validation of Extraction Protocols

24 Validation of CL4 Extraction Protocols Viral inactivation by nucleic acid extraction kits Tested Trizol LS, and AVL (RNA extraction kit) buffer against viral members from four genus types Alphavirus WEE Bunyavirus Rift Valley fever Filovirus Ebola (Zaire76) and Marburg (Musoke) Flavivirus West Nile

25 Validation of CL4 Extraction Protocols Both extraction methods were 100% successful in inactivation of any replicating viral species Rift Valley Fever (Bunyavirus) Viral load Trizol LS AVL buffer DMEM media

26 Validation of CL3 Bacterial Extraction Protocols Bacterial/Spore inactivation by commercially available nucleic acid extraction kits Tested five extraction kits (Qiagen, Biorad) B. anthracis, B. atropheaus, and S. aureus

27 Validation of CL3 Bacterial Extraction Protocols Microorganisms S.aureus (25923) B. atrophaeus (51189) B. anthracis (4889) NA Extraction Kits DNeasy Blood & Tissue DNeasy Plant S. aureus: Overnight culture B. species: 72 hrs culture (sporulating media) DNAzol Genomic Wizard

28 Enumeration of Viable Bacteria Serial ten fold dilutions Filter the dilutions and plate the filters for bacterial growth Count bacterial colonies

29 DNeasy Blood and Tissue (Qiagen) DNeasy combines the properties of a silica based membrane with simple micro spin technology - lyse cells with enzymatic lysis buffer (optional microbial inactivation of 95 C for 15 min) - DNA is selectively bound to the Dneasy membrane as contaminants pass through (Discard 1) - DNA is washed (Discards 2 and3) to remove remaining contaminants - DNA is eluted (Eluate)

30 Validation of CL3 Bacterial Extraction Kit Microorganism viability after Dneasy Blood and Tissue Treatment Bacterial counts (log 10) E.coli S.aureus B.atrophaeus B.atrophaeus with 95 C Heat inactivation B.anthracis B.anthracis with 95 C heat inactivation untreated wash 1 wash 2 wash 3 DNA Eluate sample

31 Survival of Bacterial Spores Following DNA Extraction Procedures Cristal Cizik, Diane Gordon, Jay Krishnan, and Steven Theriault Author Review Stage Demonstrated the limitations of bacterial nucleotide extraction kits Demonstrate that heat treatment above 95 o C reduces the number of viable spores, and is necessary with spores formers Filtration is needed to fully remove any spores from the solution

32 CRTI Projects

33 CRTI Projects CRTI grant RD, RD developing standards for end point decontamination Decontamination studies against biotoxin (Anthrax, Y. Pestis, Streptococcus) using gaseous disinfectant methods Design and development of new bio-indicators Evaluation of disinfectant efficacy used in the decontamination of CL3 agents Evaluation of space decontamination Methods

34 CRTI grant RD Space Decontamination (Completed 2007) Determined the effects of one cycle of VHP gaseous decontamination Determined the effects of VHP decon on building material Outdoor testing area demonstrated high background Bacillus Atrophaeus-GFP

35 CRTI grant RD Space Decontamination of Spore Forming Bacteria Developed a building material bioindicator Brick Carpet Dry Wall Concert Wood Metal Plastic Ceiling Tile Bio-Indicator

36 CRTI grant RD Developing Standards for End Point Decontamination We are currently evaluating inactivation procedures for decontamination of Biotoxins/Proteins using: Chlorine dioxide Hydrogen peroxide Formaldehyde These studies show promising results for the inactivation of Bio-toxins/Proteins on innate surfaces with and without serum load

37 Inactivation of Bio-Toxins using Gaseous Decontamination Gaseous Chlorine Dioxide exposure: 2 hours 4-12% non reducing Gel C: Control T: Test (GCD exposed) Vaporized Hydrogen Peroxide exposure: 2 hours 4-12% non-reducing gel C: Control T: Test (VHP exposed)

38 Project Objective Evaluating inactivation procedures for decontamination of bio-toxins using: Vaporous Hydrogen Peroxide (VHP) Modified VHP (mvhp) Gaseous Chlorine Dioxide (GCD) B. anthracis Toxin Neutralization assay lethal toxin (LF) Protective antigen (PA) of B. anthracis

39 Evaluation using Toxin Neutralization Assay Toxins are added to mouse macrophage tissue culture cells LF + PA = cell death LF only = viable cells PA only = viable cells MTT (2,5-diphenyl-tetrazolium bromide) is added to cells Viable cells reduction MTT = purple Dead cells no reduction of MTT = yellow Absorbance values read at 570nm

40 Evaluation of Toxin Neutralization Assay Absorbance values using fresh unexposed LF and PA PA LF PA + LF PA and LF separately do not cause cell death (purple) PA and LF together cause cell death (yellow) LF 0.12ug/ml LF 0.1 ug/ml PA 0.1 ug/ml LF LF 0.08ug/ml 0.06ug/ml LF 0.04 ug/ml PA 0.1ug/ml LF 0.1 ug/ml Media Control A B C Active Toxin Inactivated Toxin

41 VHP vs. LF Toxin Preliminary Data Control: Unexposed LF toxin & fresh PA LF and PA together cause cell death (yellow) LF & PA 10ug/ml LF & PA 5 ug/ml LF & PA 1ug/ml LF & PA 0.5ug/ml LF & PA 0.1ug/ml LF only 1ug/ml PA only 1 ug/ml Media Control A B C minute VHP exposure of LF toxin & fresh PA LF and PA together do not cause cell death (purple) Exposed LF has been detoxified LF & PA 10ug/ml LF & PA 5 ug/ml LF & PA 1ug/ml LF & PA 0.5ug/ml LF & PA 0.1ug/ml LF only 0.1ug/ml PA only 0.1 ug/ml Media Control A B C

42 Decontamination of Field Equipment and Containment Area

43 Decontamination of Field Equipment and Containment Area Utilizing gaseous decontamination methods Assay the ability gaseous methods to decontaminate field equipment and field containment areas Gaseous methods include: Vaporous Hydrogen Peroxide (VHP) Modified VHP (mvhp) Gaseous Chlorine Dioxide (GCD)

44 Decontamination of Field Equipment and Containment Area Chlorine Dioxide tablets and water to generate CD gas (no machine needed) Tested this method during a decontamination of a 12x12 chicken barn Chlorine Dioxide gas penetrated all areas inactivating all Bio-indicators These methods will now be tested in a field situation Promising results for decontamination of buildings after an natural outbreak or BT release

45 Uses in the Field

46 Methods of Generating Gaseous Chlorine Dioxide Old method: Sodium chlorite solution is mixed with an acid activator and water depending on concentration needed New method: MB-10 tablet made from solid sodium chlorite, solid inorganic acid and soluble inorganic additives

47 Methods of Generating Gaseous Chlorine Dioxide Tablets which rapidly generate activated CIO 2 solution and gas when immersed in water (Quip MB-10) Typically 90% conversion of chlorite (CIO2-) to CIO 2 8 to12 minutes depending upon size (1.5 6 grams) Tablet is stable when dry Forms reactive acidic solution in pores when immersed in water Free ClO 2 disperses into water as tablet dissolves

48 Tablet GCD technologies can be applied to self-contained containment units Space decon: Biological Safety Cabinet/Glove box

49 Gaseous Chlorine Dioxide (Present Research) Gaseous Chlorine Dioxide (GCD) is a yellowish green gas with a chlorine like odour that has long been recognized for it s disinfecting properties Mode of action is oxidation, attacks the cell wall Reacts only with reduced sulphur compounds, secondary and tertiary amines and other highly reduced organics In order to be most effective a high relative humidity of more than 80% is recommended Generated using a commercial generator

50 Objective (Present Research) To determine how a reduced relative humidity would affect the efficacy of GCD in its capacity to inactivate microorganisms. The recommended high relative humidity may not always be desirable Sensitive electronic and computer equipment During field decontamination (eg. Chicken barns)

51 Methods (Present Research) Quantitative Carrier Method 2 (QCT-2), a standardized testing method designed to imitate field conditions B.atropheaus spores were dried on stainless steel discs Exposed to 5mg/L GCD at 50%, 60%, 70% and 80% relative humidity Spores were eluted off the carriers and the eluates were cultured to determine the amount of surviving organisms and log reduction

52 Results: Summary Relative Humidity D-Value (min) 12 log Kill time (min) 50% % % % 5 60

53 Conclusions of Humidity Research The higher the relative humidity the less time required for a total kill At a lower humidity of 60% decontamination can still be achieved within a reasonable amount of time (two hours and 12 minutes) This would be desirable in situations where high humidity is difficult to maintain or damaging to equipment

54 Concluding Remarks Gaseous decontamination methods are showing promising results Large spaces Many different building materials can be treated Developing portable systems Microbial extraction kits Inactivation Protocols need to be modify to ensure all organisms are removed

55 Project Update: Using EO Water Generator as Disinfectant on Different Samples of Microorganisms

56 Objective The EO Water Generator (ORP >1000mV) Electrolyzes salt to produce 2 output water streams: 1. Alkaline Solution: (ph= 11.5) 2. Acidic Sanitizer: (ph= 2.5)

57 Recap: The EO Water Generator Reported to be an efficient disinfectant against a broad spectrum of microorganisms Inexpensive Produces pure water No added chemicals Safe Method

58 Test Microorganisms 1. E.coli (Gm ve) Complete kill at 3 min 2. S.aureus (Gm+ve) Complete kill at 3 min 3. B. atrophaeus ~4.5 log reduction at 30 min Triplicate (controls), and duplicate of tests (time periods) performed 3x ORP, ph and chlorine [ ] in acid EO water were tested before each experiment Vegetative Bacteria time periods: 30, 60 sec, and 3 min Spore time periods: 10, 20 and 30 min

59 Results: Acid EO Water vs. E. coli Using Acid EO water as a Disinfectant against E.coli Microorganism counts (log 10) Time (minutes) Tim e Log SD

60 Results: Acid EO Water vs. S. aureus Microorganism counts (log 10) Using Acid EO water as a Disinfectant against S.aureus Time (minutes) Tim e Log SD

61 Results: Acid EO Water vs. B. atrophaeus Using Acid EO Water as a Disinfectant against B. atrophaeus (51189) Time (minutes) Time Log SD Spore counts (log 10)

62 Current Experiments Candida albicans diploid fungus/yeast Time periods Started with 30 sec, 1 and 3 min, then 5, 10 and 15 min Finally: 10, 20 and 30 min (in progress) Result: the higher the time period, the higher the amount of growth

63 Results: Acid EO Water vs. C. albicans (prelim results) Time Log Acid EO water Vs. C.albicans Time (minutes) Spore counts (log 10)

64 EO Water Results Green disinfectant only contains water and NaCl Decreases bacterial and viral loads on building material Currently testing the use as a floor and bathroom cleaner HEPA filters decontamination

65 Acknowledgements Special Pathogens Program University of Manitoba Microbiology Department CRTI Office of Laboratory Security International Center of Infectious Disease Applied Biosafety Research team

66 Questions?