ABP Viral Vectors Rodents by rgj0CLO

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									                                                                                                                         Animal
                                            Inoculation of Viral Vectors in                                             Biosafety
                                            Laboratory Rodents                                                         Procedures

1. Purpose and Scope
   This Animal Biosafety Procedure (ABP) describes prudent practices, procedures, and equipment to reduce
   risk when introducing viral vectors (e.g. lentiviral, adenoviral) into laboratory rodents at Animal Biosafety Level
   2 (ABSL-2). The practices and procedures are in accordance with those described in the CDC/NIH Biosafety
                                                                  th
   in Microbiological and Biomedical Laboratories (BMBL), 5 edition
   (http://www.cdc.gov/biosafety/publications/bmbl5/index.htm), and the NIH Guidelines for Research Involving
   Recombinant DNA Molecules (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm ).

     ***Alternative practices, procedures, and equipment may be used, but they must be described in a
     user-generated standard operating procedure, and approved by EHS and the IACUC before use.***

2. Responsibilities
   The Principal Investigator will ensure that personnel are made aware of the hazards associated with the
   infectious agent and that they receive training commensurate with their activities prior to commencing ABSL-2
   experiments. Personnel will comply with the safe work practices and procedures described within this Animal
   Biosafety Procedure.

3. Administrative Controls
   3.1 General Risk Assessment for Viral Vectors
       3.1.1 Consider the nature of the vector system (e.g., HIV vs. feline immunodeficiency virus based
              systems), tropism (e.g., pseudotyping with vesicular stomatitis virus glycoprotein), and the
              potential for regeneration of replication competent virus from the vector components.
       3.1.2 The nature of the transgene (e.g., known oncogenes or genes with high oncogenic potential) may
              increase personal exposure risk when manipulating viral vectors.
       3.1.3 The inherent biological containment of the animal host, if relevant (e.g., non-permissive systems
              such as immune-competent mice do not support replication of infectious HIV)
       3.1.4 Latest generation systems obtained from reputable commercial suppliers have increased
              biosafety features. In contrast, older generation viral vector systems have fewer biosafety
              features and may be more likely to produce replication competent virus. The IBC strongly
              discourages the use of these older viral vector systems.
       3.1.5 Before use, verify the identity of plasmids obtained from colleagues or institutions.
       3.1.6 Consult the attached viral vector table for more information regarding biosafety levels and
              handling of infected animals.

     3.2 Training
         3.2.1 Receive laboratory-specific training for safe manipulation of viral vectors, and spill and exposure
                 response procedures.
         3.2.2 Complete the ABSL-2 online training module and CARE training for handling of rodents.
         3.2.3 CARE and EHS will provide additional on-site training, as necessary.

     3.3 Access and Signage
         3.3.1 Inform the facility manager prior to introducing viral vectors in rodents.
         3.3.2 Review the hazards and potential risks of the experiment, and complete IACUC module 2 before
                accessing the animal facility.
         3.3.3 The facility supervisor will post a hazard sign at the animal room. Research, EHS, animal care,
                and CARE veterinary staff will develop information contained in the sign, which will include:
                 The biohazard symbol and ABSL-2 designation
                 The name of the Principal Investigator and IACUC protocol number
                 The type/class of viral vector system
                 Potential shedding of the viral vector by the animal
                 Personal protective equipment
                 Disinfectant(s)
  Approved by: Institutional Biosafety Committee 12/13/11                                81441012-03ae-41c9-8dac-cb4df220e2d7.doc
  Last revised by: Frank A. Cantone                                                                                    Page 1 of 5
  Revision date: 10/31/11
The most recent version of this document is available electronically at: http://sp.ehs.cornell.edu/lab-research-safety/bios/animal-
research/Pages/default.aspx
                                                                                   Inoculation of Viral Vectors in Laboratory Rodents

          3.3.4     Post a hazard ID card on cages that contain inoculated animals. The card will include: the
                    biohazard symbol, name of the viral vector, and date of inoculation.

     3.4 Medical Surveillance
         3.4.1 Participate in the Animal Users Health and Safety Program (AUHSP).

4. Work Practice and Procedure Controls
   4.1 Inoculation of animals
       4.1.1 See Sections 5.1, engineering controls, and 6.1, personal protective equipment (PPE).
       4.1.2 Use an appropriate manual or physical restraint device. If the procedure or conditions of
               inoculation pose too high a risk with an awake animal (e.g., retro-orbital injections, inexperienced
               individual performing the procedure), sedate the animal prior to inoculations.
       4.1.3 Use a disinfectant-soaked cloth to wipe away excess inoculum leaking from inoculation site.

     4.2 Sharps Handling
         4.2.1 Substitute plasticware for glassware whenever possible, and implement the following safe
                practices for handling sharps:
                 Limit the use of sharps to when no other alternatives are available
                 Keep all sharps in full view at all times
                 Use only Luer-lock syringes and needles or units where the needle is integral to the syringe
                 Implement safety engineered sharps where practical
                 Dispose of sharps directly, without manipulation, in an approved sharps disposal container (i.e.,
                  do not bend, shear, break, recap, or use hands to remove needles from syringes or blades from
                  scalpels). Maintain disposal container within arm’s reach (including inside a biosafety cabinet)
                 Handle broken glass or other sharps with a secondary device such as forceps or broom and
                  dustpan- not your hands
         4.2.2 Do not recap needles. However, if recapping must be done first receive approval by EHS and the
                IACUC, and use one of the following two methods: one handed scoop technique; forceps or tongs
                to place the cap on the needle.

     4.3 Hygiene
         4.3.1 Eating, drinking, smoking, handling contact lenses, applying cosmetics, storing food for human
                consumption, and mouth pipetting are strictly prohibited in animal facilities.
         4.3.2 Wash hands thoroughly with soap and water after removing gloves. Use an alcohol-based hand
                sanitizer if sink is not readily available.
         4.3.3 If working long hours in a rodent room consider taking a full body shower to reduce the amount of
                potential allergens present on your body.

     4.4 Decontamination and Spill Response
         4.4.1 Decontaminate work surfaces and equipment (e.g., inside of biosafety cabinet, animal cages)
                with a suitable disinfectant- allow at least 5-10 minutes of contact time. Suitable disinfectants
                must be active against the targeted vector, and address factors such as environment (e.g.,
                organic      load,       surfaces)       contact     time,     application,     and       safety:
                    http://www.cfsph.iastate.edu/BRM/resources/Disinfectants/CharacteristicsSelectedDisinfectants.pdf
          4.4.2     Cover spills with absorbent towels/pads and saturate with disinfectant. Allow at least 5-10
                    minutes contact time to achieve adequate disinfection. Appropriately segregate waste in red
                    biohazard bags or sharps disposal containers and re-apply disinfectant to spill area.

     4.5 Handling of Waste
         4.5.2 Inside a biosafety cabinet, place a water soaked paper towel in the dirty cage, to generate steam
                when autoclaved, or leave water bottle in cage. Wipe exterior of cage with disinfectant before
                removing from biosafety cabinet.
         4.5.3 Dispose of sharps-related items (e.g., needles, syringes, Pasteur pipettes, blood tubes) directly in
                a sharps disposal container.
         4.5.4 Dispose of non-sharps items (e.g., gloves, intact plasticware) in a red biohazard bag.
         4.5.5 Treat infectious liquid waste with concentrated household bleach to a final volume of 10% bleach
  Approved by: Institutional Biosafety Committee 12/13/11                                81441012-03ae-41c9-8dac-cb4df220e2d7.doc
  Last revised by: Frank A. Cantone                                                                                    Page 2 of 5
  Revision date: 10/31/2011
The most recent version of this document is available electronically at: http://sp.ehs.cornell.edu/lab-research-safety/bios/animal-
research/Pages/default.aspx
                                                                                   Inoculation of Viral Vectors in Laboratory Rodents

                    and allow at least 30 minutes contact time before disposal in the sanitary waste drain- follow with
                    copious amounts of water.
          4.5.6     Place carcasses (no gloves, plastic, etc.) in bags suitable for disposal in the Waste Management
                    digester. Wipe bags with appropriate disinfectant and store all bags in a larger biohazard bag or
                    biohazard-labeled drawer in refrigerator. Alternatively, roll up carcasses in bench diapers and
                    place them directly in biohazard bags. These bags will be disposed of offsite.
          4.5.7     Animal care staff will dispose of waste and carcasses, unless other arrangements are made.

     4.6 Transport of Biohazardous Materials
         4.6.2 Transport vector preparations and contaminated samples between laboratory and animal facility
                in a sealed, secondary container with absorbent toweling, and labeled with the biohazard symbol.

     4.7 Tissue Harvest
         4.7.2 Perform tissue harvest in a certified class II biosafety cabinet- use a tray or bench diaper to
                 collect fluids. Use tape instead of pins to secure carcass.
         4.7.3 When possible, use only one sharps item (e.g., scalpel, scissors) at a time and keep in full view.
         4.7.4 Place any harvested tissue or fluids in appropriate primary containers (e.g., screw top vial,
                 sealable plastic bag), decontaminate exterior, and transport as per section 4.6. Fixed tissues
                 (e.g., 10% buffered formalin) are no longer considered biohazardous. Use appropriate personal
                 protective equipment when handling these samples and transport in a secondary container.
         4.7.5 Follow the sharps handling practices outlined in section 4.2.

5. Engineering Controls
   5.1 Biosafety Cabinet Use
       5.1.1 Perform all procedures carefully to minimize the creation of aerosols. Use a certified class II
               biosafety cabinet for: inoculation; necropsy and tissue harvest; cage changing; and manipulation
               of high concentrations or large volumes of viral vectors.
       5.1.2 Wipe cages with appropriate disinfectant when moving out of biosafety cabinet.

     5.2 Housing and Handling of Infected Animals
         5.2.1 House animals in a primary containment device appropriate for the rodent species, such as a
                ventilated micro-isolator cage or static micro-isolator cage with a filter top.
         5.2.2 Whenever possible, use forceps to transfer inoculated animals between cages.
         5.2.3 Conduct inoculations, cage changing, and other procedures in a biosafety cabinet.
         5.2.4 Infected animals may excrete viral vectors. Consult the attached viral vector table for more
                information regarding biosafety levels and handling of infected animals. In certain circumstances
                the biosafety level and containment of animals may be reduced (e.g., ABSL-2 to ABSL-1).

6. Personal Protective Equipment (PPE)
   6.1 Don the following minimum PPE before entering ABSL-2 animal rooms:
        Disposable fluid resistant, solid front gown
        Disposable gloves (nitrile- avoid latex when possible) - Use double gloves when handling the viral
         vector or when inoculating animals. Outer glove should overlay cuff of gown
        Shoe covers
   6.2 Wear additional PPE (e.g., face shield, respiratory protection, cut/bite resistant gloves) when appropriate
       engineering controls are not available, or as indicated by the hazards or experimental conditions.
   6.3 Solid toed shoes are required for entry into animal rooms.
   6.4 Change gloves frequently (or decontaminate with disinfectant) during activities to avoid contamination of
       equipment and surfaces. Remove and replace other PPE if contaminated or breached.
   6.5 Remove PPE upon exiting the animal room and dispose in red biohazard bag. First remove outer gloves,
       gown (turning inside out), shoe covers while stepping out of the room (step-over technique), and finally
       inner gloves.

7. Response to Accidental Exposures
   7.1 Personnel who sustain an overt exposure such as a splash to mucous membranes, direct contact with
   open wounds, or a sharps injury should:
  Approved by: Institutional Biosafety Committee 12/13/11                                81441012-03ae-41c9-8dac-cb4df220e2d7.doc
  Last revised by: Frank A. Cantone                                                                                    Page 3 of 5
  Revision date: 10/31/2011
The most recent version of this document is available electronically at: http://sp.ehs.cornell.edu/lab-research-safety/bios/animal-
research/Pages/default.aspx
                                                                                   Inoculation of Viral Vectors in Laboratory Rodents

              Wash exposed area with soap and water or rinse in eye wash for at least 10 minutes
              Perform first aid, if applicable
              Notify supervisor
              Seek medical evaluation at Gannett Health Services, Occupational Medicine (255-6960) as soon as
               possible after an exposure. Have MSDS or other information document readily available. After hours
               seek evaluation at Cayuga Medical Center.
              Contact Gannett Occupational Medicine if you develop symptoms suggestive of exposure to the
               hazardous agent.
              Document exposures, injuries, and illnesses in the Cornell University Injury/Illness/Exposure Report,
               http://cfp-rmps.coldfusion.cornell.edu/accinj/.

8. Emergency Phone Numbers
    Police, Fire, and Medical Emergencies: 911
    Environmental Health & Safety (EHS): 255-8200 (off hours 255-1111)
    Gannett Health Services, Occupational Medicine: 255-6960 (off hours 255-5155)
    Cornell Animal Resources and Education (CARE): 253 4378 (off hours 1-800-349-2456 for veterinary
     medical emergencies)

9. References
                                                                   th
   9.1 Biosafety in Microbiological and Biomedical Laboratories, 5 edition. 2009. Centers for Disease Control
       and Prevention, National Institutes of Health.
   9.2 National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules
   9.3 Recombinant DNA Advisory Committee (RAC) Guidance Document for Biosafety Considerations for
       Research with Lentiviral Vectors.
       http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf
   9.4 Working at Animal BSL 2. American Biological Safety Association. http://www.absa.org/ttabsl2.html




  Approved by: Institutional Biosafety Committee 12/13/11                                81441012-03ae-41c9-8dac-cb4df220e2d7.doc
  Last revised by: Frank A. Cantone                                                                                    Page 4 of 5
  Revision date: 10/31/2011
The most recent version of this document is available electronically at: http://sp.ehs.cornell.edu/lab-research-safety/bios/animal-
research/Pages/default.aspx
                                                                                       Inoculation of Viral Vectors in Laboratory Rodents


                                                              Viral Vector Table

 Viral Vector               Biosafety       Animal Biosafety Level **                                       Disinfectants
                              Level

 Murine Retrovirus               1          ABSL-1                                                          1:10 Dilution of household bleach
 (Ecotropic – only                                                                                          (recommended)
 infects mice)

 Murine Retrovirus               2          ABSL-2                                                          1:10 Dilution of household bleach
 (amphotropic or                                                                                            (recommended)
 pseudo-typed –
 can infect multiple
 hosts)

 Lentivirus                      2          ABSL-2                                                          1:10 Dilution of household bleach
                                                                                                            (recommended), 70% alcohol
                                            (At four days post-administration, non-
                                            permissive animals may be handled at
                                            ABSL-1)

 Adenovirus                      2          ABSL-2                                                          1:10 Dilution of household bleach
                                                                                                            (recommended)
                                            (At four days post-administration, animals                      Alcohol is not effective against
                                            may be handled at ABSL-1)                                       adenoviral species

 Adeno-Associated                1          ABSL-1 if no helper virus                                       1:10 Dilution of household bleach
 virus (AAV)                                                                                                (recommended)
                                 2          ABSL-2 with a known or potential helper virus

 Herpesvirus I and               2          ABSL-2                                                          1:10 Dilution of household bleach
 II                                                                                                         (recommended), 70% ethanol

 Epstein Barr                    2          ABSL-2                                                          1:10 Dilution of household bleach
                                                                                                            (recommended), 70% ethanol

 Vaccinia                        2          ABSL-2                                                           1:10 Dilution of household bleach
                                                                                                            (recommended), 70% ethanol



** Special consideration for transgenes, toxins, oncogenes, elements that alter host range, etc.




      Approved by: Institutional Biosafety Committee 12/13/11                                81441012-03ae-41c9-8dac-cb4df220e2d7.doc
      Last revised by: Frank A. Cantone                                                                                    Page 5 of 5
      Revision date: 10/31/2011
    The most recent version of this document is available electronically at: http://sp.ehs.cornell.edu/lab-research-safety/bios/animal-
    research/Pages/default.aspx

								
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