ABP Viral Vectors in Rodents
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 in Microbiological and Biomedical Laboratories (BMBL), 6th edition, and the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, found on the NIH Office of Science Policy website.
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.
The Principal Investigator will ensure that personnel are made aware of the hazards associated with the viral vectors 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 and responsibilities set forth in ABSL-2 Program Document and Biosafety Levels 1 & 2 Written Program. The PI will report spills outside of containment involving or exposures to viral vectors within 24 hours to EHS and the IBC.
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.
- 3.2.1 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 type/class of viral vector system
- Potential shedding of the viral vector by the animal
- Personal protective equipment
- 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 practices described in the table:
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 (retractable needles, needle tip shields, self-sheathing scalpels, etc.) Dispose of sharps directly, without manipulation (i.e., do not bend, shear, break, recap, or use hands to remove needles from syringes or blades from scalpels), in an approved sharps disposal container. Maintain disposal container in animal room within arm’s reach 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.
- If recapping must be done: first receive approval by EHS and the IACUC. Once approved, only use one of the following two methods: one handed scoop technique; forceps or tongs to place the cap on the needle.
- 4.2.3 If a needle/scalpel must be set down temporarily during a procedure, set the sharp end inside a clean 50-mL conical tube or equivalent to act as a protective sheath (rather than recapping) and to prevent injury.
- 4.2.1 Substitute plasticware for glassware whenever possible, and implement the following practices described in the table:
- 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.
For more information, see ACUP 713: Hygiene – Hand Washing
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: The Center for Food Security & Public Health
- 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
- 4.5.1 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.
- 4.5.2 Dispose of sharps-related items (e.g., needles, syringes, Pasteur pipettes, blood tubes) directly in a sharps disposal container.
- 4.5.3 Dispose of non-sharps items (e.g., gloves, intact plasticware) in a red biohazard bag.
- 4.5.4 Treat infectious liquid waste with concentrated household bleach to a final volume of 10% bleach and allow at least 30 minutes contact time before disposal in the sanitary waste drain- follow with copious amounts of water.
- 4.5.5 Place carcasses (no gloves, plastic, etc.) in compostable 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.
- 4.5.6 Animal care staff will dispose of waste and carcasses, unless other arrangements are made.
4.6 Transport of Biohazardous Materials
- 4.6.1 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
- 4.7.1 Perform tissue harvest in a certified class II biosafety cabinet- use a tray or bench diaper to collect fluids. Preferably, use tape instead of pins to secure carcass.
- 4.7.2 When possible, use only one sharps item (e.g., scalpel, scissors) at a time and keep in full view.
- 4.7.3 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.
- 4.7.4 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 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.
- For more information, see ACUP 715: Personal Protective Equipment
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:
Wash exposed area with soap and water or rinse in eye wash for at least 5 minutes
- Perform first aid, if applicable
- Notify supervisor
- Seek medical evaluation at Cornell Health Services, Occupational Medicine (607-255-6960) as soon as possible after an exposure. Have MSDS or other information document readily available. After hours seek evaluation at Cayuga Medical Center.
- Contact Cornell Health Services, 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.
- For more information, see ACUP 707: Animal Related Injury
8. Emergency Phone Numbers
Police, Fire, and Medical Emergencies: calling 911 from an on-campus phone or by calling 607-255-1111 from a cellular phone
- Environment, Health and Safety (EHS): 607-255-8200 (off hours 607-255-1111)
- Cornell Health Services, Occupational Medicine: 607-255-6960 (off hours 607-255-5155)
- Cornell Animal Resources and Education (CARE): 607-253-4378 (off hours 1-800-349-2456 for veterinary medical emergencies)
Viral Vector Table
|Biosafety Level||Animal Biosafety Level||Disinfectants|
|Murine Retrovirus(Ecotropic-only infects mice||1||ABSL-1||1:10 Dilution of household bleach (recommended)|
|Murine Retrovirus (amphotropic or pseudo-typed-can infect multiple hosts)||2||ABSL-2||1:10 Dilution of household bleach (recommended)|
|Lentivirus||2||ABSL-2 (At seven days post-administration, non-permissive animals may be handled at ABSL-1)||1:10 Dilution of household bleach (recommended), 70% alcohol|
|Adenovirus||2||ABSL-2 (At seven days post-administration, animals may be handled at ABSL-1)||1:10 Dilution of household bleach (recommended) Alcohol is not effective against adenoviral species|
|Adeno-Associated virus (AVV)||2||ABSL-1 if no helper virus, ABSL-2- with known or potential helper virus||1:10 Dilution of household bleach (recommended)|
|Herpesvirus I and II||2||ABSL-2||1:10 Dilution of household bleach (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|
- Cornell University BSL-2 Biosafety Manual
- Cornell University IBC
- Cornell University Institutional Biosafety Committee Guidance on The Use of Lentiviral-Based Vectors
- Biosafety in Microbiological and Biomedical Laboratories, 6th edition. 2020. Centers for Disease Control and Prevention, National Institutes of Health.
- National Institutes of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules as found on the NIH Office of Science Policy website.
- Working at Animal BSL 2. American Biological Safety Association.