IBC Guidelines on Handling Lentivirus
Introduction and Rationale
- Lentivirus vectors are a common tool used in laboratory benchtop and animal research that have the capacity for causing illness in healthy, human adults. The use of lentiviral vectors has been increasing because the vector system has attractive features; however, such research also raises biosafety issues. This document is for HIV-based lentivirus vectors, and not for non-human lentivirus vectors such as FIV, SIV, EIAV.
- To provide the MU research community with Institutional Biosafety Committee (IBC) approved guidance on the handling of lentivirus vectors in the course of research.
- The IBC requires PI provides laboratory specific training on their IBC Protocol that references these guidelines in the use of lentivirus vectors.
- Primary PI laboratory staff and animal care staff must receive training in items listed below. EHS strongly recommends documenting the training (sign-in sheet) be maintained by the PI or animal care facility.
- How agent can be transmitted
- Signs and symptoms of infection
- Treatment options
- How animal infections are conducted
- How infected animals should be handled
- Signs and symptoms infected animals will exhibit
Risks of Lentivirus Vectors
- The major risks to be considered for research with HIV-1 based lentivirus vectors are:
- potential for generation of replication-competent lentivirus (RCL), and
- potential for oncogenesis.
- These risks can be mitigated by the nature of the vector system (and its safety features) or exacerbated by the nature of the transgene insert encoded by the vector.
- In cases where murine leukemia viruses are used for pseudotyping in place of vesicular stomatitis virus glycoprotein (VSV-g) envelope, there is evidence of enhanced biosafety and a potential for BSL-1 to be recommended (Schembach, et al).
Transport of Lentivirus
- Transport all lentivirus material in a double-sealed leakproof container.
- Label the container with a biohazard symbol, the name of the agent, the amount, and the Principal Investigator's name and telephone number.
- If shipping and/or receiving adenoviral vectors off campus, please contact the Biosafety Office for guidance and training.
- Call the Biosafety Office for assistance in transporting infectious materials at 573-882-7018.
- Laboratory coats, gloves, and safety glasses or goggles must be worn.
- Materials containing lentivirus should be handled inside biological safety cabinets. If your planned laboratory procedures make use of the lentivirus within the BSC difficult, contact EHS Biosafety Staff for discussion of additional containment measures and/or personal protective equipment.
- In the event of a spill outside the BSC, contact EHS Biosafety Staff for guidance and/or assistance in spill cleanup.
- When performing centrifugation procedures, use centrifuge safety cups.
- Protect the vacuum lines and system with disinfectant traps and HEPA filter.
- Pay special attention to the possible generation of aerosols from unwanted biological materials.
Tissue Culture Experiments
- Incorporate the above precautions.
- All tissue culture work should be conducted in a biological safety cabinet with both product and personnel protection.
- A biological hazard sign indicating the use of lentivirus should be placed outside tissue culture room and on the biological safety cabinet.
- Laboratory coats used inside the tissue culture room should not be worn outside the tissue culture room.
- Materials should not be stored inside the biological safety cabinet (BSC). Take only what is needed to perform the procedure(s) and place it in the BSC upon initiation of the procedure. Upon conclusion of the procedure(s), decontaminate with virucide and remove materials from the BSC.
- Serological pipettes and pipette tips should be decontaminated in a virucide, such as a 1:10 dilution of household bleach for at least 30 minutes prior to discarding in solid biohazard unwanted material containers. For this purpose, a beaker containing a virucide may be kept inside the BSC while experimental procedures are being performed, or the vacuum flask setup maybe installed outside the cabinet and placed in secondary containers. If pipettes and pipette tips are to be re-used following decontamination, they should be rinsed in water prior to autoclaving.
- All plasticware placed inside the hood while working with the virus must be decontaminated with a virucide prior to disposal. This can be done by spraying all plasticware with a 1:10 dilution of household bleach (final concentration 0.525%).
- Upon conclusion of procedures in the BSC:
- For liquid unwanted biological materials contact the Resource Recovery Center to discuss and obtain initial approval for decontamination and disposal procedures.
- If aspirated liquid waste is 2/3 full, aspirate a fresh solution of virucide through the suction tube so that the final concentration is appropriate and allow to soak for at least 30 minutes, and empty entire contents down the drain. Refer to the MU Biological Safety Manual for additional guidance.
- Spray all BSC surfaces with a virucide, wipe down, then spray all surfaces with 70% ethanol, and wipe down. Allow the ethanol to air dry. NOTE: Spray the full interior of the BSC (work surface, sides and back of cabinet, as well as the back of the viewing glass).
- If you will dispose of unwanted biohazardous material by autoclaving: When the solid biohazard unwanted material bag is full, seal it with autoclave tape, and take the bag (double bagged and in secondary containment) to the autoclave room for sterilization. After the autoclave cycle is complete and you note the change in the autoclave tape, place the bag inside a black trash bag. The autoclaved unwanted biological material may then be disposed of within a regular sanitation pickup container in accordance with the Autoclave Procedure in the Biological Safety Manual.
- If you will dispose of unwanted biohazardous material using the University's biohazard waste vendor in either a biohazard box or bin: Close off the bag within the biohazard box or bin, assure the yellow HML lab is complete and on the bag (for bins) or box. Place the bin or box in one of the campus unwanted biological materials pickup locations. Some labs may not have direct access to a unwanted biological materials pickup location. If no convenient pickup location is available, complete a Pickup Request Form (PURF).
- Do not overfill biohazard unwanted material bags.
- Two-third's (2/3) is considered full.
- Biohazard bins or boxes MUST NOT weigh more than 40 pounds.
- Biohazard unwanted material boxes are NOT SHARPS CONTAINERS.
- Sharps must be disposed of within a closed/sealed hard plastic sharps containers.
- Sealed sharps containers may be placed inside a biohazard box.
- For additional information on sharps disposal, refer to the MU Biological Safety Manual.
Small Animal Experiments:
(During shedding period of lentivirus vector, ~ 72 hours)
Some animals, such as wild-type mice, cannot support replication of infectious HIV-1. As a result, the potential for shedding of RCL from such animals is very low (even if replication competent lentivirus were present in the original vector inoculum). In general, the initial delivery of vector should be performed under Biosafety Level 2 for Animals (ABSL-2) or under enhanced ABSL-2 containment so as to minimize the risk of autoinoculation by the investigator. However, it may be permissible to reduce the containment level at some point following vector delivery. For example, if there is no expectation of infection (see below), the site of inoculation has been thoroughly cleansed, and the bedding changed, it may be acceptable to consider reducing containment from ABSL-2 to ABSL-1 within a few days (the specific time period can be specified by the local IBC, and may vary anywhere from 1-7 days depending on local and experimental considerations). The University of Missouri IBC has recommended 72 hours of ABSL-2 containment post-injection based on this information. Animals engrafted with human cells or animal hosts that are permissive for HIV-1 replication constitute a special case, in light of their potential to support replication of infectious HIV-1. Use of lentivirus vectors in these animals requires a higher level of containment.
Assume the shedding period for lentivirus to be 72 hours minimum unless it can be demonstrated on permissive cell lines to be different for an application. It is the responsibility of the investigator to submit any viral shedding results to the EHS Biosafety Office.
- A biohazard sign indicating lentivirus vector must to be placed on animal rooms when in use and on cages with date the animals were inoculated.
- Incorporate the above laboratory experiment precautions.
- Perform all administrations and manipulations of animals inside of BSC (Class I, IIA, IIB1 or IIB2).
- Use microisolators or containment type animal housing at ABSL-2 during the initial 72 hour shedding phase of the lentivirus.
- Perform cage change-outs in BSC (Class I or II).
- Autoclave the animal cage with bedding intact before dumping and then discard bedding in a conventional manner. NOTE: When animal cages are transported outside the ABSL2 animal room for autoclaving they must be placed within a biohazard bag and sprayed with a virucidal solution prior to removal from the ABSL2 animal room.
- Spray cage racks with a virucidal solution before removal from a BSL-2 facility. Allow to air dry.