Clemson University
Biosafety Procedures and Practices for Research with Lentivirus
1. Background
Lentivirus is an increasingly popular vector system for use during in vitro and in
vivo gene delivery. The major risks that should be considered for research using
lentiviral vectors (HIV-1 and FIV based systems) are:
A. Generation of replication-competent lentivirus (RCL)
B. Potential oncogenesis
The risks associated with lentiviral systems can be minimized (or exacerbated) by
the nature of the viral vector system and the transgene insert encoded by the vector.
While the NIH Guidelines for Research Involving Recombinant DNA Molecules does
not explicity address research involving lentiviral vector systems, the Recombinant
DNA Advisory Committee (RAC) of the Office of Biotechnology Activities (OBA) has
provided guidance, based on risk assessment for HIV, that will form the basis of the
SOP.
2. Laboratory Practices
A. Agents – recombinant lentiviral vectors, including those based on HIV, SIV, or
FIV. Note the non-human lentivirus vectors have the potential to transduce
human cells and cause insertional mutagenesis.
B. Employees potentially at risk – laboratory workers handling recombinant
lentiviral vectors, animal care providers handling infected humanized mice
(see below), and animal care providers handling infected animals (nonhumanized) during the first 72 hrs following infection.
C. Approval – experiments using lentiviral vector systems require approval of
the Institutional Biosafety Committee (IBC).
D. Biosafety Level Assignment – While final biosafety level determination will
be made by the IBC, the following criteria will influence this decision:
1. The nature of the vector system and potential for RCL generation.
2. The nature of the transgene insert – genes with oncogenic potential or
human toxicity are considered increased risk).
3. Vector titer and amount.
4. Manipulations involved (for example, centrifugation or sonication, which
can generate infectious aerosols).
Please note the reduced risk of exposure can be achieved using the following
methods:
1.
Use lentiviral systems that segregate essential viral systems (like
packaging) onto four or more plasmids.
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Clemson University
Biosafety Procedures and Practices for Research with Lentivirus
2.
3.
In place of native HIV-1 envelope protein, use systems that
employ non-native Env or heterologous coat proteins (VSV-G).
Use systems that lack genes essential for viral replication (like
Tat).
BL2 or enhanced BL2 containment can be appropriate when lentiviral vector
systems are used that segregate vector and packaging onto multiple
plasmids. Additionally, PPE that reduces potential for mucosal exposure
(e.g., N-95 respirator) should be employed.
Manipulating concentrated virus or performing procedures likely to generate
aerosols (centrifugation, sonication), should be performed at enhanced BL2.
E. Work Practices
1.
Signs and labels must be in place that indicated where lentiviral
materials are used and stored.
2.
All manipulation of viral vector and infectious materials must be
performed in a certified Class II biosafety cabinet (Class IIA, IIB1, or
IIB2).
3.
Centrifugation must be performed in closed containers using sealed
rotors or safety cups.
4.
Laboratory coats, gloves, and safety glasses/goggles must be worn.
Hands must be washed after removing gloves. Avoid hand to face
contact.
5.
All contaminated waste must be decontaminated before disposal
(see below).
F. Animal Use
1. All animal experiments involving lentiviral infection will be performed at
the Godley-Snell Research Center (GSRC) in accordance with RS/SOP 30007-05 (or current version) entitled, “BSL-2 (ABSL-2) Containment in
Animal Research at GSRC”. Further, these procedures should have prior
approval of the IBC and the Institutional Animal Care and Use Committee
(IACUC). Other facilities using lentiviral viruses have to have prior
approval of the IBC and IACUC.
2. Initial delivery of the viral vector should be performed under BSL-2
conditions. Because the animals may have infectious virus on their
wound or in secretions, ABSL-2 containment must be used for the first 72
hrs post-infection. For long-term maintenance of infected rodents, ABSL1 is appropriate as lentiviral vectors cannot replicate in rodents.
3. Precaution must be taken to avoid aerosol generation when cleaning
ABSL-2 facilities and rodent housing.
4. Special training is required for all animal husbandry personnel on
lentivirus and its associated hazards.
5. Dispose of carcasses in biohazard red bags in accordance with local SOP.
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Biosafety Procedures and Practices for Research with Lentivirus
6. Rodents engrafted with human cells (or other permissive animal hosts)
will be housed in ABSL-2 facilities. Please note that the required
biosafety level may be altered, depending on the animal species, agent,
and experimental details.
G. Decontamination
The most effective germicides (with minimum of 15 min contact time)
are:
1% sodium hypochlorite
2% glutaraldeyhde
5% phenol
Lentivirus is sensitive to heat, detergents and formaldehyde.
3. References
Recombinant DNA Advisory Committee (RAC) Guidance Document, Biosafety
Considerations for Research with Lentiviral Vectors
San Diego State University, IBC, Biosafety Procedures and Practices for
Research with lentivirus
UTHHSC Guidelines for the Safe Handling of Lentiviral Vectors in Laboratory,
Animal and Human Experiments
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