Lentivirus Use at Princeton University


Lentiviruses comprise a genus of the Retroviridae family and include bovine lentiviruses (e.g., Bovine immunodeficiency virus, Jembrana disease virus); equine lentiviruses (e.g., Equine infectious anemia virus); feline lentiviruses (e.g., Feline immunodeficiency virus); Ovine/caprine lentivirus (e.g., Caprine arthritis-encephalitis virus, Ovine lentivirus, Visna virus); and Primate lentiviruses (e.g., Human immunodeficiency virus (HIV) types 1 – 3, Simian AIDS retrovirus SRV-1, Human T-cell lymphotropic virus type 4, and Simian immunodeficiency virus). 

Research with lentiviral vectors must be conducted in accordance with this guidance document and containment precautions issued by the Institutional Biosafety Committee. 

Researchers should be familiar with the properties of the lentiviral vectors used in their labs prior to handling and before an exposure occurs. Minimally, staff should understand the function of the transgene, number of plasmids used to generate the vector, host range and percentage of genome deleted.

Exposure to a lentiviral vector in the lab must be reported and treated without delay. Follow the directions in the First Aid and Exposure sections. 

Risk Summary

Lentiviral Vectors 

Most of the lentiviral vectors used in our labs are derived from HIV. They have the ability to integrate into host chromosomes, infect dividing and non-dividing cells and have high mutation rates. The major risks associated with lentivirus vectors are:

  • Potential for generation of replication-competent lentivirus (RCL)
  • Potential for oncogenesis

The potential for generation of replication competent lentivirus from HIV-based lentivirus vectors depends upon the following:

The number of recombination events necessary to reassemble a replication competent genome. Second generation vector systems use two helper plasmids to separate the cis- and trans- factors. Third and fourth generation systems use three or more helper plasmids. The risk of a recombination event is lowered when a vector with multiple plasmids is used. This means that a third-generation system provides more safety than a second-generation system.

The number of essential genes that have been deleted from the vector system. Third-generation and later vector systems express the HIV regulatory Tat protein under control of a promoter regulated by a non-physiological ligand, as in the tetracycline off system; or they do not encode Tat, which is necessary for replication of wild-type HIV-1.

Later generation lentivirus systems theoretically pose fewer risks because:

  • Vector and packaging genes are separated onto four or more plasmids, lowering the possibility of recombination when compared to a two-plasmid system.
  • Essential genes are stringently regulated or deleted (Tat)
Lentivirus Picture

Image reproduced from Addgene, the nonprofit plasmid repository (Cambridge, MA)


The envelope of wild-type HIV targets CD4 cells. Replacement of the HIV envelope gene with vesicular stomatitis virus glycoprotein (VSV-G) gene broadens the cell types that can be infected. Additionally, VSV is transmitted via the airborne route. Although the risk is unknown, the replacement of the HIV envelope glycoprotein with VSV-G could theoretically introduce the potential for aerosol risks.  

Insertional Mutagenesis

Insertional mutagenesis results from gene dysregulation at the site of the lentiviral vector integration within or near a coding region of the host genome.  

While many of the lentiviral vectors do not virus does not replicate, the transgene may integrate into the host genome. The transgene may also insert in a genetically sensitive area and induce mutational changes. 

Hazards of a lentiviral vector may include the effects of the expressed transgene such as a toxin, oncogene or inactivation of a tumor suppressor being introduced into the target cell by the vector. The onetime introduction of a gene can introduce potential problems which are very difficult to assess. 

The following tables summarize the biosafety concerns that should be considered when choosing a lentiviral vector system and compare the second generation lentivirus to third and later generation systems. 

Biosafety Consideration Lower-Risk Example Higher-Risk Example
Transgene function Protein-based fluorescence (GFP) Tumor-suppressor or oncogene (Ras, Myc)
Number of plasmids in system 4 or more plasmids 3 plasmids
Expression control elements Weak promoters Strong promoters included (CMV, SV40)
Host range Non-human tropism Broad host range (coated with VSV-G)
Concentration <1x109 infectious units/mL >1x109 infectious units/mL
Production volume <100 mL >100 mL
Percentage of genome deleted >2/3 <2/3
Source of vector system Commercial third-generation system (Addgene pRSV-Rev) PI's laboratory using a second-generation system
Animal hosts Non-permissive host Permissive host, animals engrafted with human cells
Animal manipulations Housing and husbandry (no use of sharps) Vector administration (use of sharps during injection)

Source: Schlimgen, Ryan et al. “Risks Associated With Lentiviral Vector Exposures and Prevention Strategies.” Journal of Occupational and Environmental Medicine 58.12 (2016): 1159–1166. PMC. Web. 26 Jan. 2018.

Comparison of 2nd and Later Generation Lentiviral Systems:

Feature 2nd Generation 3rd or Later Generation
Transfer Plasmid Can be packaged only by a second generation packaging system that includes Tat* Can be packaged by a system with or without the presence of Tat*
Packaging Plasmid Gag, Pol, Rev, Tat all on one plasmid Gag, Pol, Rev split between multiple plasmids. Tat is not included
Envelope Plasmid Interchangeable: usually encodes for VSV-G Interchangeable: usually encodes for VSV-G
Safety Safe. Replication incompetent: HIV genes split across 3 separate plasmids Safer. Replication incompetent: HIV genes split across 4 or more separate plasmids. Tat is eliminated.

Source: “Lentiviral Guide.” Addgene, www.addgene.org/viral-vectors/lentivirus/lenti-guide/

* Tat is a regulatory gene that is essential for HIV-1 replication and production. It is a positive regulator of transcription.

Preparing to Conduct Research with Lentivirus

  • Follow the work practices in this Exposure Control Plan.  
  • The Institutional Biosafety Committee (IBC) must review and approve of research with lentiviral vectors. If you have not yet registered with the IBC, please contact the University’s Biological Safety Officer ([email protected]).
  • The Biosafety Officer must review laboratory facilities, containment and waste disposal practices prior to initiation of research with lentivirus. 

Required Training

Intro to Biosafety Training: All research and LAR staff who handle lentiviral vectors must attend.

Laboratory Safety Training: All research staff are required to attend before they can begin laboratory work.

Laboratory-Specific Training: All new lab workers are required to receive lab-specific training from their Principal Investigator, lab manager or other designated experienced researcher. Training, which can include review of this website, should be documented in the SHIELD system.

Transmission in a Laboratory Setting

  • Direct contact with mucous membranes of the eye, nose, and mouth. Wear safety glasses, and face mask if necessary. Work in a Class II biological safety cabinet.
  • Direct contact with skin that may be abraded or chapped. Wear gloves, lab coat, closed shoes and full-length pants.
  • Accidental percutaneous injection. 
    o Eliminate sharps when possible. Use blunt tip needles when appropriate. 
    o Never re-cap needle or remove needle from syringe. 
    ​o Use of safety needles is recommended.
  • Hazard of aerosol exposure is unknown. Many lentiviral vectors are pseudotyped with the glycoprotein of Vesicular Stomatitis Virus (VSV). VSV can be transmitted by aerosol route.

Minimum Personal Protective Equipment and Engineering Controls:

Type Laboratory Vivarium
Safety Glasses
Lab Coat √ (or disposable gown)
Gloves √ (double gloves)
Disposable Gowns  
Shoe Covers  
Face Shield  
Cap/Head Cover  
Biosafety Cabinets**
Closed Centrifuge Rotors  
Closed Centrifuge Tubes  

* All waste, including soiled animal bedding that may contain lentiviral vectors must be autoclaved prior to disposal.

** Use a biosafety cabinet for all manipulations involving lentiviral vectors and infected animals, including necropsies. Consult with Biosafety Officer for exceptions.


The use of sharps including needles, blades, and glassware must be limited. 

  • Consider using engineered sharps systems like Vanish Point syringes, and blunt tip needles in place of sharp tip needles. 
  • Use plastic disposable pipettes in place of Pasteur pipettes. 
  • All disposable sharps waste must be placed directly into red sharps containers. When ¾ full, close and dispose in Regulated Medical Waste.  
  • Place reuseable sharps (with sharp ends pointing in the same direction) into a pan filled with a disinfectant, such as a 1% sodium hypochlorite solution. Allow a 30 minute soak time prior to scrubbing and sterilizing the reuseable sharps. 


Lentiviral vectors are susceptible to many disinfectants due to the presence of a lipid envelope, and are inactivated when exposed to drying environmental conditions. A 1% sodium hypochlorite solution, accelerated hydrogen peroxide and 70% ethanol are all effective against lentiviral vectors and enveloped viruses in general. The use of 70% ethanol is not recommended for spill cleanup due to evaporation.

Waste Disposal

All disposable materials that have come in contact with lentivirus must be disinfected prior to disposal into the regulated medical waste stream. Autoclave disposable materials if appropriate. Chemically disinfect items that cannot be autoclaved.


First Aid – Immediately after Exposure:

  • Eye Exposure: Rinse for 15 minutes at an eyewash station.
  • Skin Exposure: Wash the area with soap and warm water for 15 minutes. Use a safety shower for 15 minutes if a large portion of skin is affected (spill).
  • Needlestick or sharps exposure: Wash the area with soap and warm water for 15 minutes.

Medical Follow-Up: Proceed Without Delay!

  • Between the hours of 8:00AM – 4:00PM, Monday-Friday, report directly to Employee Health, University Health Services.
  • Weekends and after hours: Contact Environmental Health and Safety (EHS) at 609-258-5294.
  • Inform your Principal Investigator or supervisor immediately of an exposure or suspect infection.

Medical Treatment and Follow-Up 

Replication competent lentiviral vector

  • Follow-up to an exposure involving replication-competent lentiviral vector must occur immediately (WITHIN ONE HOUR).
  • University Health Services will follow the standard procedure for exposure to wild-type HIV and/or a bloodborne pathogen.

Replication incompetent lentiviral vector

  • UHS clinicians will advise the exposed person of the risks and benefits of initiating drug therapy to prevent insertional risks.   
  • Drug therapy should be initiated within 2 hours or less and no later than 24 hours to prevent insertional risks. Due to the unlikelihood of benefits, drug therapy may not be recommended after 72 hours. 

Additional Links

University of Cincinnati Lentiviral Vectors: http://researchcompliance.uc.edu/training/lentiviral-vectors/story_html…

Addgene Lentiviral Guide and Frequently Asked Questions: https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/

NIH Biosafety Considerations for Research with Lentiviral Vectors: https://osp.od.nih.gov/wp-content/uploads/Lenti_Containment_Guidance.pdf


Meagan Fitzpatrick
Biosafety Officer

Halina Staniszewska,
Sr. Lab Safety Specialist