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Lentiviral Vectors (3rd Generation and above) Biological Agent Reference Sheet (BARS)

Last Revised 1/17/2024

Disclaimer: Risk group, biosafety level, and all other precautions noted here are subject to change after a risk assessment by EHS.



Agent TypeRisk GroupBiosafety LevelAnimal Housing Biosafety Level
Viral VectorRG-3

BSL-2+ (enhanced)



Synonym or Cross Reference: NA


Agent Characteristics

Description: Lentiviruses are medium-sized (120 nm), enveloped viruses composed of a nucleocapsid containing two copies of single-stranded positive-sense RNA. Lentivirus is a genus of of the Retroviridae family, characterized by a long incubation period. The viruses are species-specific in host range, and several are recognized as pathogens of domestic animals, non-human primates, and humans. These viruses include HIV, SIV, SHIV and FIV among others. Most lentiviral vectors used in research laboratories consist of an HIV backbone that has been modified such that it can be used as a carrier vehicle, called a lentiviral vector, to efficiently introduce genetic material (transgenes) into both dividing and non-dividing target cell genomes. Third and 4th generation lentiviral vectors typically used today are considered to be replication incompetent.

In a third-generation Lentiviral Vector (LVV) system, only gag, pol, and rev genes remain present (tat is eliminated). The rev gene is provided in a separate plasmid. Since the HIV promoter in 5’ LTR depends on tat, a vector that lacks tat must have its wild type promoter replaced with a heterologous enhancer/promoter such as CMV or RSV to ensure transcription. Hazards of a lentiviral vector include the effects of the expressed transgene such as an oncogene, or toxin being introduced into the target cell by the vector. The onetime introduction of a gene can introduce potential problems which are hard to gauge and may be long term. In addition, for replication incompetent lentiviral vectors, while the virus does not replicate, the transgene is integrated into the host genome. The transgene may insert in a genetically sensitive area and induce mutational changes. This is called insertional risk. There is marginal risk of recombination with wild-type HIV in persons who are infected with HIV.

Third-generation systems (or higher) are currently the safest to use because the virus production is split across four (or more) plasmids.

Host Range: The host range depends upon the viral envelope glycoproteins and structural proteins involved in integration. Possible hosts include human, murine, feline, bovine, and avian. Pseudotyping lentiviral vectors with Vesicular Stomatitis Virus glycoprotein G (VSV-G) increases the host range to a broad array of cell types and species. Though helpful for research purposes, this poses an increased risk of infection for lab workers in case of exposure to VSV-G pseudotyped lentiviral vectors since these vectors will be able to target a more extensive range of cells.

Host Shedding: Unknown

Route of Exposure to Humans: Mucous Membranes, Contaminated Items, Percutaneous, Sexual, Broken skin

Infectious Dose: Unknown

Incubation Period: 1-6 months


Health Hazards

Lentiviruses may persist lifelong due to their ability to integrate into the host chromosome and the ability to evade host immunity. Later-generation lentiviral vectors still have the:

  • Potential to generate replication-competent lentivirus (RCL)
  • Potential to cause oncogenesis.

Immunizations: None available

Prophylaxis: Post-exposure prophylaxis for occupational exposure with HIV-based viral vectors may include the use of antiretroviral drugs. If an exposure occurs, seek immediate medical evaluation.

Formal medical advice is obtained during consultations with Cornell Health or primary healthcare providers as needed.


Agent Viability

Survival Outside HostDisinfection MethodsInactivation
90-99% reduction in several hours1:10 Bleach Dilution (30+ seconds) or other EHS-approved disinfectant.Heat at 56°C for 30+ minutes

For more guidance on disinfection, see disinfectant selection.


Laboratory Hazards

Risks include direct contact with skin and mucous membranes of the eye, nose, and mouth, parenteral inoculation, and ingestion.

  • High energy-creating activities (centrifugation, sonication, high-pressure systems, vortexing, tube cap popping)
  • Handling of sharps (needles, scalpels, microtome blades, broken glass, etc.)
  • Splash/droplet-creating activities (shaking incubators, liquid culturing, mechanical pipetting)
  • Equipment contamination
  • Exposed skin/uncovered wounds/broken or chapped skin


Laboratory Acquired Infection (LAI) History: None known.


Laboratory Handling Guidelines

Laboratory Biosafety Level (BSL): BSL-2

Third-generation and higher lentiviral vectors used in labs are often pseudotyped with envelope glycoprotein from vesicular stomatitis virus VSV-G. In this case, BSL2 containment is implemented since these viruses can transduce human cells.

There are still conditions when BSL2-enhanced precautions are used:

  • Oncogenic transgenes: Lentivirus vectors incorporating transgenes with oncogenic potential must be generated and used at BSL-2 enhanced containment regardless of whether second or third-generation systems are used.
  • Scale of production: Lentivirus vectors made at a level of production > 100 ml volume must be generated and used at BSL-2 enhanced containment regardless of whether second or third-generation systems are used.
  • CRISPR/Cas9: Vectors that encode both the guide RNA and the Cas9 must be used at BSL2 enhanced containment. In addition, vectors designed to knock down the expression of tumor suppressor genes (or that may knock down these genes because of off-target effects) must be used at BSL2 enhanced.


TrainingLab Engineering ControlPersonal Protective Equipment

EHS Laboratory Safety Training CULearn Course #2555


EHS Bloodborne Pathogens Training CULearn course #1070


EHS Biosafety Level 2 CULearn course #5060


Lab-specific protocol training

Biosafety Cabinet (for aerosol containment)


Centrifuge lids or safety cups; samples are loaded/unloaded inside the BSC.


Use of safety-engineered sharp

Eye Protection - For activities conducted outside of a biosafety cabinet (e.g., stereotactic injection), mucous membrane protection devices are of extreme importance.


Single gloves


Snap-front lab coat with cinch cuffs

Attenuated Strain Alternatives: Using higher-generation lentiviral vectors with additional safety measures is encouraged.

Waste Management: Regulated Medical Waste (RMW)

Shipping Guidance: Refer to EHS Biological Materials Shipping.


Animal Vivarium Guidance

Animals receiving 3rd generation or higher vectors will remain at ABSL-2 for 7 days and may be moved to ABSL-1 after subsequent cage change. Reduction to ABSL-1 may not apply if the animals contain any human cells or tissues. Take precautions to avoid creating aerosols when emptying animal waste material. Soiled cages are disinfected before washing. Experimental animals are housed separately.

Animal Housing Biosafety Level (ABSL): ABSL-2

Perform Inoculations: Biosafety Cabinet

Change Cages: Biosafety Cabinet


Exposures and Spill Procedures

In the event of exposure to a lentiviral vector it is crucial to take immediate action to ensure safety. If exposed, go to an urgent care provider including the Cayuga Medical Center Emergency Department. Cornell Health is not an urgent care. Call 911 from a campus phone or 607-255-1111 from a mobile phone for emergencies.

Print this BARS or be prepared to provide it to your medical provider and the generation(s) of lentivirus and constructs involved in the exposure.

Mucous Membranes: Flush eyes, mouth, or nose for 15 minutes at an eyewash station.

Other Exposures: Wash with soap and water for 15 minutes (open wounds, sores, chapped skin, etc.) or at least 5 minutes for areas with intact skin.

First Aid Treatment: Call emergency personnel if immediate medical care is needed (911). Stabilize the individual and provide first aid for injuries that require immediate medical care (eg, deep cuts, bleeding, etc.)

Small Spills: Notify others working in the lab. Evacuate the area and allow 30 minutes for aerosols to settle. Don proper PPE. Cover the area of the spill with paper towels and apply disinfectant, working from the perimeter toward the center. Allow 30 minutes of contact time before disposal and cleanup of spilled materials. See: spill cleanup

Large Spills: Request assistance from the EHS Spill Team by calling CUPD dispatch. Call 911 from a campus phone or 607-255-1111 from a mobile phone.

Incident Reporting: Immediately report the incident to the supervisor and complete the EHS online injury/illness report as soon as possible. If evaluated at the ER, follow up with respective campus provider next business day. Notify supervisor of incident. Complete an Accident Report Form there. By law, any exposures to recombinant DNA (which includes lentiviral vector) must be reported to the NIH, by notifying EH&S.


Physician Information

Verify that first aid was performed, and skin was washed with soap and water for no less than 5 minutes and mucous membranes flushed with clean water for 15 minutes. Any significant lentiviral or retroviral vector exposures need post exposure prophylaxis with anti-HIV treatments. Read the sections below for more information.

Document and understand the exposure: Enquire to the type of lentiviral vector (HIV versus different species of lentivirus), the generation, replication competence, transgenes such as oncogenes, knockdown or knockout genes, or toxin genes carried by the vector. Confirm what type of cells and animal tissues as these may also present a hazard. Note that cell lines supplied by commercial suppliers are not screened for all potential bloodborne pathogens. Macaque cells may pose a separate risk of macacine herpes virus 1 (herpes B virus). Confirm if the exposure route: mucocutaneous, percutaneous, or aerosol and how large the exposure was as well as when it occurred along with viral vector titer. The affected individual should be able to describe the vector generation and constructs involved in their exposure to aid in a medical evaluation. Contact the principal investigator or lab director, with the permission of the affected personnel, to assist with the risk assessment. Cornell University’s Biosafety Team is also available – 607-255-1111 – and ask for biosafety assistance.

The potential health concerns due to exposure to lentiviral vectors are the production of replication-competent lentivirus by recombination of vector components or potential oncogenesis from insertional mutagenesis. Please see the reference section information, above, about lentiviral vectors. As with HIV, exposure to intact skin is not clinically relevant and does not pose a significant risk.

Due to the relatively safe profile of newer anti-HIV drugs, the potential risks associated with HIV/LVVs, and the time-critical nature of the intervention, physicians may wish to consider immediate postexposure prophylaxis. The following treatments may be offered:

  • As soon as possible but within 72 hours, initiate a 7-day course of a NRTI (such as tenofovir) and an integrase inhibitor (such as raltegravir or dolutegravir).
  • Observation and treatment of overt effects of the exposure incident.

If no prior discussion about treatment has occurred, treatment with tenofovir and raltegravir/dolutegravir should be considered and begin within 72 hours and continued based upon a discussion and acceptance of the risks and benefits of treatment.

Testing and Follow-up. Testing for lentiviral vector exposure is generally not helpful. HIV testing may be worthwhile as if someone has undiagnosed HIV, the PEP regimen could produce HIV resistance, and there is potential risk for wild HIV recombination with the lentiviral vector. The post-exposure visit documents the exposure and so the extent of documentation if something arises down the road may be quite important. Baseline and follow-up labs are indicated for those started on medications and depend upon the medications but generally include CBC and diff, BUN/creatinine and AST + ALT.

Medication There are no studies of the benefits or risks of post-exposure prophylaxis for insertional risks for treating exposures to non-replication competent lentiviral vectors. There are no national, published guidelines or consensus on this. Experts advise rapid evaluation, within 2 hours or less - certainly by 12-24 hours. Exposure prophylaxis should be started to prevent insertional risks, particularly with a lentiviral vector carrying hazardous transgenes. After 72 hours there is no likely benefit. Exposure to cells or animal tissues that have been transduced with a lentiviral vector presents minimal risk, and there is no likely benefit to post-exposure prophylaxis, especially if the transduction occurred more than 72 hours prior.  

Recommended regimen Two drugs ; Raltegravir (Isentress) 400 mg BID with or without Tenofovir (Viread) 300 mg once daily x 7 days) or Isentress + Emtricitabine x 7 days can be used as alternative drugs (some institutions use 7 days) may be used in the event of a worrisome transgene. This is off label use. Protease inhibitors (like Kaletra) have no effect on transduction or integration of the lentiviral vector and therefore are not used for insertional hazards.

Replication-competent lentiviral vector should be treated just like a wild-type HIV BBP exposure, so use the normal HIV exposure protocol, for example, Isentress and Truvada for 28 days.

More Information

  1. Schlimgen, R., Howard, J., Wooley, D., Thompson, M., Baden, L.R., Yang, O.O., Christiani, D.C., Mostoslavsky, G., Diamond, D.V., Gilman Duane, E., Byers, K., Winters, T., Gelfand, J.A., Fujimoto, G., Hudson, T.W., Vyas, J.M. "Risks Associated with Lentiviral Vector Exposures and Prevention Strategies." Journal of Occupational and Environmental Medicine, 58(12), 1159-1166 (December 2016). DOI: 10.1097/JOM.0000000000000879
  2. Centers for Disease Control and Prevention (CDC). Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition. 2020.;
  3. National Institutes of Health (NIH). Biosafety Considerations for Research with Lentiviral Vectors. (viewed November 30, 2023)