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

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

Summary

Agent Type Risk Group Biosafety Level Animal Housing Biosafety Level
Viral Vector  RG-2 BSL-2  ABSL-1, ABSL-2

Agent Characteristics 

Risk Group:  RG-2 associated with human disease, rarely serious; preventive, or therapeutic interventions often available.

Agent Type:  Viral Vector

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 slow viruses (lente-, Latin for "slow") of the Retroviridae family, characterized by a long incubation period. The viruses are species-specific in host range and several have been recognized as pathogens of domestic animals, non-human primates, and humans.

In a third-generation 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 needs to have its wild type promoter replaced with a heterologous enhancer/promoter such as CMV or RSV to ensure transcription.

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 is dependent 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 advantageous for research purposes, this poses an increased risk of infection in case of exposure to VSV-G pseudotyped lentiviral vectors for lab workers, since these vectors will be able to target a larger range of cells

Host Shedding: Unavailable

Routes of Exposure to Humans: Mucous Membranes, Contaminated Items, Percutaneous, Broken (dry or chapped) 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. The risks from an exposure are low, if an exposure occurs, seek consultation with a medical professional within 4 and no later than 72 hours.

 

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


Agent Viability 

Survival Outside Host Disinfection Inactivation

90-99% reduction in several hours

1:10 Bleach Dilution (30+ seconds)

EHS-approved disinfectants

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, 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 reported


Laboratory Handling Guidelines 

Laboratory Biosafety Level (BSL): BSL-2

Third generation and higher lentiviral vectors used in labs are frequently pseudotyped with envelope glycoprotein from vesicular stomatitis virus VSV-G. In this case, BSL2 containment is implemented since these viruses have the capability of transducing human cells.

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

Oncogenic transgenes:

Lentivirus vectors that incorporate 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 expression of tumor suppressor genes (or that may knock down these genes because of off target effects) must be used at BSL2 enhanced.

Training

Lab Engineering Controls Personal Protective Equipment
  • Eye Protection - For activites conducted outside of a biosafety cabinet (e.g. stereotactic injection), the use of mucous membrane protection devices is of extreme importance.
  • Single gloves
  • Snap-front lab coat with cinch cuffs

Waste Management: Regulated Medical Waste (RMW)

Shipping Guidance: Refer to EHS Biological Materials Shipping 


Animal Vivarium Guidance

Animal Housing Biosafety Level (ABSL): ABSL-1, ABSL-2 Note: Animals receiving 3rd generation or higher vectors will remain at ABSL-2 for 7 days, 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 prior to washing.

Animal Biosecurity: Experimental animals are housed separately         

Perform Inoculations: In a Biosafety Cabinet

Change Cages: In a Biosafety Cabinet


Exposure and Spill Procedures 

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

Other Exposures: Wash with soap and water for 15 minutes (open wounds, sores, etc.) or a minimum of 20 seconds for areas with intact skin. See: responding to exposures.

Small Spills: Notify others working in the lab. Evacuate the area and allow 30 minutes for aerosols to settle. Don appropriate PPE. Cover 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 spill 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.

Medical Follow-Up:
Seek consultation with a medical professional within 4 hours and no later than 72 hours.

  • For students, seek medical attention at Cornell Health or local primary care provider. Call Cornell Health at 607-255-5155 (24-hour phone consultation line) or a local urgent care. 

  • For faculty and staff, seek medical evaluation with a local primary care provider or urgent care. Cornell Health does not see employees for post-exposure care. 
  • Emergencies: Call 911 from a campus phone or 607-255-1111 from a mobile phone. 

Cornell EHS would like to thank Emory University for the use of their Biological Agent Reference Sheet (BARS) format and some content.