Lentivirus and Lentiviral Vectors

Characteristics
Morphology	Family Retroviridae; Genus Lentiviridae. The Lentiviridae genus includes the primate Human Immunodeficiency Virus (HIV) types 1-2 and the non-human primate Simian Immunodeficiency Virus (SIV). HIV-derived vectors are highly efficient vehicles for in vivo gene delivery. Lentiviruses are approx. 120nm in diameter, enveloped, and contain a nucleocapsid containing two copies of single-stranded positive-sense RNA. Cis and trans-acting factors of the lentivirus are on separate plasmids depending on the Viral Vector generation. Third generation systems are currently the safest to use because the virus production is split across four plasmids.
Growth Conditions	Most often 293T cells are used as Packaging cells. Lentiviral vectors can infect dividing and non-dividing cells.

Characteristics

Morphology

Family Retroviridae; Genus Lentiviridae. The Lentiviridae genus includes the primate Human Immunodeficiency Virus (HIV) types 1-2 and the non-human primate Simian Immunodeficiency Virus (SIV). HIV-derived vectors are highly efficient vehicles for in vivo gene delivery. Lentiviruses are approx. 120nm in diameter, enveloped, and contain a nucleocapsid containing two copies of single-stranded positive-sense RNA. Cis and trans-acting factors of the lentivirus are on separate plasmids depending on the Viral Vector generation. Third generation systems are currently the safest to use because the virus production is split across four plasmids.

Growth
Conditions

Most often 293T cells are used as Packaging cells. Lentiviral vectors can infect dividing and non-dividing cells.

Health Hazards
Host Range	If the HIV envelop is replaced with Vesiculo-Stomatitis Virus (VSV)-G then a broad host-range can be achieved.
Modes of Transmission	Direct exposure to infected bodily fluids, sexual contact, splash or percutaneous injection.
Signs and Symptoms	Fever, runny nose, sore throat, diarrhea, coughing, vomiting, nausea, lethargy, myalgia, anorexia and dyspnea.
Infectious Dose	All depends on the source of the infection, so there is no baseline
Incubation Period	1-6 months

Medical Precautions/Treatment
Prophylaxis	Post exposure prophylaxis for occupational exposure with HIV-based viral vectors includes the use of anti-retroviral drugs
Vaccines	None available
Treatment	Anti-retroviral therapy is available
Surveillance	Monitor for symptoms of influenza. Confirm diagnosis with RT-PCR (favored) or point-of-care testing.
GWU Requirements	Report all incidents to the Office of Risk Management as well as the Office of Research Safety (ORS) IMMEDIATELY FOLLOWING THE OCCURRENCE.

Containment
BSL2/ABSL2	Risk Group 2 classification is applied to lentivirus. Containment Level 2 facilities, equipment, and operational practices for work with lentivirus. No open-bench work should be performed with retroviral vectors. All work should be performed inside a Biosafety Cabinet. Use of needle-safe sharps is encouraged. Centrifuge rotors must have a lid, samples should be loaded/unloaded inside the BSC and the centrifuge should be decontaminated with appropriate disinfectant after use. If the vector is replication incompetent, animals infected with retroviral vectors will remain at ABSL-2 for 72h, then moved to ABSL1. If the vector is replication competent, animals will be housed at ABSL2 for the length of the experiment.

Containment

BSL2/ABSL2

Risk Group 2 classification is applied to lentivirus. Containment Level 2 facilities, equipment, and operational practices for work with lentivirus. No open-bench work should be performed with retroviral vectors. All work should be performed inside a Biosafety Cabinet. Use of needle-safe sharps is encouraged. Centrifuge rotors must have a lid, samples should be loaded/unloaded inside the BSC and the centrifuge should be decontaminated with appropriate disinfectant after use. If the vector is replication incompetent, animals infected with retroviral vectors will remain at ABSL-2 for 72h, then moved to ABSL1. If the vector is replication competent, animals will be housed at ABSL2 for the length of the experiment.

Spill Procedures
Small Spills (<1 liter)	If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup. Lentivirus is not commonly transmitted via aerosols, but extra caution should still be taken during a spill outside of a BSC to reduce the risk of exposure. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against enveloped viruses (bleach, glutaraldehyde, hypochlorite, iodine, and phenolics), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.
Large Spills	Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Spill Procedures

Small Spills (<1 liter)

If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup.

Lentivirus is not commonly transmitted via aerosols, but extra caution should still be taken during a spill outside of a BSC to reduce the risk of exposure. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply an EPA registered disinfectant effective against enveloped viruses (bleach, glutaraldehyde, hypochlorite, iodine, and phenolics), working from the perimeter towards the center. Allow 30 minutes of contact time before disposal and cleanup of spill materials.

Large Spills

Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Exposure Procedures
Mucus Membrane	Flush eyes, mouth or nose for 15 minutes at eyewash station.
Other Exposure	Wash area with soap and water for 15 minutes.
Reporting	Report ALL injuries to the PI immediately and reported to the Office of Risk Management at [email protected] IMMEDIATELY FOLLOWING THE OCCURRENCE. Exposures that involve a bloodborne-pathogen or recombinant DNA also need to be reported to the Office of Research Safety at [email protected]. If the injury requires immediate medical attention, call GWPD at 202-994-6111 or call 911.
Medical Monitoring	Seek immediate medical evaluation, treatment, and post exposure follow-up at the Employee Health Office at GWU Hospital (900 23rd St., NW, Suite G-1090, Phone: 202-715-4275). Students should go to the Students Health Office at Marvin Center. After hours treatment can be received at the GWU hospital emergency room.

Stability
Disinfection	10% freshly prepared bleach solution or 70% ethanol
Inactivation	Heat at 56˚C for 30+ minutes
Survival Outside Host	90-99% reduction in virion infective ability after several hours outside of a host

Personal Protective Equipment (PPE)
Minimum PPE Requirements	At minimum, personnel are required to don gloves, closed toed shoes, lab coat, and appropriate face and eye protection prior to working with lentiviruses. Additional PPE may be required depending on lab specific SOPs.
Additional Precautions	Extreme care must be taken to avoid spilling and/or splashing infected materials. Lentivirus should be presumed to be in/on all equipment and devices coming in direct contact with infected materials.

References
Millipore Sigma (https://www.sigmaaldrich.com/US/en/technical-documents/technical-articl…) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) Canadian PHAC (https://www.canada.ca/en/public-health/services/laboratory-biosafety-bi…)