Retroviruses are enveloped, single-stranded RNA viruses capable of infecting dividing cells. Upon infection, the RNA genome is reverse transcribed and integrates as a DNA provirus into the chromosomal DNA of the infected cell.
Lentiviruses are a group of retroviruses that are capable of infecting non-dividing cells.
Retroviral vector characteristics include
- A cloning capacity of ~7.5kb.
- Titer production around 108.
- Long term (stable) expression.
Potential Health Hazards
Retroviruses can act as insertional mutagens, due to their ability to integrate into the host’s DNA. Transcriptional activation of host genes adjacent to the site of integration may result, a process dependent on the enhancer/promoter within the viral long terminal repeat (LTR).
Replication defective retroviruses can recombine with endogenous retro-elements, thus reestablishing or enhancing the pathogenic potential of the virus undergoing recombination.
While murine retroviruses are inactivated by human complement and are not capable of causing human disease, lentiviruses are not inactivated by human complement and can cause disease.
Modes of Transmission
Retroviral transmission can occur through non-intact skin or mucous membrane exposure, accidental parenteral inoculation, or ingestion. The hazard of aerosol exposure is unknown. HIV in particular can also be transmitted from person to person through direct exposure to infected body fluids (blood, semen).
Laboratory Acquired Infections
Occupational exposure to HIV has been documented in 57 cases of HIV seroconversion among health care workers in the United States (as of the last report in December of 2002); among those 57 cases, 26 developed AIDS. Five laboratory acquired infections with HIV have been reported as a result of splashing of infected materials, inapparent skin exposure and puncture wounds.
While needlesticks can transmit the virus, less than 1% of HIV contaminated needlesticks have resulted in infection.
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.
Survival in the general environment is poor. Drying in the environment can cause 90-99% reduction in HIV concentration within several hours.
The appropriate biosafety level (BSL) practices and facilities will depend on the host range (envelope glycoproteins) and insert characteristics of the recombinant virus. A majority of the retroviral vectors are based on the murine leukemia viruses (MLV) which is a risk group 1 agent; however, the use of viral-based vectors for gene delivery to mammalian cells has been assigned BSL2 containment by the Institutional Biosafety Committee (IBC) unless justification for a lower classification is proposed by the PI and approved by the IBC.
Biosafety level 2 practices and facilities must be used for activities involving retroviruses and retroviral vectors (for HIV, see below).
- Biohazard signs and labels must be displayed in areas and on equipment where viruses are used and stored. This includes, but is not limited to, laboratory entrance doors, biological safety cabinets, incubators, refrigerators, and freezers.
- Use a biological safety cabinet (BSC) (a.k.a. tissue culture hood) for manipulations that can generate aerosols, such as pipetting, harvesting, infecting cells, filling tubes/containers, and opening sealed centrifuge canisters. If a procedure cannot be done in a BSC and only on an open bench, use a plastic shield to prevent exposure through inhalation or splashing.
- Use aerosol containment devices when centrifuging. These include sealed canisters that fit in the centrifuge bucket, covers for the centrifuge bucket, heat sealed tubes, or sealed centrifuge rotors. Rotors should be removed and opened inside a BSC. Centrifuge tubes should be filled and opened in a BSC.
- Vacuum lines must be protected with liquid disinfectant traps and/or micron filters.
Biosafety Level 2 with Biosafety level 3 practices and containment equipment must be used for activities involving research-laboratory-scale quantities of HIV, manipulation of HIV preparations and activities that may produce aerosols.
- All work must be done in a biological safety cabinet,
- Lab doors must be closed and remain closed when work is in progress. Access is restricted to those whose presence is required while work is in progress.
- Autoclave waste items as soon as possible and before the end of the day.
- Use only disposable plastic flasks, tubes, plates, etc. for culture materials.
- Pay strict attention to sharps safety and the use of safety devices.
- Standard operation procedures (SOPs) must be developed and include biosafety precautions, an emergency plan, spill plan, etc.
Biosafety level 3 practices and facilities must be used when preparing or manipulating concentrated quantities of HIV.
- Directional airflow is provided, drawing air into the laboratory from “clean” areas and toward “contaminated” areas.
- A biosafety manual is developed specific to the laboratory and biosafety precautions are incorporated into standard operating procedures.
- Respiratory protection may be required.
- Policies and procedures are developed such that only persons who have been advised of the potential biohazards, who meet any specific entry requirements, and who comply with all entry and exit procedures may enter the lab.
Personal Protective Equipment
Personal protective equipment (PPE) includes, but is not limited to:
- Disposable gloves (nitrile, latex, etc.)
- Lab coats or gowns when working in the area. Remove when leaving the laboratory.
- Goggles or face shield for splash protection.
Precautions When Using Animals
When animals are infected with MLV or FIV based vectors, the Animal Biosafety Level of the project will be generally assigned to ABSL-1; HIV based vectors may be assigned to Animal Biosafety Level 2 or 3, depending on the proposed research. The Institutional Biosafety Committee (IBC) will ensure the appropriate Animal Biosafety Level is assigned to the project; Animal Biosafety Level 2a and 2c protocols.
Animal use requests are made to the Institutional Animal Care and Use Committee (IACUC) by submitting an Animal Care and Use Form (ACURF).
Animal cages must be labeled with a biohazard sign.
Recombinant Retroviral Research
Eye exposure - Rinse eyes in an eyewash for at least 15 minutes.
Skin exposure - Rinse skin with soap and water.
Accidental Needlestick Injury - Scrub contaminated skin with soap and water.
- Report Incidents and Seek Treatment - Report actual or suspected exposure incidents to your supervisor immediately. Seek treatment at the Worker’s Health Clinic, if necessary. If you suspect an exposure incident with HIV, immediately report to the Worker’s Health Clinic. It is located on the first floor of Boyd Tower – General Hospital. The clinic’s phone number is 353-8653. If the incident occurs after 4:30 pm, during the weekend, or on holidays, proceed to UIHC’s Emergency Treatment Center (ETC). The phone number is 356-2233.
- Symptoms – Non-specific symptoms for HIV infection including lymphadenopathy, anorexia, chronic diarrhea, weight loss and fever.
- Immunizations and Prophylaxis – No immunizations available; a series of drugs can be administered immediately after a high risk exposure to HIV that is expected to reduce the likelihood of infection.
- Incubation Period – HIV has a minimum range from 6 months to more than 7 years.
Spill and Disposal Procedures
For spills outside a biological safety cabinet, leave the area while holding your breath. Once outside the area, wash hands and face with soap and water. Do not allow anyone inside the area or room where the spill occurred. Allow 30 minutes for the aerosols to settle. Enter the room wearing required protective clothing, gently cover the spill with paper towels, and apply disinfectant starting at the perimeter and working towards the center. Allow the disinfectant to remain on the spill for at least 20 minutes before initiating spill clean up. After initial clean up, disinfect the area a second time.
For spills inside a biological safety cabinet, cover the spill with paper towels or wipes. Gently pour disinfectant over the spill area. Let the disinfectant soak for 20 minutes before cleaning up the spill. After initial clean up, disinfect area a second time.
Contaminated materials must be disposed of as biohazardous waste.
Decontaminate adjacent surfaces with bleach solution.
Disinfectants should be allowed a minimum of 20-30 minutes contact time. Use one of the following:
- Sodium hypochlorite (use 1-10% dilution of fresh bleach)
- 70% Ethanol
- 2% Glutaraldehyde, or Formaldehyde
Autoclave cultures for 30 minutes at 121°C or 250°F (15 lbs per square inch of steam pressure).
Disinfect work surfaces using an effective germicide (see above). This may be followed by an alcohol wipe to lessen the corrosive nature of the germicide.
Materials must be appropriately contained and labeled for transport within the University. Shipping infectious substances, diagnostic specimens, and/or shipping with dry ice off-campus require training and certification. See EHS’s fact sheet “Transporting and Shipping Infectious Substances”.
If you have questions, contact EHS’s Biological Safety Section at 5-8501.
Information and References
- Bloodborne Pathogens 1910.1030. Standard – 29 CFR. United States Department of Labor. Occupational Safety and Health Administration.
- Debyser, Z. 2003. A short course on virology / vectorology / gene therapy. Current Gene Therapy. 3:495-9.
- Health Canada Office of Laboratory Security. (1996, September). Infectious Substances MSDS Web Site. Human Immunodeficiency Virus. Retrieved February 2015.
- Health and Safety Executive, United Kingdom. 2000. Advisory Committee on Genetic Modification. Guidance on commonly used vectors. Part 2B-Annex III.
- U.S. Department of Health and Human Services Centers for Disease control and Prevention and National Institutes of Health. (2007 February) Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition. Retrieved February 2015.