International Biohazard SymbolBiological Safety

Research Utilizing Viral Vectors

Introduction PDF Document

More researchers, many of whom are not virologists by training, are choosing to utilize viral vectors. The following information was developed which defines terms, identifies research that requires IBC registration, and provides information about the virus/viral vectors, practices and Biosafety approaches that may be used as a starting point for a risk assessment.

Bacterial viruses (bacteriophage or phage): Recombinant bacterial viruses require registration with the IBC. However, these viruses are not generally considered harmful to humans, animals, plants, or the environment. Thus, wild type bacterial viruses do not as a rule require registration as biohazards. If a non-recombinant bacterial virus is found or suspected to cause harm, then registration would be required.

Eukaryotic viruses: All wild type and recombinant eukaryotic viruses will require registration as a biohazard. Note that field release or field sampling of viruses, although not involved with containment, do require IBC oversight and registration. The biosafety containment level may be inferred by consulting primarily the current edition of the BMBL http://www.cdc.gov/biosafety/publications/bmbl5/index.htm and secondarily the American Biological Safety Association site for Risk Group Classification for Infectious Agents (http://www.absa.org/riskgroups/) and the Material Data Safety Sheets compiled by the Public Health agency of Canada (http://www.phac-aspc.gc.ca/msds-ftss/index.html). Unfortunately many viruses, especially plant and non-zoonotic animal viruses, do not have documented formal risk analyses. In these cases, the PI will do the risk assessment based on experience or the literature, and the IBC will either concur or require modifications.

Recombinant DNA or RNA Viruses

All recombinant DNA and RNA must be registered. These include recombinant viruses from all sources, even if the PI was not involved in the manipulations, but obtained the materials from an external source. Ultimately the identity and portion of the viral backbone present, the inserted or modified recombinant nucleic acid, the host cell, and the experimental context determine the containment level. These rules also hold for cases when recombinant viruses are prepared by a collaborator or made from a commercial kit. The NIH Guidelines (http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf) and the Recombinant DNA Advisory Committee (RAC) guidance document on lentiviral vectors (http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf) explicitly dictate the containment level in most instances. The three common ones are Section III-D-3 for recombinant viruses in tissue culture systems, Section III-D-4-a for recombinant viruses in animals, and Appendices B-II-D to B-IV-D for Risk Group Classification of Various Viruses. Other NIH sections are relevant to viruses as well as to other agents. In common practice, the default biosafety level for recombinant viruses is BL-2 and ABL-2. The following are exceptions to that generality:

  1. A lower level may suffice if an incomplete virus is cultured in vitro (NIH Guidelines).
  2. A few animal and/or human viruses qualify for a lower level (NIH Guidelines).
  3. Certain recombinant viruses that are infectious to humans and carry a gene that will confer a selective growth advantage or lead to immune dysfunction require BL-2 containment and the demonstration of replication incompetency prior to the actual research (University of Illinois at Urbana-Champaign IBC and NIH Guidelines).
  4. Animals with recombinant viruses that ordinarily require ABL-2 containment can be down-graded to ABL-1 if and when the animals are considered to no longer be shedding.

A frequently encountered situation with viral vectors is to take advantage of their high specific infectivity rates, while still using them as plasmids. The replication functions are first genetically dissected from the packaging (infectivity) functions. Both functions are then transiently combined on separate molecules (in trans) to package an infectious but non-replicating recombinant molecule. The recombinant virus can be eventually lost or persist in the target cell either integrated into, or independent of, the chromosomes. However, as long as the packaging and replicating functions are kept from recombining into a single molecule, no further viral-driven replication or viral life cycles will ensue. However, if an endogenous virus is present in the host, the deficient viral vector may be rescued by this endogenous virus in trans producing a fully competent viral particle. The successful execution of this dictum has implications from both a biosafety and research standpoint. A number of strategies have been developed to minimize the occurrence of recombining the two functions.

  1. Ensure that no homologous recombination occurs by neatly dissecting the replication from the packaging functions.
  2. Minimize the probability of homologous and end-joining recombination by separating the individual packaging functions into several different plasmid molecules.
  3. Utilize a heterologous (from a different virus) packaging system to minimize the probability of homologous recombination. In this case, the host range and tissue tropism are determined by the packaging system, and the virus is said to be “pseudotyped”.
  4. Utilize a SIN (self-inactivating) derivative, which lacks the native viral 3’ enhancer/promoter. Upon integration, only the recombinant gene without the rest of the virus is transcribed and thus virus should not be able to undergo another round of replication.

However, two main risks are still in play: the virus is highly infectious, and integration into the genome can have severe consequences.

Each of the following virus information fact sheets should be considered a starting point for conducting a risk assessment. Utilizing each viral vector system brings along the biology of the virus and must be considered.

Retroviruses/Moloney Murine Leukemia Virus (MoMuLV) PDF Document

Risks of MMLV vectors: Data suggests a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis leading to cell transformation and tumor formation. Pseudotyping also increases host range and risk of infection.

Retroviruses/Lentiviruses PDF Document

Human: HIV; Animals: EIV, FIV, SIV, etc.

Risks of Lentiviral vectors: HIV-AIDS in humans.

Adenovirus PDF Document

Risks of Adenovirus vectors: Infections and, in rare cases, severe disease.

Adeno-Associated virus (AAV) PDF Document

Risks of AAV vectors: Recent events suggest that AAV vectors may be associated with insertional mutagenesis and cancer, and therefore, may not be as safe as previously thought. AAV can preferentially insert at a specific site on human chromosome 19 and remain latent. Potentially at a later time when a helper virus is present, AAV can be reactivated and produce infection. Some characteristics of AAV may increase risk, including the following: detection in embryonic tissue, as association with male infertility, and the ability to replicate in some cases without a helper virus.

Herpesvirus/Gammaherpesvirus/Epstein Barr (EB) PDF Document

Risks of EB vectors: Human cancer

Herpesvirus/Type A/Herpes simplex virus type-1 and type-2 PDF Document

Risks of HSV vectors: Infections in humans, mild to severe. Therapeutic medications are available.

Poxviridae/Vaccinia PDF Document

Risks of VV vectors: A specific strain of vaccinia has been used as the vaccine for Smallpox, a disease that has been eradicated from the world. There are many strains with varying virulence in animals as well as humans. The potential for human infection from a localized skin infection to severe systemic infection exists. Prophylactic vaccination is available and recommended (but not required) if working with certain strains but not recommended for highly attenuated strains. Certain individuals are more likely to experience severe side effects. Please consult your personal physician and the following website for advice: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1.htm

Viral Vector Table PDF Document

Viral Vector Risk Group Hazard Biosafety Level Animal Biosafety Level Disinfection
Murine Retrovirus (Ecotropic) 1 Injection, splash to face 1 ABL-1 10% bleach (recommended)
Murine Retrovirus (amphotropic or pseudotyped) 2 Injection, splash to face 2 ABL-2 (with special consideration for transgenes, toxins, oncogenes, elements that alter host range, etc.) 10% bleach (recommended)
Lentivirus 2 Injection, splash to face 2 ABL-2 (if animals or cells are permissive) 10% bleach (recommended), 2% glutaraldehyde, 4% formaldehyde, 70% ethanol
Adenovirus 2 Droplets, aerosol, injection 2 ABL-2 (49-72 hours post injection/exposure) 10% bleach (recommended), 5% phenol Note: Alcohol is not an effective disinfectant against adenovirus species.
Adeno-Associated virus (AAV) 2   2 (helper virus), 1 no helper virus ABL-1 if no helper virus, ABL-2 with a helper virus or other special considerations 10% bleach (recommended)
Herpesvirus I and II 2 Aerosols, injection 2 ABL-2 10% bleach (recommended), 70% ethanol
Epstein Barr 2 Aerosols, injection 2 ABL-2 10% bleach (recommended), 70% ethanol
Vaccinia 2 Aerosols, Splash to face (Severe ocular infection), droplets, injection 2 ABL-2 10% bleach (recommended), 70% ethanol

Questions?

Contact the Division of Research Safety, Biological Safety Section (333-2755 or via e-mail) or visit our website: http://www.drs.illinois.edu/bss/.

Other Biosafety Facts Sheets are available from the Biological Safety Section at our website: http://www.drs.illinois.edu/bss/factsheets/.

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