Viral Vectors for Gene Transfer

(Updated September 2009)

The following table lists the most common viral vectors used for gene transfer / gene therapy studies, the biosafety levels which must be employed when producing vector stocks and/or producer lines, and the appropriate disinfectants for various vector types. The currently recommended tests for replication competent virus (RCV) are also described. See footnote 10 for how to report replication competent virus (RCV) test results prior to lowering containment levels. See footnote 11 for special restrictions on the inclusion of gene inserts such as oncogenes that may require a higher containment level.



VirusBiosafety LevelTest for Replication Competent VirusDisinfectant(10)
Gammaretrovirus (ecotropic pseudotyped) BSL-1(1) Not required 70% ethanol,
70% isopropanol,
detergent,
10% bleach
Gammaretrovirus (other pseudotypes) BSL-2 Marker rescue assay(5) 70% ethanol,
70% isopropanol, detergent,
10% bleach
Adenovirus BSL-2 Polymerase chain reaction (PCR) for E1a(6) 10% bleach
Adeno-Associated virus (with adenovirus) BSL-2 Replication-competent adenovirus(7) 10% bleach
Adeno-Associated virus (adenovirus-free) BSL-1(2) Not required 10% bleach
Vaccinia virus BSL-2 Not applicable 10% bleach
Herpesvirus amplicons BSL-2 Plaque assay(8) 70% ethanol,
70% isopropanol, detergent,
10% bleach
Foamyvirus (replication competent) BSL-2 Not applicable 70% ethanol,
70% isopropanol, detergent,
10% bleach
Foamyvirus (replication defective) BSL-1(3) Not required 70% ethanol,
70% isopropanol, detergent,
10% bleach
Lentivirus (non-HIV pseudotyped) BSL-2(4) Serial transfer and ELISA for p24 antigen(9)Example Lentiviral RCV Protocol (Word) 70% ethanol,
70% isopropanol, detergent,
10% bleach
  1. Gammaretrovirus vectors pseudotyped with an ecotropic envelope (one which allows for the transduction of mouse cells but not human cells) and generated by direct plasmid transfection can be handled at a BSL-1 level. There is no requirement that the vector stocks and/or producer lines be tested prior to use in mice; however, a marker rescue assay(5) for RCV is recommended as part of any experimental design. Ecotropic producer cells generated by transduction with non-ecotropic pseudotyped producer cells must be handled at a BSL-2 level until demonstrated to be free of RCV by a marker rescue assay (5).
  2. Adeno-associated virus vector stocks generated with adenovirus-free packaging systems can be handled at a BSL-1 level, and no further testing is needed for studies in mice. Reference for adenovirus free packaging system: Allen JM, Halbert CL, Miller AD. 2000. Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6. Mol Ther 1:88-95.
  3. Foamyvirus vector stocks generated with packaging systems shown to be free of RCV by a marker rescue assay can be used in mice and handled at a BSL-1 level without further testing. Foamyvirus vectors which are replication-competent must be handled at a BSL-2 level. Reference for a marker rescue assay: Trobridge GD, Russell DW. 1998. Helper-free foamy virus vectors. Hum Gene Ther 1998 9:2517-2525. Results should be <1 infectious units/milliliter.
  4. Lentivector systems with HIV envelope gene sequences require BSL-2 with BSL-3 practices at a minimum.
  5. Gammaretrovirus vectors pseudotyped with envelopes other than ecotropic must be tested for RCV by a marker rescue assay prior to being approved for use at BSL-1/ABSL-1. The vector stock or producer line should be tested at a limit of sensitivity of 1 infectious unit per milliliter and the test should include a known positive control. Reference for a marker rescue assay: Miller AD, Buttimore C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 6:2895-2902.
  6. Adenovirus: The following conditions must be met before adenovirus vector stocks can be used in animals and handled at a BSL-1 level. First, the vector must contain less than 2/3 of the wild-type genome; currently this only includes what are known as "gutless" vectors. Second, the vector stocks must be tested for RCV by PCR for E1a prior to use. The vector stock should be tested at a limit of sensitivity of 1 in less than 106 virus particles compared to a known positive control and the results of the test must be available upon request. For murine studies, if only the second condition is met, the vector must be administered at BSL-2 and the animals held for at least one hour prior to their return to standard ABSL-1 housing. Reference for E1a PCR assay: Zhang WW, Kock PE, Roth JA. 1995. Detection of wild-type contamination in a recombinant adenoviral preparation by PCR. Biotechniques 18: 444-447.
  7. Adeno-associated virus vectors generated with adenovirus must be tested for the presence of replication-competent adenovirus after heat-inactivation. Reference for a RCV assay: Hehir KM, Armentano D, Cardoza LM, et a1. 1996. Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence. J Virol 70:8459-8467.
  8. Herpesvirus generated using amplicons must be must be tested for RCV by a plaque assay prior to approval for use at BSL-1 and ABSL-1. The vector stock should be tested at a limit of sensitivity of 1 infectious unit per milliliter and the test should include a known positive control. Herpesvirus vectors based on attenuated herpesvirus must always be handled at a BSL-2 level. Reference for a plaque assay: Strathdee CA, McLeod MR. 2000. A modular set of helper-dependent herpes simplex virus expression vectors. Mol Ther 5:479-485.
  9. Lentivirus vector stocks generated with packaging systems devoid of the HIV envelope gene must be tested for RCV by serial transfer in a cell line documented to be capable of supporting wild type HIV and ELISA assay for p24 antigen prior to approval for use at BSL-1 and ABSL-1. The vector stock should be tested at a limit of sensitivity of 1 infectious unit per milliliter. Lentivector systems with HIV envelope gene sequences require BSL-2 with BSL-3 practices at a minimum. Strictly adhere to the published protocol: Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. 1998. A third-generation lentivirus vector with a conditional packaging system. J Virol 72: 8463-8471. For questions contact the Research & Biological Safety Office at 206.221.7770. Example Lentiviral RCV Protocol (Word)
  10. In general, vector stocks and/or producer lines must be tested for RCV before approval for use at BSL-1 and ABSL-1. The following steps are required to lower containment for viral vector use.
    1. Complete the referenced test for replication competent virus.
    2. Forward the testing results to the EH&S Research & Biological Safety Office at rbso@uw.edu, Box 357165, or fax at 206-221-3068.
    3. Biocontainment will be lowered accordingly for results that meet the requirements. The PI will be notified via e-mail and the approval letter will be updated to reflect the change.
    4. EH&S will forward this information to the appropriate animal facilities manager. The certification information (virus, batch, results) must be referenced on the animal cage by the researcher when the vector is administered. See the animal facility supervisor to obtain RCV cage cards.
    5. All related animal facility standard operating procedures must be followed.
  11. Inclusion of oncogenes in integrating viral vectors typically requires a higher containment level. Oncogenes should be registered with EH&S. See the below links for lists of oncogenes.
    1. A Census of Human Cancer Genes. Supplementary Information Table S1. Nature Review Cancer. 2004; 4:177-183. oncogene list (pdf)
    2. Mouse Retrovirus Tagged Cancer Gene Database. http://rtcgd.abcc.ncifcrf.gov/mm8/easy_search.html