Frequently Asked Questions Related to Biosafety using Adeno-associated viral, Adenoviral, Retroviral and Lentiviral Vectors

NOTE: The following information pertains to all vectors packaged at vector core. If you have biosafety-related questions regarding the vectors, please contact the vector Core Facility via email at li.zhong@umassmed.edu or Guangping.gao@umassmed.edu

1. Adeno-associated viral vectors

Name? AAV serotype 1, 2, 3, 4, 5, 6, 6.2, 7, 8, 9, rh.10, rh.39, rh.43 and more novel ones of rAAV.

Source? Human or nonhuman primate.

Selectable marker? Ampicillin for the vector plasmid.

Will viral particles be able to infect human cells? Yes.

Replication competent (Y/N)? No.

If replication defective:

What genes are deleted/modified resulting in replication defect? All viral genes are deleted.

What elements of the original virus genome are retained? Only terminal repeats are retained (about 6% of the entire genome).

What is the packaging system/cell line used to produce virus stocks? 293 cells.

Helper virus used in the packaging ? (Y/N) No, helper free system is used.

Assay to test for helper virus: Not applicable


2. Adenoviral vectors

Name? Replication defective H5 Adenovirus vector.

Source of genome? Human serotype 5 adenovirus.

Selectable marker? Ampicillin for the vector plasmid.

Will viral particles be able to infect human cells? Yes.

Replication competent (Y/N)? No.

If replication defective:

What genes are deleted/modified resulting in replication defect? Early region 1 which is responsible for replication is deleted.

What is the packaging system/cell line used to produce virus stocks? 293 cells.

3. Retroviral and Lentiviral vectors

Name? VSV-G.HIV.CMVEGFP.WPRE or VSV-G.HIV.CMVLacZ.WPRE

Source of genome? Human immunodeficiency virus

Envelope glycoprotein? Vesticular stomatitis virus envelope glycoprotein

Host range (tropism)? Broad

Will viral particles be able to infect human cells? Yes.

Selectable marker? Ampicillin for the vector plasmid but none for the lentiviral vector

Replication competent (Y/N)? No.

If replication defective:

What genes are deleted/modified resulting in replication defect? All viral genes are deleted except only the 5’ and 3’ LTRs and a portion of the gag coding region totaling under 30% of the entire viral genome

What is the packaging system/cell line used to produce virus stocks? 293 cells.

How many recombination events are necessary to produce replication competent virus (RCV)? Greater than 3 recombination events necessary to produce RCV. There is no evidence that recombination events occur. Note that production of viral particles uses a well-established packaging system and established protocols, with strict BL2 level guidelines.

What assay is used to test for RCV? p24 ELISA assay using cell lines that are able to propagate wild-type HIV, such as SupT1 lymphocytes.

Frequency of testing of stocks for RCV: The UMMS vector Core Facility does not keep virus stocks, but instead prepares fresh virus from plasmid DNA. If an individual lab keeps the virus stock, every stock used for in vivo studies should be tested for RCV.