Safe and stable retroviral helper cell line and related compositions and methods

An SIV transfer vector, comprising:

• a. an SIV 5′ LTR, or a modified version thereof in which all or part of the U3 region of the 5′ LTR is replaced by a non-SIV promoter, or a derivative thereof;
• b. an SIV packaging sequence or a derivative thereof;
• c. an SIV rev-response element or a derivative thereof;
• d. one of a gene or a cloning site;
• and
• e. one of a 3′ SIV LTR, a modified version thereof in which a U3 region is inactivated, or a derivative thereof.

A method for testing for replication-competent SIV virus or recombinant versions thereof, comprising the steps of:

• a. inoculating a non-human primate with recombinant SIV virus particles to be tested;
• b. collecting peripheral blood lymphocytes from the non-human primate;
• c. co-culturing the peripheral blood lymphocytes with cells in which SIV can replicate;
• i) testing the co-culture for replication-competent virus by at least one of 1) incubating replication-competent virus indicator cells with cell-free supernatant from the co-culture and staining the sMAGI cells for LacZ expression, and
• ii) analyzing DNA extracted from the co-culture for the presence of SIV-specific nucleic acid sequences.

A method for testing for replication-competent SIV virus or recombinant versions thereof, comprising the steps of:

• a. culturing the virus with a first culture of cells in which SIV can replicate;
• b. inoculating a second culture of cells in which SIV can replicate with cell-free supernatant of the first culture;
• and
• c. testing the second culture for replication-competent virus by at least one of
• i) incubating replication-competent virus cells with cell-free supernatant from the second culture and staining the replication-competent virus indicator cells for LacZ expression;
• and
• ii) analyzing DNA extracted from cells of the second culture for the presence of SIV-specific nucleic acids.