Generation of high-titer defective HSV-1 vectors

There are two types of replication-deficient herpes simplex virus type 1 (HSV-1) vectors: those in which the foreign DNA of interest is cloned into the viral genome itself, and those that are comprised of a plasmid (amplicon) carrying minimal HSV-1 sequences that allow it to be packaged into virus particles with the aid of a helper virus. This unit describes the generation of helper virus stocks. Preparation of recombinant amplicon vector particles by transfection of amplicon and superinfection of helper virus into cells, and harvesting of packaged particles, is also delineated. Thorough characterization of the viral vector stock involves measuring (1) the helper virus plaque-forming units per ml (pfu/ml) on 2-2 cells, (2) the wild-type HSV-1 pfu/ml on Vero cells, and (3) the amplicon stock infectious vector units per ml (ivu/ml) on PC12 cells. Support protocols detail methods for determining titers of helper virus and wild-type virus by plaque assay, and of amplicon stocks by vector assay.