High throughput assay for measuring adenovirus replication kinetics

A method for measuring replication kinetics of a recombinant adenovirus, comprising:

• (i) transfecting cells with the genome of the recombinant adenovirus, or infecting cells with particles of the recombinant adenovirus,
• wherein the recombinant adenovirus comprises a recombinant adenovirus genome comprising a heterologous opening reading frame (ORF) encoding a fluorescent protein, and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF, and wherein:
• (a) the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;
• (b) the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;
• (c) the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;
• (d) the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;
• (e) the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k;
• or
• (f) the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2;
• (ii) culturing the transfected cells or infected cells for at least two days;
• (ii) measuring fluorescence at regular intervals throughout the culture period;
• and
• (iv) calculating log-slope from the fluorescence measurements, thereby measuring replication kinetics of the recombinant adenovirus.