HIGH THROUGHPUT ASSAY FOR MEASURING ADENOVIRUS REPLICATION KINETICS

1 . A recombinant adenovirus genome, comprising a heterologous open reading frame (ORF) 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:

the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;

the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;

the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;

the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;

the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k; or

the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2.

2 . The recombinant adenovirus genome of claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.

3 . The recombinant adenovirus genome of claim 2 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.

4 . The recombinant adenovirus genome of claim 3 , wherein the amino acid sequence of the self-cleaving peptide is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.

5 . The recombinant adenovirus genome of claim 3 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 12-19.

6 . The recombinant adenovirus genome of claim 1 , wherein the heterologous ORF encodes a fluorescent protein.

7 . The recombinant adenovirus genome of claim 6 , wherein the fluorescent protein is a green fluorescent protein (GFP) a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a blue fluorescent protein (BFP).

8 . The recombinant adenovirus genome of claim 7 , wherein the YFP is YPet or the RFP is mCherry.

9 . The recombinant adenovirus genome of claim 1 , comprising in the 5′ to 3′ direction:

E1B-55K-P2A-YPet;

E1B-55K-P2A-mCherry;

YPet-P2A-(DNA polymerase);

DBP-P2A-YPet;

YPet-P2A-ADP;

E3-14.7k-P2A-YPet;

YPet-P2A-E4-ORF2; or

mCherry-P2A-E4-ORF2.

10 . The recombinant adenovirus genome of claim 1 , comprising the nucleotide sequence of any one of SEQ ID NOs: 3-7, 9-11, 20 and 21.

11 . A recombinant adenovirus comprising the recombinant adenovirus genome of claim 1 .

12 . A kit comprising:

(i) the recombinant adenovirus genome of claim 1 ; and

(ii) cells, cell culture media and/or a multi-well plate.

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

(i) transfecting cells with a genome of the recombinant adenovirus, or infecting cells with particles of the recombinant adenovirus comprising a genome, wherein the recombinant adenovirus genome comprises a heterologous open 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;

(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.