AdamiForge: a modular reverse genetics tool for human adenovirus type 12 (HAdV-A12)

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• Human adenoviruses (HAdVs) encompass over 100 genotypes causing diverse infections.
• Current research predominantly focuses on HAdV-C5, with limited tools for other genotypes like HAdV-A12.
• Strain-specific characteristics impact HAdV pathogenicity and vector applications.

Purpose of the Study:

• To develop a novel reverse genetics system, AdamiForge, for engineering the less-characterized HAdV-A12.
• To create tools enabling the study of HAdV-A12, which is phylogenetically distant from HAdV-C5.
• To facilitate research on HAdV genotypes beyond HAdV-C5 for broader understanding and application.

Main Methods:

• AdamiForge divides the HAdV-A12 genome into fragments cloned into plasmids.
• Genomic fragments are released and reassembled *in vitro* using Gibson assembly.
• Reconstituted genomes are introduced into permissive cells to generate new virions.

Main Results:

• Successfully reconstituted wild-type HAdV-A12 using the AdamiForge system.
• Engineered recombinant HAdV-A12 expressing modified DNA-binding proteins (DBPs) fused to eGFP or FLAG-tag.
• Demonstrated successful detection of HAdV-A12 DBP via Western blotting and immunofluorescence using engineered tags.

Conclusions:

• AdamiForge provides a valuable reverse genetics tool for engineering HAdV-A12.
• The developed tools, including tagged DBPs, overcome limitations in antibody availability for HAdV-A12.
• This work expands the toolkit for studying diverse HAdV genotypes, crucial for understanding pathogenesis and vector development.