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

ABSTRACT Human adenoviruses (HAdVs) are a large family of viruses comprising more than 100 genotypes distributed across seven species (A to G). Depending on the genotype, they cause respiratory, ocular, urinary tract, or gastrointestinal infections. Despite the considerable diversity within the human adenovirus genus, most of our current knowledge comes from studies on HAdV-C5. The availability of experimental tools, including antibodies and reverse genetics systems, for the study of adenoviruses other than the C5 strain remains very limited. Here, we describe a reverse genetics tool to engineer HAdV-A12, named AdamiForge. HAdV-A12 is phylogenetically distant from HAdV-C5 and differs markedly in its clinical manifestations, warranting the development of dedicated experimental tools. In principle, AdamiForge is similar to the widely used reverse genetics tool for HAdV-C5 known as AdenoBuilder. Briefly, the viral genome is divided into several blocks and cloned into high-copy plasmids. These genomic fragments are released using a restriction enzyme and recombined in vitro by Gibson assembly. The reconstituted genome is then delivered into permissive cells to generate new virions. The main advantage of this approach lies in the fragmentation of the viral genome into small plasmids, which can be easily modified. In addition to the reconstitution of the wild-type virus, we engineered two recombinant viruses expressing modified versions of the viral DNA-binding protein fused either to eGFP or to a FLAG-tag. These modifications compensated for the lack of specific antibodies and enabled successful detection of the HAdV-A12 DBP by both Western blotting and immunofluorescence. IMPORTANCE Adenoviruses belong to a large family of viruses comprising seven species (A–G) and more than 100 genotypes. Beyond their role as human pathogens, adenoviruses have been extensively studied as vectors in gene therapy, vaccine development, and oncolytic virotherapy. To date, research has largely focused on a limited number of strains. However, strain-specific characteristics can substantially influence both pathogenic potential and vector performance. It is therefore essential to expand research to other genotypes and to develop new tools for the study of less-characterized strains such as human adenovirus A12.