Ec_Ou_ZC1, a new Tunavirus, reveals Cor superinfection exclusion and differential Avs susceptibility across Drexlerviridae subfamilies.

The global rise of antibiotic resistance has rekindled interest in bacteriophages (phages) as alternative therapeutics. Here, we isolated and characterized a novel Escherichia coli phage, Ec_Ou_ZC1, from the family Drexlerviridae, genus Tunavirus, sharing 94.72% genomic identity with Escherichia phage phiD2-2. Ec_Ou_ZC1 encodes an orphan methyltransferase and a Cor superinfection exclusion (SIE) protein. Heterologous expression of Cor blocked all tested FhuA-dependent Drexlerviridae phages (Ec_Ou_ZC1, T1, Bas03, Bas12, Bas13) but not T4 or T7, confirming family-specific SIE through FhuA receptor inactivation. Plaque assays against four antiviral STAND (Avs) systems revealed differential susceptibility within Drexlerviridae: Bas03 (Braunvirinae) was sensitive to SeAvs3, while Ec_Ou_ZC1, Bas05, Bas12, Bas13, and T1 (Tempevirinae and Tunavirinae) resisted all four systems, suggesting that Avs resistance depends on specific genomic features. Species-level phylogenetic analyses using concatenated single-copy orthologs robustly resolved evolutionary relationships within Tunavirus, separating Escherichia- and Shigella-infecting phages and providing a framework for predicting host range of new isolates.