Abstract
Ectromelia virus (ECTV), a member of the Orthopoxvirus genus, serves as both a causative agent of mousepox and a pivotal surrogate model for studying highly pathogenic orthopoxviruses. Although genomic data on ECTV remains limited, we report the isolation and characterization of a novel strain, ECTV-C-Tan-GD01, obtained from rodents in Guangdong Province, China. Nanopore sequencing yielded a complete genome (199 annotated genes, including one gene truncated at the C-terminus) with inverted terminal repeats (ITRs) harboring a conserved hairpin structure. Notably, a frameshift-inducing "G" deletion in the EV159 gene resulted in the truncation of a semaphorin-like protein. In vitro assays demonstrated cell-associated viral replication kinetics, with maximum titers achieved earlier in Vero/HeLa cells (72 h) than in BHK-21/CEF cells (84 h). Murine challenge experiments revealed extreme virulence (LD(50) < 1 plaque-forming unit (PFU) via intranasal/footpad routes) and hepatosplenic tropism. Furthermore, ECTV-C-Tan-GD01 exhibited utility in evaluating orthopoxvirus countermeasures: a single dose of vaccinia virus Tiantan (VTT) or non-replicating vaccinia virus Tiantan (NTV) conferred cross-protection, while tecovirimat (ST-246), cidofovir (CDV), and brincidofovir (initially CMX001) significantly reduced viral loads and pathology. This study establishes ECTV-C-Tan-GD01 as a dual-purpose resource for probing orthopoxvirus evolution and advancing therapeutic development.