Abstract
Chicken infectious anemia virus (CIAV) is a major immunosuppressive pathogen that significantly undermines the global poultry industry. In the absence of effective commercial vaccines, targeted antiviral strategies for CIAV are urgently needed. In this study, we established a novel dual-copy reverse-genetics platform and characterized the virulence phenotypes of VP3-mutant rescue viruses. The CIAV strain, designated SD15, was obtained from blood samples of clinically anemic commercial chickens. Phylogenetic analysis revealed that CIAV-SD15 shares high genetic homology (95.8 % to 98.8 %) with 40 reference strains, with VP3 carrying two low-frequency specific mutations (S25 and N67, only 0.09 % and 0.12 %, respectively). These mutations were introduced into VP3 to construct recombinant full-length DNA clones. High titers of infectious progeny viruses were successfully rescued in specific-pathogen-free (SPF) chicks via direct intramuscular injection within 10 dpi, leading to severe bone-marrow damage and hemorrhage. The rescued CIAV-SD15 (rCIAV-SD15) exhibited in vitro growth kinetics and cytopathic effects (CPE) similar to wild-type (wt) CIAV-SD15, whereas rCIAV-N67S and rCIAV-S25L/N67S displayed significantly slower replication rates. Notably, the retention of serine at position 25, combined with the N67S substitution in VP3, substantially enhances CIAV-SD15 virulence, as evidenced by the lowest immune organ indices and highest thymocyte-apoptosis rates in SPF chicks. These findings underscore the critical role of specific VP3 mutations in modulating CIAV pathogenicity. The reverse genetics platform developed here provides a foundational framework for advancing our understanding of CIAV pathogenesis.