ORF3 Depletion Attenuates SADS‐CoV Virulence and Retains Partial Maternally Mediated Protection in Suckling Mice

Swine acute diarrhea syndrome coronavirus (SADS‐CoV) is an emerging enteric coronavirus that causes severe diarrhea and high mortality in neonatal piglets. Its broad host range, tropism for human‐origin cells, and limited vaccine availability underscore the importance of defining viral determinants of pathogenicity. The accessory protein ORF3 has been linked to CoV virulence, but its role in SADS‐CoV infection in vivo and its impact on maternally transferred protection have not been fully established. Here, we used reverse genetics to generate rSADS‐ΔORF3/GFP, a recombinant virus with ORF3 replaced by GFP, and compared it with parental rSADS‐CoV in suckling BALB/c mice. ORF3 depletion markedly attenuated disease and reduced viral RNA burdens across tissues relative to parental rSADS‐CoV. Because maternally derived immunity is critical to protection against swine enteric CoV infection in newborn animals, we further evaluated whether rSADS‐ΔORF3/GFP, as a potential live attenuated vaccine (LAV), could elicit maternal protection transferred from inoculated dams to their offspring. Compared with parental rSADS‐CoV, rSADS‐ΔORF3/GFP induced lower maternal neutralizing antibody titers and provided less robust protection in challenged offspring. Nevertheless, offspring from rSADS‐ΔORF3/GFP‐inoculated dams showed improved survival, weight gain, milder intestinal pathology, and lower tissue viral RNA burdens than offspring from DMEM‐inoculated dams, indicating retained protective immunogenicity. Together, these findings support a contribution of ORF3 to SADS‐CoV virulence and demonstrate that ORF3 depletion yields an attenuated virus that retains partial maternally mediated protective efficacy. The residual immunogenicity of rSADS‐ΔORF3/GFP, combined with its reduced virulence, supports its further evaluation as a LAV candidate.