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.