Preclinical evaluation of an 8-segmented influenza D virus as a live-attenuated vaccine platform in mice

Influenza D virus (IDV) is an emerging contributor to bovine respiratory disease complex (BRDC), yet current commercial vaccines do not include IDV. To explore a vaccine specifically targeting IDV-associated respiratory disease, we generated an eight-segmented recombinant virus, rD/OK-NS2ΔNOR, by separating the NS gene into independent NS1 and NS2 segments. Here, we assessed its potential as a live-attenuated vaccine. In BALB/c mice, intranasal infection with rD/OK-NS2ΔNOR resulted in markedly reduced replication in the nasal turbinate and lungs compared with the wild-type D/OK strain, confirming replication-limited phenotype in vivo. Intranasal primary and booster immunization induced virus-specific serum IgG and nasal IgA antibody responses, whereas inactivated-virus controls elicited limited antibody responses under the conditions tested. Following challenge with wild-type D/OK, viral replication in both the upper and lower respiratory tract was markedly suppressed to levels below the limit of detection in rD/OK-NS2ΔNOR-immunized mice. These results demonstrate that the eight-segmented rD/OK-NS2ΔNOR efficiently elicits both systemic and mucosal immunity and limit viral replication upon homologous challenge in a mouse model. This study provides preclinical proof of concept for developing a live-attenuated IDV vaccine platform, supporting further evaluation in natural hosts.