Differential tissue tropism and transmission efficiency of two dominant influenza D clades with overlapping but distinct receptor binding fine specificities in ferrets

Influenza D virus (IDV) utilizes bovines as a primary reservoir causing periodical spillover to pigs and other hosts. In this study, we utilized ferrets to study IDV with a focus on the role of the Hemagglutinin-Esterase-Fusion (HEF) protein in the replication, tissue tropism, and transmission of two dominant clades of IDV– swine D/OK, and bovine D/660. In addition to swine D/OK, we rescued a chimeric virus (D/OK 660HEF ) expressing the bovine D/660 HEF using reverse genetic system. Two isogenic IDVs differing only in the HEF protein were characterized in ferrets with respect to viral shedding, tissue tropism, transmission, and pathogenesis. Ferrets intranasally infected with D/OK and D/OK 660HEF showed similar levels of viral shedding but exhibited slight differences in transmission efficiency to contact sentinel ferrets. Specifically, D/OK replicated mostly in the upper respiratory tract and transmitted to 2/3 naive ferrets, while D/OK 660HEF replicated in both upper and lower respiratory tract (trachea) but transmitted only to 1/3 naive ferrets. Both direct inoculated and contact sentinel ferrets seroconverted at 14 days post-infection, which indicated an association with viral replication fitness and transmission efficiency. Distinct receptor fine specificities plus six amino acid mutations in the receptor binding domain of the HEF protein between swine D/OK and bovine D/660 viruses may explain the different tissue tropism and transmission efficiency observed between these two viruses. Furthermore, while no detectable virus titers were observed in the lungs and intestines of ferrets, fluorescent RNAscope probe-based in-situ hybridization assay detected viral RNAs in these tissues. Finally, deep-sequencing revealed ferret-adapted mutations in PB1, PB2, and M segments that have not appeared in natural IDV isolates from bovines or pigs which need further characterization. Taken together, results of this study demonstrate that IDV is optimized for replication and spread in mammals and subtle mutations in HEF protein may affect viral tropism and transmission efficiency.