RECEPTOR USAGE AND INFECTION BIOLOGY OF INFLUENZA D VIRUS

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated by the traditional hemagglutination assay coupled with sialoglycan microarray and functional assays that IDV can recognize and bind both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac). Agricultural animals such as cattle can produce both Neu5,9Ac2 and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. The bifunctional enzyme, glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) is a master regulator of de-novo sialic acid synthesis while 9-O-Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize in more detail, the role of human and non-human 9-O-acetylated sialic acids in IDV infection and determine which form of 9-O-acetylated sialic acids drives IDV entry and productive infection, we took advantage of cells either deficient in 9-O-acetylated sialic acid or completely devoid of sialic acid, and synthetic receptor analog-based feeding experiment in combination with the single-round and multi-round IDV infection assays. The data from our study indicated that IDV can utilize human Neu5,9Ac2 or non-human Neu5Gc9Ac as a functional receptor for infection. Ferret is a gold standard model for the study of human influenza viruses. Ferrets, like humans, can only synthesize Neu5,9Ac2 and lack Neu5Gc9Ac. We used ferrets to study the replication fitness and transmission of the influenza D virus and to evaluate its zoonotic potential. We demonstrated that IDV can utilize Neu5,9Ac2 for infection and transmission. Our studies provide compelling evidence that IDV has acquired the unique ability to infect and transmit in agricultural animals such as bovines that are relatively enriched in non-human Neu5Gc9Ac as well as in ferrets only expressing human Neu5,9Ac2. Results of this work provide the foundation for further investigation of infection biology and zoonosis of influenza D virus