From its initial isolation in the USA in 2011 to the present, influenza D virus (IDV) has been detected in cattle and swine populations worldwide. IDV has exceptional thermal and acid stability and a broad host range. The virus utilizes cattle as its natural reservoir and amplification host with periodic spillover to other mammalian species, including swine. IDV infection can cause mild to moderate respiratory illnesses in cattle and has been implicated as a contributor to bovine respiratory disease (BRD) complex, which is the most common and costly disease affecting the cattle industry. Bovine and swine IDV outbreaks continue to increase globally, and there is increasing evidence indicating that IDV may have the potential to infect humans. This review discusses recent advances in IDV biology and epidemiology, and summarizes our current understanding of IDV pathogenesis and zoonotic potential.

Influenza D virus (IDV), with bovines as a primary host, circulates widely in cattle populations across North America and Eurasia. Here we report the identification of a novel IDV group with broad antigenicity in U.S. bovine herds, which is genetically different from previously known lineages of IDV.

Immunization with an insect cell lysate/baculovirus mixture containing recombinant porcine epidemic diarrhea virus (PEDV) spike protein induced high levels of neutralizing antibodies in both mice and piglets. However, immunization of piglets with this vaccine resulted in enhancement of disease symptoms and virus replication in vaccine recipients exposed to PEDV challenge. Thus, these observations demonstrate a previously unrecognized challenge of PEDV vaccine research, which has important implications for coronavirus vaccine development.

Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other hosts. We have previously demonstrated that IDV binds both 9- O -acetylated N -acetylneuraminic acid (Neu5,9Ac ₂ ) and 9- O -acetylated N -glycolylneuraminic acid (Neu5Gc9Ac). Bovines produce both Neu5,9Ac ₂ and Neu5Gc9Ac, while humans are genetically unable to synthesize Neu5Gc9Ac. 9- O -Acetylation of sialic acids is catalyzed by CASD1 via a covalent acetyl-enzyme intermediate. To characterize the role of Neu5,9Ac ₂ and Neu5Gc9Ac in IDV infection and determine which form of 9- O -acetylated sialic acids drives IDV entry, we took advantage of a CASD1 knockout (KO) MDCK cell line and carried out feeding experiments using synthetic 9- O -acetyl sialic acids in combination with the single-round and multi-round IDV infection assays. The data from our studies show that (i) CASD1 KO cells are resistant to IDV infection and lack of IDV binding to the cell surface is responsible for the failure of IDV replication; (ii) feeding CASD1 KO cells with Neu5,9Ac ₂ or Neu5Gc9Ac resulted in a dose-dependent rescue of IDV infectivity; and (iii) diverse IDVs replicated robustly in CASD1 KO cells fed with either Neu5,9Ac ₂ or Neu5Gc9Ac at a level similar to that in wild-type cells with a functional CASD1. These data demonstrate that IDV can utilize Neu5,9Ac ₂ - or non-human Neu5Gc9Ac-containing glycan receptor for infection. Our findings provide evidence that IDV has acquired the ability to infect and transmit among agricultural animals that are enriched in Neu5Gc9Ac, in addition to posing a zoonotic risk to humans expressing only Neu5,9Ac ₂ . IMPORTANCE: Influenza D virus (IDV) has emerged as a multiple-species-infecting pathogen with bovines as a primary reservoir. Little is known about the functional receptor that drives IDV entry and promotes its cross-species spillover potential among different hosts. Here, we demonstrated that IDV binds exclusively to 9- O -acetylated N -acetylneuraminic acid (Neu5,9Ac ₂ ) and non-human 9- O -acetylated N -glycolylneuraminic acid (Neu5Gc9Ac) and utilizes both for entry and infection. This ability in effective engagement of both 9- O- acetylated sialic acids as functional receptors for infection provides an evolutionary advantage to IDV for expanding its host range. This finding also indicates that IDV has the potential to emerge in humans because Neu5,9Ac ₂ is ubiquitously expressed in human tissues, including lung. Thus, results of our study highlight a need for continued surveillance of IDV in humans, as well as for further investigation of its biology and cross-species transmission mechanism.

Influenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022-2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022-2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.