Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

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 viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.