Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals

Abstract

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

Host Range, Biology, and Species Specificity of Seven-Segmented Influenza Viruses—A Comparative Review on Influenza C and D

“…However, a tissue microarray involving nasal tissue, pharynx, upper and lower trachea, and lung tissues with recombinant HEF protein (HEF S57A) with deficient esterase activity showed high binding to the epithelium of nasal, pharyngeal, upper and lower trachea in cattle, and only to the pharyngeal and nasal epithelium in pigs. In the case of sheep, and goats, the protein histochemical staining was comparable with almost the same level of binding to the nasal and pharyngeal epithelium, whereas the binding was mostly localized in the submucosal glands of nasal and pharyngeal tissue in horses [167].…”

SARS-CoV-2 Spike N-Terminal Domain Engages 9-O-Acetylated α2–8-Linked Sialic Acids

“…Recombinant SARS-CoV-2 spike protein NTD (GenBank: MN908947.3; AA319-541), OC43 NTD (GenBank: L14643.1; AA15-302), and HKU1 NTD (GenBank: DQ339101; AA14-294) were cloned using Gibson assembly from cDNAs encoding codon-optimized open reading frames of full-length SARS-CoV-2 spike, as previously described . Influenza D (OK/D) HEF construct has been described previously . The pCD5 expression vector was adapted so that after the signal sequence, the SARS-COV-2, OC43, and HKU1 NTD-encoding cDNAs are cloned in frame with a GCN4 trimerization motif (KQIEDKIEEIESKQKKIENEIARIKK), a TEV cleavage site, fluorescent reporter open reading frame, , and the Strep-Tag II (WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany.…”

“…43 Influenza D (OK/D) HEF construct has been described previously. 82 The pCD5 expression vector was adapted so that after the signal sequence, the SARS-COV-2, OC43, and HKU1 NTD-encoding cDNAs are cloned in frame with a GCN4 trimerization motif (KQIEDKIEEIESKQKKIENEIARIKK), a TEV cleavage site, fluorescent reporter open reading frame, 83,84 and the Strep-Tag II (WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany. Protein Expression and Purification.…”

The mammary glands of cows abundantly display receptors for circulating avian H5 viruses

“…We used the classical H5 derived from A/Indonesia/05/2005 (H5IN), A/Vietnam/1203/2004 (H5VN), and the Y161A (H5VN Y161A) mutant that confers binding to Neu5Gc (34). To detect 9-O-acetylated structures, we employed an enzymatically inactive influenza D virus hemagglutinin esterase fusion protein (D/OK) (12). We also used commonly employed plant lectins SNA and MAL-II (biotinylated, Vector laboratories) to determine the distribution of α2,3 and α2,6 linked SIAs.…”