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. 2022 Sep 27;14(10):2125.
doi: 10.3390/v14102125.

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle

Aspen M Workman  1 , Tara G McDaneld  1 , Gregory P Harhay  1 , Subha Das  2 , John Dustin Loy  3 , Benjamin M Hause  2
Affiliations

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle

Aspen M Workman et al. Viruses. .

Abstract

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

Keywords: bovine coronavirus; bovine respiratory disease; calf diarrhea; hemagglutinin-esterase; variant.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alignment of variant HE proteins with HE from the reference BCoV Mebus genome. Panel A. HE protein map with domains annotated according to [28] with the signal peptide (SP) colored white, the lectin domain colored green, the esterase domain colored blue, and the membrane-proximal (MP) domain colored pink. The R3 loop and the receptor binding domain (RBD) are also annotated. Panel B. Alignment of 12 HE variant proteins from this study with the HE-insertion variant from China (GenBank accession no. MN982199, isolate BCoV-China/SWUN/A10/2018) and the reference Mebus sequence. Amino acids divergent from the reference Mebus sequence are shown while homologous amino acids are represented as a dot.
Figure 2
Figure 2
SplitsTree NeighborNet phylogenetic network of 192 BCoV genomes. The complete coding region of 192 genomes was aligned using MAFFT and analyzed with SplitsTree6. A list of the genomes used in this analysis can be found in Table S2. HE-insertion variants are in red and the single HE-deletion variant is in blue. The three vaccine strains are marked with a star.
Figure 3
Figure 3
SplitsTree NeighborNet phylogenetic networks of 192 BCoV HE and spike genes. One hundred and ninety-two HE genes (A) or spike genes (B) were deduplicated, aligned using MAFFT, and analyzed with SplitsTree6. A list of the genomes used in this analysis can be found in Table S2.
Figure 4
Figure 4
SplitsTree NeighborNet phylogenetic networks of HE gene sequences used in recombination detection. (A) NeighborNet of 491 unique Betacoronavirus HE gene sequences. GenBank accession OK391229 was removed from phylogenetic analysis due to poor sequence quality. (B) NeighborNet of 256 unique BCoV HE gene sequences. Stars represent the HE gene sequences flagged as potential recombinant sequences by RDP5. HE-insertion variants are colored red and the single HE-deletion variant is blue.
Figure 5
Figure 5
Functional Characterization of HE gene length variants. The full-length (HE424), insertional variant (HE428), or deletion variant (HE420) hemagglutinin-esterase genes were expressed in baculovirus. Esterase activity (panel A) and ability to bind 9-O-Ac-Sia (panel B) were measured with uninfected cells (Sf9) and cells infected with a baculovirus encoding an unrelated gene (Baculovirus) included as negative controls. A. The esterase activity between different HE variants was measured at 30 min after the start of the reaction. Error bars represent the standard deviation. The data were analyzed using a one-way ANOVA that showed significant variations (p = 0.0002). Tukey’s multiple comparison analyses showed that esterase activity of all HE variants were significantly higher than both Baculovirus and Sf9 negative controls (p < 0.05). B. The ability to bind the 9-O-Ac-Sia was assayed on bovine submaxillary mucin. Binding to ligand was significantly greater for all HE variants than Sf9 and Baculovirus controls (p < 0.05).
Figure 6
Figure 6
Structural modeling of HE gene length variants. Structural models of the homodimeric hemagglutinin-esterase protein (HE424) and mutants containing a 4-amino acid insertion (HE428) or deletion (HE420) in the receptor binding domain. A. Domains of HE424 were colored according to [28] with the lectin domain colored green, the esterase domain colored blue, and the membrane-proximal domain colored pink. The smaller panels zoom in on the R3 loop with the residues in the receptor binding domain (RBD) colored red. B. Sequences of the HE protein variants in the region containing the R3 loop.
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