Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding

Abstract

Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.

Keywords: coronavirus; glycan receptor; guinea fowl; receptor affinity; spike protein; tissue tropism.

FIG 1

(Immuno)histological analyses of guinea fowl intestinal tract. Shown are representative images of duodenum and colon from a guinea fowl presenting with peracute enteritis in 2014 after samples were stained with H&E or antibodies against the M protein of infectious bronchitis virus, known to cross-react with GfCoV M protein in immunohistochemistry (IHC). Black arrowheads indicate inflammatory cells, and white arrowheads indicate viral protein expression.

FIG 2

Molecular phylogenetic analysis by maximum likelihood method comparing GfCoV (partial) spike sequences. Phylogenetic tree was based on the Kimura two-parameter model, in which bootstrap values are shown next to the branches. The analysis involved 22 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 893 nucleotide positions in the final data set. Evolutionary analyses were conducted in MEGA, version 6. *, partial S1 sequence of GfCoV.

FIG 3

Binding of GfCoV S1 to the enteric coronavirus glycan receptor diLacNAc. Concentration-dependent binding of GfCoV S1 proteins to Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc in ELISA. The N-terminal domain of the S1 protein of IBV strain M41 was used as a negative control (10). 1, significant difference between GfCoV S1 and IBV-M41; 2, significant difference between GfCoV/2014 S1 and GfCoV/2011 S1 (P < 0.001).

FIG 4

Glycan binding specificity of guinea fowl S1 proteins. (A) Schematic representation of selected glycan structures present on the glycan array; numbers correspond to those shown in the graphs. Numbers 1 to 4 represent glycans ending with galactose, numbers 5 to 8 represent glycans capped with alpha-2,3 -linked sialic acids, and numbers 9 to 12 represent glycans capped with alpha-2,6-linked sialic acids. Yellow circle, galactose; blue square, GlcNAc; green circle, mannose. (B and C) Glycan receptor specificity of GfCoV S1 proteins and lectins ECA, MAL1, and SNA in glycan array assay (Broszeit and de Vries, submitted). RFU, relative fluorescent units.

FIG 5

Glycan binding affinity of guinea fowl S1 proteins. (A and B) Glycan binding of GfCoV/2014 S1 and GfCoV/2011 S1 is shown as a heat map with 5-fold dilutions (100 µg/ml to 4 µg/ml) of the proteins applied to glycan array slides that were scanned at different laser intensities. RFU, relative fluorescent units. Glycan numbers correspond to schematic representations shown in Fig. 4A.

FIG 6

Binding of GfCoV S1 proteins to guinea fowl duodenum and colon without and with enzymatic pretreatment of the tissues. (A and B) Spike histochemistry was performed on uninfected, healthy duodenum and colon tissues without and with pretreatment of enzymes (AUNA and/or galactosidase) before application of GfCoV/2014 S1 and GfCoV/2011 S1. Binding of proteins was visualized by red staining.