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.

Since the emergence of low pathogenic avian influenza (LPAI) H9N2 viruses in Morocco in 2016, severe respiratory problems have been encountered in the field. Infectious bronchitis virus (IBV) is often detected together with H9N2, suggesting disease exacerbation in cases of co-infections. This hypothesis was therefore tested and confirmed in laboratory conditions using specificpathogen-free chickens. Most common field vaccine programmes were then tested to compare their efficacies against these two co-infecting agents. IBV γCoV/chicken/Morocco/ I38/2014 (Mor-IT02) and LPAI virus A/chicken/Morocco/SF1/2016 (Mor-H9N2) were thus inoculated to commercial chickens. We showed that vaccination with two heterologous IBV vaccines (H120 at day one and 4/91 at day 14 of age) reduced the severity of clinical signs as well as macroscopic lesions after simultaneous experimental challenge. In addition, LPAI H9N2 vaccination was more efficient at day 7 than at day 1 in limiting disease post simultaneous challenge. RESEARCH HIGHLIGHTS. Simultaneous challenge with IBV and AIV H9N2 induced higher pathogenicity in SPF birds than inoculation with IBV or AIV H9N2 alone. . Recommended vaccination programme in commercial broilers to counter Mor-IT02 IBV and LPAIV H9N2 simultaneous infections: IB live vaccine H120 (d1), AIV H9N2 inactivated vaccine (d7), IB live vaccine 4-91 (d14).