Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses.

A species barrier for the influenza A virus is the differential expression of sialic acid, which can either be α2,3-linked for avians or α2,6-linked for human viruses. The influenza A virus hosts also express other species-specific sialic acid derivatives. One major modification at C-5 is N-glycolyl (NeuGc), instead of N-acetyl (NeuAc). N-glycolyl is mammalian specific and expressed in pigs and horses, but not in humans, ferrets, seals, or dogs. Hemagglutinin (HA) adaptation to either N-acetyl or N-glycolyl is analyzed on a sialoside microarray containing both α2,3- and α2,6-linkage modifications on biologically relevant N-glycans. Binding studies reveal that avian, human, and equine HAs bind either N-glycolyl or N-acetyl. Structural data on N-glycolyl binding HA proteins of both H5 and H7 origin describe this specificity. Neuraminidases can cleave N-glycolyl efficiently, and tissue-binding studies reveal strict species specificity. The exclusive manner in which influenza A viruses differentiate between N-glycolyl and N-acetyl is indicative of selection.

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

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.