Wild birds as reservoirs for diverse and abundant gamma- and deltacoronaviruses.

Wild birds interconnect all parts of the globe through annual cycles of migration with little respect for country or continental borders. Although wild birds are reservoir hosts for a high diversity of gamma- and deltacoronaviruses, we have little understanding of the ecology or evolution of any of these viruses. In this review, we use genome sequence and ecological data to disentangle the evolution of coronaviruses in wild birds. Specifically, we explore host range at the levels of viral genus and species, and reveal the multi-host nature of many viral species, albeit with biases to certain types of avian host. We conclude that it is currently challenging to infer viral ecology due to major sampling and technical limitations, and suggest that improved assay performance across the breadth of gamma- and deltacoronaviruses, assay standardization, as well as better sequencing approaches, will improve both the repeatability and interpretation of results. Finally, we discuss cross-species virus transmission across both the wild bird - poultry interface as well as from birds to mammals. Clarifying the ecology and diversity in the wild bird reservoir has important ramifications for our ability to respond to the likely future emergence of coronaviruses in socioeconomically important animal species or human populations.

Detection and Characterization of New Coronavirus in Bottlenose Dolphin, United States, 2019.

We characterized novel coronaviruses detected in US bottlenose dolphins (BdCoVs) with diarrhea. These viruses are closely related to the other 2 known cetacean coronaviruses, Hong Kong BdCoV and beluga whale CoV. A deletion in the spike gene and insertions in the membrane gene and untranslated regions were found in US BdCoVs (unrelated to severe acute respiratory syndrome coronavirus 2).

miRNA repertoire and host immune factor regulation upon avian coronavirus infection in eggs.

Avian infectious bronchitis virus (IBV) is a coronavirus with great economic impact on the poultry industry, causing an acute and highly contagious disease in chickens that primarily affects the respiratory and reproductive systems. The cellular regulation of IBV pathogenesis and the host immune responses involved remain to be fully elucidated. MicroRNAs (miRNAs) have emerged as a class of crucial regulators of numerous cellular processes, including responses to viral infections. Here, we employed a high-throughput sequencing approach to analyze the miRNA composition of the spleen and the lungs of chicken embryos upon IBV infection. Compared to healthy chicken embryos, 13 and six miRNAs were upregulated in the spleen and the lungs, respectively, all predicted to influence viral transcription, cytokine production, and lymphocyte functioning. Subsequent downregulation of NFATC3, NFAT5, SPPL3, and TGFB2 genes in particular was observed only in the spleen, demonstrating the biological functionality of the miRNAs in this lymphoid organ. This is the first study that describes the modulation of miRNAs and the related host immune factors by IBV in chicken embryos. Our data provide novel insight into complex virus-host interactions and specifically highlight components that could affect the host's immune response to IBV infection.

Avian coronavirus infection induces mannose-binding lectin production in dendritic cell precursors of chicken lymphoid organs.

The aim of this immunocytochemical study was to compare mannose-binding lectin (MBL) production induced by avian coronavirus in the spleen and caecal tonsil (CT). One-day-old specific-pathogen-free (SPF) chickens were experimentally infected with six QX field isolates and the H120 vaccine strain. In the negative control birds, the spleen was MBL negative, while the CT showed scattered MBL-positive cells in close proximity and within the surface epithelium and germinal centre (GC)-like cell clusters. MBL was detectable in the ellipsoid-associated cells (EACs) and cell clusters in the periarterial lymphoid sheath (PALS) by 7 days post infection (dpi). In both organs, the MBL-positive cells occupy antigen-exposed areas, indicating that GC formation depends on resident precursors of dendritic cells. The majority of MBL-positive EACs express the CD83 antigen, providing evidence that coronavirus infection facilitated the maturation of dendritic cell precursors. Surprisingly, co-localisation of MBL and CD83 was not detectable in the CT. In the spleen (associated with circulation), the EACs producing MBL and expressing CD83 are a common precursor of both follicular (FDC) and interdigitating dendritic cells (IDC). In the CT (gut-associated lymphoid tissue, GALT) the precursors of FDC and IDC are MBL-producing cells and CD83-positive cells, respectively. In the CT the two separate precursors of lymphoid dendritic cells provide some 'autonomy' for the GALT.

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.

Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity.

Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.