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Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.

Additional file 6. Virus titers of each strain in different passages.

Additional file 15. Summary of the histopathological findings in the small intestine of the colostrums-deprived neonatal piglets vaccinated with a live attenuated monovalent vaccine strain (PRG942V-80) or filtered supernatant of a mixture of homogenized small intestine and feces sampled from each passage.

Additional file 16. Summary of the histopathological findings in the small intestine of the colostrums-deprived neonatal piglets vaccinated with a live attenuated monovalent vaccine strain (K71V-80) or filtered supernatant of a mixture of homogenized small intestine and feces sampled from each passage.

Additional file 1. Comparison of genotype constellation of porcine 174-1 (G8P[7]), PRG942 (G9P[23]), and K71 (G5P[7]) strains with other known reference genotypes.

Additional file 11. Comparison of full-length amino acid sequences of 11 genomic segments of 174-1V-80 (G8P[7]) vaccine strain with its different passages. The full-length amino acid sequences of the 11 genomic segments of the 80th-passage attenuated 174-1V-80 vaccine strain was compared with those of the 20th-, 40th-, and 60th-passage attenuated strains and the original virulent strain.

Additional file 5. Summary of safety test results for a live attenuated porcine rotavirus monovalent vaccine strains 174-1V-80, PRG942V-80, and K71V-80 in mice, guinea pigs, and pigs.

Additional file 4. Summary of the histopathological findings in the small intestine of the colostrums-deprived neonatal piglets inoculated with each virulent strains (174-1, PRG942, and K71), or immunized with each live attenuated monovalent or trivalent vaccines and then challenged with each corresponding original virulent strain(s).

Additional file 12. Comparison of full-length amino acid sequences of 11 genomic segments of PRG942V-80 (G9P[23]) vaccine strain with its different passages. The full-length amino acid sequences of the 11 genomic segments of the 80th-passage attenuated PRG942V-80 vaccine strain was compared with those of the 20th-, 40th-, and 60th-passage attenuated strains and the original virulent strain.

Additional file 14. Summary of the histopathological findings in the small intestine of the colostrums-deprived neonatal piglets vaccinated with a live attenuated monovalent vaccine strain (174-1V-80) or filtered supernatant of a mixture of homogenized small intestine and feces sampled from each passage.

Additional file 2. Oligonucleotide primers for sequencing or for 5′ and 3′ RACE PCRs of all eleven genomic segments of the porcine 174-1, PRG942, and K71 strains and their passages. Listed in the table are the primer pairs used to generate the full-length sequence of Korean porcine 174-1, PRG942, and K71 rotavirus strains. Also indicated are the gene-specific primers used for 5′ and 3′ RACE PCR.

Additional file 3. Full-length ORF nucleotide sequence identities (%) of ORFa in 11 genomic segments between three Korean porcine 174-1, PRG942, and K71 strains and other known strains representative of corresponding or neighbor genotypes. The nucleotide sequences of open reading frame of the virulent and attenuated porcine 174-1, PRG942, and K71 rotavirus strains were compared with known RVA strains. The values represent the nucleotide similarity of porcine 174-1, PRG942, and K71with the reference strains.

Additional file 13. Comparison of full-length amino acid sequences of 11 genomic segments of K71V-80 (G9P[23]) vaccine strain with its different passages. The full-length amino acid sequences of the 11 genomic segments of the 80th-passage attenuated K71V-80 vaccine strain was compared with those of the 20th-, 40th-, and 60th-passage attenuated strains and the original virulent strain.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.

International audience; Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs) and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract which are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked-fucose and α1,3/4-linked-fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked-fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.

Additional file 1. Sequences of siRNAs against target molecules and scrambled siRNA used in this study.

International audience; Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs) and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract which are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked-fucose and α1,3/4-linked-fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked-fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.