Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to an understanding of pathogenesis and to development of intervention strategies. The fourth CoV genus, , evolutionarily related to the, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine deltacoronovirus (PDCoV) and alphacoronovirus (AlphaCoV) (transmissible gastroenteritis virus [TGEV]) based upon three lines of evidence. First, the soluble S1 protein of PDCoV bound to the surface of target porcine cell lines known to express pAPN as efficiently as TGEV-S1, which could be blocked by soluble pAPN pretreatment. Second, both PDCoV-S1 and TGEV-S1 physically recognized and interacted with pAPN by coimmunoprecipitation in pAPN cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and to PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells than TGEV-S1, suggesting that there may be differences between these two viruses in the virus-binding regions in pAPN. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection. The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by a species-specific receptor of the host. Among the four genera of the , a couple of functional receptors for the representative members in the genera and have been identified, whereas receptors for and , which are believed to originate from birds, are still unknown. Porcine coronaviruses, including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets, have posed a serious threat to the pork industry in Asia and North America. Here, we report that PDCoV employs the alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of the PDCoV receptor provides another example of the expanded host range of CoV and paves the way for further investigation of PDCoV-host interaction and pathogenesis.

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host-cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated that overexpression of APN orthologues from various species including mice and felines in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo , distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1 B ) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both the domain A (S1 A ) and S1 B were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1 A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1 A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. Importance Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro . Here we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1 A and S1 B ) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1 B domain critical for APN binding, and demonstrating neutralization activity of S1 A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV’s great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

A novel swine enteric alphacoronavirus, swine acute diarrhoea syndrome coronavirus (SADS‐CoV), related to Rhinolophus bat CoV HKU2 in the subgenus Rhinacovirus emerged in southern China in 2017, causing diarrhoea in newborn piglets, and critical questions remain about the pathogenicity, cross‐species transmission and potential animal reservoirs. Our laboratory's previous research has shown that SADS‐CoV can replicate in various cell types from different species, including chickens. Here, we systematically explore the susceptibility of chickens to a cell‐adapted SADS‐CoV strain both in vitro and in vivo. First, evidence of SADS‐CoV replication in primary chicken cells, including cytopathic effects, immunofluorescence staining, growth curves and structural protein expression, was proven. Furthermore, we observed that SADS‐CoV replicated in chicken embryos without causing gross lesions and that experimental infection of chicks resulted in mild respiratory symptoms. More importantly, SADS‐CoV shedding and viral distribution in the lungs, spleens, small intestines and large intestines of infected chickens were confirmed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The genomic sequence of the original SADS‐CoV from the pig source sample in 2017 was determined to have nine nucleotide differences compared to the cell‐adapted strain used; among these were three nonsynonymous mutations in the spike gene. These results collectively demonstrate that chickens are susceptible to SADS‐CoV infection, suggesting that they are a potential animal reservoir. To our knowledge, this study provides the first experimental evidence of cross‐species infection in which a mammalian alphacoronavirus is able to infect an avian species.

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