Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Susceptibility to mice and potential evolutionary characteristics of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in suckling piglets and has the potential for cross-species transmission, posing a threat to animal and human health. However, the susceptibility profile of different species of mice to PDCoV infection and its evolutionary characteristics are still unclear. In the current study, we found that BALB/c and Kunming mice are susceptible to PDCoV. Our results showed that there were obvious lesions in intestinal and lung tissues from the infected mice. PDCoV RNAs were detected in the lung, kidney, and intestinal tissues from the infected mice of both strains, and there existed wider tissue tropism in the PDCoV-infected BALB/c mice. The RNA and protein levels of aminopeptidase N from mice were relatively high in the kidney and intestinal tissues and obviously increased after PDCoV infection. The viral-specific IgG and neutralizing antibodies against PDCoV were detected in the serum of infected mice. An interesting finding was that two key amino acid mutations, D138H and Q641K, in the S protein were identified in the PDCoV-infected mice. The essential roles of these two mutations for PDCoV-adaptive evolution were confirmed by cryo-electron microscope structure model analysis. The evolutionary characteristics of PDCoV among Deltacoronaviruses (δ-CoVs) were further analyzed. δ-CoVs from multiple mammals are closely related based on the phylogenetic analysis. The codon usage analysis demonstrated that similar codon usage patterns were used by most of the mammalian δ-CoVs at the global codon, synonymous codon, and amino acid usage levels. These results may provide more insights into the evolution, host ranges, and cross-species potential of PDCoV.

Comparative genomic analysis reveals varying levels of mammalian adaptation to coronavirus infections.

Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is of zoonotic origin. Evolutionary analyses assessing whether coronaviruses similar to SARS-CoV-2 infected ancestral species of modern-day animal hosts could be useful in identifying additional reservoirs of potentially dangerous coronaviruses. We reasoned that if a clade of species has been repeatedly exposed to a virus, then their proteins relevant for viral entry may exhibit adaptations that affect host susceptibility or response. We perform comparative analyses across the mammalian phylogeny of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2, in order to uncover evidence for selection acting at its binding interface with the SARS-CoV-2 spike protein. We uncover that in rodents there is evidence for adaptive amino acid substitutions at positions comprising the ACE2-spike interaction interface, whereas the variation within ACE2 proteins in primates and some other mammalian clades is not consistent with evolutionary adaptations. We also analyze aminopeptidase N (APN), the receptor for the human coronavirus 229E, a virus that causes the common cold, and find evidence for adaptation in primates. Altogether, our results suggest that the rodent and primate lineages may have had ancient exposures to viruses similar to SARS-CoV-2 and HCoV-229E, respectively.

Describing variability in pig genes involved in coronavirus infections for a One Health perspective in conservation of animal genetic resources.

Coronaviruses silently circulate in human and animal populations, causing mild to severe diseases. Therefore, livestock are important components of a "One Health" perspective aimed to control these viral infections. However, at present there is no example that considers pig genetic resources in this context. In this study, we investigated the variability of four genes (ACE2, ANPEP and DPP4 encoding for host receptors of the viral spike proteins and TMPRSS2 encoding for a host proteinase) in 23 European (19 autochthonous and three commercial breeds and one wild boar population) and two Asian Sus scrofa populations. A total of 2229 variants were identified in the four candidate genes: 26% of them were not previously described; 29 variants affected the protein sequence and might potentially interact with the infection mechanisms. The results coming from this work are a first step towards a "One Health" perspective that should consider conservation programs of pig genetic resources with twofold objectives: (i) genetic resources could be reservoirs of host gene variability useful to design selection programs to increase resistance to coronaviruses; (ii) the described variability in genes involved in coronavirus infections across many different pig populations might be part of a risk assessment including pig genetic resources.

Aminopeptidase N Expression, Not Interferon Responses, Determines the Intestinal Segmental Tropism of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.

CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance.

Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.