Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown once again that coronavirus (CoV) in animals are potential sources for epidemics in humans. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen of swine with a worldwide distribution. Here, we implemented and described an approach to analyze the epidemiology of PDCoV following its emergence in the pig population. We performed an integrated analysis of full genome sequence data from 21 newly sequenced viruses, along with comprehensive epidemiological surveillance data collected globally over the last 15 years. We found four distinct phylogenetic lineages of PDCoV, which differ in their geographic circulation patterns. Interestingly, we identified more frequent intra- and interlineage recombination and higher virus genetic diversity in the Chinese lineages compared with the USA lineage where pigs are raised in different farming systems and ecological environments. Most recombination breakpoints are located in the ORF1ab gene rather than in genes encoding structural proteins. We also identified five amino acids under positive selection in the spike protein suggesting a role for adaptive evolution. According to structural mapping, three positively selected sites are located in the N-terminal domain of the S1 subunit, which is the most likely involved in binding to a carbohydrate receptor, whereas the other two are located in or near the fusion peptide of the S2 subunit and thus might affect membrane fusion. Finally, our phylogeographic investigations highlighted notable South-North transmission as well as frequent long-distance dispersal events in China that could implicate human-mediated transmission. Our findings provide new insights into the evolution and dispersal of PDCoV that contribute to our understanding of the critical factors involved in CoVs emergence.

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, Deltacoronavirus , evolutionarily related to the Gammacoronavirus , 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. IMPORTANCE 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 Coronavirinae , a couple of functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptors for Gammacoronavirus and Deltacoronavirus , 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.

Infections with bat-origin coronaviruses (CoVs) (severe acute respiratory syndrome CoV [SARS-CoV] and Middle East respiratory syndrome CoV [MERS-CoV]) have caused severe illness in humans after “host jump” events. Recently, a novel bat-HKU2-like CoV named swine acute diarrhea syndrome CoV (SADS-CoV) has emerged in southern China, causing lethal diarrhea in newborn piglets. It is important to assess the species barriers of SADS-CoV infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. An in vitro susceptibility study revealed a broad species tropism of SADS-CoV, including various rodent and human cell lines. We established a mouse model of SADS-CoV infection, identifying its active replication in splenic dendritic cells, which suggests that SADS-CoV has the potential to infect rodents. These findings highlight the potential cross-species transmissibility of SADS-CoV, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-HKU2-origin CoV.