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17The emergence of a novel coronavirus, SARS-CoV-2, resulted in a pandemic. Here, we used X-ray 18 structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from 19 SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, 20 and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins 21 known to serve or not serve as receptor allows to define residues important for binding. From the 20 22 amino acids in ACE2 that contact S up to seven can be replaced and ACE2 can still function as the SARS-23CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery 24 of the binding site, while changes of the invariable residues prevent S-binding or infection of the 25 respective animal. Some ACE2 proteins even tolerate the loss or the acquisition of N-glycosylation sites 26 located near the S-interface. Of note, Pigs and dogs, which are not or not effectively infected and have 27 only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. 28Comparison of the RBD of S of SARS-CoV-2 with viruses from Bat-CoV-RaTG13 and Pangolin-CoV 29 revealed that the latter contains only one substitution, whereas the Bat-CoV-RaTG13 exhibits five. 30However, ACE2 of pangolin exhibit seven changes relative to human ACE2, a similar number of 31 substitutions is present in ACE2 of bats, raccoon, and civet suggesting that SARS-CoV-2 may not 32 especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new 33 insight into the receptor usage and animal source/origin of SARS-CoV-2. 34 IMPORTANCE 35 SARS-CoV-2 is threatening people worldwide and there are no drugs or vaccines available to mitigate 36 its spread. The origin of the virus is still unclear and whether pets and livestock can be infected and 37 transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host 38 receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis 39 provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from 40 species known to support SARS-CoV-2 infection tolerate many amino acid changes indicating that the 41 species barrier might be low. An exception are dogs and especially pigs, which, however, revealed 42

Abstract 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.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based realtime PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 10 2 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.