Sample collection

Bats were captured and sampled in their natural habitat in Guangdong province (Extended Data Fig. 1) as described previously4. Faecal swab samples were collected in viral transport medium (VTM) composed of Hank’s balanced salt solution at pH 7.4 containing BSA (1%), amphotericin (15 μg ml−1), penicillin G (100 units ml−1) and streptomycin (50 μg ml−1). Stool samples from sick pigs were collected in VTM. When appropriate and feasible, intestinal samples were also taken from deceased animals. Samples were aliquoted and stored at –80 °C until use. Blood samples were collected from recovered sows and workers on the farms who had close contact with sick pigs. Serum was separated by centrifugation at 3,000g for 15 min within 24 h of collection and preserved at 4 °C. Human serum collection was approved by the Medical Ethics Committee of the Wuhan School of Public Health, Wuhan University and Hummingbird IRB. Human, pigs and bats were sampled without gender or age preference unless indicated (for example, piglets or sows). No statistical methods were used to predetermine sample size.

Virus isolation

The following cells were used for virus isolation in this study: Vero (cultured in DMEM and 10% FBS); Rhinolophus sinicus primary or immortalized cells generated in our laboratory (all cultured in DMEM/F12 and 15% FBS): kidney primary cells (RsKi9409), lung primary cells (RsLu4323), lung immortalized cells (RsLuT), brain immortalized cells (RsBrT) and heart immortalized cells (RsHeT); and swine cell lines: two intestinal porcine enterocytes cell lines, IPEC (RPMI1640 and 10% FBS) and SIEC (DMEM and 10% FBS), three kidney cell lines PK15, LLC-PK1 (DMEM and 10% FBS for both) and IBRS (MEM and 10% FBS), and one pig testes cell line, ST (DMEM and 10% FBS). All cell lines were tested free of mycoplasma contamination, species were confirmed and authenticated by microscopic morphologic evaluation. None of the cell lines was on the list of commonly misidentified cell lines (by the ICLAC).

Cultured cell monolayers were maintained in their respective medium. PCR-positive pig faecal samples or the supernatant from homogenized pig intestine (in 200 μl VTM) were spun at 8,000g for 15 min, filtered and diluted 1:2 with DMEM supplemented with 16 μg ml−1 trypsin before addition to the cells. After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics (below) and 16 μg ml−1 trypsin. The cells were incubated at 37 °C and observed daily for cytopathic effect (CPE). Four blind passages (three-day interval between every passage) were performed for each sample. After each passage, both the culture supernatant and cell pellet were examined for the presence of virus by RT–PCR using the SADS-CoV primers listed in Supplementary Table 2. Penicillin (100 units ml−1) and streptomycin (15 μg ml−1) were included in all tissue culture media.

RNA extraction, S1 gene amplification and qPCR

Whenever commercial kits were used, the manufacturer’s instructions were followed without modification. RNA was extracted from 200 μl of swab samples (bat), faeces or homogenized intestine (pig) with the High Pure Viral RNA Kit (Roche). RNA was eluted in 50 μl of elution buffer and used as the template for RT–PCR. Reverse transcription was performed using the SuperScript III kit (Thermo Fisher Scientific).

To amplify S1 genes from bat samples, nested PCR was performed with primers designed based on HKU2-CoV (GenBank accession number NC_009988.1)19 (Supplementary Table 2). The 25-μl first-round PCR mixture contained 2.5 μl 10× PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl 2 , 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Thermo Fisher Scientific) and 1 μl cDNA. The 50-μl second-round PCR mixture was identical to the first-round PCR mixture except for the primers. Amplification of both rounds was performed as follows: 94 °C for 5 min followed by 60 cycles at 94 °C for 30 s, 50 °C for 40 s, 72 °C for 2.5 min, and a final extension at 72 °C for 10 min. PCR products were gel-purified and sequenced.

For qPCR analysis, primers based on SADS-CoV RdRp and N genes were used (Supplementary Table 2). RNA extracted from above was reverse-transcribed using PrimeScript RT Master Mix (Takara). The 10 μl qPCR reaction mix contained 5 μl 2× SYBR premix Ex TaqII (Takara), 0.4 μM of each primer and 1 μl cDNA. Amplification was performed as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and a melting curve step.

Luciferase immunoprecipitation system assay

The SADS-CoV S1 gene was codon-optimized for eukaryotic expression, synthesized (GenScript) and cloned in frame with the Renilla luciferase gene (Rluc) and a Flag tag in the pREN2 vector21. pREN2-S1 plasmids were transfected into Cos-1 cells using Lipofectamine 2000 (Thermo Fisher Scientific). At 48 h post-transfection, cells were collected, lysed and a luciferase assay was performed to determine Rluc expression for both the empty vector (pREN2) and the pREN2-S1 construct. For testing of unknown pig or human serum samples, 1 μl of serum was incubated with 10 million units of Rluc alone (vector) or Rluc-S1, respectively, together with 3.5 μl of a 30% protein A/G UltraLink resin suspension (Pierce, Thermo Fisher Scientific). After extensive washing to remove unbounded luciferase-tagged antigens, the captured luciferase amount was determined using the commercial luciferase substrate kit (Promega). The ratio of Rluc-S1:Rluc (vector) was used to determine the specific S1 reactivity of pig and human sera. Commercial Flag antibody (Thermo Fisher Scientific) was used as the positive control, and various pig sera (from uninfected animals in China or Singapore; or pigs infected with PEDV, TGEV or Nipah virus) were used as a negative control.

Protein expression and antibody production

The N gene from SADSr-CoV 3755 (GenBank accession number MF094702), which shares a 98% amino acid sequence identity to the SADS-CoV N protein, was inserted into pET-28a+ (Novagen) for prokaryotic expression. Transformed Escherichia coli were grown at 37 °C for 12–18 h in medium containing 1 mM IPTG. Bacteria were collected by centrifugation and resuspended in 30 ml of 5 mM imidazole and lysed by sonication. The lysate, from which N protein expression was confirmed with an anti-His-tag antibody, was applied to Ni2+ resin (Thermo Fisher Scientific). The purified N protein, at a concentration of 400 μg ml−1, was used to immunize rabbits for antibody production following published methods27. After immunization and two boosts, rabbits were euthanized and sera were collected. Rabbit anti-N protein serum was used 1:10,000 for subsequent western blots.

Amplification, cloning and expression of human and swine genes

Construction of expression clones for human ACE2 in pcDNA3.1 has been described previously5, 28. Human DPP4 was amplified from human cell lines. Human APN (also known as ANPEP) was commercially synthesized. Swine APN (also known as ANPEP), DPP4 and ACE2 were amplified from piglet intestine. Full-length gene fragments were amplified using specific primers (provided upon request). Human ACE2 was cloned into pCDNA3.1 fused with a His tag. Human APN and DPP4, swine APN, DPP4 and ACE2 were cloned into pCAGGS fused with an S tag. Purified plasmids were transfected into HeLa cells. After 24 h, expression human or swine genes in HeLa cells was confirmed by immunofluorescence assay using mouse anti-His tag or mouse anti-S tag monoclonal antibodies (produced in house) followed by Cy3-labelled goat anti-mouse/rabbit IgG (Proteintech Group).

Pseudovirus preparation

The codon-humanized S genes of SADS-CoV or MERS-CoV cloned into pcDNA3.1 were used for pseudovirus construction as described previously5, 28. In brief, 15 μg of each pHIV-Luc plasmid (pNL4.3.Luc.R-E-Luc) and the S-protein-expressing plasmid (or empty vector control) were co-transfected into 4 × 106 HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scientific). After 4 h, the medium was replaced with fresh medium. Supernatants were collected 48 h after transfection and clarified by centrifugation at 3,000g, then passed through a 0.45-μm filter (Millipore). The filtered supernatants were stored at −80 °C in aliquots until use. To evaluate the incorporation of S proteins into the core of HIV virions, pseudoviruses in supernatant (20 ml) were concentrated by ultracentrifugation through a 20% sucrose cushion (5 ml) at 80,000g for 90 min using a SW41 rotor (Beckman). Pelleted pseudoviruses were dissolved in 50 μl phosphate-buffered saline (PBS) and examined by electron microscopy.

Pseudovirus infection

HeLa cells transiently expressing APN, ACE2 or DPP4 were prepared using Lipofectamine 2000 (Thermo Fisher Scientific). Pseudoviruses prepared above were added to HeLa cells overexpressing APN, ACE2 or DPP4 24 h after transfection. The unabsorbed viruses were removed and replaced with fresh medium at 3 h after infection. The infection was monitored by measuring the luciferase activity conferred by the reporter gene carried by the pseudovirus, using the Luciferase Assay System (Promega) as follows: cells were lysed 48 h after infection, and 20 μl of the lysates was taken for determining luciferase activity after the addition of 50 μl of luciferase substrate.

Examination of known CoV receptors for SADS-CoV entry/infection

HeLa cells transiently expressing APN, ACE2 or DPP4 were prepared using Lipofectamine 2000 (Thermo Fisher Scientific) in a 96-well plate, with mock-transfected cells as controls. SADS-CoV grown in Vero cells was used to infect HeLa cells transiently expressing APN, ACE2 or DPP4. The inoculum was removed after 1 h of absorption and washed twice with PBS and supplemented with medium. SARS-related-CoV WIV167 and MERS-CoV HIV-pseudovirus were used as positive control for human/swine ACE2 or human/swine DPP4, respectively. After 24 h of infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. SARS-related-CoV WIV16 replication was detected using rabbit antibody against the SARS-related-CoV Rp3 N protein (made in house, 1:100) followed by Cy3-conjugated goat anti-rabbit IgG (1:50, Proteintech)7. SADS-CoV replication was monitored using rabbit antibody against the SADSr-CoV 3755 N protein (made in house, 1:50) followed by FITC-conjugated goat anti-rabbit IgG (1:50, Proteintech). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus). Infection of MERS-CoV HIV-pseudovirus was monitored by luciferase 48 h after infection.

High-throughput sequencing, pathogen screening and genome assembly

Tissue from the small intestine of deceased pigs was homogenized and filtered through 0.45-μm filters before nucleic acid extraction and ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (New England Biolabs). Metagenomics analysis of both RNA and DNA viruses was performed. For RNA virus screening, the sequencing library was constructed using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific). For DNA virus screening, NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs) was used for library preparation. Both libraries were sequenced on an Ion S5 sequencer (Thermo Fisher Scientific). An analysis pipeline was applied to the sequencing data, which included the following analysis steps: (1) raw data quality filtering; (2) host genomic sequence filtering; (3) BLASTn search against the virus nucleotide database using BLAST; (4) BLASTx search against the virus protein database using DIAMOND v.0.9.0; (5) contig assembling and BLASTx search against the virus protein database. For whole viral genome sequencing, amplicon primers (provided upon request) were designed using the Thermo Fisher Scientific online tool with the HKU2-CoV and the SADS-CoV farm A genomes as references, and the sequencing libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina and sequenced on an MiSeq sequencer. PCR and Sanger sequencing was performed to fill gaps in the genome. Genome sequences were assembled using CLC Genomic Workbench v.9.0. 5′-RACE was performed to determine the 5′-end of the genomes using SMARTer RACE 5′/3′ Kit (Takara). Genomes were annotated using Clone Manager Professional Suite 8 (Sci-Ed Software).

Phylogenetic analysis

SADS-CoV genome sequences and other representative coronavirus sequences (obtained from GenBank) were aligned using MAFFT v.7.221. Phylogenetic analyses with full-length genome, S gene and RdRp were performed using MrBayes v.3.2. Markov chain Monte Carlo was run for 20–50 million steps using the GTR+G+I model (general time reversible model of nucleotide substitution with a proportion of invariant sites and γ-distributed rates among sites). The first 10% was removed as burn-in. The association between phylogenies and phenotypes (for example, host species and farms) was assessed by BaTS beta-build2, with the trees obtained in the previous step used as input. For SADS-CoVs, a median-joining network analysis was performed using PopART v.1.7, with ɛ = 0. Phylogenetic analysis of the 33 full-length SADS-CoV genome sequences was performed using RAxML v.8.2.11, with GTRGAMMA as the nucleotide substitution model and 1,000 bootstrap replicates. The maximum likelihood tree was used to test the molecular clock using TempEst v.1.5. Potential genetic recombination events in our datasets were detected using RDP v.4.72.

Animal infection studies

Experiments were carried out strictly in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The use of animals in this study was approved by the South China Agricultural University Committee of Animal Experiments (approval number 201004152).

Two different animal challenge experiments were conducted. Pigs were used without gender preference. In the first experiment, which was conducted before the virus was isolated, we used three-day old specific pathogen-free (SPF) piglets of the same breeding line, cared for at a SPF facility, fed with colostrum (except one). These piglets were bred and reared to be free of PEDV, CSFV, SIV, PCV2 and PPV infections, and were routinely tested for viral infections using PCR. We also conducted NGS to further confirm that these were animals were free of infection of the above viruses before the animal experiment, and to demonstrate that the animals were free of SADS-CoV infection. The intestinal tissue samples from healthy and diseased animals (intestinal samples excised from euthanized piglets, then ground to make slurry for the inoculum and NGS was performed to confirm no other pig pathogens were found in the samples), were used to feed two groups of 5 (control) and 7 (infection) animals, respectively. For the second experiment, isolated SADS-CoV was used to infect healthy piglets from a farm in Guangdong, which had been free of diarrheal disease for a number of weeks. These piglets were from the same breed as those on SADS-affected farms, to eliminate potential host factor differences and to more accurately reproduce the conditions that occurred during the outbreak in the region. Both groups of piglets were cared for at a known pig disease-free facility. Again, qPCR and NGS were used to make sure that there was no other known swine diarrhoea virus present in the virus inoculum or any of the experimental animals. Two groups (6 for each group) of three-day old piglets were inoculated with SADS-CoV culture supernatant or normal cell culture medium as control. NGS and qPCR were used to confirm that there were no other known swine pathogens in the inoculum.

For both experiments, animals were recorded daily for signs of diseases, such as diarrhoea, weight loss and death. Faecal swabs were collected daily from all animals and screened for known swine diarrhoea viruses by qPCR. Weight loss was calculated as the percentage weight loss compared the original weight at day 0 with a threshold of >5%. It is important to point out that piglets when they are three days old tend to suffer from diarrhoea and weight loss when they are taken away from sows and the natural breast-feeding environment even without infection. At experimental endpoints, piglets were humanely euthanized and necropsies performed. Pictures were taken to record gross pathological changes to the intestines. Ileal, jejunal and duodenal tissues were taken from selected animals and stored at –80 °C for further analysis.

Haematoxylin and eosin and immunohistochemistry analysis

Frozen (–80 °C) small intestinal tissues including duodenum, jejunum and ileum taken from the experimentally infected pigs were pre-frozen at –20 °C for 10 min. Tissues were then embedded in optimal cutting temperature (OCT) compound and cut into 8-μm sections using the Cryotome FSE machine (Thermo Fisher Scientific). Mounted microscope slides were fixed with paraformaldehyde and stained with haematoxylin and eosin for histopathological examination.

For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Sequence data that support the findings of this study have been deposited in GenBank with accession codes MF094681–MF094688, MF769416–MF769444, MF094697–MF094701, MF769406–MF769415 and MG557844. Raw sequencing data that support the findings of this study have been deposited in the Sequence Read Achieve (SRA) with accession codes SRR5991648, SRR5991649, SRR5991650, SRR5991651, SRR5991652, SRR5991654, SRR5991655, SRR5991656, SRR5991657, SRR5991658 and SRR5995595.