Sampling

Bats were trapped in their natural habitat as described previously5. Throat and faecal swab samples were collected in viral transport medium (VTM) composed of Hank’s balanced salt solution, pH 7.4, containing BSA (1%), amphotericin (15 μg ml−1), penicillin G (100 U ml−1) and streptomycin (50 μg ml−1). To collect fresh faecal samples, clean plastic sheets measuring 2.0 by 2.0 m were placed under known bat roosting sites at about 18:00 h each evening. Relatively fresh faecal samples were collected from sheets at approximately 05:30–06:00 the next morning and placed in VTM. Samples were transported to the laboratory and stored at −80 °C until use. All animals trapped for this study were released back to their habitat after sample collection. All sampling processes were performed by veterinarians with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH05210201) and EcoHealth Alliance under an inter-institutional agreement with University of California, Davis (UC Davis protocol no. 16048).

RNA extraction, PCR and sequencing

RNA was extracted from 140 μl of swab or faecal samples with a Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was eluted in 60 μl RNAse-free buffer (buffer AVE, Qiagen), then aliquoted and stored at −80 °C. One-step RT–PCR (Invitrogen) was used to detect coronavirus sequences as described previously15. First round PCR was conducted in a 25-μl reaction mix containing 12.5 μl PCR 2× reaction mix buffer, 10 pmol of each primer, 2.5 mM MgSO4, 20 U RNase inhibitor, 1 μl SuperScript III/ Platinum Taq Enzyme Mix and 5 μl RNA. Amplification of the RdRP-gene fragment was performed as follows: 50 °C for 30 min, 94 °C for 2 min, followed by 40 cycles consisting of 94 °C for 15 s, 62 °C for 15 s, 68 °C for 40 s, and a final extension of 68 °C for 5 min. Second round PCR was conducted in a 25-μl reaction mix containing 2.5 μl PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl 2 , 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl first round PCR product. The amplification of RdRP-gene fragment was performed as follows: 94 °C for 5 min followed by 35 cycles consisting of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 40 s, and a final extension of 72 °C for 5 min.

To amplify the RBD region, one-step RT–PCR was performed with primers designed based on available SARS-CoV or bat SL-CoVs (first round PCR primers; F, forward; R, reverse: CoVS931F-5′-VWGADGTTGTKAGRTTYCCT-3′ and CoVS1909R-5′-TAARACAVCCWGCYTGWGT-3′; second PCR primers: CoVS951F-5′-TGTKAGRTTYCCTAAYATTAC-3′ and CoVS1805R-5′-ACATCYTGATANARAACAGC-3′). First-round PCR was conducted in a 25-μl reaction mix as described above except primers specific for the S gene were used. The amplification of the RBD region of the S gene was performed as follows: 50 °C for 30 min, 94 °C for 2 min, followed by 35 cycles consisting of 94 °C for 15 s, 43 °C for 15 s, 68 °C for 90 s, and a final extension of 68 °C for 5 min. Second-round PCR was conducted in a 25-μl reaction mix containing 2.5 μl PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl 2 , 0.5 mM dNTP, 0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl first round PCR product. Amplification was performed as follows: 94 °C for 5 min followed by 40 cycles consisting of 94 °C for 30 s, 41 °C for 30 s, 72 °C for 60 s, and a final extension of 72 °C for 5 min.

PCR products were gel purified and cloned into pGEM-T Easy Vector (Promega). At least four independent clones were sequenced to obtain a consensus sequence for each of the amplified regions.

Sequencing full-length genomes

Degenerate coronavirus primers were designed based on all available SARS-CoV and bat SL-CoV sequences in GenBank and specific primers were designed from genome sequences generated from previous rounds of sequencing in this study (primer sequences will be provided upon request). All PCRs were conducted using the One-Step RT–PCR kit (Invitrogen). The 5′ and 3′ genomic ends were determined using the 5′ or 3′ RACE kit (Roche), respectively. PCR products were gel purified and sequenced directly or following cloning into pGEM-T Easy Vector (Promega). At least four independent clones were sequenced to obtain a consensus sequence for each of the amplified regions and each region was sequenced at least twice.

Sequence analysis and databank accession numbers

Routine sequence management and analysis was carried out using DNAStar or Geneious. Sequence alignment and editing was conducted using ClustalW, BioEdit or GeneDoc. Maximum Likelihood phylogenetic trees based on the protein sequences were constructed using a Poisson model with bootstrap values determined by 1,000 replicates in the MEGA5 software package.

Sequences obtained in this study have been deposited in GenBank as follows (accession numbers given in parenthesis): full-length genome sequence of SL-CoV RsSHC014 and Rs3367 (KC881005, KC881006); full-length sequence of WIV1 S (KC881007); RBD (KC880984-KC881003); ACE2 (KC8810040). SARS-CoV sequences used in this study: human SARS-CoV strains Tor2 (AY274119), BJ01 (AY278488), GZ02 (AY390556) and civet SARS-CoV strain SZ3 (AY304486). Bat coronavirus sequences used in this study: Rs672 (FJ588686), Rp3 (DQ071615), Rf1 (DQ412042), Rm1 (DQ412043), HKU3-1 (DQ022305), BM48-31 (NC_014470), HKU9-1 (NC_009021), HKU4 (NC_009019), HKU5 (NC_009020), HKU8 (DQ249228), HKU2 (EF203067), BtCoV512 (NC_009657), 1A (NC_010437). Other coronavirus sequences used in this study: HCoV-229E (AF304460), HCoV-OC43 (AY391777), HCoV-NL63 (AY567487), HKU1 (NC_006577), EMC (JX869059), FIPV (NC_002306), PRCV (DQ811787), BWCoV (NC_010646), MHV (AY700211), IBV (AY851295).

Amplification, cloning and expression of the bat ACE2 gene

Construction of expression clones for human and civet ACE2 in pcDNA3.1 has been described previously29. Bat ACE2 was amplified from a R. sinicus (sample no. 3357). In brief, total RNA was extracted from bat rectal tissue using the RNeasy Mini Kit (Qiagen). First-strand complementary DNA was synthesized from total RNA by reverse transcription with random hexamers. Full-length bat ACE2 fragments were amplified using forward primer bAF2 and reverse primer bAR2 (ref. 29). The ACE2 gene was cloned into pCDNA3.1 with KpnI and XhoI, and verified by sequencing. Purified ACE2 plasmids were transfected to HeLa cells. After 24 h, lysates of HeLa cells expressing human, civet, or bat ACE2 were confirmed by western blot or immunofluorescence assay.

Western blot analysis

Lysates of cells or filtered supernatants containing pseudoviruses were separated by SDS–PAGE, followed by transfer to a nitrocellulose membrane (Millipore). For detection of S protein, the membrane was incubated with rabbit anti-Rp3 S fragment (amino acids 561–666) polyantibodies (1:200), and the bound antibodies were detected by alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (1:1,000). For detection of HIV-1 p24 in supernatants, monoclonal antibody against HIV p24 (p24 MAb) was used as the primary antibody at a dilution of 1:1,000, followed by incubation with AP-conjugated goat anti-mouse IgG at the same dilution. To detect the expression of ACE2 in HeLa cells, goat antibody against the human ACE2 ectodomain (1:500) was used as the first antibody, followed by incubation with horseradish peroxidase-conjugated donkey anti-goat IgG (1:1,000).

Virus isolation

Vero E6 cell monolayers were maintained in DMEM supplemented with 10% FCS. PCR-positive samples (in 200 μl buffer) were gradient centrifuged at 3,000–12,000g, and supernatant were diluted 1:10 in DMEM before being added to Vero E6 cells. After incubation at 37 °C for 1 h, inocula were removed and replaced with fresh DMEM with 2% FCS. Cells were incubated at 37 °C for 3 days and checked daily for cytopathic effect. Double-dose triple antibiotics penicillin/streptomycin/amphotericin (Gibco) were included in all tissue culture media (penicillin 200 IU ml−1, streptomycin 0.2 mg ml−1, amphotericin 0.5 μg ml−1). Three blind passages were carried out for each sample. After each passage, both the culture supernatant and cell pellet were examined for presence of virus by RT–PCR using primers targeting the RdRP or S gene. Virions in supernatant (10 ml) were collected and fixed using 0.1% formaldehyde for 4 h, then concentrated by ultracentrifugation through a 20% sucrose cushion (5 ml) at 80,000g for 90 min using a Ty90 rotor (Beckman). The pelleted viral particles were suspended in 100 μl PBS, stained with 2% phosphotungstic acid (pH 7.0) and examined using a Tecnai transmission electron microscope (FEI) at 200 kV.

Virus infectivity detected by immunofluorescence assay

Cell lines used for this study and their culture conditions are summarized in Extended Data Table 5. Virus titre was determined in Vero E6 cells by cytopathic effect (CPE) counts. Cell lines from different origins and HeLa cells expressing ACE2 from human, civet or Chinese horseshoe bat were grown on coverslips in 24-well plates (Corning) incubated with bat SL-CoV-WIV1 at a multiplicity of infection = 10 for 1 h. The inoculum was removed and washed twice with PBS and supplemented with medium. HeLa cells without ACE2 expression and Vero E6 cells were used as negative and positive controls, respectively. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at 4 °C. ACE2 expression was detected using goat anti-human ACE2 immunoglobulin (R&D Systems) followed by FITC-labelled donkey anti-goat immunoglobulin (PTGLab). Virus replication was detected using rabbit antibody against the SL-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI. Staining patterns were examined using a FV1200 confocal microscope (Olympus).

Virus infectivity detected by real-time RT–PCR

Vero E6, A549, PK15, RSKT and HeLa cells with or without expression of ACE2 of different origins were inoculated with 0.1 TCID 50 WIV-1 and incubated for 1 h at 37 °C. After removing the inoculum, the cells were cultured with medium containing 1% FBS. Supernatants were collected at 0, 12, 24 and 48 h. RNA from 140 μl of each supernatant was extracted with the Viral RNA Mini Kit (Qiagen) following manufacturer’s instructions and eluted in 60 μl buffer AVE (Qiagen). RNA was quantified on the ABI StepOne system, with the TaqMan AgPath-ID One-Step RT–PCR Kit (Applied Biosystems) in a 25 μl reaction mix containing 4 μl RNA, 1 × RT–PCR enzyme mix, 1 × RT–PCR buffer, 40 pmol forward primer (5′-GTGGTGGTGACGGCAAAATG-3′), 40 pmol reverse primer (5′-AAGTGAAGCTTCTGGGCCAG-3′) and 12 pmol probe (5′-FAM-AAAGAGCTCAGCCCCAGATG-BHQ1-3′). Amplification parameters were 10 min at 50 °C, 10 min at 95 °C and 50 cycles of 15 s at 95 °C and 20 s at 60 °C. RNA dilutions from purified WIV-1 stock were used as a standard.

Serum neutralization test

SARS patient sera were inactivated at 56 °C for 30 min and then used for virus neutralization testing. Sera were diluted starting with 1:10 and then serially twofold diluted in 96-well cell plates to 1:40. Each 100 μl serum dilution was mixed with 100 μl viral supernatant containing 100 TCID5 0 of WIV1 and incubated at 37 °C for 1 h. The mixture was added in triplicate wells of 96-well cell plates with plated monolayers of Vero E6 cells and further incubated at 37 °C for 2 days. Serum from a healthy blood donor was used as a negative control in each experiment. CPE was observed using an inverted microscope 2 days after inoculation. The neutralizing antibody titre was read as the highest dilution of serum which completely suppressed CPE in infected wells. The neutralization test was repeated twice.

Recombination analysis

Full-length genomic sequences of SL-CoV Rs3367 or RsSHC014 were aligned with those of selected SARS-CoVs and bat SL-CoVs using Clustal X. The aligned sequences were preliminarily scanned for recombination events using Recombination Detection Program (RDP) 4.0 (ref. 19). The potential recombination events suggested by RDP owing to their strong P values (<10–20) were investigated further by similarity plot and bootscan analyses implemented in Simplot 3.5.1. Phylogenetic origin of the major and minor parental regions of Rs3367 or RsSHC014 were constructed from the concatenated sequences of the essential ORFs of the major and minor parental regions of selected SARS-CoV and SL-CoVs. Two genome regions between three estimated breakpoints (20,827–26,553 and 26,554–28,685) were aligned independently using ClustalX and generated two alignments of 5,727 base pairs and 2,133 base pairs. The two alignments were used to construct maximum likelihood trees to better infer the fragment parents. All nucleotide numberings in this study are based on Rs3367 genome position.