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Link to original content: https://pubmed.ncbi.nlm.nih.gov/24172901
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. 2013 Nov 28;503(7477):535-8.
doi: 10.1038/nature12711. Epub 2013 Oct 30.

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

Xing-Yi Ge  1 , Jia-Lu Li, Xing-Lou Yang, Aleksei A Chmura, Guangjian Zhu, Jonathan H Epstein, Jonna K Mazet, Ben Hu, Wei Zhang, Cheng Peng, Yu-Ji Zhang, Chu-Ming Luo, Bing Tan, Ning Wang, Yan Zhu, Gary Crameri, Shu-Yi Zhang, Lin-Fa Wang, Peter Daszak, Zheng-Li Shi
Affiliations

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

Xing-Yi Ge et al. Nature. .

Abstract

The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Phylogenetic tree based on amino acid sequences of the S RBD region and the two parental regions of bat SL-CoV Rs3367 or RsSHC014.
a, SARS-CoV S protein amino acid residues 310–520 were aligned with homologous regions of bat SL-CoVs using the ClustalW software. A maximum-likelihood phylogenetic tree was constructed using a Poisson model with bootstrap values determined by 1,000 replicates in the MEGA5 software package. The RBD sequences identified in this study are in bold and named by the sample numbers. The key amino acid residues involved in interacting with the human ACE2 molecule are indicated on the right of the tree. SARS-CoV GZ02, BJ01 and Tor2 were isolated from patients in the early, middle and late phase, respectively, of the SARS outbreak in 2003. SARS-CoV SZ3 was identified from Paguma larvata in 2003 collected in Guangdong, China. SL-CoV Rp3, Rs672 and HKU3-1 were identified from R. sinicus collected in China (respectively: Guangxi, 2004; Guizhou, 2006; Hong Kong, 2005). Rf1 and Rm1 were identified from R. ferrumequinum and R. macrotis, respectively, collected in Hubei, China, in 2004. Bat SARS-related CoV BM48-31 was identified from R. blasii collected in Bulgaria in 2008. Bat CoV HKU9-1 was identified from Rousettus leschenaultii collected in Guangdong, China in 2005/2006 and used as an outgroup. All sequences in bold and italics were identified in the current study. Filled triangles, circles and diamonds indicate samples with co-infection by two different SL-CoVs. ‘–’ indicates the amino acid deletion. b, Phylogenetic origins of the two parental regions of Rs3367 or RsSHC014. Maximum likelihood phylogenetic trees were constructed from alignments of two fragments covering nucleotides 20,827–26,533 (5,727 nucleotides) and 26,534 –28,685 (2,133 nucleotides) of the Rs3367 genome, respectively. For display purposes, the trees were midpoint rooted. The taxa were annotated according to strain names: SARS-CoV, SARS coronavirus; SARS-like CoV, bat SARS-like coronavirus. The two novel SL-CoVs, Rs3367 and RsSHC014, are in bold and italics. PowerPoint slide
Figure 2
Figure 2. Electron micrograph of purified virions.
Virions from a 10-ml culture were collected, fixed and concentrated/purified by sucrose gradient centrifugation. The pelleted viral particles were suspended in 100 μl PBS, stained with 2% phosphotungstic acid (pH 7.0) and examined directly using a Tecnai transmission electron microscope (FEI) at 200 kV. PowerPoint slide
Figure 3
Figure 3. Analysis of receptor usage of SL-CoV-WIV1 determined by immunofluorescence assay and real-time PCR.
Determination of virus infectivity in HeLa cells with and without the expression of ACE2. b, bat; c, civet; h, human. ACE2 expression was detected with goat anti-humanACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SL-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red), merged triple-stained images and real-time PCR results, respectively. (n = 3); error bars represent standard deviation. PowerPoint slide
Figure 4
Figure 4. Analysis of host range of SL-CoV-WIV1 determined by immunofluorescence assay and real-time PCR.
Virus infection in A549, RSKT, Vero E6 and PK-15 cells. Virus replication was detected as described for Fig. 3. The columns (from left to right) show staining of nuclei (blue), virus replication (red), merged double-stained images and real-time PCR results, respectively. n = 3; error bars represent s.d. PowerPoint slide
Extended Data Figure 1
Extended Data Figure 1. Sequence alignment of CoV S protein RBD.
SARS-CoV S protein (amino acids 310–520) is aligned with homologous regions of bat SL-CoVs using ClustalW. The newly discovered bat SL-CoVs are indicated with a bold vertical line on the left. The key amino acid residues involved in the interaction with human ACE2 are numbered on the top of the aligned sequences.
Extended Data Figure 2
Extended Data Figure 2. Alignment of CoV S protein S1 sequences.
Alignment of S1 sequences (amino acids 1–660) of the two novel bat SL-CoV S proteins with those of previously reported bat SL-CoVs and human and civet SARS-CoVs. The newly discovered bat SL-CoVs are boxed in red. SARS-CoV GZ02, BJ01 and Tor2 were isolated from patients in the early, middle and late phase, respectively, of the SARS outbreak in 2003. SARS-CoV SZ3 was identified from P. larvata in 2003 collected in Guangdong, China. SL-CoV Rp3, Rs 672 and HKU3-1 were identified from R. sinicus collected in Guangxi, Guizhou and Hong Kong, China, respectively. Rf1 and Rm1 were identified from R. ferrumequinum and R. macrotis, respectively, collected in Hubei Province, China. Bat SARS-related CoV BM48-31 was identified from R. blasii collected in Bulgaria.
Extended Data Figure 3
Extended Data Figure 3. Complete RdRP sequence phylogeny.
Phylogenetic tree of bat SL-CoVs and SARS-CoVs on the basis of complete RdRP sequences (2,796 nucleotides). Bat SL-CoVs RsSHC014 and Rs3367 are highlighted by filled circles. Three established coronaivirus genera, Alphacoronavirus, Betacoronavirus and Gammacoronavirus are marked as α, β and γ, respectively. Four CoV groups in the genus Betacoronavirus are indicated as A, B, C and D, respectively. MHV, murine hepatitis virus; PHEV, porcine haemagglutinating encephalomyelitis virus; PRCV, porcine respiratory coronavirus; FIPV, feline infectious peritonitis virus; IBV, infectious bronchitis coronavirus; BW, beluga whale coronavirus.
Extended Data Figure 4
Extended Data Figure 4. Sequence phylogeny of the complete S protein of SL-CoVs and SARS-CoV.
Phylogenetic tree of bat SL-CoVs and SARS-CoVs on the basis of complete S protein sequences (1,256 amino acids). Bat SL-CoVs RsSHC014 and Rs3367 are highlighted by filled circles. Bat CoV HKU9 was used as an outgroup.
Extended Data Figure 5
Extended Data Figure 5. Detection of potential recombination events.
a, b, Similarity plot (a) and bootscan analysis (b) detected three recombination breakpoints in the bat SL-CoV Rs3367 or SHC014 genome. The three breakpoints were located at the ORF1b (nt 20,827), M (nucleotides 26,553) and N (nucleotides 28,685) genes, respectively. Both analyses were performed with an F84 distance model, a window size of 1,500 base pairs and a step size of 300 base pairs.
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References

    1. Ksiazek TG, et al. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 2003;348:1953–1966. doi: 10.1056/NEJMoa030781. - DOI - PubMed
    1. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012;367:1814–1820. doi: 10.1056/NEJMoa1211721. - DOI - PubMed
    1. Anthony SJ, et al. Coronaviruses in bats from Mexico. J. Gen. Virol. 2013;94:1028–1038. doi: 10.1099/vir.0.049759-0. - DOI - PMC - PubMed
    1. Raj VS, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013;495:251–254. doi: 10.1038/nature12005. - DOI - PMC - PubMed
    1. Li W, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science. 2005;310:676–679. doi: 10.1126/science.1118391. - DOI - PubMed

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