The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about 280 m2, is divided into the auxiliary working area and the protection area with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3 Laboratory, as the main auxiliary facility of the P4 Laboratory, plays a significant role in biosafety cluster platforms.
The scope of pathogenic activities that have been applied to be engaged in by the Laboratory is: the operation of infected materials that have not cultivated successfully for Ebola, SARS, Nipah, Marburg, Lassa fever virus, and Crimean-Congo hemorrhagic fever virus; pathogen cultivation, transformation, detection, susceptibility testing for mycobacterium tuberculosis and bacillus anthracis, and the experiment of small-sized animals' infection with mycobacterium tuberculosis; the isolation and cultivation, virus amplification and collection & use, detection of virus antigen and antibody, serological neutralization test and small-sized animal infection experiments for TBEV, CHIKV, MERS coronavirus.
The facilities to be built include test facility for fulminant disease pathogens such as Cellular Level Biosafety Level 4 Laboratory, emerging disease research facility and fulminant disease pathogen storage facility ( that is, BSL-4, BSL-3 and BSL-2 , ordinary laboratory, animal feeding room and relevant supporting facilities)
【 在 CBI 的大作中提到: 】 : :武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进 入分类的安全程度最低的ordinary laboratory, animal feeding room and relevant :supporting facilities. : :对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实 验并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。 : :P3 : :The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about :280 m2, is divided into the auxiliary working area and the protection area :with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized :animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3
【 在 CBI (史迪威) 的大作中提到: 】 : 武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进入 : 分类的安全程度最低的ordinary laboratory, animal feeding room and relevant : supporting facilities. : 对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实验 : 并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。 : P3 : The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about : 280 m2, is divided into the auxiliary working area and the protection area : with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized : animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3 : ...................
【 在 CBI (史迪威) 的大作中提到: 】 : 武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进入 : 分类的安全程度最低的ordinary laboratory, animal feeding room and relevant : supporting facilities. : 对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实验 : 并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。 : P3 : The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about : 280 m2, is divided into the auxiliary working area and the protection area : with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized : animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3 : ...................
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.
请看他们自己在2013年11月号发表在nature 上的研究报告是怎么说的吧！
Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor
Xing-Yi Ge, 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
Nature volume 503, pages535–538(2013)Cite this article
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 history1. An ongoing outbreak of Middle East respiratory syndrome coronavirus2 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 viruses3,4,5, 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 Africa5,6,7,8, 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)9,10. 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.
Main The 2002–3 pandemic of SARS1 and the ongoing emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV)2 demonstrate that CoVs are a significant public health threat. SARS-CoV was shown to use the human ACE2 molecule as its entry receptor, and this is considered a hallmark of its cross-species transmissibility11. The receptor binding domain (RBD) located in the amino-terminal region (amino acids 318–510) of the SARS-CoV spike (S) protein is directly involved in binding to ACE2 (ref. 12). However, despite phylogenetic evidence that SARS-CoV evolved from bat SL-CoVs, all previously identified SL-CoVs have major sequence differences from SARS-CoV in the RBD of their S proteins, including one or two deletions6,9. Replacing the RBD of one SL-CoV S protein with SARS-CoV S conferred the ability to use human ACE2 and replicate efficiently in mice9,13. However, to date, no SL-CoVs have been isolated from bats, and no wild-type SL-CoV of bat origin has been shown to use ACE2.
We conducted a 12-month longitudinal survey (April 2011–September 2012) of SL-CoVs in a colony of Rhinolophus sinicus at a single location in Kunming, Yunnan Province, China (Extended Data Table 1). A total of 117 anal swabs or faecal samples were collected from individual bats using a previously published method5,14. A one-step reverse transcription (RT)-nested PCR was conducted to amplify the RNA-dependent RNA polymerase (RdRP) motifs A and C, which are conserved among alphacoronaviruses and betacoronaviruses15.
Twenty-seven of the 117 samples (23%) were classed as positive by PCR and subsequently confirmed by sequencing. The species origin of all positive samples was confirmed to be R. sinicus by cytochrome b sequence analysis, as described previously16. A higher prevalence was observed in samples collected in October (30% in 2011 and 48.7% in 2012) than those in April (7.1% in 2011) or May (7.4% in 2012) (Extended Data Table 1). Analysis of the S protein RBD sequences indicated the presence of seven different strains of SL-CoVs (Fig. 1a and Extended Data Figs 1 and 2). In addition to RBD sequences, which closely matched previously described SL-CoVs (Rs672, Rf1 and HKU3)5,8,17,18, two novel strains (designated SL-CoV RsSHC014 and Rs3367) were discovered. Their full-length genome sequences were determined, and both were found to be 29,787 base pairs in size (excluding the poly(A) tail). The overall nucleotide sequence identity of these two genomes with human SARS-CoV (Tor2 strain) is 95%, higher than that observed previously for bat SL-CoVs in China (88–92%)5,8,17,18 or Europe (76%)6 (Extended Data Table 2 and Extended Data Figs 3 and 4). Higher sequence identities were observed at the protein level between these new SL-CoVs and SARS-CoVs (Extended Data Tables 3 and 4). To understand the evolutionary origin of these two novel SL-CoV strains, we conducted recombination analysis with the Recombination Detection Program 4.0 package19 using available genome sequences of bat SL- CoV strains (Rf1, Rp3, Rs672, Rm1, HKU3 and BM48-31) and human and civet representative SARS-CoV strains (BJ01, SZ3, Tor2 and GZ02). Three breakpoints were detected with strong P values (<10 8722="" 20="" and="" supported="" by="" similarity="" plot="" and="" bootscan="" analysis="" Extended="" Data="" Fig.="" 5a="" b="" .="">Breakpoints were located at nucleotides 20,827, 26,553 and 28,685 in the Rs3367 (and RsSHC014) genome, and generated recombination fragments covering nucleotides 20,827–26,533 (5,727 nucleotides) (including partial open reading frame (ORF) 1b, full-length S, ORF3, E and partial M gene) and nucleotides 26,534–28,685 (2,133 nucleotides) (including partial ORF M, full-length ORF6, ORF7, ORF8 and partial N gene). Phylogenetic analysis using the major and minor parental regions suggested that Rs3367, or RsSHC014, is the descendent of a recombination of lineages that ultimately lead to SARS-CoV and SL-CoV Rs672 (Fig. 1b).
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. figure1 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.
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Full size image The most notable sequence differences between these two new SL-CoVs and previously identified SL-CoVs is in the RBD regions of their S proteins. First, they have higher amino acid sequence identity to SARS-CoV (85% and 96% for RsSHC014 and Rs3367, respectively). Second, there are no deletions and they have perfect sequence alignment with the SARS-CoV RBD region (Extended Data Figs 1 and 2). Structural and mutagenesis studies have previously identified five key residues (amino acids 442, 472, 479, 487 and 491) in the RBD of the SARS-CoV S protein that have a pivotal role in receptor binding20,21. Although all five residues in the RsSHC014 S protein were found to be different from those of SARS-CoV, two of the five residues in the Rs3367 RBD were conserved (Fig. 1 and Extended Data Fig. 1).
Despite the rapid accumulation of bat CoV sequences in the last decade, there has been no report of successful virus isolation6,22,23. We attempted isolation from SL-CoV PCR-positive samples. Using an optimized protocol and Vero E6 cells, we obtained one isolate which caused cytopathic effect during the second blind passage. Purified virions displayed typical coronavirus morphology under electron microscopy (Fig. 2). Sequence analysis using a sequence-independent amplification method14 to avoid PCR-introduced contamination indicated that the isolate was almost identical to Rs3367, with 99.9% nucleotide genome sequence identity and 100% amino acid sequence identity for the S1 region. The new isolate was named SL-CoV-WIV1.
Figure 2: Electron micrograph of purified virions. figure2 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&# 8201;7.0) and examined directly using a Tecnai transmission electron microscope (FEI) at 200 kV.
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Full size image To determine whether WIV1 can use ACE2 as a cellular entry receptor, we conducted virus infectivity studies using HeLa cells expressing or not expressing ACE2 from humans, civets or Chinese horseshoe bats. We found that WIV1 is able to use ACE2 of different origins as an entry receptor and replicated efficiently in the ACE2-expressing cells (Fig. 3). This is, to our knowledge, the first identification of a wild-type bat SL-CoV capable of using ACE2 as an entry receptor.
Figure 3: Analysis of receptor usage of SL-CoV-WIV1 determined by immunofluorescence assay and real-time PCR. figure3 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.
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Full size image To assess its cross-species transmission potential, we conducted infectivity assays in cell lines from a range of species. Our results (Fig. 4 and Extended Data Table 5) indicate that bat SL-CoV-WIV1 can grow in human alveolar basal epithelial (A549), pig kidney 15 (PK-15) and Rhinolophus sinicus kidney (RSKT) cell lines, but not in human cervix (HeLa), Syrian golden hamster kidney (BHK21), Myotis davidii kidney (BK), Myotis chinensis kidney (MCKT), Rousettus leschenaulti kidney (RLK) or Pteropus alecto kidney (PaKi) cell lines. Real-time RT–PCR indicated that WIV1 replicated much less efficiently in A549, PK-15 and RSKT cells than in Vero E6 cells (Fig. 4).
Figure 4: Analysis of host range of SL-CoV-WIV1 determined by immunofluorescence assay and real-time PCR. figure4 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.
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Full size image To assess the cross-neutralization activity of human SARS-CoV sera against WIV1, we conducted serum-neutralization assays using nine convalescent sera from SARS patients collected in 2003. The results showed that seven of these were able to completely neutralize 100 tissue culture infectious dose 50 ( TCID50) WIV1 at dilutions of 1:10 to 1:40, further confirming the close relationship between WIV1 and SARS-CoV.
Our findings have important implications for public health. First, they provide the clearest evidence yet that SARS-CoV originated in bats. Our previous work provided phylogenetic evidence of this5, but the lack of an isolate or evidence that bat SL-CoVs can naturally infect human cells, until now, had cast doubt on this hypothesis. Second, the lack of capacity of SL-CoVs to use of ACE2 receptors has previously been considered as the key barrier for their direct spillover into humans, supporting the suggestion that civets were intermediate hosts for SARS-CoV adaptation to human transmission during the SARS outbreak24. However, the ability of SL-CoV-WIV1 to use human ACE2 argues against the necessity of this step for SL-CoV-WIV1 and suggests that direct bat-to-human infection is a plausible scenario for some bat SL-CoVs. This has implications for public health control measures in the face of potential spillover of a diverse and growing pool of recently discovered SARS-like CoVs with a wide geographic distribution.
Our findings suggest that the diversity of bat CoVs is substantially higher than that previously reported. In this study we were able to demonstrate the circulation of at least seven different strains of SL-CoVs within a single colony of R. sinicus during a 12-month period. The high genetic diversity of SL-CoVs within this colony was mirrored by high phenotypic diversity in the differential use of ACE2 by different strains. It would therefore not be surprising if further surveillance reveals a broad diversity of bat SL-CoVs that are able to use ACE2, some of which may have even closer homology to SARS-CoV than SL-CoV-WIV1. Our results—in addition to the recent demonstration of MERS-CoV in a Saudi Arabian bat25, and of bat CoVs closely related to MERS-CoV in China, Africa, Europe and North America3,26,27— suggest that bat coronaviruses remain a substantial global threat to public health.
Finally, this study demonstrates the public health importance of pathogen discovery programs targeting wildlife that aim to identify the ‘known unknowns’—previously unknown viral strains closely related to known pathogens. These programs, focused on specific high-risk wildlife groups and hotspots of disease emergence, may be a critical part of future global strategies to predict, prepare for, and prevent pandemic emergence28.
Methods Summary Throat and faecal swabs or fresh faecal samples were collected in viral transport medium as described previously14. All PCR was conducted with the One-Step RT–PCR kit (Invitrogen). Primers targeting the highly conserved regions of the RdRP gene were used for detection of all alphacoronaviruses and betacoronaviruses as described previously15. Degenerate primers were designed on the basis of all available genomic sequences of SARS-CoVs and SL-CoVs and used for amplification of the RBD sequences of S genes or full- length genomic sequences. Degenerate primers were used for amplification of the bat ACE2 gene as described previously29. 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. PCR-positive faecal samples (in 200 μl buffer) were gradient centrifuged at 3,000–12, 000g and supernatant diluted at 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 and checked daily for cytopathic effect. Cell lines from different origins were grown on coverslips in 24-well plates and inoculated with the novel SL-CoV at a multiplicity of infection of 10. Virus replication was detected at 24 h after infection using rabbit antibodies against the SL-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated goat anti- rabbit IgG.
Online Methods 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&# 8201;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&# 8201;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 MgCl2, 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&# 8201;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 MgCl2, 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&# 8201;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 TCID50 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&# 8201;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 TCID50of 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. https://www.nature.com/articles/nature12711
武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进入分类的安全程度最低的ordinary laboratory, animal feeding room and relevant
supporting facilities.
对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实验并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。
P3
The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about
280 m2, is divided into the auxiliary working area and the protection area
with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized
animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3
Laboratory, as the main auxiliary facility of the P4 Laboratory, plays a
significant role in biosafety cluster platforms.
The scope of pathogenic activities that have been applied to be engaged in
by the Laboratory is: the operation of infected materials that have not
cultivated successfully for Ebola, SARS, Nipah, Marburg, Lassa fever virus, and Crimean-Congo hemorrhagic fever virus; pathogen cultivation,
transformation, detection, susceptibility testing for mycobacterium
tuberculosis and bacillus anthracis, and the experiment of small-sized
animals' infection with mycobacterium tuberculosis; the isolation and
cultivation, virus amplification and collection & use, detection of virus
antigen and antibody, serological neutralization test and small-sized animal infection experiments for TBEV, CHIKV, MERS coronavirus.
这些动物就包括这coronavirus! 而且这是已经经过实验感染的动物。
至于弄进来的原始蝙蝠呀什么的动物， 连P-2，P-1 管理级别都没有。 直接列入安全管理分级以外的动物饲养室了。
The facilities to be built include test facility for fulminant disease
pathogens such as Cellular Level Biosafety Level 4 Laboratory, emerging
disease research facility and fulminant disease pathogen storage facility (
that is, BSL-4, BSL-3 and BSL-2 , ordinary laboratory, animal feeding room
and relevant supporting facilities)
这些可都是中科院网站介绍的武汉病毒研究所的情况，应该比较真实可信。大家赶紧看看并备份吧！
http://lssf.cas.cn/en/facilities-view.jsp?id=ff8080814ff56599014ff59e677e003d
是否得到国际认证？
我们要问，弄进来的大量实验用蝙蝠等带毒动物到底是怎么饲养和管理的？ 和其它动物到底怎么区隔的？ 动物死了后到底怎么销毁的？
这里有一大堆问题需要武汉病毒所出来给全国人民解说清楚！
武汉病毒研究所的P4国家实验室，是和武汉大学共建的。 研究所的所长是武汉大学副
校长的继任年轻老婆， 在美国硕士毕业。 到底这一任命有什么令人欣慰的子丑寅卯
，大家需要知道中间的真相到底是什么！
这个问题其实现在用脚趾头都能想明白。 带天然病毒的蝙蝠在千里之外的云南高原。
自己压根就到不了武汉。 而武汉人又不吃蝙蝠， 只有病毒所才弄了大量的蝙蝠过来搞研究。 所以，除了病毒所，没有哪个傻逼能神经病兮兮莫名其妙的从云南贩运蝙蝠到
武汉玩。
搞研究需要大量的蝙蝠，这蝙蝠运到了病毒所以后，怎么管理，问题就多了。 按照中
国科学院网站上对武汉病毒所的介绍，分离成功的病毒保管是P4级别的安全管理。 未
分离完成的半成品和感染过病毒的动物是 p3 级别的安全管理。 至于其它被用于研究
的动物，连P2， P1 管理都没有，就是一般的动物饲养室。那么，武汉病毒所对那些贩运来的蝙蝠到底怎么管理的？ 能告诉大家嘛？
再说蝙蝠是能飞的动物，带着病毒飞走几只能知道嘛？死了，让猫或者狗吃了，又如何说得明白？ 所以，病毒所应当好好给全国人民说清楚，讲明白！
不管什么立场，这才是正确的探讨问题的帖子
刚在另外一个帖子发过，再贴一次：
先前不知道武汉还有个p4实验室，p4实验灭菌消毒很严格，实验室泄露不大可能。但是这种病毒有潜伏期，做相关研究的，如果有疫苗的话，都会给打疫苗；唯一concern的
是，他们在收集实验材料，提取RNA的时候，是否在p4条件下进行。另外p4实验室的气
流每隔一段时间都要进行负压检查和病原检查，就是检测病毒等在实验室气流中的散布，这个需要非常专业的疾控梯队负责。
P3也好P4也罢，法国人只能负责硬件跟规则制度。到最后的执行，国人的不守规则是出了名的，天知道哪里会掉链子。
【 在 CBI 的大作中提到: 】
:
:武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进
入分类的安全程度最低的ordinary laboratory, animal feeding room and relevant :supporting facilities.
:
:对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实
验并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。
:
:P3
:
:The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about :280 m2, is divided into the auxiliary working area and the protection area :with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized
:animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3
不是说动物尸体焚烧处理，因环境部门罚款，后来外包了么？
病毒所，就tm卖B所长，去科罗拉多卖B，2年下崽，名曰留学，其实是卖B，除了卖B，
学了nothing。
回来在后，在草的烂雕指导下，获得博士，接下来2年教授，3年主任，就一投机的女流氓，病毒跑出来，奇怪吗。
各种垃圾废物严格分类收集，这是肯定的。
然后统一交给垃圾工人，也是肯定的。
很想知道有几个垃圾处理工人，
有哪几个是p4级的垃圾工人、至少拥有本科学位？
【 在 CBI (史迪威) 的大作中提到: 】
: 武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进入
: 分类的安全程度最低的ordinary laboratory, animal feeding room and relevant : supporting facilities.
: 对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实验
: 并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。
: P3
: The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about
: 280 m2, is divided into the auxiliary working area and the protection area
: with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized : animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3
: ...................
你不是学生物的就不要造生物的谣
谈一谈个人的看法。欢迎讨论，请勿置顶。谢绝谩骂。
病毒源现在还无法确定，但是国内舆论（官方的民间的）的导向很显然一直专注于三个方面：1.武汉华南海鲜市场 2.野生动物，蝙蝠，竹鼠和蛇等 3. 人们胡乱吃野味。只
有海外的报道可以看到对武汉病毒所作为病毒源头的怀疑。正常人都应该承认至少这种怀疑是合理的，如果说病毒的源头很大可能是野生动物携带，那么武汉市可能的源头至少有三个：
1.华南海鲜市场和各类野味交易市场（食材用途）
2.武汉市动物园和各种娱乐用野生动物场所（娱乐用途）
3. 涉及到野生动物科研的场所（包含武汉病毒所饲养的各类科研用野生动物）（科研
用途）
如果你怀疑官媒的关于目前感染人数和死亡人数的报道（湖北省10W张床位应对3000例
确认感染），那么你也有理由怀疑病毒的源头未必是官方舆论只允许报道的华南海鲜市场的野生动物及其违规食用。
武汉病毒所公开的中文信息提及自身具备符合科研标准的活体野生动物的科研管理场所，但是并未提到蝙蝠。野生的老鼠、兔子、和猴子这些都明确写在其中文网页信息中。也很容易理解，具备这样规模的科研机构每年甚至每个月都会有各类实验用野生动物的采集、购买、饲养、实验和处理活动。如果人们要怀疑各类野味市场的（食材用途）野生动物能够携带病毒，因为都是自野外捕获很有可能接触蝙蝠食用过的食物甚至是沾染蝙蝠的粪便。那么同样有理由怀疑这一类科研用野生动物也会携带病毒。同样，武汉动物园之类的娱乐场所也具备病毒源的疑点。
武汉病毒所内并不饲养蝙蝠是合理的，因为蝙蝠的生存、生活特性会给实验室饲养带来很多问题。所以，研究机构（除非是专门研究蝙蝠的机构）可能不会大费周章从原产地运送大批活体蝙蝠到实验室进行研究。那么病毒所内关于各类蝙蝠的科研是如何进行的呢？看一下国内官方的正规报道显示，2018年武汉病毒所某教授的科研课题组远赴广东调查广东猪瘟问题，最后在养猪场附近的山洞中找到了病毒源（又是一款新型冠状病毒），恰好也是一种蝙蝠。那么在荒郊野外进行科研比如病毒基因测序是不可能的，所以团队花大量时间针对蝙蝠昼伏夜出的习性进行蝙蝠样本采集，比如蝙蝠的毛发、唾液、甚至粪便等等。之后肯定会将采集到的样本带回研究所进行各种研究，然后发表论文。论文提及仅就广东猪流感的研究就涉及到591个蝙蝠样本。而某教授自非典SARS之后就
致力于各类蝙蝠导致的病毒研究，甚至远赴国外采集样本。具体该团队以及整个研究所这些年进行了多少样本采集，而这些样本在武汉是如何保存、运输和销毁的，就没有调查报道了。
关于某教授的猪流感病毒（新型冠状病毒），其官方报道显示已经研究出有效针对性的病毒疫苗，同时强调该病毒并不存在传染人的可能性。可惜我不是学病毒学的，也没有任何生物背景。从有限的对SARS病毒的了解，一些生物学者的分析结论是，通常野生动物身上携带的病毒不会传染人类，但是在极其特殊的自然条件下病毒可能发生变异感染人类。所以，从常识和逻辑上看这是自相矛盾的，如果你相信病毒会变异感染人类，那有什么理由相信猪流感病毒（新型冠状病毒）就不会变异感染人类？如果你相信并不存在传染人类的可能，那么SARS又是如何成功地变异传染人类的呢？也许只有真正做病毒研究的人能解答吧。
最后，当然也可以用一句话全盘否定以上对病毒研究所的怀疑，那就是，P4最高等级病毒研究所一定是严格遵守国际通行标准进行生物实验和病毒研究的。实验样本也严格遵照程序运输、保管、实验和销毁的。根本就不可能存在实验生物体流入野味市场或者娱乐用途、甚至是病毒泄露的可能。然而从YOUTUBE上就有关于该病毒所某教授的科研团
队如何进行野外样本采集的介绍（这个视频的上载时间和动机也很诡异），有该科研团队进行野外作业的照片和介绍。其中也提到，病毒从动物身上传染人的可能性非常小，因此在野外采集蝙蝠样本的时候也只做简单防护。如果科研人员也只是做简单的防护，那为什么集中舆论要公众相信病毒就一定是从野味市场里传染给人的呢？科研机构其实也同样存在这样的风险。封城、断路、隔离、组织全国医护呼吁正能量诚然是解决问题的方法，但是公众同样需要知道真相。只有真相知道了，才是解决此类问题的根本途径。把非典和武汉肺炎中公众所承受的苦难根源解释为公众自身的过失，然后各种舆论导向，这根本就是不负责任的。
【 在 CBI (史迪威) 的大作中提到: 】
: 武汉病毒所有几个级别的实验室level， 依次是安全曾级最高的P4， P3，P2 和不进入
: 分类的安全程度最低的ordinary laboratory, animal feeding room and relevant : supporting facilities.
: 对于corona virus等完成实验的病毒，是列被如 p-4 级别管理。 而对于正在进行实验
: 并已经感染的动物和材料， 并不列入p-4管理，而是列入p3级别来管理的。
: P3
: The National Biosafety Laboratory, Wuhan (NBL3), covering an area of about
: 280 m2, is divided into the auxiliary working area and the protection area
: with its core working area, 140 m2, made up of 3 cell labs, 1 small-sized : animal lab, 1 medium-sized animal lab, and 1 dissecting room. The P3
: ...................
武汉实验室在野外采取动物的程序是否是学术界惯例？如果是的话，任何一个从事类似病毒研究的研究所都有所谓嫌疑。
【 在 leewalk (妖蛾子也飞不过沧海) 的大作中提到: 】
: 谈一谈个人的看法。欢迎讨论，请勿置顶。谢绝谩骂。
: 病毒源现在还无法确定，但是国内舆论（官方的民间的）的导向很显然一直专注于三个
: 方面：1.武汉华南海鲜市场 2.野生动物，蝙蝠，竹鼠和蛇等 3. 人们胡乱吃野味。只
: 有海外的报道可以看到对武汉病毒所作为病毒源头的怀疑。正常人都应该承认至少这种
: 怀疑是合理的，如果说病毒的源头很大可能是野生动物携带，那么武汉市可能的源头至
: 少有三个：
: 1.华南海鲜市场和各类野味交易市场（食材用途）
: 2.武汉市动物园和各种娱乐用野生动物场所（娱乐用途）
: 3. 涉及到野生动物科研的场所（包含武汉病毒所饲养的各类科研用野生动物）（科研
: 用途）
: ...................
常识上讲是的,但是这个事情现在来看水是太深了......
2019年10月18日,约翰霍普金斯大学School of Public Health,世界经济论坛和比尔盖
茨基金会共同举办了一个会议. 会议讨论的竟然是如果在全球范围内大规模爆发冠状病毒感染和传播应该如何应对. 与会的中国人(可能没有实际参会,但是利用多媒体远程影像语音)就是目前中科院院士,中国疾控中心主任高福. 高福是山西人, 山西农业大学毕业, 牛津博士, 哈佛博士后. 高福的主要研究方向为病原微生物跨种间传播机制与分子免疫学. https://baike.baidu.com/item/%E9%AB%98%E7%A6%8F/15610
这个会议的录像就在油管 youtube.com/watch?v=AoLw-Q8X174&t=60s
会议的网站上现在有一个声明,大概意思是宣称此次会议议题与武汉肺炎新型冠状病毒
爆发纯属巧合.而就在同一天,武汉正在开始世界军人运动会...
这个时间点一个月前, 2019年9月18日,武汉天河机场进行口岸突发事件应急处置演练活动场景，以实战形式，模拟了机场口岸通道发现1例新型冠状病毒感染的处置全过程https://m.weibo.cn/status/4418347615555173?
这个时间点两个月后,2020年1月1日,8名散布武汉肺炎谣言者竟然被查处并且中央台亲
自做辟谣报道...
https://www.weibo.com/tv/v/IrHX9bvRg?fid=1034:4466038041935899
一切都是巧合...
【 在 simadong (simadong) 的大作中提到: 】
: 武汉实验室在野外采取动物的程序是否是学术界惯例？如果是的话，任何一个从事类似
: 病毒研究的研究所都有所谓嫌疑。
水木有个人冒泡
他多年前在武汉病毒所读研究生
那位石正丽组的管理看起来很松散
拿网兜装着蝙蝠...
【 在 CBI (史迪威) 的大作中提到: 】
: 这个问题其实现在用脚趾头都能想明白。 带天然病毒的蝙蝠在千里之外的云南高原。
: 自己压根就到不了武汉。 而武汉人又不吃蝙蝠， 只有病毒所才弄了大量的蝙蝠过来搞
: 研究。 所以，除了病毒所，没有哪个傻逼能神经病兮兮莫名其妙的从云南贩运蝙蝠到
: 武汉玩。
: 搞研究需要大量的蝙蝠，这蝙蝠运到了病毒所以后，怎么管理，问题就多了。 按照中
: 国科学院网站上对武汉病毒所的介绍，分离成功的病毒保管是P4级别的安全管理。 未
: 分离完成的半成品和感染过病毒的动物是 p3 级别的安全管理。 至于其它被用于研究
: 的动物，连P2， P1 管理都没有，就是一般的动物饲养室。那么，武汉病毒所对那些贩
: 运来的蝙蝠到底怎么管理的？ 能告诉大家嘛？
: 再说蝙蝠是能飞的动物，带着病毒飞走几只能知道嘛？死了，让猫或者狗吃了，又如何
: ...................
我其实蛮同意这个猜想的
这事本来水就很深。是武汉病毒所泄露(有意无意)，或是
被CIA线人恶意投毒都有可能。
人类的残忍和邪恶不是华人那些傻白甜大妈和脑残轮子
所能想象的，多读读历史就知道了。
当年蒙古人往黑海港口投鼠疫尸体，造成欧洲3个世纪鼠疫
肆虐，杀了2500多万人。
一战德国使用细菌武器，才有了战后日内瓦签订
的禁止使用细菌武器的协定。
二战日本臭名昭著的黑太阳731，用东北人做人体实验，
就都是史实。
【 在 leewalk (妖蛾子也飞不过沧海) 的大作中提到: 】
: 常识上讲是的,但是这个事情现在来看水是太深了......
: 2019年10月18日,约翰霍普金斯大学School of Public Health,世界经济论坛和比尔盖
: 茨基金会共同举办了一个会议. 会议讨论的竟然是如果在全球范围内大规模爆发冠状病
: 毒感染和传播应该如何应对. 与会的中国人(可能没有实际参会,但是利用多媒体远程影
: 像语音)就是目前中科院院士,中国疾控中心主任高福. 高福是山西人, 山西农业大学毕
: 业, 牛津博士, 哈佛博士后. 高福的主要研究方向为病原微生物跨种间传播机制与分子
: 免疫学. https://baike.baidu.com/item/%E9%AB%98%E7%A6%8F/15610
: 这个会议的录像就在油管 youtube.com/watch?v=AoLw-Q8X174&t=60s
: 会议的网站上现在有一个声明,大概意思是宣称此次会议议题与武汉肺炎新型冠状病毒
: 爆发纯属巧合.而就在同一天,武汉正在开始世界军人运动会...
: ...................
【 在 leewalk (妖蛾子也飞不过沧海) 的大作中提到: 】
: 常识上讲是的,但是这个事情现在来看水是太深了......
: 2019年10月18日,约翰霍普金斯大学School of Public Health,世界经济论坛和比尔盖
: 茨基金会共同举办了一个会议. 会议讨论的竟然是如果在全球范围内大规模爆发冠状病
: 毒感染和传播应该如何应对. 与会的中国人(可能没有实际参会,但是利用多媒体远程影
: 像语音)就是目前中科院院士,中国疾控中心主任高福. 高福是山西人, 山西农业大学毕
: 业, 牛津博士, 哈佛博士后. 高福的主要研究方向为病原微生物跨种间传播机制与分子
: 免疫学. https://baike.baidu.com/item/%E9%AB%98%E7%A6%8F/15610
: 这个会议的录像就在油管 youtube.com/watch?v=AoLw-Q8X174&t=60s
: 会议的网站上现在有一个声明,大概意思是宣称此次会议议题与武汉肺炎新型冠状病毒
: 爆发纯属巧合.而就在同一天,武汉正在开始世界军人运动会...
: ...................
MERS是2015年的啊
【 在 leewalk (妖蛾子也飞不过沧海) 的大作中提到: 】
: 常识上讲是的,但是这个事情现在来看水是太深了......
: 2019年10月18日,约翰霍普金斯大学School of Public Health,世界经济论坛和比尔盖
: 茨基金会共同举办了一个会议. 会议讨论的竟然是如果在全球范围内大规模爆发冠状病
: 毒感染和传播应该如何应对. 与会的中国人(可能没有实际参会,但是利用多媒体远程影
: 像语音)就是目前中科院院士,中国疾控中心主任高福. 高福是山西人, 山西农业大学毕
: 业, 牛津博士, 哈佛博士后. 高福的主要研究方向为病原微生物跨种间传播机制与分子
: 免疫学. https://baike.baidu.com/item/%E9%AB%98%E7%A6%8F/15610
: 这个会议的录像就在油管 youtube.com/watch?v=AoLw-Q8X174&t=60s
: 会议的网站上现在有一个声明,大概意思是宣称此次会议议题与武汉肺炎新型冠状病毒
: 爆发纯属巧合.而就在同一天,武汉正在开始世界军人运动会...
: ...................
MERS是2015年的啊
我相信武汉那边肯定有好多蝙蝠病毒，刚发了个云南的蝙蝠，挂在bioarix上了
我在武汉呆了六年，没听说过武汉喜欢吃野味
武汉好像也没有蝙蝠
"武汉人又不吃蝙蝠",您老有啥证据，海鲜市场卖的都是给外地人吃的吗？
【 在 CBI (史迪威) 的大作中提到: 】
: 这个问题其实现在用脚趾头都能想明白。 带天然病毒的蝙蝠在千里之外的云南高原。
: 自己压根就到不了武汉。 而武汉人又不吃蝙蝠， 只有病毒所才弄了大量的蝙蝠过来搞
: 研究。 所以，除了病毒所，没有哪个傻逼能神经病兮兮莫名其妙的从云南贩运蝙蝠到
: 武汉玩。
: 搞研究需要大量的蝙蝠，这蝙蝠运到了病毒所以后，怎么管理，问题就多了。 按照中
: 国科学院网站上对武汉病毒所的介绍，分离成功的病毒保管是P4级别的安全管理。 未
: 分离完成的半成品和感染过病毒的动物是 p3 级别的安全管理。 至于其它被用于研究
: 的动物，连P2， P1 管理都没有，就是一般的动物饲养室。那么，武汉病毒所对那些贩
: 运来的蝙蝠到底怎么管理的？ 能告诉大家嘛？
: 再说蝙蝠是能飞的动物，带着病毒飞走几只能知道嘛？死了，让猫或者狗吃了，又如何
: ...................
首先 你怎么证明武汉海鲜市场的情况是真的
第二 你拿出武汉人吃蝙蝠的证据
第三 柳叶刀说了有相当案例并非来自于海鲜市场，那么海鲜市场可能就未必是原发地
武汉病毒所发的武汉病毒的文章可是实打实的，而且几个蝙蝠病毒都是来自于不同地方的蝙蝠
我是武大的本科，生物
微生物的硕士，方向病毒
我们班一半湖北，10%是武汉的
我周围没有认识一个人吃野味
而且你现在查了这么久，你至少应该知道，是谁谁谁吃的蝙蝠，在海鲜市场被感染的吧
叫什么？多大年龄？ 你证明你的说法，那你就要提供证据
他们病毒所研究冠状病毒和蝙蝠，是他们自己发的文章
能教授看罢，泪如雨下，都是女人，俺咋就这么不幸，11公你这个王八蛋。
【 在 yctsua (dt) 的大作中提到: 】
: 病毒所，就tm卖B所长，去科罗拉多卖B，2年下崽，名曰留学，其实是卖B，除了卖B，
: 学了nothing。
: 回来在后，在草的烂雕指导下，获得博士，接下来2年教授，3年主任，就一投机的女流
: 氓，病毒跑出来，奇怪吗。
所以要立刻抓捕武汉病毒所的所有人员，由国安，公安立案调查。老虎凳辣椒水都整齐了，不怕这帮生物千老不招供
【 在 mstp (再试一次) 的大作中提到: 】
: 这事本来水就很深。是武汉病毒所泄露(有意无意)，或是
: 被CIA线人恶意投毒都有可能。
: 人类的残忍和邪恶不是华人那些傻白甜大妈和脑残轮子
: 所能想象的，多读读历史就知道了。
: 当年蒙古人往黑海港口投鼠疫尸体，造成欧洲3个世纪鼠疫
: 肆虐，杀了2500多万人。
: 一战德国使用细菌武器，才有了战后日内瓦签订
: 的禁止使用细菌武器的协定。
: 二战日本臭名昭著的黑太阳731，用东北人做人体实验，
: 就都是史实。
病毒所恨死本站琐男了，宣布以后一律不收有本站id的
这些是历史发生的，但是现在发生的有太多漏洞。各种细思恐极。
在明明知道有冠状病毒爆发的风险和8名医生(据说是来自于三个不同的武汉医生群)的
爆料之后还要刻意进行辟谣，同时放出病毒不存在人传人的传播等信息，武汉市继续社区大聚会，各种返乡。封城警告提前8小时通知等等。这到底是出于维稳还是恶意，都
已经突破了人类底线。
而且如果仔细看中纪委的关于华南海鲜市场的报道，第一个问题就是中纪委怎么也能发关于病毒的通告？华南海鲜市场有没有病毒这跟中纪委有什么关系？
ccdi.gov.cn/yaowen/202001/t20200127_210367.html
根据这份中纪委报道，几乎言之凿凿华南海鲜市场就是病毒源。“2019年12月31日，根据中国疾控中心要求，病毒病所选派专家组赴武汉参加疫情防控。于2020年1月1日上午赴华南海鲜市场，针对病例相关商户及相关街区集中采集环境样本515份，运送至病毒
病所进行检测。1月12日，病毒病所专家再次在华南海鲜市场采集野生动物贩卖商铺相
关标本70份，并转运至实验室进行检测。两批华南海鲜市场的样本共计585份，PCR检测结果显示其中33份标本为新型冠状病毒核酸阳性。这些阳性样本分布在市场上的22个摊位和1个垃圾车，其中93.9%(31/33)阳性标本分布在华南海鲜市场的西区。"
这份报道的诡异之处就在于，如果病毒标本采样，1月1日的515份样品居然不够检测到
足够统计分析合理的结论（我可以理解成一份显示新冠病毒的样品都没有），而必须在1月12日再去专门采集野生动物贩卖商铺的70个样本。
而根据武汉本地报道，楚天都市报的记者恰好也是1月1日去海鲜市场采访，”正遇到一队乘坐公务车到达现场的工作组。工作组几名戴着口罩的工作人员对市场内多户商户进行了调查，询问市场消毒、商户生病等情况。记者询问工作组的所属单位时，被工作人员婉拒。“这些人应该正好是疾控中心派出的所谓专家组。而该记者报道，采访当日海鲜市场依然在营业，而且采访商户说在1月1日前晚2时许（应该是1月1日凌晨），已有
政府相关部门派出的人员对市场进行了消毒。
http://www.cnr.cn/hubei/yaowen/20200101/t20200101_524921188.shtml
非常矛盾的地方就在于，海鲜市场1月1日凌晨进行了消毒，1月1日白天正常营业。同一天，湖北记者采访海鲜市场竟然意外遇到国家疾控中心在采集样本。为什么消毒完了去采集样本？这还有什么意义吗？而且巧合的又是同一天，武汉警方宣布抓住8名非典病
情造谣者。1月2号更是中央台13频道面向全国辟谣。
所以可以推测出来，所谓的33份标本为新型冠状病毒核酸阳性基本全部是从1月12日“
再去专门采集野生动物贩卖商铺的70个样本。”得来。
令人匪夷所思的地方是，前期515个样本在1月1日凌晨消毒之后的上午去采样，花了12
天不到就检测完了。估计是没有发现冠状病毒。因为逻辑上讲，如果发现了就没必要再去采集。之后1月12日又去采集70个而且是专门去采集野生动物贩卖商铺，疾控中心怎
么就突然知道一定是野生动物商铺有病毒样本呢？
更难以想象的是直到武汉病情爆发之后才报道出就是在华南海峡市场发现了新型冠状病毒核酸阳性。1月12号到1月27号这中间有至少的两周的时间，中国疾控中心竟然无法从70个样本中找到任何一个新冠病毒核酸阳性并且报道出来警告公众？直到病情爆发才发现吗? 并且中纪委专门要确认华南海鲜市场就是病毒源。
一切太匪夷所思。。。
官方媒体就是要大众相信病毒源就是出自华南海鲜市场的野生动物商铺. 而且样本是在灾情大白于天下之前采集, 大白于天下之后公示! 期间隔了至少两周的时间...
欲加之罪,何患无辞....
【 在 mstp (再试一次) 的大作中提到: 】
: 这事本来水就很深。是武汉病毒所泄露(有意无意)，或是
: 被CIA线人恶意投毒都有可能。
: 人类的残忍和邪恶不是华人那些傻白甜大妈和脑残轮子
: 所能想象的，多读读历史就知道了。
: 当年蒙古人往黑海港口投鼠疫尸体，造成欧洲3个世纪鼠疫
: 肆虐，杀了2500多万人。
: 一战德国使用细菌武器，才有了战后日内瓦签订
: 的禁止使用细菌武器的协定。
: 二战日本臭名昭著的黑太阳731，用东北人做人体实验，
: 就都是史实。
好笑。你以为知识分子能有多大胆量这也敢隐瞒
【 在 zhetian (叶凡) 的大作中提到: 】
: 所以要立刻抓捕武汉病毒所的所有人员，由国安，公安立案调查。老虎凳辣椒水都整齐
: 了，不怕这帮生物千老不招供
【 在 leewalk (妖蛾子也飞不过沧海) 的大作中提到: 】
: 这些是历史发生的，但是现在发生的有太多漏洞。各种细思恐极。
: 在明明知道有冠状病毒爆发的风险和8名医生(据说是来自于三个不同的武汉医生群)的
: 爆料之后还要刻意进行辟谣，同时放出病毒不存在人传人的传播等信息，武汉市继续社
: 区大聚会，各种返乡。封城警告提前8小时通知等等。这到底是出于维稳还是恶意，都
: 已经突破了人类底线。
: 而且如果仔细看中纪委的关于华南海鲜市场的报道，第一个问题就是中纪委怎么也能发
: 关于病毒的通告？华南海鲜市场有没有病毒这跟中纪委有什么关系？
: ccdi.gov.cn/yaowen/202001/t20200127_210367.html
: 根据这份中纪委报道，几乎言之凿凿华南海鲜市场就是病毒源。“2019年12月31日，根
: 据中国疾控中心要求，病毒病所选派专家组赴武汉参加疫情防控。于2020年1月1日上午
: ...................
如果是真的，这科研还能信吗？最搞笑的是还根据这些样本发了一系列文章！那些玩数据，整天对着一堆假数据各种作，连病毒是什么样的，怎么做，风险度有多高都不清楚，在那瞎叨叨，还故作高深。M的，不知道自己在吃着带血的馒头
事实上，如果我们稍微用心去看看病毒研究所的研究轨迹和报道记录，就会发现很多东西可以佐证大家的怀疑和判断。
2017年12月29日， 就是距离这次疫情大爆发刚好2年前， 中央电视台有一个专题报道
叫做：
+++++ 【13年不懈追踪 中国科学家寻获SARS病毒源头】 +++++
在这个长篇报道中，图文并茂的介绍了武汉病毒研究所在全中国各地找蝙蝠，抓蝙蝠，研究蝙蝠的经过和结果。 其中明确的提到，在病毒研究所，不光石正丽团队在研究蝙
蝠的所携带的病毒，还有其它团队也在做与蝙蝠有关的病毒研究。
而仅仅一个石正丽团队，就对分属不同科属的408只蝙蝠进行了抗体和核酸检测。 愿文是这么说的：
“通过检测不同科属的408只蝙蝠进行抗体、核酸的监测，目标逐渐清晰，最终在菊头
蝠身上找到了和SARS病毒相似的冠状病毒。菊头蝠因有结构复杂的马蹄形鼻叶而得名，是狂犬病等许多动物源病毒的重要宿主。这一发现刊载于当年的国际权威学术期刊《科学》杂志，引发多方关注。”
那么，这408只蝙蝠又是这么弄到实验室的呢，他们是这么说的：
中国科学院武汉病毒研究所 研究员 石正丽：贵州广西广东湖北湖南河南，到处跑，哪里说有蝙蝠洞我们就往哪里去。由于SARS属于致病性高的烈性病毒，05年之后，随着疫情远去，国内的研究者也少了很多。 但石正丽和她团队并没有停止追踪病毒源头的脚
步，他们深入中国西南、华南、华中等地， 在全国各地寻找蝙蝠病毒样本。最远到过
西藏墨脱、云南西双版纳，深山老林、荒郊野岭的蝙蝠洞，几乎都被他们找遍了。
中国科学院武汉病毒研究所 助理研究员 胡犇：有蝙蝠的地方，一个不太好找，
二它路途也比较遥远，地势也比较险峻，有时候是没有路的，得翻山越岭，跋山
涉水。
中国科学院武汉病毒研究所 博士生 罗东升：在广东抓蝙蝠的时候，当时洞积水
很深，进去的时候，鞋也没有脱，直接水就快到腰了，里面蝙蝠很多，多到拿着
网，就可以撞到网里面，有那种洞还需要匍匐进去，要匍匐个一两百米进去。
直到2011年，在云南的一个蝙蝠洞里，首次检测到了和SARS病毒更相近的SARS样
冠状病毒S基因。2013 年，中科院武汉病毒研究所实验室，从样品中分离出第一
株蝙蝠SARS 样冠状病毒的活病毒，更相近的S基因，让这株病毒能够使用和SARS病
毒相同的受体，并能够感染人的细胞。它被以武汉病毒研究所的英文简称命名
“WIV1”，以彰显这一发现的重要价值。这个成果刊载于2013年11月的《自然》杂
志。“钥匙”终于找到了。但这个小山洞揭开的秘密，远比研究者预想得更多。
到此，我们得出了两个结论：
1. 病毒研究所在全国各地到处抓蝙蝠往武汉运。 而且一折腾就是13个年头。这13年里，到底往武汉运了多少只蝙蝠，我们不得而知，但保守估计至少要比408只多很多。 特别是在云南抓回来的蝙蝠，带有明显的和sars类似的冠状病毒。也就是今天到处传染祸害全中国和世界的corona virus.
2. 抓回来蝙蝠，检测到了 corona virus 后，分离出了病毒，并且证明可以不需要所
谓的中间宿体，直接就可以实现从蝙蝠到人的传染。对此，还以武汉病毒研究所的英文简称 WIV1 做了命名。并分别在science 和 nature 上发了两个paper. 就不知道他们
是怎么完成从蝙蝠到人体的活体传播的实际测试实验，证明他们的WIV1研究结论的？！
——————————————————————————————————————这里是中央电视台的报道链接，估计很快会被删掉，http://m.news.cctv.com/2017/12/29/ARTI641hBlhVaFRe0fnOIidO171229.shtml
上面这个2017年的报道，说 2013年从云南蝙蝠发现的病毒，是不是前些天刚公布的
Bat-RatG13?
如果是Bat-RatG13， 那明显更接近这个2019年的冠状病毒，而不是SARS病毒， 说找到SARS源头并不成立
如果不是Bat-RatG13， 那就是武汉病毒所还藏着一个非常接近SARS的病毒没有公布
一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过其它受体/宿体，而直接传染给人。 他们在发paper 时明确强调的核心就是：中国的
bats corona virus 可以直接传染给人，不需要经过其它中间动物/宿体。
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.
请看他们自己在2013年11月号发表在nature 上的研究报告是怎么说的吧！
Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor
Xing-Yi Ge, 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
Nature volume 503, pages535–538(2013)Cite this article
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
history1. An ongoing outbreak of Middle East respiratory syndrome
coronavirus2 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 viruses3,4,5,
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 Africa5,6,7,8, 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)9,10. 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.
Main
The 2002–3 pandemic of SARS1 and the ongoing emergence of the Middle East
respiratory syndrome coronavirus (MERS-CoV)2 demonstrate that CoVs are a
significant public health threat. SARS-CoV was shown to use the human ACE2
molecule as its entry receptor, and this is considered a hallmark of its
cross-species transmissibility11. The receptor binding domain (RBD) located in the amino-terminal region (amino acids 318–510) of the SARS-CoV spike (S) protein is directly involved in binding to ACE2 (ref. 12). However,
despite phylogenetic evidence that SARS-CoV evolved from bat SL-CoVs, all
previously identified SL-CoVs have major sequence differences from SARS-CoV in the RBD of their S proteins, including one or two deletions6,9. Replacing the RBD of one SL-CoV S protein with SARS-CoV S conferred the ability to
use human ACE2 and replicate efficiently in mice9,13. However, to date, no
SL-CoVs have been isolated from bats, and no wild-type SL-CoV of bat origin has been shown to use ACE2.
We conducted a 12-month longitudinal survey (April 2011–September 2012) of SL-CoVs in a colony of Rhinolophus sinicus at a single location in Kunming, Yunnan Province, China (Extended Data Table 1). A total of 117 anal swabs or faecal samples were collected from individual bats using a previously
published method5,14. A one-step reverse transcription (RT)-nested PCR was
conducted to amplify the RNA-dependent RNA polymerase (RdRP) motifs A and C, which are conserved among alphacoronaviruses and betacoronaviruses15.
Twenty-seven of the 117 samples (23%) were classed as positive by PCR and
subsequently confirmed by sequencing. The species origin of all positive
samples was confirmed to be R. sinicus by cytochrome b sequence analysis, as described previously16. A higher prevalence was observed in samples
collected in October (30% in 2011 and 48.7% in 2012) than those in April (7.1% in 2011) or May (7.4% in 2012) (Extended Data Table 1). Analysis of the S protein RBD sequences indicated the presence of seven different strains of SL-CoVs (Fig. 1a and Extended Data Figs 1 and 2). In addition to RBD
sequences, which closely matched previously described SL-CoVs (Rs672, Rf1
and HKU3)5,8,17,18, two novel strains (designated SL-CoV RsSHC014 and Rs3367) were discovered. Their full-length genome sequences were determined, and
both were found to be 29,787 base pairs in size (excluding the poly(A) tail). The overall nucleotide sequence identity of these two genomes with human
SARS-CoV (Tor2 strain) is 95%, higher than that observed previously for bat SL-CoVs in China (88–92%)5,8,17,18 or Europe (76%)6 (Extended Data Table 2 and Extended Data Figs 3 and 4). Higher sequence identities were observed at the protein level between these new SL-CoVs and SARS-CoVs (Extended Data
Tables 3 and 4). To understand the evolutionary origin of these two novel SL-CoV strains, we conducted recombination analysis with the Recombination
Detection Program 4.0 package19 using available genome sequences of bat SL-
CoV strains (Rf1, Rp3, Rs672, Rm1, HKU3 and BM48-31) and human and civet
representative SARS-CoV strains (BJ01, SZ3, Tor2 and GZ02). Three
breakpoints were detected with strong P values (<10 8722="" 20="" and="" supported="" by="" similarity="" plot="" and="" bootscan="" analysis="" Extended="" Data="" Fig.="" 5a="" b="" .="">Breakpoints were located at nucleotides 20,827, 26,553 and 28,685 in the
Rs3367 (and RsSHC014) genome, and generated recombination fragments covering nucleotides 20,827–26,533 (5,727 nucleotides) (including partial open
reading frame (ORF) 1b, full-length S, ORF3, E and partial M gene) and
nucleotides 26,534–28,685 (2,133 nucleotides) (including partial ORF M,
full-length ORF6, ORF7, ORF8 and partial N gene). Phylogenetic analysis
using the major and minor parental regions suggested that Rs3367, or
RsSHC014, is the descendent of a recombination of lineages that ultimately
lead to SARS-CoV and SL-CoV Rs672 (Fig. 1b).
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.
figure1
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.
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The most notable sequence differences between these two new SL-CoVs and
previously identified SL-CoVs is in the RBD regions of their S proteins.
First, they have higher amino acid sequence identity to SARS-CoV (85% and 96% for RsSHC014 and Rs3367, respectively). Second, there are no deletions and they have perfect sequence alignment with the SARS-CoV RBD region (Extended Data Figs 1 and 2). Structural and mutagenesis studies have previously
identified five key residues (amino acids 442, 472, 479, 487 and 491) in the RBD of the SARS-CoV S protein that have a pivotal role in receptor
binding20,21. Although all five residues in the RsSHC014 S protein were
found to be different from those of SARS-CoV, two of the five residues in
the Rs3367 RBD were conserved (Fig. 1 and Extended Data Fig. 1).
Despite the rapid accumulation of bat CoV sequences in the last decade,
there has been no report of successful virus isolation6,22,23. We attempted isolation from SL-CoV PCR-positive samples. Using an optimized protocol and Vero E6 cells, we obtained one isolate which caused cytopathic effect during the second blind passage. Purified virions displayed typical coronavirus
morphology under electron microscopy (Fig. 2). Sequence analysis using a
sequence-independent amplification method14 to avoid PCR-introduced
contamination indicated that the isolate was almost identical to Rs3367,
with 99.9% nucleotide genome sequence identity and 100% amino acid sequence identity for the S1 region. The new isolate was named SL-CoV-WIV1.
Figure 2: Electron micrograph of purified virions.
figure2
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&#
8201;7.0) and examined directly using a Tecnai transmission electron
microscope (FEI) at 200 kV.
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To determine whether WIV1 can use ACE2 as a cellular entry receptor, we
conducted virus infectivity studies using HeLa cells expressing or not
expressing ACE2 from humans, civets or Chinese horseshoe bats. We found that WIV1 is able to use ACE2 of different origins as an entry receptor and
replicated efficiently in the ACE2-expressing cells (Fig. 3). This is, to
our knowledge, the first identification of a wild-type bat SL-CoV capable of using ACE2 as an entry receptor.
Figure 3: Analysis of receptor usage of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR.
figure3
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.
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To assess its cross-species transmission potential, we conducted infectivity assays in cell lines from a range of species. Our results (Fig. 4 and
Extended Data Table 5) indicate that bat SL-CoV-WIV1 can grow in human
alveolar basal epithelial (A549), pig kidney 15 (PK-15) and Rhinolophus
sinicus kidney (RSKT) cell lines, but not in human cervix (HeLa), Syrian
golden hamster kidney (BHK21), Myotis davidii kidney (BK), Myotis chinensis kidney (MCKT), Rousettus leschenaulti kidney (RLK) or Pteropus alecto kidney (PaKi) cell lines. Real-time RT–PCR indicated that WIV1 replicated much
less efficiently in A549, PK-15 and RSKT cells than in Vero E6 cells (Fig. 4).
Figure 4: Analysis of host range of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR.
figure4
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.
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To assess the cross-neutralization activity of human SARS-CoV sera against
WIV1, we conducted serum-neutralization assays using nine convalescent sera from SARS patients collected in 2003. The results showed that seven of these were able to completely neutralize 100 tissue culture infectious dose 50 (
TCID50) WIV1 at dilutions of 1:10 to 1:40, further confirming the close
relationship between WIV1 and SARS-CoV.
Our findings have important implications for public health. First, they
provide the clearest evidence yet that SARS-CoV originated in bats. Our
previous work provided phylogenetic evidence of this5, but the lack of an
isolate or evidence that bat SL-CoVs can naturally infect human cells, until now, had cast doubt on this hypothesis. Second, the lack of capacity of SL-CoVs to use of ACE2 receptors has previously been considered as the key
barrier for their direct spillover into humans, supporting the suggestion
that civets were intermediate hosts for SARS-CoV adaptation to human
transmission during the SARS outbreak24. However, the ability of SL-CoV-WIV1 to use human ACE2 argues against the necessity of this step for SL-CoV-WIV1 and suggests that direct bat-to-human infection is a plausible scenario for some bat SL-CoVs. This has implications for public health control measures in the face of potential spillover of a diverse and growing pool of recently discovered SARS-like CoVs with a wide geographic distribution.
Our findings suggest that the diversity of bat CoVs is substantially higher than that previously reported. In this study we were able to demonstrate the circulation of at least seven different strains of SL-CoVs within a single colony of R. sinicus during a 12-month period. The high genetic diversity of SL-CoVs within this colony was mirrored by high phenotypic diversity in the differential use of ACE2 by different strains. It would therefore not be
surprising if further surveillance reveals a broad diversity of bat SL-CoVs that are able to use ACE2, some of which may have even closer homology to
SARS-CoV than SL-CoV-WIV1. Our results—in addition to the recent
demonstration of MERS-CoV in a Saudi Arabian bat25, and of bat CoVs closely related to MERS-CoV in China, Africa, Europe and North America3,26,27—
suggest that bat coronaviruses remain a substantial global threat to public health.
Finally, this study demonstrates the public health importance of pathogen
discovery programs targeting wildlife that aim to identify the ‘known
unknowns’—previously unknown viral strains closely related to known
pathogens. These programs, focused on specific high-risk wildlife groups and hotspots of disease emergence, may be a critical part of future global
strategies to predict, prepare for, and prevent pandemic emergence28.
Methods Summary
Throat and faecal swabs or fresh faecal samples were collected in viral
transport medium as described previously14. All PCR was conducted with the
One-Step RT–PCR kit (Invitrogen). Primers targeting the highly conserved
regions of the RdRP gene were used for detection of all alphacoronaviruses
and betacoronaviruses as described previously15. Degenerate primers were
designed on the basis of all available genomic sequences of SARS-CoVs and SL-CoVs and used for amplification of the RBD sequences of S genes or full-
length genomic sequences. Degenerate primers were used for amplification of the bat ACE2 gene as described previously29. 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. PCR-positive faecal
samples (in 200 μl buffer) were gradient centrifuged at 3,000–12,
000g and supernatant diluted at 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 and checked daily for cytopathic effect. Cell lines from different
origins were grown on coverslips in 24-well plates and inoculated with the
novel SL-CoV at a multiplicity of infection of 10. Virus replication
was detected at 24 h after infection using rabbit antibodies against
the SL-CoV Rp3 nucleocapsid protein followed by Cy3-conjugated goat anti-
rabbit IgG.
Online Methods
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&#
8201;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&#
8201;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 MgCl2, 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&#
8201;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 MgCl2, 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&#
8201;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 TCID50 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&#
8201;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 TCID50of 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.
https://www.nature.com/articles/nature12711