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niman

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  1. Materials and methods Ethics statement All sampling procedures were performed by veterinarians with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH05210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People’s Republic of China. Sampling Bat samplings were conducted ten times from April 2011 to October 2015 at different seasons in their natural habitat at a single location (cave) in Kunming, Yunnan Province, China. All members of field teams wore appropriate personal protective equipment, including N95 masks, tear-resistant gloves, disposable outerwear, and safety glasses. Bats were trapped and fecal swab samples were collected as described previously [9]. Clean plastic sheets measuring 2.0 by 2.0 m were placed under known bat roosting sites at about 18:00 h each evening for collection of fecal samples. Fresh fecal pellets were collected from sheets early in the next morning. Each sample (approximately 1 gram of fecal pellet) was collected in 1ml of viral transport medium composed of Hank's balanced salt solution at pH7.4 containing BSA (1%), amphotericin (15 μg/ml), penicillin G (100 units/ml), and streptomycin (50 μg/ml), and were stored at -80°C until processing. Bats trapped for this study were released back into their habitat. RNA extraction, PCR screening and sequencing Fecal swab or pellet samples were vortexed for 1 min, and 140 μl of supernatant was collected from each sample after centrifuge at 3000 rpm under 4°C for 1min. Viral RNA was extracted with Viral RNA Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was eluted in 60 μl of buffer AVE (RNase-free water with 0.04% sodium azide, Qiagen), aliquoted, and stored at -80°C. One-step hemi-nested RT-PCR (Invitrogen) was employed to detect the presence of coronavirus sequences as described previously using a set of primers that target a 440-nt fragment in the RNA-dependent RNA polymerase gene (RdRp) of all known alpha- and betacoronaviruses [20]. For the first round PCR, the 25 μl reaction mix contained 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 template. The amplification was performed as follows: 50°C for 30 min, 94°C for 2 min, followed by 40 cycles consisting of 94°C for 15 sec, 52°C for 30 sec, 68°C for 40 sec, and a final extension of 68°C for 5 min. For the second round PCR, the 25 μl reaction mix contained 2.5 μl PCR reaction buffer, 5 pmol of each primer, 50 mM MgCl2, 0.5mM dNTP, 0.1 μl Platinum Taq Enzyme (Invitrogen) and 1 μl product of the first round PCR. The amplification was performed as follows: 94°C for 3 min followed by 35 cycles consisting of 94°C for 30 sec, 52°C for 30 sec, 72°C for 40 sec, and a final extension of 72°C for 7 min. The RBD region was amplified using the one-step nested RT-PCR method previously described [17]. PCR products were gel purified and sequenced with an ABI Prism 3730 DNA analyzer (Applied Biosystems, USA). PCR products with low concentration or generating heterogeneity in the sequencing chromatograms were cloned into pGEM-T Easy Vector (Promega) for sequencing. The positive samples in this study were termed using the abbreviated name of bat species plus the sample ID number (e.g. Rs4081). To confirm the bat species of individual sample, PCR amplification of cytochrome b (Cytob) or NADH dehydrogenase subunit 1 (ND1) gene was performed using DNA extracted from the feces or swabs [38,39]. Sequencing of full-length genomes Full genomic sequences of 11 SARSr-CoVs were determined by One-step PCR (Invitrogen) amplification of overlapping genomic fragments with degenerate primers designed by multiple alignment of available SARS-CoV and bat SARSr- CoV sequences deposited in GenBank, and additional specific primers designed from the results of previous rounds of sequencing in this study. Primer sequences are available upon request. Sequences of the 5’ and 3’ genomic ends were obtained by 5’ and 3’ RACE (Roche), respectively. PCR products with expected size were gel-purified and subjected directly to sequencing. Each fragment was sequenced at least twice. The sequencing chromatogram of each product was thoroughly examined and sequence heterogeneity was not observed. For some fragments with low concentration of amplicons, the PCR products were cloned into pGEM-T Easy Vector (Promega) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. Co-presence of sequences of distinct SARSr-CoVs was not found in any of the amplicons. The sequences of overlapping genomic fragments were assembled to obtain the full-length genome sequences, with each overlapping sequence longer than 100 bp. Evolution analysis Full-length genome sequences of the 15 SARSr-CoVs detected from bats in the cave surveyed in this study were aligned with those of selected SARS-CoVs using MUSCLE [40]. The aligned sequences were scanned for recombination events by Recombination Detection Program (RDP) [41]. The potential recombination events suggested by strong P values (<10−20) were further confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [42]. Phylogenetic trees based on nucleotide sequences were constructed using the Maximum Likelihood algorithm under the LG model with bootstrap values determined by 1000 replicates in the PhyML (version 3.0) software package [43]. Virus isolation The Vero E6 cell line was kindly provided by Australian Animal Health Laboratory, CSIRO (Geelong, Australia). Vero E6 monolayer was maintained in DMEM medium supplemented with 10% fetal calf serum (FCS). Fecal samples (in 200 μl buffer) were gradient centrifuged at 3,000–12,000 g, and the supernatant was diluted 1:10 in DMEM before being added to Vero E6 cells. After incubation at 37°C for 1 h, the inoculum was removed and replaced with fresh DMEM medium with 2% FCS. The cells were incubated at 37°C and checked daily for cytopathic effect. All tissue culture media were supplemented with triple antibiotics penicillin/ streptomycin/amphotericin (Gibco) (penicillin 200 IU/ml, streptomycin 0.2 mg/ml, amphotericin 0.5 μg/ml). Three blind passages were carried out for each sample. After each passage, both the culture supernatant and cell pellet were examined for presence of SARSr-CoV by RT-PCR using specific primers targeting the RdRp or S gene. The viruses which caused obvious cytopathic effect and could be detected in three blind passages by RT-PCR were further confirmed by electron microscopy. Construction of recombinant viruses Recombinant viruses with the S gene of the novel bat SARSr-CoVs and the backbone of the infectious clone of SARSr-CoV WIV1 were constructed using the reverse genetic system described previously [23] (S9 Fig). The fragments E and F were re-amplified with primer pairs (FE, 5’-AGGGCCCACCTGGCACTGGTAAGAGTCATTTTGC-3’, R-EsBsaI, 5’-ACTGGTCTCTTCGTTTAGTTATTAACTAAAATATCACTAGACACC-3’) and (F-FsBsaI, 5’-TGAGGTCTCCGAACTTATGGATTTGTTTATGAG-3’, RF, 5’-AGGTAGGCCTCTAGGGCAGCTAAC-3’), respectively. The products were named as fragment Es and Fs, which leave the spike gene coding region as an independent fragment. BsaI sites (5’-GGTCTCN|NNNN-3’) were introduced into the 3’ terminal of the Es fragment and the 5’ terminal of the Fs fragment, respectively. The spike sequence of Rs4231 was amplified with the primer pair (F-Rs4231-BsmBI, 5’-AGTCGTCTCAACGAACATGTTTATTTTCTTATTCTTTCTCACTCTCAC-3’ and R-Rs4231-BsmBI, 5’-TCACGTCTCAGTTCGTTTATGTGTAATGTAATTTGACACCCTTG-3’). The S gene sequence of Rs7327 was amplified with primer pair (F-Rs7327-BsaI, 5’-AGTGGTCTCAACGAACATGAAATTGTTAGTTTTAGTTTTTGCTAC-3’ and R-Rs7327-BsaI, 5’- TCAGGTCTCAGTTCGTTTATGTGTAATGTAATTTAACACCCTTG-3’). The fragment Es and Fs were both digested with BglI (NEB) and BsaI (NEB). The Rs4231 S gene was digested with BsmBI. The Rs7327 S gene was digested with BsaI. The other fragments and bacterial artificial chromosome (BAC) were prepared as described previously. Then the two prepared spike DNA fragments were separately inserted into BAC with Es, Fs and other fragments. The correct infectious BAC clones were screened. The chimeric viruses were rescued as described previously [23]. Determination of virus infectivity by immunofluorescence assay The HeLa cell line was kindly provided by Australian Animal Health Laboratory, CSIRO (Geelong, Australia). HeLa cells expressing human ACE2 were constructed as described previously [17]. HeLa cells expressing human ACE2 and Vero E6 cells were cultured on coverslips in 24-well plates (Corning) incubated with the newly isolated or recombinant bat SARSr-CoVs at a multiplicity of infection (MOI) = 1.0 for 1h. The inoculum was removed and the cells were washed twice with PBS and supplemented with medium. Vero E6 cells without virus inoculation and HeLa cells without ACE2 were used as negative control. Twenty-four hours after infection, cells were rinsed with PBS and fixed with 4% formaldehyde in PBS (pH7.4) at 4°C for 20 min. ACE2 expression was detected by using goat anti-human ACE2 immunoglobulin followed by FITC-labelled donkey anti-goat immunoglobulin (PTGLab). Virus replication was detected by using rabbit antibody against the nucleocapsid protein of bat SARSr-CoV Rp3 followed by Cy3-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI. Staining patterns were observed under an FV1200 confocal microscope (Olympus). Determination of virus replication in Vero E6 cells by plaque assay Vero E6 cells were infected with WIV1, Rs4874, WIV1-Rs4231S, and WIV1-Rs7327S at an MOI of 1.0 and 0.01. After incubation for an hour, the cells were washed with DHanks for three times and supplied with DMEM containing 2% FCS. Samples were collected at 0, 10, 27, and 48 h post infection. The viral titers were determined by plaque assay. Determination of virus replication in HeLa cells expressing human ACE2 by quantitative RT-PCR HeLa cells expressing human ACE2 were inoculated with WIV1, Rs4874, WIV1-Rs4231S, and WIV1-Rs7327S at an MOI of 1.0, and were incubated for 1h at 37°C. After the inoculum was removed, the cells were supplemented with medium containing 1% FBS. Supernatants were collected at 0, 12, 24 and 48h. Virus titers were determined using quantitative RT-PCR targeting the partial N gene with a standard curve which expresses the correlation between Ct value and virus titer (shown as TCID50/ml). The standard curve was made using RNA dilutions from the purified Rs4874 virus stock (with a titer of 2.15 × 106 TCID50/ml). For qPCR, RNA was extracted from 140 μl of each supernatant with Viral RNA Mini Kit (Qiagen) following manufacturer’s instructions and eluted in 60 μl AVE buffer. The PCR was performed 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’-GTGGTGGTGACGGCA AAATG-3’), 40 pmol reverse primer (5’-AAGTGAAGCTTCTGGGCCAG-3’) and 12 pmol probe (5’-FAM-AAAGAGCTCAGCCCCAGATG-BHQ1-3’). The amplification was performed as follows: 50°C for 10 min, 95°C for 10 min followed by 50 cycles consisting of 95°C for 15 sec and 60°C for 20 sec. Plasmids The ORF8 genes of bat SARSr-CoV WIV1 and Rf4092 and the ORF8a gene of bat SARSr-CoV Rs4084 were amplified by PCR from the viral RNA extracted from the isolated virus or fecal samples. The ORF8 gene of SARS-CoV GZ02 and bat SARSr-CoV Rf1, and the ORF8a gene of SARS-CoV Tor2 were synthesized by Tsingke Biological Technology Co., Ltd (Wuhan, China). All genes were cloned into the pCAGGS vector constructed with a C-terminal HA tag. Expression of the proteins was confirmed by Western blotting using a mAb against the HA tag. Five tandem copies of the ATF6 consensus binding sites were synthesized and inserted into the pGL3-Basic vector to construct the luciferase reporter plasmid 5×ATF6-GL3, in which the luciferase gene is under the control of the c-fos minimal promoter and the ATF6 consensus binding sites. Luciferase reporter assay HeLa cells in 24-well plates were transfected using Lipofectamine 3000 reagent (Life Technologies) following the manufacturer’s instruction. Cells per well were co-transfected with 600ng of the 5×ATF6-GL3 reporter plasmid, with 300ng of each expression plasmid of SARS-CoV and SARSr-CoV ORF8 or empty vector and 20ng of pRL-TK (Promega) which served as an internal control. The cells were incubated for 24h, and were treated with or without 2μg/ml tunicamycin for 16h. Cells were harvested and lysed. Luciferase activity was determined using a dual-luciferase assay system (Promega). The experiment was performed in triplicate wells. Quantification of apoptotic cells 293T cells in 12-well plates were transfected using Lipofectamine 3000 reagent (Life Technologies) following the manufacturer’s instruction. Cells per well were transfected with 3μg of the expression plasmid of SARS-CoV Tor2 or SARSr-CoV Rs4084 ORF8a, or the empty vector. 24h post transfection, apoptotic cells were quantified by using the Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection Kit (Yeasen Biotech, Shanghai) in accordance with the manufacturer’s instruction. Apoptosis was analyzed by flow cytometry. The experiment was performed in triplicate wells. Accession numbers The complete genome sequences of bat SARS-related coronavirus strains As6526, Rs4081, Rs4084, Rf4092, Rs4231, Rs4237, Rs4247, Rs4255, Rs4874, Rs7327 and Rs9401 have been deposited in the GenBank database with the accession numbers from KY417142 to KY417152, respectively.
  2. Discussion Genetically diverse SARSr-CoVs have been detected in various horseshoe bat species across a wide geographic range in China in the past decade [9–12,14,29]. However, most bat SARSr-CoVs show considerable genetic distance to SARS-CoV, particularly in the highly variable S1, ORF8 and ORF3 regions [10,25]. Recently, several novel SARSr-CoVs have been described to be more closely related to SARS-CoV, either in the S gene or in ORF8. The S proteins of RsSHC014, Rs3367, WIV1 and WIV16, which were reported in our previous studies, shared 90% to 97% aa sequence identities to those of human/civet SARS-CoVs [17,18]. Another strain from Rhinolophus affinis in Yunnan termed LYRa11 showed 90% aa sequence identity to SARS-CoV in the S gene [13]. In addition, two studies have described 4 novel SARSr-CoVs (YNLF_31C/34C and GX2013/YN2013) which possessed a full-length ORF8 with substantially higher similarity to that of SARS-CoV [22,30]. These findings provide strong genetic evidence for the bat origin of SARS-CoV with regard to the S gene or ORF8. However, all of these SARSr-CoVs were distinct from SARS-CoV in at least one other gene, suggesting that none of them was the immediate progenitor of SARS-CoV. Moreover, these SARSr-CoVs were discovered in bat populations from physically distinct locations. The site of origin of the true progenitor of SARS-CoV and the evolutionary origin of SARS-CoV have until now remained elusive. In the current study, we have identified a bat habitat potentially important for SARSr-CoV evolution where a series of recombination events have likely occurred among different SARSr-CoV strains, which provides new insights into the origin of SARS-CoV. SARS first emerged in Guangdong province in late 2002 [7]. However, SARSr-CoVs discovered in bats from neighboring areas of Guangdong to date have shown phylogenetic disparity from SARS-CoV especially in the S gene [9,10,14], suggesting SARS-CoV may have originated from another region. Our analysis of the phylogeny of SARS-CoVs and all known bat SARSr-CoVs using the nt sequence of their non-structural ORF1a and ORF1b genes, which constitute the majority of the genome, shows that SARSr-CoV evolution is strongly correlated with their geographical origin, but not host species. It is noteworthy that SARSr-CoVs detected in Yunnan are more closely related to SARS-CoV than strains from other regions in China. This finding implies that Yunnan, or southwestern China, is more likely to be the geographical source of SARS-CoV than other regions in China, but data from more extensive surveillance are yet needed to support this inference. In our longitudinal surveillance of SARSr-CoVs in a single cave in Yunnan where we discovered Rs3367, RsSHC014, WIV1 and WIV16, the CoV prevalence in fecal samples varied among different sampling time. Generally, a higher prevalence was observed in autumn (September and October) than in spring and early summer (April and May). This may be due to the establishment of a susceptible subpopulation of newborn bats which had not developed their own immunity after the parturition period [31]. Another factor may be the changes in the composition of bat species in the cave at different sampling dates. For example, in September 2012 when the CoV prevalence reached 51.3%, the majority of samples were from R. sinicus, but in May 2015 when only 3 out of the 145 samples tested positive, Aselliscus stoliczkanus was the predominant bat species in the cave. We failed to amplify the RBD sequences from 15 of the 64 SARSr-CoV positive samples. Most of these samples had comparatively low viral concentration (< 107 copies/g) (S8 Fig), as revealed by our previous quantitative studies [32]. The unsuccessful amplification of RBD in some samples with high viral concentration was probably because of the more divergent sequences in this region of these SARSr-CoV genomes. In this cave, we have now obtained full-length genome sequences of additional 11 novel SARSr-CoVs from bats. Our findings suggest the co-circulation of different bat SARSr-CoVs highly similar to SARS-CoV in the most variable S1 (NTD and RBD), ORF8 and ORF3 regions, respectively, in this single location. In the ORF1a, ORF1b, E, M and N genes, the SARSr-CoVs circulating in this cave also shared > 98% aa sequence identities with human/civet SARS-CoVs. Thus, all of the building blocks of the SARS-CoV genome were present in SARSr-CoVs from this single location in Yunnan during our sampling period. Furthermore, strains closely related to different representative bat SARSr-CoVs from other provinces (e.g. Rs672, HKU3 and Rf1) in the RBD region were also detected there. Therefore, this cave could be regarded as a rich gene pool of bat SARSr-CoVs, wherein concurrent circulation of a high diversity of SARSr-CoV strains has led to an unusually diverse assemblage of SARSr-CoVs. During our 5-year surveillance in this single cave, we first reported Rs3367 and WIV1 in 2013, with RBD sequence closely resembling that of SARS-CoV [17]. More recently, we discovered WIV16 which had an RBD almost identical to WIV1’s but shared much higher similarity with SARS-CoV than WIV1 in the NTD region of S1, making it the closest SARSr-CoV to the epidemic strains identified to date [18]. In this study, we found a novel strain Rs4231 from the same location sharing almost identical NTD sequence with WIV16 but distinct from it in the RBD, with evidence of a recombination event. Our recombination analysis indicated that a recombination event may have taken place at the junction between the coding region of NTD and RBD in the Rs4231 and WIV1 genomes and resulted in WIV16. Recombination at this genomic position also happened among other SARSr-CoVs relatively distant to SARS-CoV found in this location (e.g. Rs4081 and Rs4247, S5 Fig). The frequent recombination at this hotspot in the S gene increased the genetic diversity of SARSr-CoVs harbored in these bat populations and might have been responsible for the generation of the S gene of the direct progenitor strain of SARS-CoV. The genomes of SARS-CoVs from patients during the early epidemic phase and civet SARS-CoVs all contained a single full-length ORF8 [3,7]. We have found that a number of bat SARSr-CoVs from this cave possessed a complete ORF8 highly similar to that of early human/civet SARS-CoV (>97% nt sequence identity), represented by strain Rf4092 (S3C Fig). This provided further evidence for the source of human SARS-CoV ORF8 in bats [22,30]. In contrast, the ORF8 was split into overlapping ORF8a and ORF8b in most human SARS-CoV strains from later-phase patients due to the acquisition of a 29-nt deletion [8,26]. In this study, we have discovered for the first time a bat SARSr-CoV with ORF8a and ORF8b highly similar to the later-phase human SARS-CoVs, though the split of ORF8 in the bat SARSr-CoV and that in human SARS-CoV were two independent events. Our recombination analysis suggests that this strain, Rs4084, likely acquired its ORF8 from Rf4092 through recombination, followed by the development of the 5-nt deletion which led to the splitting. It suggests that ORF8 region in bat SARSr-CoV genomes is prone to deletions as in human SARS-CoV [3,25]. Finally, the recombination analysis suggests that an ancestral strain of SARS-CoV SZ3 would have been generated if the recombination around ORF8 had occurred between the lineages that led to WIV16 and Rf4092. Taken together, the evidence of recombination events among SARSr-CoVs harbored by bats in this single location suggests that the direct progenitor of SARS-CoV may have originated as a result of a series of recombination within the S gene and around ORF8. This could have been followed by the spillover from bats to civets and people either in the region, or during movement of infected animals through the wildlife trade. However, given the paucity of data on animal trade prior to the SARS outbreak, the likely high geographical sampling bias in bat surveillance for SARSr-CoVs in southern China, and the possibility that other caves harbor similar bat species assemblages and a rich diversity of SARSr-CoVs, a definite conclusion about the geographical origin of SARS-CoV cannot be drawn at this point. R. sinicus are regarded as the primary natural host of SARS-CoV, as all SARSr-CoVs highly homologous to SARS-CoV in the S gene were predominantly found in this species. However, it is noted that two SARSr-CoVs previously reported from R. ferrumequinum showed the closest phylogenetic position to SARS-CoV in the ORF1a/1b trees. These strains were discovered in another location in Yunnan 80 km from the cave surveyed in the current study [22]. This information also supports the speculation that SARS-CoV may have originated from this region. Nonetheless, since the correlation between the host species and the phylogeny of SARSr-CoV ORF1ab seems limited, more SARSr-CoV sequences need to be obtained from different Rhinolophus bat species in both locations in Yunnan, and from other locations in southern China. In particular, it will be important to assess whether R. ferrumequinum played a more important role in the evolution of SARS-CoV ORF1ab. The cave we studied is located approximately 60 km from the city of Kunming. Beside a number of rhinolophid and hipposiderid species from which SARSr-CoVs have been detected, other bats like myotis were also present there. The temperature in the cave is around 22–25°C and the humidity around 85%-90%. The physical nature of the cave is not unique, but it does appear to host a particularly dense population of bats in the reproductive season. Similar caves co-inhabited by bat populations of different species are not rare in other areas in Yunnan. We propose that efforts to study the ecology, host species diversity, and viral strain populations of these caves may provide critical information on what drives SARSr-CoV evolution. Our previous studies demonstrated the capacity of both WIV1 and WIV16 to use ACE2 orthologs for cell entry and to efficiently replicate in human cells [17,18]. In this study, we confirmed the use of human ACE2 as receptor of two novel SARSr-CoVs by using chimeric viruses with the WIV1 backbone replaced with the S gene of the newly identified SARSr-CoVs. Rs7327’s S protein varied from that of WIV1 and WIV16 at three aa residues in the receptor-binding motif, including one contact residue (aa 484) with human ACE2. This difference did not seem to affect its entry and replication efficiency in human ACE2-expressing cells. A previous study using the SARS-CoV infectious clone showed that the RsSHC014 S protein could efficiently utilize human ACE2 [33], despite being distinct from SARS-CoV and WIV1 in the RBD (S1 Fig). We examined the infectivity of Rs4231, which shared similar RBD sequence with RsSHC014 but had a distinct NTD sequence, and found the chimeric virus WIV1-Rs4231S also readily replicated in HeLa cells expressing human ACE2 molecule. The novel live SARSr-CoV we isolated in the current study (Rs4874) has an S gene almost identical to that of WIV16. As expected, it is also capable of utilizing human ACE2. These results indicate that diverse variants of SARSr-CoV S protein without deletions in their RBD are able to use human ACE2. In contrast, our previous study revealed that the S protein of a R. sinicus SARSr-CoV with deletions (Rp3) failed to use human, civet and bat ACE2 for cell entry [34]. In this study, in addition to Rs4231 and Rs7327, we also constructed infectious clones with the S gene of Rs4081, Rf4075, Rs4085, Rs4235 and As6526, which all contained the deletions in their RBD. These 7 strains, plus Rs4874 and the previously studied WIV1 and RsSHC014, could represent all types of S variants of SARSr-CoVs in this location (S3A Fig). However, none of the strains with deletions in the RBD could be rescued from Vero E6 cells. Therefore, the two distinct clades of SARSr-CoV S gene may represent the usage of different receptors in their bat hosts. The full-length ORF8 protein of SARS-CoV is a luminal endoplasmic reticulum (ER) membrane-associated protein that induces the activation of ATF6, an ER stress-regulated transcription factor that activates the transcription of ER chaperones involved in protein folding [35]. We amplified the ORF8 genes of Rf1, Rf4092 and WIV1, which represent three different genotypes of bat SARSr-CoV ORF8 (S3C Fig), and constructed the expression plasmids. All of the three ORF8 proteins transiently expressed in HeLa cells can stimulate the ATF6-dependent transcription. Among them, the WIV1 ORF8, which is highly divergent from the SARS-CoV ORF8, exhibited the strongest activation. The results indicate that the variants of bat SARSr-CoV ORF8 proteins may play a role in modulating ER stress by activating the ATF6 pathway. In addition, the ORF8a protein of SARS-CoV from the later phase has been demonstrated to induce apoptosis [28]. In this study, we have found that the ORF8a protein of the newly identified SARSr-CoV Rs4084, which contained an 8-aa insertion compared with the SARS-CoV ORF8a, significantly triggered apoptosis in 293T cells as well. Compared with the 154-aa ORF3b of SARS-CoV, the ORF3b proteins of all previously identified bat SARSr-CoVs were smaller in size due to the early translation termination. However, for the first time, we discovered an ORF3b without the C-terminal truncation in a bat SARSr-CoV, Rs7327, which differed from the ORF 3b of SARS-CoV GZ02 strain at only one aa residue. The SARS-CoV ORF3b antagonizes interferon function by modulating the activity of IFN regulatory factor 3 (IRF3) [27]. As previous studies suggested, the nuclear localization signal-containing C-terminal may not be required for the IFN antagonist activity of ORF3b [36]. Our previous studies also demonstrated that the ORF3b protein of a bat SARSr-CoV, termed Rm1, which was C-terminally truncated to 56 aa and shared 62% aa sequence identity with SARS-CoV, still displayed the IFN antagonist activity [37]. It is very interesting to investigate in further studies whether Rs7327’s ORF3b and other versions of truncated ORF3b such as WIV1 and WIV16 also show IFN antagonism profiles. As a whole, our findings from a 5-year longitudinal study conclusively demonstrate that all building blocks of the pandemic SARS-CoV genome are present in bat SARSr-CoVs from a single location in Yunnan. The data show that frequent recombination events have happened among those SARSr-CoVs in the same cave. While we cannot rule out the possibility that similar gene pools of SARSr-CoVs exist elsewhere, we have provided sufficient evidence to conclude that SARS-CoV most likely originated from horseshoe bats via recombination events among existing SARSr-CoVs. In addition, we have also revealed that various SARSr-CoVs capable of using human ACE2 are still circulating among bats in this region. Thus, the risk of spillover into people and emergence of a disease similar to SARS is possible. This is particularly important given that the nearest village to the bat cave we surveyed is only 1.1 km away, which indicates a potential risk of exposure to bats for the local residents. Thus, we propose that monitoring of SARSr-CoV evolution at this and other sites should continue, as well as examination of human behavioral risk for infection and serological surveys of people, to determine if spillover is already occurring at these sites and to design intervention strategies to avoid future disease emergence.
  3. Induction of apoptosis by the ORF8a of the newly identified bat SARSr-CoV We conducted transient transfection to examine whether the ORF8a of SARSr-CoV Rs4084 triggered apoptosis. As shown in Fig 9B, 11.76% and 9.40% of the 293T cells transfected with the SARSr-CoV Rs4084-ORF8a and SARS-CoV Tor2-ORF8a expression plasmid underwent apoptosis, respectively. In contrast, transfection with the empty vector resulted in apoptosis in only 2.79% of the cells. The results indicate that Rs4084 ORF8a has an apoptosis induction activity similar to that of SARS-CoV [28].
  4. Activation of activating transcription factor 6 (ATF6) by the ORF8 proteins of different bat SARSr-CoVs The induction of the ATF6-dependent transcription by the ORF8s of SARS-CoV and bat SARSr-CoVs were investigated using a luciferase reporter, 5×ATF6-GL3. In HeLa cells transiently transfected with the expression plasmids of the ORF8s of bat SARSr-CoV Rf1, Rf4092 and WIV1, the relative luciferase activities of the 5×ATF6-GL3 reporter was enhanced by 5.56 to 9.26 folds compared with cells transfected with the pCAGGS empty vector, while it was increased by 4.42 fold by the SARS-CoV GZ02 ORF8. As a control, the treatment with tunicamyxin (TM) stimulated the transcription by about 11 folds (Fig 9A). The results suggests that various ORF8 proteins of bat SARSr-CoVs can activate ATF6, and those of some strains have a stronger effect than the SARS-CoV ORF8. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 9. Functional characterization of diverse ORF8 and ORF8a proteins of bat SARSr-CoVs. (A) The ORF8 proteins of SARS-CoV and bat SARSr-CoVs induces the ATF6-dependent transcriptional activity. HeLa cells were transiently transfected with the pcAGGS expression plasmids of the ORF8 of SARS-CoV GZ02, bat SARSr-CoV Rf1, WIV1 and Rf4092 and the reporter plasmid 5×ATF6-GL3 for 40h. Control cells were co-transfected with the reporter plasmid and the empty pCAGGS vector for 24h, and treated with or without TM (2μg/ml) for an additional 16h. The cell lysates were harvested for dual luciferase assay and data are shown as the average values from triplicate wells. (B) The ORF8a proteins of SARS-CoV and bat SARSr-CoV triggered apoptosis. 293T cells were transfected with the expression plasmids of the ORF8a of SARS-CoV Tor2 and bat SARSr-CoV Rs4084 and a pcAGGS vector control for 24h. Apoptosis was analyzed by flow cytometry after annexin V staining and the percentage of apoptotic cells were calculated. Data are shown as the average values from triplicate cells. Error bars indicate SDs. * P<0.05. https://doi.org/10.1371/journal.ppat.1006698.g009
  5. Rescue of bat SARSr-CoVs and virus infectivity experiments In the current study, we successfully cultured an additional novel SARSr-CoV Rs4874 from a single fecal sample using an optimized protocol and Vero E6 cells [17]. Its S protein shared 99.9% aa sequence identity with that of previously isolated WIV16 and it was identical to WIV16 in RBD. Using the reverse genetics technique we previously developed for WIV1 [23], we constructed a group of infectious bacterial artificial chromosome (BAC) clones with the backbone of WIV1 and variants of S genes from 8 different bat SARSr-CoVs. Only the infectious clones for Rs4231 and Rs7327 led to cytopathic effects in Vero E6 cells after transfection (S7 Fig). The other six strains with deletions in the RBD region, Rf4075, Rs4081, Rs4085, Rs4235, As6526 and Rp3 (S1 Fig) failed to be rescued, as no cytopathic effects was observed and viral replication cannot be detected by immunofluorescence assay in Vero E6 cells (S7 Fig). In contrast, when Vero E6 cells were respectively infected with the two successfully rescued chimeric SARSr-CoVs, WIV1-Rs4231S and WIV1-Rs7327S, and the newly isolated Rs4874, efficient virus replication was detected in all infections (Fig 7). To assess whether the three novel SARSr-CoVs can use human ACE2 as a cellular entry receptor, we conducted virus infectivity studies using HeLa cells with or without the expression of human ACE2. All viruses replicated efficiently in the human ACE2-expressing cells. The results were further confirmed by quantification of viral RNA using real-time RT-PCR (Fig 8). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 7. Infection of Vero E6 cells by bat SARSr-CoV WIV1, Rs4874, WIV1-Rs4231S and WIV1-Rs7327S. (A) The successful infection was confirmed by immunofluorescent antibody staining using rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein. The columns (from left to right) show staining of nuclei (blue), virus replication (red), and both nuclei and virus replication (merged double-stain images). (B) The growth curves in Vero E6 cells with a MOI of 1.0 and 0.01. https://doi.org/10.1371/journal.ppat.1006698.g007 Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 8. Analysis of receptor usage by immunofluorescence assay (A) and real-time PCR (B). Virus infectivity of Rs4874, WIV1-Rs4231S and WIV1-Rs7327S was determined in HeLa cells with and without the expression of human ACE2. ACE2 expression was detected with goat anti-human ACE2 antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG. Virus replication was detected with rabbit antibody against the SARSr-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-phenylindole).The columns (from left to right) show staining of nuclei (blue), ACE2 expression (green), virus replication (red) and the merged triple-stained images, respectively. https://doi.org/10.1371/journal.ppat.1006698.g008
  6. Phylogenetic analysis Phylogenetic trees were constructed using the nt sequences of nonstructural protein gene ORF1a and ORF1b. Unlike the high genetic diversity in the S gene, nearly all SARSr-CoVs from the bat cave we surveyed were closely clustered, and showed closer phylogenetic relationship to SARS-CoV than the majority of currently known bat SARSr-CoVs discovered from other locations, except YNLF_31C and 34C which were recently reported in greater horseshoe bats from another location in Yunnan [22] (Fig 6). The phylogeny of SARSr-CoVs in ORF1a and ORF1b appeared to be associated with their geographical distribution rather than with host species. Regardless of different host bat species, SARS-CoV and SARSr-CoVs detected in bats from southwestern China (Yunnan, Guizhou and Guangxi province) formed one clade, in which SARSr-CoV strains showing closer relationship to SARS-CoV were all from Yunnan. SARSr-CoVs detected in southeastern, central and northern provinces, such as Hong Kong, Hubei and Shaanxi, formed the other clade which was phylogenetically distant to human and civet SARS-CoVs (Fig 6 and S6 Fig). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. Phylogenetic trees based on nucleotide sequences of ORF1a (A) and ORF1b (B). The trees were constructed by the maximum likelihood method using the LG model with bootstrap values determined by 1000 replicates. Only bootstraps > 50% are shown. The scale bars represent 0.03 (A) and 0.02 (B) substitutions per nucleotide position. Rs, Rhinolophus sinicus; Rf, Rhinolophus ferremequinum; Rm, Rhinolophus macrotis; Ra, Rhinolophus affinis; Rp, Rhinolophus pusillus; As, Aselliscus stoliczkanus; Cp, Chaerephon plicata. SARSr-CoVs detected in bats from the single cave surveyed in this study are in bold. Sequences detected in southwestern China are indicated in red. https://doi.org/10.1371/journal.ppat.1006698.g006
  7. Recombination analysis The full-length genome sequences of all 15 SARSr-CoVs from the surveyed cave were screened for evidence of potential recombination events. Both similarity plot and bootscan analyses revealed frequent recombination events among these SARSr-CoV strains. It was suggested that WIV16, the closest progenitor of human SARS-CoV known to date [18], was likely to be a recombinant strain from three SARSr-CoVs harbored by bats in the same cave, namely WIV1, Rs4231 and Rs4081, with strong P value (<10−30). Breakpoints were identified at genome positions nt 18391, 22615 and 28160 (Fig 5A). In the genomic region between nt 22615 and 28160, which contained the region encoding the RBD and the S2 subunit of the S protein, WIV16 was highly similar to WIV1, sharing 99% sequence identity. In contrast, in the region between nt 18391 and 22615, which covered a part of ORF1b and the region encoding the NTD of the S gene, WIV16 showed substantially closer relationship to Rs4231. Meanwhile, the ORF1ab sequences upstream from nt 18391 of WIV16 displayed the highest genetic similarity (99.8% nt sequence identity) to that of Rs4081. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. Detection of potential recombination events by similarity plot and boot scan analysis. (A) Full-length genome sequence of SARSr-CoV WIV16 was used as query sequence and WIV1, Rs4231 and Rs4081 as reference sequences. (B) Full-length genome sequence of SARS-CoV SZ3 was used as query sequence and SARSr-CoV WIV16, Rf4092 and Rs4081 as reference sequences. All analyses were performed with a Kimura model, a window size of 1500 base pairs, and a step size of 150 base pairs. The gene map of query genome sequences are used to position breakpoints. https://doi.org/10.1371/journal.ppat.1006698.g005 Evidence of recombination event was also detected in the genome of the novel SARSr-CoV Rs4084, which had a unique genome organization with split ORF8a and 8b. The previously reported strain RsSHC014 and the newly identified strain Rf4092 were suggested to be the major and minor parent of Rs4084, respectively (P value < 10−80). The breakpoint was located at nt 26796 (S5 Fig). In the region downstream of the breakpoint including ORF8, Rs4084 showed closet genetic relationship with Rf4092, sharing 98.9% nt sequence identity, while it shared the highest nt sequence identity (99.4%) with RsSHC014 in the majority of its genome upstream from the breakpoint. When civet SARS-CoV SZ3 was used as the query sequence in similarity plot and bootscan analysis, evidence for recombination events was also detected (Fig 5B). In the region between the two breakpoints at the genome positions nt 21161 and nt 27766, including the S gene, closer genetic relationship between SZ3 and WIV16 was observed. However, from position nt 27766 towards the 3’ end of its genome, a notably close genetic relationship was observed between SZ3 and Rf4092 instead. Throughout the non-structural gene, moreover, SZ3 shared a similarly high sequence identity with WIV16 and Rf4092. It indicates that civet SARS-CoV was likely to be the descendent from a recombinant of the precursors of WIV16 and Rf4092, or that the SARSr-CoVs found in this cave, like WIV16 or Rf4092, may have been the descendants of the SARS-CoV lineage.
  8. Co-circulation of different bat SARSr-CoVs with S, ORF8 and ORF3 sequences similar to those in SARS-CoV at a single location The primary difference between SARS-CoV and most bat SARSr-CoVs is located in S gene. The S protein is functionally divided into two subunits, denoted S1 and S2, which is responsible for receptor binding and cellular membrane fusion, respectively. S1 consists of two domains, the N-terminal domain (NTD) and C-terminal domain (CTD) which is also known as the RBD in SARS-CoV [24]. SARS-CoV and bat SARSr-CoVs share high sequence identity in the S2 region in contrast to the S1 region. Among the 15 SARSr-CoVs identified from bats in the surveyed cave, six strains with deletions in their RBD regions (Rs4081, Rs4237, Rs4247, Rs4255, Rf4092 and As6526) showed 78.2% to 80.2% aa sequence identity to SARS-CoV in the S protein, while the other nine strains without deletions were much more closely related to SARS-CoV, with 90.0% (Rs4084) to 97.2% (Rs4874) aa sequence identity. These nine SARSr-CoVs can be further divided into four genotypes according to their S1 sequences (Fig 2😞 RsSHC014/Rs4084 showed more genetic differences from SARS-CoV in both NTD and RBD regions; The RBD sequences of SARSr-CoV Rs7327, Rs9401 and previously reported WIV1/Rs3367 closely resembled that of SARS-CoV. However, they were distinct from SARS-CoV but similar to RsSHC014 in NTD. In contrast, we found a novel SARSr-CoV, termed Rs4231, which shared highly similar NTD, but not RBD sequence with SARS-CoV (Figs 2 and 3). Its S protein showed 94.6% to 95% aa sequence identity to those of human and civet SARS-CoVs (S1 Table). Strains with both NTD and RBD highly homologous to those of SARS-CoV were also present in this cave. In addition to WIV16 which we described previously [18], Rs4874 was also found to have the S protein closest to SARS-CoV S (> 97% aa sequence identity) of all the bat SARSr-CoVs reported to date (Figs 2 and 3). In addition to the SARSr-CoVs subjected to full-length genome sequencing, we also obtained the RBD and NTD sequences from other samples collected in this cave. The sequences with high identity to SARS-CoV RBD were amplified from 10 more R. sinicus samples. SARSr-CoVs with this genotype of RBD were detected in different seasons throughout the five years. Strains containing the NTD similar to SARS-CoV were only found in 2013 (S2 Table). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 3. Amino acid sequence comparison of the S1 subunit (corresponding to aa 1–660 of the spike protein of SARS-CoV). The receptor-binding domain (aa 318–510) of SARS-CoV and the homologous region of bat SARSr-CoVs are indicated by the red box. The key aa residues involved in the interaction with human ACE2 are numbered on top of the aligned sequences. 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 civets in 2003. SARSr-CoV Rs 672 and YN2013 were identified from R. sinicus collected in Guizhou and Yunnan Province, respectively. SARSr-CoV Rf1 and JL2012 were identified from R. ferrumequinum collected in Hubei and Jilin Province, respectively. WIV1, WIV16, RsSHC014, Rs4081, Rs4084, Rs4231, Rs4237, Rs4247, Rs7327 and Rs4874 were identified from R.sinicus, and Rf4092 from R. ferrumequinum in the cave surveyed in this study. https://doi.org/10.1371/journal.ppat.1006698.g003 ORF8 is another highly variable gene among different SARS-CoV and SARSr-CoV strains [25,26]. We aligned the ORF8 nt sequences of the representative SARSr-CoVs discovered in this surveillance with those of other SARSr-CoVs and SARS-CoVs (Fig 4). Though WIV16, WIV1, Rs4231 and RsSHC014 were genetically closer to SARS-CoV in S gene, they contained a single 366-nt ORF8 without the 29-nt deletion present in most human SARS-CoVs and showed only 47.1% to 51.0% nt sequence identity to human and civet SARS-CoVs. However, the ORF8 of strain Rf4092 from R. ferrumequinum exhibited high similarity to that of civet SARS-CoV. It possessed a single long ORF8 of the same length (369 nt) as that of civet SARS-CoV strain SZ3, with only 10 nt mutations and 3 aa mutations detected (Fig 4). Similar ORF8 sequences were also amplified from other 7 samples collected in the cave during 2011 to 2013, from both R. ferrumequinum and R. sinicus (S2 Table). The ORF8 of Rs4084 was highly similar to Rf4092’s but was split into two overlapping ORFs, ORF8a and ORF8b, due to a short 5-nt deletion (Figs 2 and 4). The position of start codons and stop codons of the two ORFs were consistent with those in most human SARS-CoV strains. Excluding the 8-aa insertion, Rs4084 and SARS-CoV strain BJ01 displayed identical aa sequence of ORF8a, and only three different aa residues were observed between their ORF8b (Fig 4). To our knowledge, Rs4084 was the first bat SARSr-CoV reported that resembled the late human SARS-CoVs in both ORF8 gene organization and sequence. Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 4. Alignment of nucleotide sequences of ORF8 or ORF8a/8b. The start codons and stop codons of ORF8, 8a and 8b are marked with black boxes and the forward and reverse arrows, respectively. The deletion responsible for the split ORF8a and 8b in human SARS-CoV BJ01, Tor2 and bat SARSr-CoV Rs4084 is marked with red boxes. See the legend for Fig 3 for the origin of various sequences used in this alignment. https://doi.org/10.1371/journal.ppat.1006698.g004 Another key difference between SARS-CoV and bat SARSr-CoV genomes is the ORF3 coding region [10,17,21]. We analyzed the ORF3a sequences amplified from 42 samples and found that most of the SARSr-CoVs closely related to SARS-CoV in the S gene shared higher ORF3a sequence similarity (96.4% to 98.9% aa identity) with SARS-CoV (S3 Fig and S2 Table). The ORF3b of SARS CoV, sharing a large part of its coding sequence with the ORF3a, encodes a 154-aa protein [27], but it is truncated to different extents at the C-terminal in previously described bat SARSr-CoVs including WIV1 and WIV16 (S4 Fig). In the current study, we identified a non-truncated ORF3b for the first time (Rs7327), which maintained the nuclear localization signal at its C-terminal. Moreover, it shared 98.1% aa sequence identity with SARS-CoV strain Tor2 with only three aa substitutions (S4 Fig). Thus, Rs7327 is the bat SARSr-CoV most similar to SARS-CoV in the ORF3 region known to date.
  9. Genomic characterization of the novel SARSr-CoVs Based on the diversity of RBD sequences, 11 novel SARSr-CoV strains named by abbreviation of bat species and sample ID (Rs4081, Rs4084, Rs4231, Rs4237, Rs4247, Rs4255, Rs4874, Rs7327, Rs9401, Rf4092 and As6526) were selected for full-length genomic sequencing based on sample abundance, genotype of RBD as well as sampling time. For each RBD genotype and each time of sampling, at least one representative strain was selected. The genome size of these novel SARSr-CoVs ranged from 29694 to 30291 nucleotides (nt). This gave a total of 15 full-length genomes of bat SARSr-CoVs from this single location (13 from R.sinicus, and one each from R. ferrumequinum and A. stoliczkanus), including our previously reported strains, Rs3367, RsSHC014, WIV1 and WIV16 [17,18]. The genomes of all 15 SARSr-CoVs circulating in this single cave shared 92.0% to 99.9% nt sequence identity. The overall nt sequence identity between these SARSr-CoVs and human and civet SARS-CoVs is 93.2% to 96%, significantly higher than that observed for bat SARSr-CoVs reported from other locations in China (88–93%) [9,10,12,14,21,22]. The genome sequence similarity among the 15 SARSr-CoVs and SARS-CoV SZ3 strain was examined by Simplot analysis (Fig 1). The 15 SARSr-CoVs are highly conserved and share a uniformly high sequence similarity to SARS-CoV in the non-structural gene ORF1a (96.6% to 97.1% nt sequence identity, 98.0% to 98.3% aa sequence identity) and ORF1b (96.1% to 96.6% nt sequence identity, 99.0% to 99.4% aa sequence identity). In contrast, a considerable genetic diversity is shown in the S gene (corresponding to SZ3 genome position 21477 to 25244) and ORF8 (corresponding to SZ3 genome position 27764 to 28132) (Fig 1). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. Similarity plot based on the full-length genome sequence of civet SARS CoV SZ3. Full-length genome sequences of all SARSr-CoV detected in bats from the cave investigated in this study were used as reference sequences. The analysis was performed with the Kimura model, a window size of 1500 base pairs and a step size of 150 base pairs. https://doi.org/10.1371/journal.ppat.1006698.g001 The 11 novel SARSr-CoVs identified from this single location generally shared similar genome organization with SARS-CoV and other bat SARSr-CoVs. In our previous study, we identified an additional ORF termed ORFx present between ORF6 and ORF7 in strain WIV1 and WIV16 [18,23]. In this study, ORFx was also found in the genomes of Rs7327 and Rs4874. Compared with that of WIV1 and WIV16, the length of ORFx in Rs7327 and Rs4874 was extended to 510 nt due to a deletion of 2 nt in a poly-T sequence that resulted in a shift of reading frame (Fig 2 and S2 Fig). Download: PPT PowerPoint slide PNG larger image TIFF original image Fig 2. Schematic diagram illustrating the genomic regions or ORFs with most variation between different SARS-CoV and SARSr-CoV isolates. Coding regions of the N-terminal domain (NTD) and receptor-binding domain (RBD) of the spike protein, ORF3a/b and ORF8 (8a/b) in bat SARSr-CoV genomes highly similar to those in SARS CoV genome are indicated with black boxes or arrows while the hollow boxes or arrows represent corresponding regions with less sequence similarity to those of SARS-CoV. The deletions in the RBD of some SARSr-CoVs are indicated by two vertical lines. https://doi.org/10.1371/journal.ppat.1006698.g002
  10. Results Continued circulation of diverse SARSr-CoVs in bats from a single location We have carried out a five-year longitudinal surveillance (April 2011 to October 2015) on SARSr-CoVs in bats from a single habitat in proximity to Kunming city, Yunnan province, China, which was mainly inhabited by horseshoe bats. A total of 602 alimentary specimens (anal swabs or feces) were collected and tested for the presence of CoVs by a Pan-CoV RT-PCR targeting the 440-nt RdRp fragment that is conserved among all known α- and β-CoVs [20]. In total, 84 samples tested positive for CoVs. Sequencing of the PCR amplicons revealed the presence of SARSr-CoVs in the majority (64/84) of the CoV-positive samples (Table 1). Host species identification by amplification of either Cytb or ND1 gene suggested that most (57/64) of the SARSr-CoV positive samples were from Rhinolophus sinicus, while the remaining 7 samples were from Rhinolophus ferrumequinum, Rhinolophus affinis and from Aselliscus stoliczkanus which belongs to the family Hipposideridae. Download: PPT PowerPoint slide PNG larger image TIFF original image Table 1. Summary of SARSr-CoV detection in bats from a single habitat in Kunming, Yunnan. https://doi.org/10.1371/journal.ppat.1006698.t001 Based on the preliminary analysis of the partial RdRp sequences, all of the 64 bat SARSr-CoV sequences showed high similarity among themselves and with other reported bat SARSr-CoVs and SARS-CoVs from humans and civets. To understand the genetic diversity of these bat SARSr-CoVs, the most variable region of the SARSr-CoV S gene, corresponding to the receptor-binding domain (RBD) of SARS-CoV, were amplified and sequenced. Due to low viral load in some samples, RBD sequences were successfully amplified only from 49 samples. These RBD sequences displayed high genetic diversity and could be divided into two large clades, both of which included multiple genotypes. Clade 1 strains shared an identical size and higher amino acid (aa) sequence identity with SARS-CoV RBD, while clade 2 had a shorter size than SARS-CoV S due to two deletions (5 and 12–13 aa, respectively) (S1 Fig). Co-infections by two strains of different clades were detected in two samples, Rs3262 and Rs4087 (S1 Fig).
  11. Introduction Severe Acute Respiratory Syndrome (SARS) is a severe emerging viral disease with high fatality characterized by fever, headache and severe respiratory symptoms including cough, dyspnea and pneumonia [1]. Due to its high transmissibility among humans, after its first emergence in southern China in late 2002, it rapidly led to a global pandemic in 2003 and was marked as one of the most significant public health threats in the 21st century [2,3]. The causative agent, SARS coronavirus (SARS-CoV), has been previously assigned to group 2b CoV and is now a member of the lineage B of genus Betacoronavirus in the family Coronaviridae [4]. It shares similar genome organization with other coronaviruses, but exhibits a unique genomic structure which includes a number of specific accessory genes, including ORF3a, 3b, ORF6, ORF7a, 7b, ORF8a, 8b and 9b [5,6]. Masked palm civets (Paguma larvata) were initially hypothesized to be the animal origin of SARS-CoV [7,8]. However, since a large number of genetically diverse SARS-related coronaviruses (SARSr-CoV) have been detected in multiple species of horseshoe bats (genus Rhinolophus) from different areas of China and Europe in the aftermath of SARS, it is prevailingly considered that SARS-CoV originated in horseshoe bats with civets acting as the intermediate amplifying and transmitting host [9–16]. Recently we have reported four novel SARSr-CoVs from Chinese horseshoe bats that shared much higher genomic sequence similarity to the epidemic strains, particularly in their S gene, of which two strains (termed WIV1 and WIV16) have been successfully cultured in vitro [17,18]. These newly identified SARSr-CoVs have been demonstrated to use the same cellular receptor (angiotensin converting enzyme-2 [ACE-2]) as SARS-CoV does and replicate efficiently in primary human airway cells [17–19]. Despite the cumulative evidence for the emergence of SARS-CoV from bats, all bat SARSr-CoVs described so far are clearly distinct from SARS-CoV in the S gene and/or one or more accessory genes such as ORF3 and ORF8, suggesting they are likely not the direct ancestor of SARS-CoV. Thus a critical gap remains in our understanding of how and where SARS-CoV originated from bat reservoirs. Previously, we reported a number of bat SARSr-CoVs with diverse S protein sequences from a single cave in Yunnan Province, including the four strains mentioned above most closely related to SARS-CoV [17,18]. Here we report the latest results of our 5-year longitudinal surveillance of bat SARSr-CoVs in this single location and systematic evolutionary analysis using full-length genome sequences of 15 SARSr-CoV strains (11 novel ones and 4 from previous studies). Efficiency of human ACE2 usage and the functions of accessory genes ORF8 and 8a were also evaluated for some of the newly identified strains.
  12. Author summary Increasing evidence has been gathered to support the bat origin of SARS coronavirus (SARS-CoV) in the past decade. However, none of the currently known bat SARSr-CoVs is thought to be the direct ancestor of SARS-CoV. Herein, we report the identification of a diverse group of bat SARSr-CoVs in a single cave in Yunnan, China. Importantly, all of the building blocks of SARS-CoV genome, including the highly variable S gene, ORF8 and ORF3, could be found in the genomes of different SARSr-CoV strains from this single location. Based on the analysis of full-length genome sequences of the newly identified bat SARSr-CoVs, we speculate that the direct ancestor of SARS-CoV may have arisen from sequential recombination events between the precursors of these bat SARSr-CoVs prior to spillover to an intermediate host. In addition, we found bat SARSr-CoV strains with different S proteins that can all use the receptor of SARS-CoV in humans (ACE2) for cell entry, suggesting diverse SARSr-CoVs capable of direct transmission to humans are circulating in bats in this cave. Our current study therefore offers a clearer picture on the evolutionary origin of SARS-CoV and highlights the risk of future emergence of SARS-like diseases.
  13. Abstract A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.
  14. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus Ben Hu , Lei-Ping Zeng , Xing-Lou Yang , Xing-Yi Ge, Wei Zhang, Bei Li, Jia-Zheng Xie, Xu-Rui Shen, Yun-Zhi Zhang, Ning Wang, Dong-Sheng Luo, Xiao-Shuang Zheng, Mei-Niang Wang, Peter Daszak, Lin-Fa Wang, Jie Cui , Zheng-Li Shi [ view less ] https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006698 Published: November 30, 2017 https://doi.org/10.1371/journal.ppat.1006698
  15. Wuhan scientists describe SARS-like bat coronovirus which have extensive recombination and ACE2 receptor binding.
  16. Tonight at 10 PM ET Update on Wuhan novel coronavirus sequence
  17. Chinese scientists obtain genetic sequence of mysterious virus, a key step in containment efforts By HELEN BRANSWELL @HelenBranswell JANUARY 9, 2020 Health officials monitor passengers arriving in Bangkok from Wuhan, China.LAUREN DECICCA/GETTY IMAGES Chinese scientists have recovered a previously unknown virus from an infected individual and generated a full genetic sequence of it, a key step in efforts to learn more about the cause of an outbreak of unusual pneumonia in the city of Wuhan, state-run media reported Thursday. Fragments of the same virus were picked up in testing of 15 patients among the 59 who have been identified as infected with the mysterious pneumonia. The speed of the findings is impressive; the first case in this outbreak became ill less than a month ago. ADVERTISEMENT “Preliminary identification of a novel virus in a short period of time is a notable achievement and demonstrates China’s increased capacity to manage new outbreaks,” the World Health Organization’s representative in China, Dr. Gauden Galea, said in a statement. NEWSLETTERS Sign up for Daily Recap A roundup of STAT's top stories of the day. Privacy Policy But more work is needed to confirm whether the virus is the cause of the outbreak and, if it is, to identify what animal species transmitted the virus to people and whether there are other cases elsewhere, Galea said. The report on CCTV appears to be the first official confirmation from Chinese authorities that they believe a new virus is responsible for this outbreak — specifically a coronavirus, a type of virus in the family that includes SARS and MERS. When viewed under an electron microscope, it exhibits the crown-like halo that gives coronaviruses their name, said Xu Jianguo, identified by CCTV as the leader of the preliminary assessment of the test results and a member of the Chinese Academy of Engineering. The outbreak is occurring in Wuhan, about 700 miles south of Beijing. Wuhan is a leading center for virology research in China, and infectious diseases experts watching this outbreak have predicted scientists there would quickly find the cause. Still, they cautioned that this discovery does not mean the outbreak is over or the threat has passed. “Everybody’s assuming … that this coronavirus is the cause. We don’t know that. They’re assuming that market was the origin. We don’t know that. And we don’t know how many other cases are walking around right now,” said Peter Daszak, president of EcoHealth Alliance, a non-profit organization that works in this sphere. The first known case in the Wuhan outbreak became noticeably ill on Dec. 12, according to a statement released Sunday by the Wuhan Municipal Health Commission. The discovery of an outbreak and identification of a new virus in a period of less than one month is exceptional, experts said. “I am stunned by the timeline and speed of this isolation and characterization, if it’s all true,” said Matthew Frieman, a coronavirus expert at the University of Maryland School of Medicine. STAT Plus: Exclusive analysis of biopharma, health policy, and the life sciences. The community of scientists who research emerging infectious diseases has been speculating for days that the source of the outbreak was a new coronavirus, a class of viruses that has shown itself adept at making the leap from animals to people. Coronaviruses originate in bats but are able to infect a number of mammals. Confirmation that scientists have the genetic sequence of the virus will increase pressure on China to release at least part of that sequence, so that health facilities around the world know what to look for as they try to detect possible cases from this outbreak and prevent spread elsewhere. During the 2003 SARS outbreak, infected travelers spread the virus from China to Hong Kong and from there to Vietnam, Singapore, Taiwan, and Toronto, Canada. In the United States, the Centers for Disease Control and Prevention on Wednesday instructed doctors to ask patients with severe respiratory infections whether they have been to Wuhan. Hong Kong and other countries in Southeast Asia are isolating any such cases until they can be tested for influenza, rhinoviruses, and other viruses that cause colds and flu. This type of very broad net will draw in many people who were in or near Wuhan and who have a respiratory tract infection but who have nothing to do with this outbreak — especially in the middle of flu season. That will eat up resources and the time of doctors, laboratories, and sick people who don’t need to be isolated. “If the Chinese truly have sequenced the virus and they’ve demonstrated that it’s present in other patients, that’s means there’s a PCR diagnostic test available. And the Chinese need to make that available to the rest of the world immediately,” said Ralph Baric, a coronavirus expert at the University of North Carolina. “And the longer they wait, the more likely the scientific community will go from a positive response to a negative response, in terms of how China is handling this outbreak,” he said. The CCTV report did not provide details on the type of coronavirus that has been discovered — for instance if it is one of several SARS-like viruses that have been found in bats in China, and if so, how genetically different it is from SARS. A number of these SARS-like viruses are able to infect human tissue cells in the laboratory, suggesting they might be able to spill over into people if given the right circumstances. Xu said more research on the new virus is needed. There has been limited information about the nature of the illness in people who have been infected. Chinese authorities have said no one has died from this infection; seven, however, were in critical condition as of Sunday. In a statement that same day, the WHO said that the main symptom was fever; some patients had difficulty breathing. The CCTV report also did not indicate whether scientists investigating the outbreak had identified the source of the virus — which will be key to any effort to determine if the virus is spreading in other locations as well. Baris said coronaviruses could jump to people directly from bats, which are eaten in China. But this virus could have used what is known as an intermediate host — an animal species that becomes infected with a bat virus that then transmits it to people. Daszak said he believes efforts to look for the virus in animals have not started. “There are probably a dozen to two dozen target species that you would go after to do a wildlife investigation. I don’t know if they’re doing that,” he said, adding that EcoHealth Alliance hopes to partner with Chinese researchers on the work “once the politics have died down.” China was roundly criticized for its early bungling of the SARS outbreak and it is widely believed authorities there have no wish to be embarrassed like that again. The capacity of the country to respond to infectious diseases outbreaks increased markedly in the aftermath of SARS. During that outbreak, it was determined that palm civets, a wild animal eaten as a delicacy in southern China, were transmitting the virus. Chinese authorities ordered a widespread culling of civets to help stop the outbreak. The Wuhan outbreak has been linked to a large seafood market that also sells the meat of exotic animals for consumption. The market was closed and decontaminated on Jan. 1. But it is important to know if other markets are selling infected animals, said Malik Peiris, a microbiologist at the University of Hong Kong. “If it can jump once, then it will jump again,” Peiris said of the virus. He was one of the scientists who first identified the coronavirus that caused the 2003 SARS outbreak, which infected more than 8,000 people and killed nearly 800. The WHO said Wednesday that Chinese authorities believe the virus “does not transmit readily between people.” Earlier statements from the Wuhan Municipal Health Authority said there has been no person-to-person spread, but disease experts challenged that claim, saying it is impossible to rule out at this stage in the exploration of a new disease. “I don’t know how you know that at all,” Frieman said of China’s claim there is no person-to-person transmission. He noted the number of cases reported makes it seem unlikely that animal-to-human transmission is the only way this virus spreads. There have been at least one or two clusters of cases within families that have raised suspicions of limited person-to-person spread, a source familiar with the outbreak told STAT. News of the pneumonia cases first emerged on Dec. 30, when the local health authority told hospitals to be on the lookout for cases. The next day Chinese authorities informed the WHO that they were dealing with what looked like an outbreak caused by an unknown virus. About the Author Helen Branswell Senior Writer, Infectious Disease Helen Branswell covers issues broadly related to infectious diseases, including outbreaks, preparedness, research, and vaccine development. [email protected] @HelenBranswell https://www.statnews.com/2020/01/09/chinese-scientists-obtain-genetic-sequence-of-mysterious-virus-a-key-step-in-containment-efforts/
  18. Scientists urge China to quickly share data on virus linked to pneumonia outbreak By Dennis Normile, Jon Cohen, Kai KupferschmidtJan. 9, 2020 , 3:15 PM Chinese scientists have identified a novel coronavirus—a pathogen family responsible for two other new diseases since 2003—as the likely cause of the outbreak of an unusual viral pneumonia in the central city of Wuhan that has sickened dozens of people since mid-December 2019. The researchers have sequenced the virus and developed a test to identify it, according to a news report today by CCTV, China’s state-owned broadcaster. Although the link between the agent and the disease needs to be confirmed, many scientists praised the discovery, which they say is a testimony of China’s prowess in virology. But they are urging the country to quickly share much more information about the new agent, the disease it causes, and how it appears to spread. “The virologists in China are some of the best in the world, they work extremely quickly, extremely efficiently,” says Peter Daszak, president of the EcoHealth Alliance. “They have a lot more information than we know about right now. I hope that information comes out very soon.” The outbreak, Daszak says, is “a chance for China to show that they are doing 21st century public health as well as 21st century virology.” “I think they really should share the sequence data, so that we can all make sure we can test for this virus if we get travelers from this region,” adds virologist Marion Koopmans of the Erasmus Medical Center. At least 59 people in Wuhan have fallen ill with the mysterious agent since mid-December, according to the Wuhan Municipal Health Commission; no deaths have been reported, and so far, there have been no reported cases of human-to-human transmission. Many cases have been linked to a seafood market in the city that also sells other live animals. China first reported the unusual cases to the World Health Organization (WHO) on 31 December 2019. (News of the discovery of the coronavirus was first reported yesterday by The Wall Street Journal.) CCTV says a virus isolated from one patient showed the spiked surface typical of coronaviruses under electron microscopy. Scientists sequenced the virus, according to the report, and then used nucleic acid testing to identify the virus in 14 additional patients. Separately, Xinhua news today identified the leader of the investigation as Xu Jianguo. Although the agency did not give his affiliation, Xu is apparently at China’s National Institute for Communicable Disease Control and Prevention, a part of the Chinese Center for Disease Control and Prevention. Xu told Xinhua that researchers are continuing their work to confirm that the coronavirus is the culprit. “Preliminary identification of a novel virus in a short period of time is a notable achievement and demonstrates China’s increased capacity to manage new outbreaks,” Gauden Galea, WHO’s representative to China, wrote in a press statement today. Scientists around the world share that sentiment, but they would like to know more. Chinese researchers “are to be congratulated on identifying the causative agent quickly,” says Malik Peiris, a virologist at the University of Hong Kong. “Now it is important that they share specific diagnostic RT-PCR [reverse transcription polymerase chain reaction] tests with WHO and the global public health community.” “What I really want to see is information on the epidemiology and pathology,” Daszak says, “so that we can all have confidence that, No. 1, this coronavirus is the cause of this outbreak, and No. 2, that it is contained and that they have been able to trace back all the potential cases, isolate them, test them. Every day that we don’t get all that information is a risk of further spread in my opinion.” Chinese researchers needn’t worry that sharing the information would preclude publication of the new virus in a prestigious journal, says Christian Drosten of Charité University Hospital: “No journal will reject a paper because this sequence has been made public.” The news reports have been careful to call the findings preliminary. During the 2003 outbreak of severe acute respiratory syndrome (SARS), Chinese authorities and scientists were embarrassed when premature reports that chlamydia was the culprit turned out to be incorrect; it later turned out to be a new coronavirus. “I can understand why politicians and scientists have to be really cautious in claiming [this virus] is the causative agent,” says Wang Linfa, an emerging disease specialist at the Duke-NUS Medical School. A key step in nailing the link is to replicate the symptoms in laboratory animals, Wang says, but that can take weeks or months. Wang says the parallels between the Wuhan outbreak and SARS are interesting. Both emerged in winter, with initial cases tied to exposure to animals sold at live animal markets. (With SARS, the intermediary host proved to be civets sold at the markets.) But there are big differences as well. SARS proved to spread relatively easily among humans and eventually caused 774 deaths in 37 countries before it was contained. The pneumonia cases in Wuhan so are far milder than SARS and there appears to be no human-to-human transmission—although some researchers aren’t so sure. “I don’t understand how you can get so many cases without human-to-human transmission,” Daszak says. “This is something I have a red flag on.” Koopmans remains to be convinced as well. The other key difference between now and 2003 is that China’s scientific expertise has grown rapidly. “The lab capacity, the clinical capacity, the outbreak capacity is orders of magnitude better now,” Daszak says. Back then, China also denied it had a problem, and actively tried to cover up the outbreak. It is not doing so now, but it hasn’t exactly been generous with information either, Koopmans says. “The communication has been better than with SARS but not perfect.” “What they’re trying to do is wrestle with when you actually announce publicly without getting egg on your face,” Daszak says, “because it would have been embarrassing to announce that a week ago and then find out that it was an incidental finding.” Scientists assume the Wuhan patients were infected by some animal sold at the market. Pinpointing the exact species is key, Peiris says. “There may be other markets where a similar virus may be circulating and it will be important to test such markets to preemptively contain similar outbreaks.” Wang suspects Chinese investigators have tried to collect samples from animals and have swabbed walls and cages: “The fact that we have not seen anything in the media suggests the issue is sensitive or the results [are] not conclusive.” The new illness again demonstrates that live animal markets should no longer be allowed, says Robert Webster, a leading influenza researcher at St. Jude Children’s Research Hospital. “There’s a host of coronaviruses out there,” he says. “You keep putting live animals in contact with humans and this is going to happen from time to time. So far we’ve got lucky that none have led to widespread human-to-human transmission.” But whatever animal spread the virus at the market likely picked it up from a natural reservoir elsewhere, scientists say. “If I were to bet, I’d bet it’s from bats,” says Wang, who led one of two teams that traced the SARS virus back to cave-dwelling horseshoe bats in Yunnan province. The coronavirus that causes Middle East respiratory syndrome (MERS), which emerged in 2012, has been traced back to wild bats as well. “Bats carry so many coronaviruses and they mutate rapidly,” Wang says. In Hong Kong, 48 people who traveled to Wuhan in recent weeks have presented with symptoms of either fever and respiratory infection or pneumonia; Singapore and South Korea have isolated sick travelers from Wuhan as well. None of them has been found to be infected with the suspected virus so far. Posted in: Asia/Pacific Health doi:10.1126/science.aba8414 https://www.sciencemag.org/news/2020/01/scientists-urge-china-quickly-share-data-virus-linked-pneumonia-outbreak
  19. Pneumonia of Unknown Cause in China Warning - Level 3, Avoid Nonessential Travel Alert - Level 2, Practice Enhanced Precautions Watch - Level 1, Practice Usual Precautions Key Points There is a cluster of cases of pneumonia in Wuhan, China. The cause is not yet known, nor is the mode of transmission. The cluster is centered at the Wuhan South China Seafood City (also called the South China Seafood Wholesale Market and the Hua Nan Seafood Market). The market has been closed since January 1, 2020, for cleaning and disinfection. Travelers to Wuhan, China, should avoid living or dead animals, animal markets, and contact with sick people. The situation is evolving. This notice will be updated as more information becomes available. What is the current situation? There is a cluster of cases of pneumonia in Wuhan, China. As of January 5, 2020, local, provincial, and national health commissions in China have reported a total of 59 cases with no deaths. The cluster is centered at the Wuhan South China Seafood City (also called the South China Seafood Wholesale Market and the Hua Nan Seafood Market). In addition to seafood, the market sells chickens, bats, marmots, and other wild animals. The market has been closed since January 1, 2020, for cleaning and disinfection. Health authorities in China are monitoring more than 150 close contacts for illness. To date, there have been no reports of spread from person to person or to health care workers. Symptoms include fever and difficulty breathing. Though the cause of this cluster is unknown, there is concern that it is a virus. Local authorities have reported negative laboratory results for seasonal influenza, avian influenza, adenovirus, and two specific coronaviruses known to cause respiratory illness (severe acute respiratory syndrome [SARS] and Middle East respiratory syndrome [MERS]). What can travelers do to protect themselves? Travelers to Wuhan should Avoid animals (alive or dead), animal markets, and products that come from animals (such as uncooked meat). Avoid contact with sick people. Wash hands often with soap and water. If you traveled to Wuhan and feel sick, you should Stay home. Except for seeking medical care, avoid contact with others. Don’t travel while sick. Seek medical care right away. Before you go to a doctor’s office or emergency room, call ahead and tell the doctor about your recent travel and your symptoms. Cover your mouth and nose with a tissue or your sleeve (not your hands) when coughing or sneezing. Clinician Information As of this posting, case-patients in the cluster reportedly have had fever, difficulty breathing, and bilateral lung infiltrates on chest radiograph. For patients with similar respiratory symptoms who recently traveled to Wuhan, consider pneumonia related to the cluster and notify infection control personnel and your local health department immediately. Although the etiology and transmission dynamics have yet to be determined, CDC recommends a cautious approach to symptomatic patients with a history of travel to Wuhan. Ask such patients to don a surgical mask as soon as they are identified. Conduct their evaluation in a private room with the door closed. Personnel entering the room to evaluate the patient should use contact precautions and wear an N95 disposable facepiece respirator. For patients admitted for inpatient care, implement contact and airborne isolation precautions, in addition to standard precautions, until further information becomes available. For additional infection control guidance see: https://www.cdc.gov/infectioncontrol/guidelines/isolation/index.html. https://wwwnc.cdc.gov/travel/notices/watch/pneumonia-china
  20. WHO Statement Regarding Cluster of Pneumonia Cases in Wuhan, China 9 January 2020 Statement China 中文 Chinese authorities have made a preliminary determination of a novel (or new) coronavirus, identified in a hospitalized person with pneumonia in Wuhan. Chinese investigators conducted gene sequencing of the virus, using an isolate from one positive patient sample. Preliminary identification of a novel virus in a short period of time is a notable achievement and demonstrates China’s increased capacity to manage new outbreaks. Initial information about the cases of pneumonia in Wuhan provided by Chinese authorities last week – including the occupation, location and symptom profile of the people affected – pointed to a coronavirus (CoV) as a possible pathogen causing this cluster. Chinese authorities subsequently reported that laboratory tests ruled out SARS-CoV, MERS-CoV, influenza, avian influenza, adenovirus and other common respiratory pathogens. Coronaviruses are a large family of viruses with some causing less-severe disease, such as the common cold, and others more severe disease such as MERS and SARS. Some transmit easily from person to person, while others do not. According to Chinese authorities, the virus in question can cause severe illness in some patients and does not transmit readily between people. Globally, novel coronaviruses emerge periodically in different areas, including SARS in 2002 and MERS in 2012. Several known coronaviruses are circulating in animals that have not yet infected humans. As surveillance improves more coronaviruses are likely to be identified. China has strong public health capacities and resources to respond and manage respiratory disease outbreaks. In addition to treating the patients in care and isolating new cases as they may be identified, public health officials remain focused on continued contact tracing, conducting environmental assessments at the seafood market, and investigations to identify the pathogen causing the outbreak. In the coming weeks, more comprehensive information is required to understand the current status and epidemiology of the outbreak, and the clinical picture. Further investigations are also required to determine the source, modes of transmission, extent of infection and countermeasures implemented. WHO continues to monitor the situation closely and, together with its partners, is ready to provide technical support to China to investigate and respond to this outbreak. The preliminary determination of a novel virus will assist authorities in other countries to conduct disease detection and response. Over the past week, people with symptoms of pneumonia and reported travel history to Wuhan have been identified at international airports. WHO does not recommend any specific measures for travellers. WHO advises against the application of any travel or trade restrictions on China based on the information currently available. https://www.who.int/china/news/detail/09-01-2020-who-statement-regarding-cluster-of-pneumonia-cases-in-wuhan-china
  21. https://www.ft.com/content/7ac8b004-3285-11ea-9703-eea0cae3f0de China says pneumonia outbreak linked to coronavirus State media says Sars-type virus likely cause as concern grows ahead of lunar new year holidays Health officials in Bangkok check passengers arriving from Wuhan, China © Lauren DeCicca/Getty Share on Twitter (opens new window) Share on Facebook (opens new window) Share on LinkedIn (opens new window) Save Tom Hancock in Shanghai 5 HOURS AGOPrint this page6 A pneumonia outbreak that has infected more than 50 people in the Chinese city of Wuhan was caused by a coronavirus, which is the same kind of pathogen involved in the deadly Sars outbreak in 2003, Chinese state media said on Thursday. The outbreak, which comes ahead of the lunar new year holidays in late January when millions of Chinese will be travelling to see their families, has caused alarm in the region. The virus has prompted widespread concern on Chinese social media and triggered memories of the 2003 outbreak of severe acute respiratory syndrome, or Sars, that infected more than 8,000 people worldwide and killed more than 700, including almost 300 in Hong Kong. The World Health Organization said, in a statement issued on Thursday, the Chinese authorities believed the disease “does not transmit readily between people”, but noted that it could cause severe illness in some patients. Chinese health officials said on Sunday that they had identified 59 cases, with seven patients in critical condition. There has been no further statement on the possible spread of the disease in China, nor on the health of those who were severely affected. However, state television said on Thursday that eight of those who had been hospitalised had been treated in Wuhan, “cured and discharged”. Hong Kong, activated its “serious” response mechanism to the disease last week and like other parts of the region has ramped up screening at airports. Hong Kong legislators have called for more transparency from mainland Chinese health authorities about the outbreak. Officials in China covered up the Sars outbreak for weeks before the growing death toll forced them to reveal the epidemic. International criticism of Beijing’s handling of the outbreak led to wide-ranging reforms to disease control methods in the country. Sophia Chan, Hong Kong’s food and health secretary said on Wednesday that there had been 38 suspected cases in the territory. However, she said none had been confirmed as related to the Wuhan outbreak, adding that 21 patients had already been discharged from hospital. Recommended Anjana Ahuja Pneumonia outbreak stirs debate over costly virus hunting The virus broke out between December 12 and 29. Some of those infected were employed at a market selling seafood and live animals in Wuhan that has since been closed for disinfection, according to Chinese disease control officials. “The reported link to a wholesale fish and live animal market could indicate an exposure link to animals,” the WHO said earlier this week. Coronaviruses in humans usually cause relatively harmless respiratory infections. However, two are deadly — Sars and Middle East respiratory syndrome (Mers), which have each caused hundreds of deaths. Additional reporting by Nicolle Liu in Hong Kong https://www.ft.com/content/7ac8b004-3285-11ea-9703-eea0cae3f0de
  22. Wuhan pneumonia outbreak: Mystery illness 'caused by coronavirus' 9 January 2020 Share Image copyrightGETTY IMAGES Scientists believe they may have found the cause of the mystery pneumonia that has infected almost 60 people in the Chinese city of Wuhan since December. Preliminary tests indicated the illness may be caused by a new coronavirus, lead scientist Xu Jianguo told the official Xinhua news agency. The World Health Organisation (WHO) had also said a coronavirus could be to blame for the outbreak. So far, 59 cases have been reported, seven of which are considered critical. Mr Xu, who is leading efforts to identify the cause, said they had found the "new type" of coronavirus by testing infected blood samples and throat swabs collected from 15 people. Mystery virus probed in Wuhan The people who risked their lives to stop the virus The coronavirus can cause anything from the common cold to much more severe diseases. Notably, it caused an epidemic of the potentially deadly, flu-like Sars virus back in 2002-3, which killed more than 700 people around the world after originating in China. However, Gauden Galea, the WHO representative to China, said "further investigations" were required to "determine the source, modes of transmission, extent of infection and countermeasures implemented". Singapore's airport says it will begin temperature screening travellers from Wuhan and Hong Kong health officials say they are also implementing checks on passengers. Authorities in Hong Kong have also stepped up the disinfection of trains and aeroplanes, AFP news agency reports. Fears the virus could be spread were further stoked by the fact it struck just before China's peak travel season, when hundreds of millions of people are set to travel for Chinese New Year later this month. But it is still unclear how the illness is transmitted, with health officials saying no cases of human-to-human transmission had been confirmed as yet. https://www.bbc.com/news/world-asia-china-51047576
  23. China's pneumonia outbreak caused by mystery new respiratory virus, authorities confirm Save Commuters wear protection masks inside a subway train in Hong Kong, which is on alert after reports of a new virus in Wuhan, China CREDIT: ANDY WONG/AP Anne Gulland, global health security deputy editor Sarah Newey 9 JANUARY 2020 • 11:07AM Follow Amystery new virus has been identified as the cause of a cluster of nearly 60 pneumonia cases in China which have put health authorities around the world on high alert. The infections, caused by a novel coronavirus, are reminiscent of the deadly severe acute respiratory syndrome (SARS) epidemic that spread from China in 2002, killing almost 800 people. But the Chinese authorities said yesterday that, although the virus was new, it did not appear to be spreading from human to human as SARS did. Xu Jianguo, the scientist who led the team that analysed the samples, said preliminary results showed that the newly detected virus - in the same family as both SARS and Middle East respiratory syndrome (MERS) - was different from other known human coronavirus species. Jonathan Ball, professor of molecular virology at the University of Nottingham, said that new viruses always posed a risk. “There’s always been a threat from coronaviruses,” he told The Telegraph. “These are viruses that are very widespread in lots of different animals and have shown the ability in the past to jump from animals into humans.” “It seems like this [new] virus is jumping into humans but not spreading on. But the worry is that if you have a new virus that is exploring a human host it’s possible that they might mutate and spread more easily in humans.” Other experts have added that it is too early to tell how dangerous China’s novel coronavirus is. “The biggest thing to ascertain is how transmissible the virus is between humans,” said Sarah Gilbert, professor of vaccinology at the Jenner Institute at Oxford University. “This will affect how easily the virus can be contained.” Laboratory tests on samples from patients found 15 positive results of a new type of coronavirus, which was isolated from one patient's samples, said Dr Xu. Coronaviruses are a large family of viruses that infect both animals and people. Six human coronaviruses species have so far been identified. SARS and MERS have proved the most serious to date, both causing widespread outbreaks and deaths. SARS first emerged in China in 2002, although it was initially covered up by authorities, and spread to 37 countries, infecting 8,000 people and killing 774. MERS emerged in the Middle East in 2012 and has infected around 2,500 people and led to 850 deaths. As of January 5, a total of 59 cases of the unexplained pneumonia had been reported in Wuhan, a city with a population of roughly 11 million and the capital of Hubei province. According to Chinese state media eight people have now been discharged from hospital. The Wuhan health commission said the infection broke out between December 12 and 29, with some of the patients employed at a city seafood market since closed for disinfection. The World Health Organization (WHO) confirmed the Chinese findings and said that the identification of a novel virus in a short period of time was a “notable achievement”. “According to Chinese authorities, the virus in question can cause severe illness in some patients and does not transmit readily between people,” said Dr Gauden Galea, WHO representative to China. He added that the WHO does not recommend any travel or travel or trade restrictions on China based on currently available information. But Hong Kong and Singapore are among the neighbouring territories which have introduced disease surveillance measures for travellers arriving from Wuhan. The picture remains far from complete and Dr Xu said that further research will be conducted to better understand the new coronavirus. It may take years for researchers to develop medicines and vaccines. https://www.telegraph.co.uk/global-health/science-and-disease/chinas-pneumonia-outbreak-caused-mystery-new-respiratory-virus/
  24. Reports indicate that some of the patients were vendors at the Wuhan South China Seafood City (South China Seafood Wholesale Market) where, in addition to seafood, chickens, bats, marmots, and other wild animals are sold, suggesting a possible zoonotic origin to the outbreak. https://emergency.cdc.gov/han/han00424.asp
  25. Published Date: 2020-01-08 23:19:25 Subject: PRO/AH/EDR> Undiagnosed pneumonia - China (HU) (07): official confirmation of novel coronavirus Archive Number: 20200108.6878869 UNDIAGNOSED PNEUMONIA - CHINA (HUBEI) (07): OFFICIAL CONFIRMATION OF NOVEL CORONAVIRUS ************************************************************************************** A ProMED-mail post http://www.promedmail.org ProMED-mail is a program of the International Society for Infectious Diseases http://www.isid.org [1] Date: 9 Jan 2020 Source; CCTV / Xinhua [in Chinese, machine translation] Preliminary progress in pathogen identification of unexplained viral pneumonia in Wuhan Original title: Experts say that the new coronavirus is an unknown cause of viral pneumonia in Wuhan ---------------------------------- A few days ago, on the issue of the pathogenic identification of unexplained viral pneumonia epidemic in Wuhan, the reporter interviewed Xu Jianguo, the leader of the preliminary assessment of pathogenic test results and a member of the Chinese Academy of Engineering. He said that the expert group believed that the pathogen of this unexplained case of viral pneumonia was initially determined to be a new coronavirus. Question 1: At present, what progress has been made in the pathogenic identification of unexplained viral pneumonia in Wuhan? Xu Jianguo [response]: As of 21:00 on [7 Jan 2020], a new coronavirus was detected in the laboratory, and the entire genome sequence of the virus was obtained. A total of 15 positive results of the new coronavirus were detected by nucleic acid detection methods. From 1 positive patient. The virus was isolated from the samples and showed a typical coronavirus appearance under an electron microscope. [Does this refer to 15 samples from 1 single patient? Or initially from 1 patient?] The expert group believes that the pathogen of this unexplained case of viral pneumonia was initially identified as a new type of coronavirus. Question 2: How is the pathogen identification carried out? Xu Jianguo [response]: The tissue laboratory used genomic sequencing, nucleic acid detection, virus isolation and other methods to perform pathogenic tests on patients' alveolar lavage fluid, throat swabs, blood and other samples. Question 3: What procedures are needed to identify the pathogen? Xu Jianguo [response]: To confirm the cause of an epidemic disease, usually the following points must be met: (1) Suspicious pathogens must be found in patients, and pathogenic nucleic acids can be detected in clinical samples of patients; (2) From clinical samples of patients The pathogen can be successfully isolated; (3) The isolated pathogen can cause the same disease symptoms after infecting the host animal. The patient's serum antibody titer during the recovery period increased 4-fold, which can help identify the pathogen. Finding the nucleic acid, genomic and antibody evidence of the pathogen from the patient can be done in a short time. Scientific research, such as pathogen isolation and pathogenicity identification, can take weeks. The development of specific drugs and vaccines against a new pathogen may take years to complete. Question 4: What is the next step? Xu Jianguo [response]: The expert group believes that the pathogen of this unexplained case of viral pneumonia is initially determined as a new type of coronavirus. The next step is to conduct expert research and judgement in combination with etiology research, epidemiological investigation and clinical manifestations. Question 5: What is a coronavirus? Xu Jianguo [response]: Coronavirus is a type of pathogen that mainly causes respiratory and intestinal diseases. There are many regularly arranged protrusions on the surface of this type of virus particle, and the entire virus particle is like an emperor's crown, hence the name "coronavirus". In addition to humans, coronaviruses can infect many mammals such as pigs, cattle, cats, dogs, marten, camels, bats, mice, hedgehogs, and various birds. So far, there are 6 known human coronaviruses. Four of these coronaviruses are more common in the population and are less pathogenic, generally causing only minor respiratory symptoms similar to the common cold. The other 2 coronaviruses - Severe Acute Respiratory Syndrome Coronavirus and Middle East Respiratory Syndrome Coronavirus, also known as SARS Coronavirus and MERS Coronavirus, can cause severe respiratory diseases. The new coronavirus that caused the epidemic is different from the human coronaviruses that have been discovered, and further understanding of the virus requires further scientific research. [Editor: Zhang Yuling Responsible editor: Wang Jingdong] [Byline: Qu Ting] -- Communicated by: ProMED-mail <[email protected]> ****** [2] Date: 8 Jan 2020 Source: NY Times [Edited] https://www.nytimes.com/2020/01/08/health/china-pneumonia-outbreak-virus.html China Identifies New Virus Causing Pneumonia-Like Illness ----------------------- The new coronavirus doesn't appear to be readily spread by humans, but researchers caution that more study is needed. Researchers in China have identified a new virus that is behind a mysterious pneumonialike illness that has sickened 59 people in Wuhan and caused a panic in the central Chinese region. There's no evidence that the virus, a coronavirus, is readily spread by humans, and it has not been tied to any deaths. But health officials in China and internationally are watching it carefully, as it comes from the same region where the deadly SARS epidemic broke out in the early 2000s. Experts said the pathogen that caused these unexplained pneumonia cases had been "initially identified" as a new coronavirus, China's state broadcaster, China Central Television, said on Thursday [9 Jan 2020]. They detected this virus among 15 of the people. The new coronavirus "is different from previous human coronaviruses that were previously discovered, and more scientific research is needed for further understanding," CCTV said in its report. Coronaviruses are a large family of viruses that infect animals and people. Some cause only the symptoms known as the common cold -although many other viruses also do that. The Wuhan government confirmed on [31 Dec 2020] that health authorities were treating dozens of cases of pneumonia of unknown cause. Symptoms of the new illness include high fever, difficulty breathing and lung lesions, the Wuhan health commission has said. No deaths have been reported, but 7 people have become critically ill. On Wednesday [8 Jan 2020], the local health commission said 8 people had been discharged. The revelations set off a panic in a country where the memory of the SARS outbreak remains fresh. SARS, a dangerous respiratory disease and also a coronavirus, spread from southern China in 2003 and infected more than 3000 people, killing 774. The new illness appeared just weeks before the Spring Festival, the country's biggest holiday, when hundreds of millions of people travel. The authorities have urged the public to be on alert for pneumonia-like symptoms like fevers, body aches and breathing difficulties. Until Thursday's [9 Jan 2020] announcement, it was not clear what was causing the illnesses in Wuhan. The World Health Organization said Wednesday [8 Jan 2020] that it had concluded that it was most likely a coronavirus. "More comprehensive information is required to confirm the pathogen," the W.H.O. said in a statement. Early reports on ProMED, a disease-alert service, said there was no evidence of human-to-human transmission. Last weekend, laboratory tests in China ruled out SARS; the deadly Middle East Respiratory Syndrome, or MERS; the flu; bird flu; adenoviruses; and other common pathogens that cause pneumonia. The initial cases of pneumonia were linked to workers at a market in Wuhan that sold live fish, animals and birds. Workers disinfected and shut down the market in Wuhan after the city health department said it traced many of the cases to it. Health authorities in Asia have stepped up screenings and isolated patients with flulike symptoms who have traveled to Wuhan. In Hong Kong, 8 people with fever and respiratory symptoms who had traveled recently to Wuhan were hospitalized as of Wednesday [8 Jan 2020]. In South Korea, the authorities said on Wednesday [8 Jan 2020] that they had put a Chinese woman under isolated treatment after she was found to have pneumonia after trips to China, including Wuhan. In Singapore, the authorities placed a Chinese girl with pneumonia in isolation because she had traveled to Wuhan, then said on Sunday [5 Jan 2020] that doctors had found that the child had a common childhood viral illness. Officials in Hong Kong have installed additional thermal imaging systems at its airport to monitor passengers coming from Wuhan, scanning for people with fevers. SARS is believed to have jumped to humans from live-animal markets. It was eventually traced to civet cats, raccoon dogs and some other species that were raised and slaughtered for the exotic food trade. The virus normally circulates in bats, and the animals may have gotten it from them, possibly from eating food contaminated by bat droppings. Most outbreaks of MERS, which appeared in 2012, have been traced to people who raise or sell camels, which are kept in the Middle East for meat, milk, racing, hauling cargo and as pets. Like SARS, it can jump from person to person, particularly in hospitals. Some patients infected many others after they were put on machines to help them breathe - the mechanisms helped spew viral particles into the air as they exhaled. Virologists want to know which patients tend to get the virus, what symptoms they show, what animals - if any - the virus has been found in and what behavior helped the virus jump from animals to humans. And they want to quickly figure out what countermeasures, such as antiviral drugs or breathing machines, help treat the illness. [Bylines: Sui-Lee Wee and Donald G. McNeil Jr.] -- Communicated by: ProMED-mail <[email protected]> [The above interview is further confirmation that the Wuhan Institute of Virology expert group identified a novel coronavirus [CoV]. The fact that this novel coronavirus was identified in 15 people is highly suggestive that at least 15 of the 59 suspected cases may have been infected by the same CoV. A question that comes to mind is whether Hong Kong could identify the novel coronavirus in those suspected cases with history of travel to Wuhan who presented with febrile respiratory illnesses but did not have etiologic agents identified (see Undiagnosed pneumonia - China (06): (HU) Hong Kong surveillance, USA CDC alert 20200108.6876648 and Undiagnosed pneumonia - China (04): (HU) Hong Kong surveillance 20200106.6874277 for details.). An updated line listing of suspected cases followed by Hong Kong can be found at: https://www.chp.gov.hk/files/pdf/enhanced_sur_pneumonia_wuhan_eng.pdf We await further information from knowledgeable sources on the detailed sequence(s) of the novel coronavirus and where on the phylogenetic tree this novel coronavirus sits, i.e., which other known coronaviruses are close relatives. There are many questions that remain, including whether this virus has been identified in any of the wildlife from this market. And more clinical and epidemiologic details on the suspected and presumably confirmed cases. And are other "undiagnosed pneumonia" cases in Wuhan being tested for this novel coronavirus? Making the sequence available as quickly as possible would allow diagnostic testing to be performed elsewhere and allow us to know whether this virus has appeared elsewhere in humans or other species. A map of China showing locations of major cities in China can be found at https://www.chinadiscovery.com/china-maps/city-maps.html. The HealthMap/ProMED map of China: http://healthmap.org/promed/p/155 - Mod.MPP] See Also Undiagnosed pneumonia - China (06): (HU) Hong Kong surveillance, USA CDC alert 20200108.6876648 Undiagnosed pneumonia - China (05): (HU) novel coronavirus identified 20200108.6877694 Undiagnosed pneumonia - China (04): (HU) Hong Kong surveillance 20200106.6874277 Undiagnosed pneumonia - China (03): (HU) updates, SARS, MERS ruled out, WHO, RFI 20200105.6872267 Undiagnosed pneumonia - China (02): (HU) updates, other country responses, RFI 20200103.6869668 Undiagnosed pneumonia - China (01): (HU) wildlife sales, market closed, RFI 20200102.6866757 2019 ---- Undiagnosed pneumonia - China: (HU) RFI 20191230.6864153 .................................................mpp/lm/mpp https://promedmail.org/promed-post/?id=6878869
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