We showed that severe acute respiratory syndrome coronavirus 2 is probably a novel recombinant virus. Its genome is closest to that of severe acute respiratory syndrome-related coronaviruses from ...horseshoe bats, and its receptor-binding domain is closest to that of pangolin viruses. Its origin and direct ancestral viruses have not been identified.
Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the ...29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission.
Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination.
Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs ...(SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species.
•313 SARSr-CoV genomes have been sequenced (274 from human, 18 civets and 47 bats).•SARSr-CoV genomes of bats in Yunnan possess highest nt identity to those from civets.•The origin of human SARS-CoV was probably a result of multiple recombination events.•Recombination from a number of SARSr-CoV ancestors in different horseshoe bat species.
We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of ...Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (βCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B βCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by βCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A βCoVs.
While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A βCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A βCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A βCoVs.
While a number of human coronaviruses are believed to be originated from ancestral viruses in bats, it remains unclear if bat coronaviruses are ready to cause direct bat-to-human transmission. Here, ...we report the isolation of a MERS-related coronavirus, Tylonycteris-bat-CoV-HKU4, from lesser bamboo bats. Tylonycteris-bat-CoV-HKU4 replicates efficiently in human colorectal adenocarcinoma and hepatocarcinoma cells with cytopathic effects, and can utilize human-dipeptidyl-peptidase-4 and dromedary camel-dipeptidyl-peptidase-4 as the receptors for cell entry. Flow cytometry, co-immunoprecipitation and surface plasmon resonance assays show that Tylonycteris-bat-CoV-HKU4-receptor-binding-domain can bind human-dipeptidyl-peptidase-4, dromedary camel-dipeptidyl-peptidase-4, and Tylonycteris pachypus-dipeptidyl-peptidase-4. Tylonycteris-bat-CoV-HKU4 can infect human-dipeptidyl-peptidase-4-transgenic mice by intranasal inoculation with self-limiting disease. Positive virus and inflammatory changes were detected in lungs and brains of infected mice, associated with suppression of antiviral cytokines and activation of proinflammatory cytokines and chemokines. The results suggest that MERS-related bat coronaviruses may overcome species barrier by utilizing dipeptidyl-peptidase-4 and potentially emerge in humans by direct bat-to-human transmission.
The discovery of Hp-BatCoV HKU25 bridges the evolutionary gap between MERS-CoV and existing bat viruses, and suggests that bat viruses may have evolved to generate MERS-CoV through modulation of the ...spike protein for binding to hDPP4.
Abstract
Although bats are known to harbor Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses, the role of bats in the evolutionary origin and pathway remains obscure. We identified a novel MERS-CoV-related betacoronavirus, Hp-BatCoV HKU25, from Chinese pipistrelle bats. Although it is closely related to MERS-CoV in most genome regions, its spike protein occupies a phylogenetic position between that of Ty-BatCoV HKU4 and Pi-BatCoV HKU5. Because Ty-BatCoV HKU4 but not Pi-BatCoV HKU5 can use the MERS-CoV receptor human dipeptidyl peptidase 4 (hDPP4) for cell entry, we tested the ability of Hp-BatCoV HKU25 to bind and use hDPP4. The HKU25-receptor binding domain (RBD) can bind to hDPP4 protein and hDPP4-expressing cells, but it does so with lower efficiency than that of MERS-RBD. Pseudovirus assays showed that HKU25-spike can use hDPP4 for entry to hDPP4-expressing cells, although with lower efficiency than that of MERS-spike and HKU4-spike. Our findings support a bat origin of MERS-CoV and suggest that bat CoV spike proteins may have evolved in a stepwise manner for binding to hDPP4.
Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ...ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.
Highlights • HRV was detected in 7.5% of archived NPA specimens from ICU patients. • Pulmonary and extrapulmonary complications of HRV were common in ICU patients. • There was a high rate of seizure ...among patients with HRV infection
Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen ...detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in Escherichia coli. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID
50
)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID
50
/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.
While dromedaries are the immediate animal source of Middle East Respiratory Syndrome (MERS) epidemic, viruses related to MERS coronavirus (MERS-CoV) have also been found in bats as well as ...hedgehogs. To elucidate the evolution of MERS-CoV-related viruses and their interspecies transmission pathway, samples were collected from different mammals in China. A novel coronavirus related to MERS-CoV,
hedgehog coronavirus HKU31 (
-HedCoV HKU31), was identified from two Amur hedgehogs. Genome analysis supported that
-HedCoV HKU31 represents a novel species under
, being most closely related to
CoV from European hedgehogs in Germany, with 79.6% genome sequence identity. Compared to other members of
,
-HedCoV HKU31 possessed unique non-structural proteins and putative cleavage sites at ORF1ab. Phylogenetic analysis showed that
-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012 were closely related to NeoCoV and BatCoV PREDICT from African bats in the spike region, suggesting that the latter bat viruses have arisen from recombination between CoVs from hedgehogs and bats. The predicted HKU31 receptor-binding domain (RBD) possessed only one out of 12 critical amino acid residues for binding to human dipeptidyl peptidase 4 (hDPP4), the MERS-CoV receptor. The structural modeling of the HKU31-RBD-hDPP4 binding interphase compared to that of MERS-CoV and
bat CoV HKU4 (
-BatCoV HKU4) suggested that HKU31-RBD is unlikely to bind to hDPP4. Our findings support that hedgehogs are an important reservoir of
, with evidence of recombination with viruses from bats. Further investigations in bats, hedgehogs and related animals are warranted to understand the evolution of MERS-CoV-related viruses.
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