Hon, C. et al. In the variable-loop region, RaTG13 diverges considerably with the TMRCA, now outside that of SARS-CoV-2 and the Pangolin Guangdong 2019 ancestor, suggesting that RaTG13 has acquired this region from a more divergent and undetected bat lineage. All four of these breakpoints were also identified with the tree-based recombination detection method GARD35. =0.00025. The shaded region corresponds to the Sprotein. Evol. Unfortunately, a response that would achieve containment was not possible. MC_UU_1201412). Developed by the Centre for Genomic Pathogen Surveillance. On first examination this would suggest that that SARS-CoV-2 is a recombinant of an ancestor of Pangolin-2019 and RaTG13, as proposed by others11,22. 68, 10521061 (2019). Avian influenza a virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures. Lemey, P., Minin, V. N., Bielejec, F., Pond, S. L. K. & Suchard, M. A. 874850). 87, 62706282 (2013). Adv. Evidence of the recombinant origin of a bat severe acute respiratory syndrome (SARS)-like coronavirus and its implications on the direct ancestor of SARS coronavirus. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Given that these pangolin viruses are ancestral to the progenitor of the RaTG13/SARS-CoV-2 lineage, it is more likely that they are also acquiring viruses from bats. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Extended Data Fig. BFRs were concatenated if no phylogenetic incongruence signal could be identified between them. The coronavirus genome that these researchers had assembled, from pangolin lung-tissue samples, contained some gene regions that were ninety-nine per cent similar to equivalent parts of the SARS . PLoS Pathog. Because coronaviruses are known to be highly recombinant, we used three different approaches to identify non-recombinant regions for use in our Bayesian time-calibrated phylogenetic inference. CNN . Relevant bootstrap values are shown on branches, and grey-shaded regions show sequences exhibiting phylogenetic incongruence along the genome. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. From this perspective, it may be useful to perform surveillance for more closely related viruses to SARS-CoV-2 along the gradient from Yunnan to Hubei. Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China. PubMed https://doi.org/10.1038/s41564-020-0771-4, DOI: https://doi.org/10.1038/s41564-020-0771-4. At present, we analyzed the diversity of SARS-CoV-2 viral genomes in India to know the evolutionary patterns of viruses in the country through their pangolin lineage and GISAID-Clade. Nevertheless, the viral population is largely spatially structured according to provinces in the south and southeast on one lineage, and provinces in the centre, east and northeast on another (Fig. 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. 92, 433440 (2020). Posada, D., Crandall, K. A. 36)gives a putative recombination-free alignment that we call non-recombinant alignment3 (NRA3) (see Methods). This underscores the need for a global network of real-time human disease surveillance systems, such as that which identified the unusual cluster of pneumonia in Wuhan in December 2019, with the capacity to rapidly deploy genomic tools and functional studies for pathogen identification and characterization. It is available as a command line tool and a web application. Centre for Genomic Pathogen Surveillance. PubMed Central Microbiol. https://doi.org/10.1093/molbev/msaa163 (2020). Several of the recombinant sequences in these trees show that recombination events do occur across geographically divergent clades. Specifically, using a formal Bayesian approach42 (see Methods), we estimate a fast evolutionary rate (0.00169 substitutions per siteyr1, 95% highest posterior density (HPD) interval (0.00131,0.00205)) for SARS viruses sampled over a limited timescale (1year), a slower rate (0.00078 (0.00063,0.00092) substitutions per siteyr1) for MERS-CoV on a timescale of about 4years and the slowest rate (0.00024 (0.00019,0.00029) substitutions per siteyr1) for HCoV-OC43 over almost five decades. J. Virol. 35, 247251 (2018). The rate of genome generation is unprecedented, yet there is currently no coherent nor accepted scheme for naming the expanding . You are using a browser version with limited support for CSS. COVID-19 lineage names can be confusing to navigate; there are many aliases and if you want to catch them all to examine further in data analyses it helps to Allen O'Brien on LinkedIn: #r #rstudio #rstats #pangolin #covid19 #datascience #epidemiology (2020) with additional (and higher quality) snake coding sequence data and several miscellaneous eukaryotes with low genomic GC content failed to find any meaningful clustering of the SARS-CoV-2 with snake genomes (a). & Bedford, T. MERS-CoV spillover at the camelhuman interface. Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? Mol. The 2009 influenza pandemic and subsequent outbreaks of MERS-CoV (2012), H7N9 avian influenza (2013), Ebola virus (2014) and Zika virus (2015) were met with rapid sequencing and genomic characterization. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Region A has been shortened to A (5,017nt) based on potential recombination signals within the region. Thank you for visiting nature.com. & Andersen, K. G. The evolution of Ebola virus: insights from the 20132016 epidemic. The first available sequence data6 placed this novel human pathogen in the Sarbecovirus subgenus of Coronaviridae7, the same subgenus as the SARS virus that caused a global outbreak of >8,000 cases in 20022003. Kosakovsky Pond, S. L., Posada, D., Gravenor, M. B., Woelk, C. H. & Frost, S. D. W. Automated phylogenetic detection of recombination using a genetic algorithm. Figure 1 (top) shows the distribution of all identified breakpoints (using 3SEQs exhaustive triplet search) by the number of candidate recombinant sequences supporting them. Alexandre Hassanin, Vuong Tan Tu, Gabor Csorba, Nicola F. Mller, Kathryn E. Kistler & Trevor Bedford, Jack M. Crook, Ivana Murphy, Diana Bell, Simon Pollett, Matthew A. Conte, Irina Maljkovic Berry, Yatish Turakhia, Bryan Thornlow, Russell Corbett-Detig, Nature Microbiology These datasets were subjected to the same recombination masking approach as NRA3 and were characterized by a strong temporal signal (Fig. obtained the genome sequences of 10 SARS-CoV-2 virus strains through nanopore sequencing of nasopharyngeal swabs in Malta and analyzed the assembled genome with pangolin software, and the results showed that these virus strains were assigned to B.1 lineage, indicating that SARS-CoV-2 was widely spread in Europe (Biazzo et al., 2021). is funded by The National Natural Science Foundation of China Excellent Young Scientists Fund (Hong Kong and Macau; no. Nucleotide positions for phylogenetic inference are 147695, 9621,686 (first tree), 3,6259,150 (second tree, also BFR B), 9,26111,795 (third tree, also BFR C), 12,44319,638 (fourth tree) and 23,63124,633, 24,79525,847, 27,70228,843 and 29,57430,650 (fifth tree). However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. Influenza viruses reassort17 but they do not undergo homologous recombination within RNA segments18,19, meaning that origins questions for influenza outbreaks can always be reduced to origins questions for each of influenzas eight RNA segments. 17, 15781579 (1999). c, Maximum likelihood phylogenetic trees rooted on a 2007 virus sampled in Kenya (BtKy72; root truncated from images), shown for five BFRs of the sarbecovirus alignment. Posterior means with 95% HPDs are shown in Supplementary Information Table 2. performed codon usage analysis. Evol. Software package for assigning SARS-CoV-2 genome sequences to global lineages. RegionB showed no PI signals within the region, except one including sequence SC2018 (Sichuan), and thus this sequence was also removed from the set. These are in general agreement with estimates using NRR2 and NRA3, which result in divergence times of 1982 (19482009) and 1948 (18791999), respectively, for SARS-CoV-2, and estimates of 1952 (19061989) and 1970 (19321996), respectively, for the divergence time of SARS-CoV from its closest known bat relative. Mol. There is a 90% DNA match between SARS CoV 2 and a coronavirus in pangolins. Combining regions A, B and C and removing the five named sequences gives us putative NRR1, as an alignment of 63sequences. P.L. All custom code used in the manuscript is available at https://github.com/plemey/SARSCoV2origins. and X.J. 2). Although the human ACE2-compatible RBD was very likely to have been present in a bat sarbecovirus lineage that ultimately led to SARS-CoV-2, this RBD sequence has hitherto been found in only a few pangolin viruses. 6, eabb9153 (2020). Chernomor, O. et al. Novel Coronavirus (2019-nCoV) Situation Report 1, 21 January 2020 (World Health Organization, 2020). 4), but also by markedly different evolutionary rates. 3). RegionsB and C span nt3,6259,150 and 9,26111,795, respectively. B.W.P. Holmes, E. C., Rambaut, A. 3) clusters with viruses from provinces in the centre, east and northeast of China. By 2009, however, rapid genomic analysis had become a routine component of outbreak response. Pink, green and orange bars show BFRs, with regionA (nt 13,29119,628) showing two trimmed segments yielding regionA (nt13,29114,932, 15,40517,162, 18,00919,628). S. China corresponds to Guangxi, Yunnan, Guizhou and Guangdong provinces. Across a large region of the virus genome, corresponding approximately to ORF1b, it did not cluster with any of the known bat coronaviruses indicating that recombination probably played a role in the evolutionary history of these viruses5,7. To gauge the length of time this lineage has circulated in bats, we estimate the time to the most recent common ancestor (TMRCA) of SARS-CoV-2 and RaTG13. Evol. Duchene, S., Holmes, E. C. & Ho, S. Y. W. Analyses of evolutionary dynamics in viruses are hindered by a time-dependent bias in rate estimates. The estimated divergence times for the pangolin virus most closely related to the SARS-CoV-2/RaTG13 lineage range from 1851 (17301958) to 1877 (17461986), indicating that these pangolin lineages were acquired from bat viruses divergent to those that gave rise to SARS-CoV-2. A counting renaissance: combining stochastic mapping and empirical Bayes to quickly detect amino acid sites under positive selection. CAS In case of DRAGEN COVID Lineage tool, the minimum accepted alignment score was set to 22 and results with scores <22 were discarded. 13, e1006698 (2017). We extracted a total of 2189 full-length SARS-CoV-2 viral genomes from various states of India from the EpiCov repository of the GISAID initiative on 12 June 2020. # File containing the ID of the samples, the Sequence of the haplotype, the Continent, the country, the Region, the Data, the Lineage of Pangolin and Nextstrain clade, and the haplotype number # In this order # Could be obtained from the database Given what was known about the origins of SARS, as well as identification of SARS-like viruses circulating in bats that had binding sites adapted to human receptors29,30,31, appropriate measures should have been in place for immediate control of outbreaks of novel coronaviruses. Slider with three articles shown per slide. Split diversity in constrained conservation prioritization using integer linear programming. To examine temporal signal in the sequenced data, we plotted root-to-tip divergence against sampling time using TempEst39 v.1.5.3 based on a maximum likelihood tree. 3) to examine the sensitivity of date estimates to this prior specification. Liu, P. et al. The divergence time estimates for SARS-CoV-2 and SARS-CoV from their respective most closely related bat lineages are reasonably consistent among the three approaches we use to eliminate the effects of recombination in the alignment. Lam, H. M., Ratmann, O. RegionB is 5,525nt long. NTD, N-terminal domain; CTD, C-terminal domain. Green boxplots show the TMRCA estimate for the RaTG13/SARS-CoV-2 lineage and its most closely related pangolin lineage (Guangdong 2019). performed recombination analysis for non-recombining alignment3, calibration of rate of evolution and phylogenetic reconstruction and dating. Wong, A. C. P., Li, X., Lau, S. K. P. & Woo, P. C. Y. Lond. Extended Data Fig. The red and blue boxplots represent the divergence time estimates for SARS-CoV-2 (red) and the 2002-2003 SARS-CoV (blue) from their most closely related bat virus, with the light- and dark-colored versions based on the HCoV-OC43 and MERS-CoV centered priors, respectively. 5). Are you sure you want to create this branch? is funded by the MRC (no. Unlike other viruses that have emerged in the past two decades, coronaviruses are highly recombinogenic14,15,16. Since the release of Version 2.0 in July 2020, however, it has used the 'pangoLEARN' machine-learning-based assignment algorithm to assign lineages to new SARS-CoV-2 genomes. In December 2019, a cluster of pneumonia cases epidemiologically linked to an open-air live animal market in the city of Wuhan (Hubei Province), China1,2 led local health officials to issue an epidemiological alert to the Chinese Center for Disease Control and Prevention and the World Health Organizations (WHO) China Country Office. Intraspecies diversity of SARS-like coronaviruses in Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in humans. We find that the sarbecovirusesthe viral subgenus containing SARS-CoV and SARS-CoV-2undergo frequent recombination and exhibit spatially structured genetic diversity on a regional scale in China. We thank A. Chan and A. Irving for helpful comments on the manuscript. Using these breakpoints, the longest putative non-recombining segment (nt1,88521,753) is 9.9kb long, and we call this region NRR2. We say that this approach is conservative because sequences and subregions generating recombination signals have been removed, and BFRs were concatenated only when no PI signals could be detected between them. The extent of sarbecovirus recombination history can be illustrated by five phylogenetic trees inferred from BFRs or concatenated adjacent BFRs (Fig. Due to the absence of temporal signal in the sarbecovirus datasets, we used informative prior distributions on the evolutionary rate to estimate divergence dates. Below, we report divergence time estimates based on the HCoV-OC43-centred rate prior for NRR1, NRR2 and NRA3 and summarize corresponding estimates for the MERS-CoV-centred rate priors in Extended Data Fig. All authors contributed to analyses and interpretations. 4 TMRCAs for SARS-CoV and SARS-CoV-2. Conducting analogous analyses of codon usage bias as Ji et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Biol. As informative rate priors for the analysis of the sarbecovirus datasets, we used two different normal prior distributions: one with a mean of 0.00078 and s.d. 4), that region and shorter BFRs were not included in combined putative non-recombinant regions. Because the SARS-CoV-2 S protein has been implicated in past recombination events or possibly convergent evolution12, we specifically investigated several subregions of the Sproteinthe N-terminal domain of S1, the C-terminal domain of S1, the variable-loop region of the C-terminal domain, and S2. Concurrent evidence also proposed pangolins as a potential intermediate species for SARS-CoV-2 emergence and suggested them as a potential reservoir species11,12,13. Intragenomic rearrangements involving 5-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, Crystal structure of the CoV-Y domain of SARS-CoV-2 nonstructural protein 3, Association of underlying comorbidities and progression of COVID-19 infection amongst 2586 patients hospitalised in the National Capital Region of India: a retrospective cohort study, Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus, Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence. To employ phylogenetic dating methods, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Virology 507, 110 (2017). This statement informs us of the possibility that a virus has spilled over from a very rare and shy reptile-looking mammal . D.L.R. 3). Biol. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Sequence similarity. A single 3SEQ run on the genome alignment resulted in 67 out of 68sequences supporting some recombination in the past, with multiple candidate breakpoint ranges listed for each putative recombinant. In other words, a true breakpoint is less likely to be called as such (this is breakpoint-conservative), and thus the construction of a non-recombining region may contain true recombination breakpoints (with insufficient evidence to call them as such). In such cases, even moderate rate variation among long, deep phylogenetic branches will substantially impact expected root-to-tip divergences over a sampling time range that represents only a small fraction of the evolutionary history40. Researchers in the UK had just set the scientific world . It performs: K-mer based detection Map/align, variant calling Consensus sequence generation Lineage/clade analysis using Pangolin and NextClade Access the DRAGEN COVID Lineage App on BaseSpace Sequence Hub PubMed 5. One study suggests that over a century ago, one lineage of coronavirus circulating in bats gave rise to SARS-CoV-2, RaTG13 and a Pangolin coronavirus known as Pangolin-2019, Live Science . PubMed Central Duchene, S. et al. 5 (NRR1) are conservative in the sense that NRR1 is more likely to be non-recombinant than NRR2 or NRA3. Effect of closure of live poultry markets on poultry-to-person transmission of avian influenza A H7N9 virus: an ecological study. Many Git commands accept both tag and branch names, so creating this branch may cause unexpected behavior. Biol. All sequence data analysed in this manuscript are available at https://github.com/plemey/SARSCoV2origins. SARS-CoV-2 itself is not a recombinant of any sarbecoviruses detected to date, and its receptor-binding motif, important for specificity to human ACE2 receptors, appears to be an ancestral trait shared with bat viruses and not one acquired recently via recombination. Python 379 102 pangoLEARN Public Store of the trained model for pangolin to access. Eden, J.-S., Tanaka, M. M., Boni, M. F., Rawlinson, W. D. & White, P. A. Recombination within the pandemic norovirus GII.4 lineage. Early detection via genomics was not possible during Southeast Asias initial outbreaks of avian influenza H5N1 (1997 and 20032004) or the first SARS outbreak (20022003). Our approach resulted in similar posterior rates using two different prior means, implying that the sarbecovirus data do inform the rate estimate even though a root-to-tip temporal signal was not apparent. Membrebe, J. V., Suchard, M. A., Rambaut, A., Baele, G. & Lemey, P. Bayesian inference of evolutionary histories under time-dependent substitution rates. Bioinformatics 30, 13121313 (2014). In regionA, we removed subregion A1 (ntpositions 3,8724,716 within regionA) and subregion A4 (nt1,6422,113) because both showed PI signals with other subregions of regionA. 62,63), the GTR+ model and 100bootstrap replicateswas inferred for each BFR >500nt. & Boni, M. F. Improved algorithmic complexity for the 3SEQ recombination detection algorithm. It is RaTG13 that is more divergent in the variable-loop region (Extended Data Fig. Anderson, K. G. nCoV-2019 codon usage and reservoir (not snakes v2). 2, bottom) show that SARS-CoV-2 is unlikely to have acquired the variable loop from an ancestor of Pangolin-2019 because these two sequences are approximately 1015% divergent throughout the entire Sprotein (excluding the N-terminal domain). ISSN 2058-5276 (online). Using both prior distributions, this results in six highly similar posterior rate estimates for NRR1, NRR2 and NRA3, centred around 0.00055 substitutions per siteyr1. Lancet 383, 541548 (2013). Mol. We compiled a dataset including 27human coronavirus OC43 virus genomes and ten related animal virus genomes (six bovine, three white-tailed deer and one canine virus). Published. 11,12,13,22,28)a signal that suggests recombinationthe divergence patterns in the Sprotein do not show evidence of recombination between the lineage leading to SARS-CoV-2 and known sarbecoviruses. Extended Data Fig. This long divergence period suggests there are unsampled virus lineages circulating in horseshoe bats that have zoonotic potential due to the ancestral position of the human-adapted contact residues in the SARS-CoV-2 RBD. The ongoing pandemic spread of a new human coronavirus, SARS-CoV-2, which is associated with severe pneumonia/disease (COVID-19), has resulted in the generation of tens of thousands of virus genome sequences. These residues are also in the Pangolin Guangdong 2019 sequence. We considered (1) the possibility that BFRs could be combined into larger non-recombinant regions and (2) the possibility of further recombination within each BFR. Bioinformatics 28, 32483256 (2012). 82, 18191826 (2008). After removal of A1 and A4, we named the new region A. Time-measured phylogenetic reconstruction was performed using a Bayesian approach implemented in BEAST42 v.1.10.4. Li, X. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Even before the COVID-19 pandemic, pangolins have been making headlines. Scientists defined the pangolin lineage of this variant to be B.1.1.523 and it was originally recognized as a variant under monitoring on July 14, 2021. The presence of SARS-CoV-2-related viruses in Malayan pangolins, in silico analysis of the ACE2 receptor polymorphism and sequence similarities between the Receptor Binding Domain (RBD) of the spike proteins of pangolin and human Sarbecoviruses led to the proposal of pangolin as intermediary. Virus Evol. 1 Phylogenetic relationships in the C-terminal domain (CTD). All three approaches to removal of recombinant genomic segments point to a single ancestral lineage for SARS-CoV-2 and RaTG13. J. Med Virol. The most parsimonious explanation for these shared ACE2-specific residues is that they were present in the common ancestors of SARS-CoV-2, RaTG13 and Pangolin Guangdong 2019, and were lost through recombination in the lineage leading to RaTG13. Two other bat viruses (CoVZXC21 and CoVZC45) from Zhejiang Province fall on this lineage as recombinants of the RaTG13/SARS-CoV-2 lineage and the clade of Hong Kong bat viruses sampled between 2005 and 2007 (Fig. The web application was developed by the Centre for Genomic Pathogen Surveillance. Coronavirus: Pangolins found to carry related strains. We aimed to analyze 3 naso-oropharyngeal swab samples collected between August and December 2021 to describe the amino acid changes present in the sequence reads that may have a role in the emergence of new . 1a-c ), has the third-highest number of confirmed COVID-19 cases in the state of So. Proc. Abstract. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. We infer time-measured evolutionary histories using a Bayesian phylogenetic approach while incorporating rate priors based on mean MERS-CoV and HCoV-OC43 rates and with standard deviations that allow for more uncertainty than the empirical estimates for both viruses (see Methods). In outbreaks of zoonotic pathogens, identification of the infection source is crucial because this may allow health authorities to separate human populations from the wildlife or domestic animal reservoirs posing the zoonotic risk9,10. Sibling lineages to RaTG13/SARS-CoV-2 include a pangolin sequence sampled in Guangdong Province in March 2019 and a clade of pangolin sequences from Guangxi Province sampled in 2017. Furthermore, the other key feature thought to be instrumental in the ability of SARS-CoV-2 to infect humansa polybasic cleavage site insertion in the Sproteinhas not yet been seen in another close bat relative of the SARS-CoV-2 virus. For the current pandemic, the novel pathogen identification component of outbreak response delivered on its promise, with viral identification and rapid genomic analysis providing a genome sequence and confirmation, within weeks, that the December 2019 outbreak first detected in Wuhan, China was caused by a coronavirus3. Syst. J. Virol. Bryant, D. & Moulton, V. Neighbor-Net: an agglomerative method for the construction of phylogenetic networks. We used an uncorrelated relaxed clock model with log-normal distribution for all datasets, except for the low-diversity SARS data for which we specified a strict molecular clock model. & Muhire, B. RDP4: Detection and analysis of recombination patterns in virus genomes. USA 113, 30483053 (2016). A tag already exists with the provided branch name. 90, 71847195 (2016). Ge, X. et al. Katoh, K., Asimenos, G. & Toh, H. in Bioinformatics for DNA Sequence Analysis (ed. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Suchard, M. A. et al. Of importance for future spillover events is the appreciation that SARS-CoV-2 has emerged from the same horseshoe bat subgenus that harbours SARS-like coronaviruses. Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China. Pangolin was developed to implement the dynamic nomenclature of SARS-CoV-2 lineages, known as the Pango nomenclature. For coronaviruses, however, recombination means that small genomic subregions can have independent origins, identifiable if sufficient sampling has been done in the animal reservoirs that support the endemic circulation, co-infection and recombination that appear to be common. SARS-CoV-2 and RaTG13 are also exceptions because they were sampled from Hubei and Yunnan, respectively. The research leading to these results received funding (to A.R. CAS The difficulty in inferring reliable evolutionary histories for coronaviruses is that their high recombination rate48,49 violates the assumption of standard phylogenetic approaches because different parts of the genome have different histories.