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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. Cov-Lineages Boxes show 95% HPD credible intervals. Bioinformatics 22, 26882690 (2006). A new coronavirus associated with human respiratory disease in China. J. Virol. The SARS-CoV divergence times are somewhat earlier than dates previously estimated15 because previous estimates were obtained using a collection of SARS-CoV genomes from human and civet hosts (as well as a few closely related bat genomes), which implies that evolutionary rates were predominantly informed by the short-term SARS outbreak scale and probably biased upwards. 1c). The command line tool is open source software available under the GNU General Public License v3.0. First, we took an approach that relies on identification of mosaic regions (via 3SEQ14 v.1.7) that are also supported by PI signals19. Anderson, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. & Garry, R. F. The proximal origin of SARS-CoV-2. In Extended Data Fig. A new SARS-CoV-2 variant (B.1.1.523) capable of escaping immune protections SARS-like WIV1-CoV poised for human emergence. & Holmes, E. C. Recombination in evolutionary genomics. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estimated as 1948 (95% highest posterior density (HPD): 18791999), 1969 (95% HPD: 19302000) and 1982 (95% HPD: 19482009), indicating that the lineage giving rise to SARS-CoV-2 has been circulating unnoticed in bats for decades. 82, 48074811 (2008). Mol. J. Virol. We use three bioinformatic approaches to remove the effects of recombination, and we combine these approaches to identify putative non-recombinant regions that can be used for reliable phylogenetic reconstruction and dating. Med. Evol. 36) (RDP, GENECONV, MaxChi, Bootscan, SisScan and 3SEQ) and considered recombination signals detected by more than two methods for breakpoint identification. 16, e1008421 (2020). However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. 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. Five example sequences with incongruent phylogenetic positions in the two trees are indicated by dashed lines. 206298/Z/17/Z. Yuan, J. et al. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. ac, Root-to-tip (RtT) divergence as a function of sampling time for the three coronavirus evolutionary histories unfolding over different timescales (HCoV-OC43 (n=37; a) MERS (n=35; b) and SARS (n=69; c)). Zhang, Y.-Z. It is available as a command line tool and a web application. 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 . More evidence Pangolin not intermediary in transmission of SARS-CoV-2 In our second stage, we wanted to construct non-recombinant regions where our approach to breakpoint identification was as conservative as possible. The Artic Network receives funding from the Wellcome Trust through project no. 2, vew007 (2016). Global epidemiology of bat coronaviruses. TMRCA estimates for SARS-CoV-2 and SARS-CoV from their respective most closely related bat lineages are reasonably consistent for the different data sets and different rate priors in our analyses. In this study, we report the case of a child with severe combined immu presenting a prolonged severe acute respiratory syndrome coronavirus 2 infection. The presence in pangolins of an RBD very similar to that of SARS-CoV-2 means that we can infer this was also probably in the virus that jumped to humans. Proc. Pangolin relies on a novel algorithm called pangoLEARN. & Muhire, B. RDP4: Detection and analysis of recombination patterns in virus genomes. Wong, A. C. P., Li, X., Lau, S. K. P. & Woo, P. C. Y. 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. J. Virol. In our analyses of the sarbecovirus datasets, we incorporated the uncertainty of the sampling dates when exact dates were not available. The key to successful surveillance is knowing which viruses to look for and prioritizing those that can readily infect humans47. D.L.R. wrote the first draft of the manuscript, and all authors contributed to manuscript editing. and X.J. 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. Means and 95% HPD intervals are 0.080 [0.0580.101] and 0.530 [0.3040.780] for the patristic distances between SARS-CoV-2 and RaTG13 (green) and 0.143 [0.1090.180] and 0.154 [0.0930.231] for the patristic distances between SARS-CoV-2 and Pangolin 2019 (orange). 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. These rate priors are subsequently used in the Bayesian inference of posterior rates for NRR1, NRR2, and NRA3 as indicated by the solid arrows. Add entries for pangolin-data/-assignment 1.18.1.1 (, Really add a document on testing strategy. Maciej F. Boni, Philippe Lemey, Andrew Rambaut or David L. Robertson. Holmes, E. C., Rambaut, A. Effect of closure of live poultry markets on poultry-to-person transmission of avian influenza A H7N9 virus: an ecological study. Abstract. Pangolin was developed to implement the dynamic nomenclature of SARS-CoV-2 lineages, known as the Pango nomenclature. 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). Extensive diversity of coronaviruses in bats from China. SARS-CoV-2 and RaTG13 are also exceptions because they were sampled from Hubei and Yunnan, respectively. is funded by The National Natural Science Foundation of China Excellent Young Scientists Fund (Hong Kong and Macau; no. Proc. Another similarity between SARS-CoV and SARS-CoV-2 is their divergence time (4070years ago) from currently known extant bat virus lineages (Fig. CAS 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. Evol. The Sichuan (SC2018) virus appears to be a recombinant of northern/central and southern viruses, while the two Zhejiang viruses (CoVZXC21 and CoVZC45) appear to carry a recombinant region from southern or central China. Virological.org http://virological.org/t/ncov-2019-codon-usage-and-reservoir-not-snakes-v2/339 (2020). In the presence of time-dependent rate variation, a widely observed phenomenon for viruses43,44,52, slower prior rates appear more appropriate for sarbecoviruses that currently encompass a sampling time range of about 18years. Google Scholar. B., Weaver, S. & Sergei, L. Evidence of significant natural selection in the evolution of SARS-CoV-2 in bats, not humans. A counting renaissance: combining stochastic mapping and empirical Bayes to quickly detect amino acid sites under positive selection. 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. PubMed Phylogenies of subregions of NRR1 depict an appreciable degree of spatial structuring of the bat sarbecovirus population across different regions (Fig. M.F.B. Researchers in the UK had just set the scientific world . PureBasic 53 13 constellations Public Python 42 17 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. A distinct name is needed for the new coronavirus. Emergence of SARS-CoV-2 through recombination and strong purifying selection. Because there is no single accepted method of inferring breakpoints and identifying clean subregions with high certainty, we implemented several approaches to identifying three classic statistical signals of recombination: mosaicism, phylogenetic incongruence and excessive homoplasy51. These shy, quirky but cute mammals are one of the most heavily trafficked yet least understood animals in the world. All custom code used in the manuscript is available at https://github.com/plemey/SARSCoV2origins. Boni, M. F., Posada, D. & Feldman, M. W. An exact nonparametric method for inferring mosaic structure in sequence triplets. Using the most conservative approach (NRR1), the divergence time estimate for SARS-CoV-2 and RaTG13 is 1969 (95% HPD: 19302000), while that between SARS-CoV and its most closely related bat sequence is 1962 (95% HPD: 19321988); see Fig. It allows a user to assign a SARS-CoV-2 genome sequence the most likely lineage (Pango lineage) to SARS-CoV-2 query sequences. Posada, D., Crandall, K. A. Nature 503, 535538 (2013). Coronavirus: Pangolins may have spread the disease to humans Slider with three articles shown per slide. Regions AC were further examined for mosaic signals by 3SEQ, and all showed signs of mosaicism. Boni, M. F., Zhou, Y., Taubenberger, J. K. & Holmes, E. C. Homologous recombination is very rare or absent in human influenza A virus. Calibration of priors can be performed using other coronaviruses (SARS-CoV, MERS-CoV and HCoV-OC43), but estimated rates vary with the timescale of sample collection. Microbiol. Trends Microbiol. PDF How COVID-19 Variants Get Their Name - doh.wa.gov . Aside from RaTG13, Pangolin-CoV is the most closely related CoV to SARS-CoV-2. 1 Phylogenetic relationships in the C-terminal domain (CTD). Instead, similarity in codon usage metrics between the SARS-CoV-2 and eukaryotes analyzed was correlated with coding sequence GC content of the eukaryote, with more similar codon usage being identified in eukaryotes with low GC content similar to that of the coronavirus (b). Collectively our analyses point to bats being the primary reservoir for the SARS-CoV-2 lineage. 4. RegionsB and C span nt3,6259,150 and 9,26111,795, respectively. 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 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. Ji, W., Wang, W., Zhao, X., Zai, J. Note that six of these sequences fall under the terms of use of the GISAID platform. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. The canine viral genome was excluded from the Bayesian phylogenetic analyses because temporal signal analyses (see below) indicated that it was an outlier. Dudas, G., Carvalho, L. M., Rambaut, A. Nat. We focused on these three non-recombining regions/alignments for divergence time estimation; this avoids inappropriate modelling of evolutionary processes with recombination on strictly bifurcating trees, which can result in different artefacts such as homoplasies that inflate branch lengths and lead to apparently longer evolutionary divergence times. This statement informs us of the possibility that a virus has spilled over from a very rare and shy reptile-looking mammal . 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. Evol. Biol. Root-to-tip divergence as a function of sampling time for non-recombinant regions NRR1 and NRR2 and recombination-masked alignment set NRA3. https://doi.org/10.1038/s41564-020-0771-4, DOI: https://doi.org/10.1038/s41564-020-0771-4. Avian influenza a virus (H7N7) epidemic in The Netherlands in 2003: course of the epidemic and effectiveness of control measures. Divergence time estimates based on the three regions/alignments where the effects of recombination have been removed. Lond. 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. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Our most conservative approach attempted to ensure that putative NRRs had no mosaic or phylogenetic incongruence signals. Because these subclades had different phylogenetic relationships in regionD (Supplementary Fig. GARD identified eight breakpoints that were also within 50nt of those identified by 3SEQ. Many Git commands accept both tag and branch names, so creating this branch may cause unexpected behavior. Note that breakpoints can be shared between sequences if they are descendants of the same recombination events. BEAST inferences made use of the BEAGLE v.3 library68 for efficient likelihood computations. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist Unfortunately, a response that would achieve containment was not possible. Trends Microbiol. We thank all authors who have kindly deposited and shared genome data on GISAID. We used TreeAnnotator to summarize posterior tree distributions and annotated the estimated values to a maximum clade credibility tree, which was visualized using FigTree. Biol. Despite the high frequency of recombination among bat viruses, the block-like nature of the recombination patterns across the genome permits retrieval of a clean subalignment for phylogenetic analysis. 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 . Aiewsakun, P. & Katzourakis, A. Time-dependent rate phenomenon in viruses. J. Med. Evol. Genet. [12] 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. 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. Genetics 172, 26652681 (2006). Two exceptions can be seen in the relatively close relationship of Hong Kong viruses to those from Zhejiang Province (with two of the latter, CoVZC45 and CoVZXC21, identified as recombinants) and a recombinant virus from Sichuan for which part of the genome (regionB of SC2018 in Fig. & Andersen, K. G. Pandemics: spend on surveillance, not prediction. Novel Coronavirus (2019-nCoV) Situation Report 1, 21 January 2020 (World Health Organization, 2020). Next, we (1) collected all breakpoints into a single set, (2) complemented this set to generate a set of non-breakpoints, (3) grouped non-breakpoints into contiguous BFRs and (4) sorted these regions by length. Biol. Combining regions A, B and C and removing the five named sequences gives us putative NRR1, as an alignment of 63sequences. Liu, P. et al. Center for Infectious Disease Dynamics, Department of Biology, Pennsylvania State University, University Park, PA, USA, Department of Microbiology, Immunology and Transplantation, KU Leuven, Rega Institute, Leuven, Belgium, Department of Biological Sciences, Xian Jiaotong-Liverpool University, Suzhou, China, State Key Laboratory of Emerging Infectious Diseases, School of Public Health, The University of Hong Kong, Hong Kong SAR, China, Department of Biology, University of Texas Arlington, Arlington, TX, USA, Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK, MRC-University of Glasgow Centre for Virus Research, Glasgow, UK, You can also search for this author in Bruen, T. C., Philippe, H. & Bryant, D. A simple and robust statistical test for detecting the presence of recombination. RegionsAC had similar phylogenetic relationships among the southern China bat viruses (Yunnan, Guangxi and Guizhou provinces), the Hong Kong viruses, northern Chinese viruses (Jilin, Shanxi, Hebei and Henan provinces, including Shaanxi), pangolin viruses and the SARS-CoV-2 lineage. 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. N. China corresponds to Jilin, Shanxi, Hebei and Henan provinces, and the N. China clade also includes one sequence sampled in Hubei Province in 2004. 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. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage - Nature 94, e0012720 (2020). 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. PubMed An initial genomic sequence analysis found that the reemergence of COVID-19 in New Zealand was caused by a SARS-CoV-2 from the (now ancestral) lineage B.1.1.1 of the pangolin nomenclature ( 17 ). Evol. This provides compelling support for the SARS-CoV-2 lineage being the consequence of a direct or nearly-direct zoonotic jump from bats, because the key ACE2-binding residues were present in viruses circulating in bats. Methods Ecol. We compiled a set of 69SARS-CoV genomes including 58 sampled from humans and 11 sampled from civets and raccoon dogs. Except for specifying that sequences are linear, all settings were kept to their defaults. 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. 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). Region A has been shortened to A (5,017nt) based on potential recombination signals within the region. Extended Data Fig. All four of these breakpoints were also identified with the tree-based recombination detection method GARD35. If the latter still identified non-negligible recombination signal, we removed additional genomes that were identified as major contributors to the remaining signal. Extended Data 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. Researchers have found that SARS-CoV-2 in humans shares about 90.3% of its genome sequence with a coronavirus found in pangolins (Cyranoski, 2020). Biazzo et al. By mid-January 2020, the virus was spreading widely within Hubei province and by early March SARS-CoV-2 was declared a pandemic8. Conducting analogous analyses of codon usage bias as Ji et al. Cell 181, 223227 (2020). A dynamic nomenclature proposal for SARS-CoV-2 lineages to - PubMed The consistency of the posterior rates for the different prior means also implies that the data do contribute to the evolutionary rate estimate, despite the fact that a temporal signal was visually not apparent (Extended Data Fig. ISSN 2058-5276 (online). Mol. Extended Data Fig. Sarbecovirus, HCoV-OC43 and SARS-CoV data were assembled from GenBank to be as complete as possible, with sampling year as an inclusion criterion. DRAGEN COVID Lineage App This app aligns reads to a SARS-CoV-2 reference genome and reports coverage of targeted regions. Nature 558, 180182 (2018). A., Lytras, S., Singer, J. After removal of A1 and A4, we named the new region A. 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. G066215N, G0D5117N and G0B9317N)) and by the European Unions Horizon 2020 project MOOD (no. Individual sequences such as RpShaanxi2011, Guangxi GX2013 and two sequences from Zhejiang Province (CoVZXC21/CoVZC45), as previously shown22,25, have strong phylogenetic recombination signals because they fall on different evolutionary lineages (with bootstrap support >80%) depending on what region of the genome is being examined. and D.L.R. 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. Pangolins: What are they and why are they linked to Covid-19? - Inverse Phylogenetic Assignment of Named Global Outbreak Lineages 88, 70707082 (2014). Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China. Posterior distributions were approximated through Markov chain Monte Carlo sampling, which were run sufficiently long to ensure effective sampling sizes >100. Preprint at https://doi.org/10.1101/2020.04.20.052019 (2020). 4 we compare these divergence time estimates to those obtained using the MERS-CoV-centred rate priors for NRR1, NRR2 and NRA3. (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). The relatively fast evolutionary rate means that it is most appropriate to estimate shallow nodes in the sarbecovirus evolutionary history. These authors contributed equally: Maciej F. Boni, Philippe Lemey. 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. We thank originating laboratories at South China Agricultural University (Y. Shen, L. Xiao and W. Chen; no. cov-lineages/pangolin - GitHub Nature 583, 282285 (2020). master 4 branches 94 tags Code AngieHinrichs Add entries for pangolin-data/-assignment 1.18.1.1 ( #512) ad16752 4 days ago 990 commits .github/ workflows Update pangolin.yml 7 months ago docs docs need guide tree now 3 years ago pangolin Martin, D. P., Murrell, B., Golden, M., Khoosal, A. Mol. 21, 15081514 (2015). Nature 538, 193200 (2016). The rate of genome generation is unprecedented, yet there is currently no coherent nor accepted scheme for naming the expanding . Mol. The S1 protein of Pangolin-CoV is much more closely related to SARS-CoV-2 than to RaTG13. The authors declare no competing interests. 25, 3548 (2017). Patino-Galindo, J. B.W.P. Which animal did the novel coronavirus come from? | Live Science Posterior means with 95% HPDs are shown in Supplementary Information Table 2. 95% credible interval bars are shown for all internal node ages. 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. Stegeman, A. et al. D.L.R. Duchene, S. et al. Coronavirus: Pangolins found to carry related strains - BBC News The shaded region corresponds to the Sprotein. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Correspondence to And this genotype pattern led to creating a new Pangolin lineage named B.1.640.2, a phylogenetic sister group to the old B.1.640 lineage renamed B.1.640.1. Sorting these breakpoint-free regions (BFRs) by length results in two segments >5kb: an ORF1a subregion spanning nucleotides (nt) 3,6259,150 and the first half of ORF1b spanning nt13,29119,628 (sequence numbering given in Source Data, https://github.com/plemey/SARSCoV2origins). 23, 18911901 (2006). Curr. J. Virol. 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). Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? 13, e1006698 (2017). The web application was developed by the Centre for Genomic Pathogen Surveillance. "This is an extremely interesting . 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. Visual exploration using TempEst39 indicates that there is no evidence for temporal signal in these datasets (Extended Data Fig. Adv. Posterior means (horizontal bars) of patristic distances between SARS-CoV-2 and its closest bat and pangolin sequences, for the spike proteins variable loop region and CTD region excluding the variable loop. Li, X. et al. & Bedford, T. MERS-CoV spillover at the camelhuman interface. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. 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. Google Scholar. This boundary appears to be rarely crossed. PLoS Pathog. 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. Current Overview on Disease and Health Research Vol. 6 Extended Data Fig. RegionC showed no PI signals within it. Specifically, progenitors of the RaTG13/SARS-CoV-2 lineage appear to have recombined with the Hong Kong clade (with inferred breakpoints at 11.9 and 20.8kb) to form the CoVZXC21/CoVZC45-lineage. This new approach classifies the newly sequenced genome against all the diverse lineages present instead of a representative select sequences. Removal of five sequences that appear to be recombinants and two small subregions of BFRA was necessary to ensure that there were no phylogenetic incongruence signals among or within the three BFRs.