Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic

“2.8 (Stamatakis, 2014, 2006), GTR+Γ model, 100 bootstrap replicates – was inferred for each BFR longer than 500nt.”

“The latter was reconstructed using IQTREE (Nguyen et al., 2014 ) under a General-Time Reversible (GTR) model with a discrete gamma distribution to model among-site rate variation.”

“…thought to be instrumental to SARS-CoV-2’s ability to infect humans – a polybasic cleavage site insertion in the Spike protein – has been seen in another close bat relative of the SARS-CoV-2 virus (Zhou et al., 2020a); however, the sequence is different and it is likely an independent event.”

“In December 2019, a cluster of pneumonia cases epidemiologically linked to an open-air wet market in city of Wuhan (Hubei Province), China (Li et al., 2020a; Zhou et al., 2020b) led local health officials to issue an epidemiological alert to the Chinese Center for Disease Control and Prevention…”

“2.8 (Stamatakis, 2014, 2006), GTR+Γ model, 100 bootstrap replicates – was inferred for each BFR longer than 500nt.”

“Across a large region of its genome, corresponding approximately to ORF1b , it did not cluster with any of the known bat coronaviruses indicating that recombination likely played a role in the evolutionary history of these viruses (Lu et al., 2020; Wu et al.”

“When the first genome sequence of SARS-CoV-2, Wuhan-Hu-1, was released on January 10 2020 on Virological.org by a consortium led by Yong-Zhen Zhang (Wu et al., 2020a) it enabled the immediate analyses of its ancestry.”

“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.”

“Influenza viruses reassort (Webster et al., 1992) but they do not undergo homologous recombination within RNA segments (Boni et al., 2008, 2010), meaning that ‘origins’ questions for influenza outbreaks can always be reduced to origins questions for each of influenza’s eight RNA segments.”

“Interestingly, despite the lineage leading to SARS-CoV-2 acquiring residues in its Spike (S) protein's receptorbinding domain (RBD) permitting use of human ACE2 (Wan et al., 2020) and being closer to the pangolin virus than RaTG13 in this region (Lam et al., 2020 ) -a signal that suggests recombination -the divergence…”

“Specifically, using a formal Bayesian approach (Suchard et al., 2018) (see Methods ), we estimate a fast evolutionary rate (0.00169 subst/site/yr, 95% highest posterior density (HPD) interval [0.00131,0.00205]) for SARS viruses sampled over a limited time scale (1 year), a slower rate (0.00078…”

“Using a third consensus-based approach for identifying recombinant regions in individual sequences – with six different recombination detection methods in RDP5 (Martin et al., 2015) – gives a putative recombination-free alignment that we call non-recombinant alignment 3 ( NRA3 ) (see Methods ).”

“The key difficulty in inferring reliable evolutionary histories for coronaviruses is that their high recombination rate (Graham and Baric, 2010; Su et al., 2016) violates the assumption of standard phylogenetic approaches because different parts of the genome will have different origins.”

“In order to examine temporal signal in the sequenced data, we plotted root-totip divergence against sampling time using TempEst (Rambaut et al., 2016) based on a maximum likelihood tree.”

“Recent evidence has also identified pangolins as potential intermediate species for SARS-CoV-2 emergence or as a potential reservoir species themselves (Lam et al., 2020; Xiao et al., 2020).”

“Our results indicate the presence of a single lineage circulating in bats with properties that allowed it to infect human cells, as previously described for the bat sarbecoviruses related to the first SARSCoV lineage (Ge et al., 2013; Menachery et al., 2015, 2016).”

“Sequences were aligned by MAFTT (Katoh et al., 2009), with a final alignment length of 30927, and used in the analyses below.”

“%) incompatible splits in Neighbor-Nets (Bryant and Moulton, 2004), and (iii) a near-complete reduction of mosaic signal as identified by 3SEQ.”

“All four of these breakpoints were also identified with the tree-based recombination detection method GARD (Kosakovsky Pond et al., 2006).”

“We demonstrate that the sarbecoviruses circulating in horseshoe bats have complex recombination histories 5 / 25 permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al…”

“A hypothesis of snakes as intermediate hosts of SARS-CoV-2 was posited during the early epidemic phase (Ji et al., 2020), but we found no evidence of this (Anderson, 2020; Robertson, 2020); see Supplementary Section 3.”

“Early detection via genomics and sequencing was not possible during SE Asia's initial outbreaks of avian influenza H5N1 (1997, (2003) (2004) (Ge et al., 2013; Menachery et al., 2015 Menachery et al., , 2016 , appropriate measures should have been in place for outbreaks of novel coronaviruses.”

“…have acted as an intermediate species facilitating transmission to humans, the evidence is consistent with the virus having evolved in bats resulting in bat sarbecoviruses that can replicate in the upper respiratory tract of both humans and pangolins (Zhang and Holmes, 2020; Zhou et al., 2020a).”

“As 3SEQ is the most powerful the mosaic methods (Boni et al., 2007) , we used 3SEQ to identify the best supported breakpoint history for each potential child (recombinant) sequence in the data set.”

“The key difficulty in inferring reliable evolutionary histories for coronaviruses is that their high recombination rate (Graham and Baric, 2010; Su et al., 2016) violates the assumption of standard phylogenetic approaches because different parts of the genome will have different origins.”

“Unlike other viruses that have emerged in the past two decades, coronaviruses are highly recombinogenic (Forni et al., 2017; Hon et al., 2008; Lam et al., 2018).”

“Recent evidence has also identified pangolins as potential intermediate species for SARS-CoV-2 emergence or as a potential reservoir species themselves (Lam et al., 2020; Xiao et al., 2020).”

“Early detection via genomics and sequencing was not possible during SE Asia's initial outbreaks of avian influenza H5N1 (1997, (2003) (2004) (Ge et al., 2013; Menachery et al., 2015 Menachery et al., , 2016 , appropriate measures should have been in place for outbreaks of novel coronaviruses.”

“Individual sequences such as RpShaanxi2011, Guangxi GX2013, and two sequences from Zhejiang province (CoVZXC21/CoVZC45), as previously shown (Li et al., 2020b; Zhou et al., 2020a), show strong phylogenetic recombination signals, as they fall on different evolutionary lineages (with bootstrap…”

“With horseshoe bats currently the most plausible source for SARS-CoV-2, it is important to consider that sarbecoviruses circulate in a variety of horseshoe bat species with often widely overlapping species ranges (Wong et al., 2019 ).”

“When the first genome sequence of SARS-CoV-2, Wuhan-Hu-1, was released on January 10 2020 on Virological.org by a consortium led by Yong-Zhen Zhang (Wu et al., 2020a) it enabled the immediate analyses of its ancestry.”

“Over relatively shallow time-scales, such differences can primarily be explained by varying selective pressure with mildly deleterious variants being eliminated more strongly by purifying selection over longer time scales (Holmes, 2009; Holmes et al., 2016; Membrebe et al., 2019).”

“phylogenetic incongruence and excessive homoplasy (Posada et al., 2002) .”

“In outbreaks of zoonotic pathogens, identification of the infection source is crucial, as this may allow health authorities to separate human populations from the wild-life or domestic-animal reservoirs posing the zoonotic risk (Stegeman et al., 2004; Yu et al., 2013).”

“This is evidence for numerous recombination events occurring in the evolutionary history of the sarbecoviruses ; however, identifying all past events and their temporal order (Eden et al., 2013) is challenging.”

“BEAST inferences made use of the BEAGLEv3 library (Yres et al., 2019) for efficient likelihood computations.”

“, due to shared ancestral recombination events (all Dunn-Sidak corrected p < 4 × 10-4, 3SEQ (Lam et al., 2018)).”

“5 / 25 permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al., 2020a).”

“These differences reflect the fact that rate estimates can vary considerably with the time scale of measurement, a frequently observed phenomenon in viruses known as time-dependent evolutionary rates (Aiewsakun and Katzourakis, 2016; Duchene et al., 2014; Membrebe et al., 2019).”

“The key to successful surveillance is that we know which human-adapted viral phenotypes to look for (Holmes et al., 2018).”

“We demonstrate that the sarbecoviruses circulating in horseshoe bats have complex recombination histories 5 / 25 permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al…”

“As a proxy, it would be possible to model the long-term purifying selection dynamics as a major source of time-dependent rates rates (Duchene et al., 2014; Aiewsakun and Katzourakis, 2016; Membrebe et al., 2019), but this is beyond the scope of the current study.”

“Evolutionary rate estimation can be profoundly affected by the presence of recombination (Schierup and Hein, 1999).”

“We demonstrate that the sarbecoviruses circulating in horseshoe bats have complex recombination histories 5 / 25 permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al…”

“permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al., 2020a).”

“First, we took an approach that relies on identifying mosaic regions (via 3SEQ) that are also supported by phylogenetic incongruence (PI) signals (Boni et al., 2010) .”

“As a proxy, it would be possible to model the long-term purifying selection dynamics as a major source of time-dependent rates rates (Duchene et al., 2014; Aiewsakun and Katzourakis, 2016; Membrebe et al., 2019), but this is beyond the scope of the current study.”

“In such cases, even moderate rate variation among long deep phylogenetic branches will significantly impact expected root-to-tip divergences over a sampling time range that represents only a small fraction of the evolutionary history (Trova et al., 2015).”

“These differences reflect the fact that rate estimates can vary considerably with the time scale of measurement, a frequently observed phenomenon in viruses known as time-dependent evolutionary rates (Aiewsakun and Katzourakis, 2016; Duchene et al., 2014; Membrebe et al., 2019).”

“We demonstrate that the sarbecoviruses circulating in horseshoe bats have complex recombination histories 5 / 25 permitting generation of novel viral variants, as reported by others (He et al., 2014; Hon et al., 2008; Hu et al., 2017; Li et al., 2020b; Lin et al., 2017; Wang et al., 2017; Yuan et al., 2010; Zhou et al…”

“There is a dizzying diversity of RBD genotypes and phenotypes among sarbecoviruses within bat reservoirs (Boni et al., 2020; Demogines et al., 2012; Frank et al., 2020; Hu et al., 2017; Latinne et al., 2020; Letko et al., 2020; MacLean et al., 2020).”