*Naturally occurring indels in multiple coronavirus spikes* *Introduction* Proponents of theories for the unnatural origin of SARS-CoV-2 have asserted that the 12 nucleotide insert in the SARS-CoV-2 spike gene, which results in acquisition of a furin cleavage site, may have arisen by laboratory manipulation (Relman, 2020; Segreto and Deigin, 2020; Seyran et al., 2020; Sirotkin and Sirotkin, 2020). Here, wecompile evidence demonstrating that that insertion/deletion (indel) events at the S1/S2 and S2' protease cleavage sites of the spike precursor are commonly occurring natural features of coronavirus evolution. We also identifyheretofore undescribed similarities in the S1/S2 and S2' junctions of multiple diverse coronavirus spikes that provide further evidence again a laboratory origin of SARS-CoV-2. *Methods* The _Orthocoronaviridae_ includes four genuses, _Alpha-_, _Beta-_, _Gamma-_ and the newly described_Deltacoronavirus (Taxonomy)_. The _Betacoronavirus_ genus is further subdivided into six subgenuses, including the three subgenuses _Sarbecoviruses_, _Merbecoviruses and Embecoviruses_ with human pathogens. Weperformed an alignment of the S1/S2 junction of representative alpha-, beta-, and gammacoronaviruses (Fig. 1). Tofacilitate the identification of insertions we aligned conserved amino acids and other features that flank the S1/S2junctions, and included viruses that appear to be ancestral to the subgenuses where known. O-linked glycosylation sites were predicted by Net-O-Glyc v. 4.0 (Steentoft et al., 2013).*Results* While recent analyses suggest the existence of as yet unsampled sarbecovirus lineages, it is likely that SARS-CoV-2 and recently described coronaviruses isolated from pangolins share common ancestors with the bat sarbecovirus RaTG13 (Boni et al., 2020). As previously discussed (Andersen et al., 2020; Coutard et al., 2020;Gallaher, 2020a), alignment of the spike protein of SARS-CoV-2 with spike proteins of RaTG13 and other sarbecoviruses demonstrates that the 12 base insertion in the SARS-CoV-2 spike gene adds 4 amino acids (PRRA)at the S1/S2 junction and converts a monobasic cleavage site (R) to a minimal furin (polybasic) cleavage site(RRAR) (Fig. 1A). As previously described (Zhou et al., 2020), alignment of the spike of newly detected RmYN02 with other sarbecovirus spikes demonstrates that the S1/S2 junction of sarbecoviruses is variable. The neuralnetwork prediction algorithm Net-O-Glyc predicts that the S1/S2 junction of SARS-CoV-2 spike contains 3 O-linked glycans. The _Merbecovirus_ subgenus of the _Betacoronaviridae_ includes bat coronaviruses HKU4 and HKU5 as well asMiddle Eastern Respiratory Syndrome corononavirus (MERS-CoV), which infects camels and humans. Phylogenicanalyses place HKU4 at a basal position leading separately to the HKU5 and MERS-CoV lineages (Lau et al.,2013). Relative to the HKU4 spike, the HKU5 spike has an insertion of 3 amino acids (RFR) at the S1/S2 junction (Fig. 1B). This insertion generates an optimal furin cleavage site (RFRR). The S1/S2 junction of the HKU5 spike ispredicted to contain two O-linked glycans. Relative to HKU4, the MERS-CoV spike displays an insertion of 6 amino acids (LTPRSV). The insertion in the MERS-CoV spike produces a minimal furin cleavage site (RSVR), albeit without predicted O-linked glycans. The _Embecovirus_ subgenus of the _Betacoronaviridae_ includes the seasonal coronaviruses OC43 and HKU1.Of additional importance is Betacoronavirus 1, an embecovirus notable for its spread to a large number of diverse animal species (Corman et al., 2018). Betacoronavirus 1 is represented here by a bovine coronavirus. The rat embecovirus HKU24 is ancestral to each of these viruses (Lau et al., 2015). HKU24, OC43 and Betacoronavirus 1spikes have optimal furin cleavage sites (Fig. 1A). The junctional sequences in Betacoronavirus 1 and OC43 spikes are predicted to contain 4 and 2 O-linked glycans, respectively. Relative to HKU24 spike some variants of HKU1 spike have an insert of 6 amino acids (PSSSS) near the S1/S2 junction. Other variants have inserts of 2 amino acids (PS). It is unclear whether the HKU1 spike inserts have occurred independently or sequentially. HKU1 S1/S2junctions are predicted to contain 2 or 3 O-linked glycans. Mouse hepatitis virus (MHV), a well-studied embecovirus, further illustrates the natural variability in the S1/S2junction of betacornavirus spikes, with examples of monobasic, minimal furin and optimal furin cleavage sites existing in spike of various isolates (Fig. 1B). As with certain other betacoronaviruses, various serine and threonineresidues at the MHV spike S1/S2 junctions are predicted O-linked glycosylation sites. The MHV spike, like other coronavirus spikes undergoes additional proteolytic cleavages, including cleavage at a site referred to as S2'(Belouzard et al., 2009; Millet and Whittaker, 2014). Cleavage at S2' exposes a fusion peptide that interacts with ahost cell membrane permitting fusion with the viral envelope. Compared to the spike of ancestral embecovirus HKU24 the S2' site of MHV spike contains insertions of variable lengths, although deletions may also contribute tovariability at the S2' site in spike of different MHV variants. Furin cleavage sites are present in spikes of other coronaviruses. The S1/S2 junctions of feline alphacoronavirusspikes bear similarities to the corresponding region in sarbeco- and merbecovirus spikes. Feline alphacoronavirusesare divided into two different types (Jaimes et al., 2020). Type I feline coronavirus spikes have an optimal furin cleavage site, whereas Type II feline coronavirus spikes lack either a monobasic or polybasic cleavage site at theS1/S2 junction (Fig. 1C). Relative to Type II feline coronavirus spikes, the optimal furin cleavage sites in Type Ispikes are included in 17 or 18 amino acid insertion at the S1/S2 junction. The modification adds 2 or 3 predicted O-linked glycosylation sites. Type I feline coronavirus spikes have a monobasic cleavage site at S2', whereas feline coronaviruses have three amino acid indel at S2'. In some cases (for example strain KUK-HL) the S2' junction contains a minimal furin cleavage site.Gammacoronavirus spikes, including infectious bursal disease virus contain optimal furin cleavage sites at theS1/S2 junction (Fig. 1D). Some avian gammacoronavirus spikes have predicted O-linked glycans at the S1/S2cleavage site. Avian gammacoronavirus spikes display sequence variability at the S2' cleavage site; most are monobasic, but infectious bursal disease strain Beaudette spike has acquired an optimal furin cleavage site. The S2'site of Infectious bursal disease virus variant SES_15SK spike provides an example of a predicted O-linked glycosylation site that is associated with a monobasic cleavage site. Because the evolutionary history ofgammacoronaviruses is not well-described, it cannot be inferred whether or not evolution of their S1/S2 or S2' spike cleavage sites has involved insertions or deletions. *Discussion* Furin cleavage sites have been generated naturally in the spike proteins of members of at least 3 of 4orthocoronavirus genuses, including three betacoronavirus subgenuses, via insertions and/or deletions. Both theS1/S2 and S2' junction s of coronavirus spike genes are hot spots for RNA recombination. Furthermore, deletions, but not insertions, in the S1/S2 junction appear to arise commonly on serial passage of SARS-CoV-2 in cell culture, and have also been detected as quasi-species in infected humans (Lau et al., 2020; Liu et al., 2020). These observation stand in direct contrast to proponents of theories that SARS-CoV-2 has a nonnatural origin. A recent analysis by one of us (WRG) suggests that this variability is facilitated by short oligonucleotide "breakpointsequences" that direct recombination to certain positions in the genome (Gallaher, 2020b).Further evidence for the natural origin of the furin cleavage site in the SARS-CoV-2 spike is the observation that the indels in other coronavirus spikes often encode sequences with a propensity for O-linked glycosylation. As noted previously, computational prediction of O-linked glycosylation sites does not ensure that these sites are utilized bySARS-CoV-2 (Andersen et al., 2020). The sites may be used only in some cell types or under specific conditions ornot utilized at all. This caveat also applies to the current analyses. However, the frequency that the O-glycosylation sites are predicted across spike proteins from various genuses suggests that their presence in not due to chance.Mucin-like domains which contain O-linked glycans are characterized by an abundance of serines, theonines andprolines. These amino acids form turns in protein structures. Turns potentially contribute to accessibility of furin cleavage sites, and therefore may be under positive selection. The pattern of proline and serine/threonine residues in or near insertions of the polybasic residues, as noted inSARS-CoV-2, MERS-CoV, HKU1 and Type I feline coronaviruses, has not to our knowledge been previously discussed. With notable exceptions, including a predicted mucin-like patch in the carboxy terminal domain ofembecovirus spikes, predicted O-glycans are rare in coronavirus spikes other than near cleavage sites. Whilecomputational algorithms such as Net-O-Glyc are capable of determining sites that are likely to be O-glycosylated,this pattern would not have been obvious to anyone constructing SARS-CoV-2 in a laboratory, either for gain-of-function research or nefarious purposes.Previously one of us (WRG) presented additional strong evidence that the furin cleavage site insertion inSARS-CoV2 was generated via a natural process (Gallaher, 2020c). Although the 12 base insertion preserves the reading frame, the insertion is out-of-frame. It is highly implausible that any scientist attempting to insert a furin cleavage site would do so by making an out-of-frame insertion. Previous studies that introduced furin cleavage sitesin SARS-CoV and MERS-CoV spike genes did not introduce insertions (Follis et al., 2006; Yang et al., 2015). Nor isit likely that any laboratorian would have engineered a change in the SARS-CoV-2 spike that purposefully resulted inprediction of O-linked glycan sites.