Fauci Intelligence Community Release — page 45

"lineage B" betacoronaviruses, although other human betacoronaviruses, including HKU1 (lineage A), have them and predicted O-linked glycans​13​. Given the level of genetic variation in the spike it is likely that SARS-CoV-2-like viruses with partial or full polybasic cleavage sites will be discovered in other species. The functional consequence of the polybasic cleavage site in SARS-CoV-2 is unknown and it will be important to determine its impact on transmissibility and pathogenesis in animal models. Experiments with SARS-CoV have shown that insertion of a furin cleavage site at the S1/S2 junction enhances cell-cell fusion without affecting virus entry​14​. In addition, efficient cleavage of the MERS-CoV spike enables MERS-like coronaviruses from bats to infect human cells​15​. In avian influenza viruses, rapid replication and transmission in highly dense chicken populations selects for the acquisition of polybasic cleavage sites in the haemagglutinin (HA) protein​16​, which serves a similar function as the coronavirus spike protein. Acquisition of polybasic cleavage sites in HA, by insertion or recombination, converts low pathogenicity avian influenza viruses into highly pathogenic forms​16​. The acquisition of polybasic cleavage sites by HA has also been observed after repeated passage in cell culture or through animals​17​. The function of the predicted O-linked glycans is unclear, but they could create a "mucin-like domain" shielding epitopes or key residues on the SARS-CoV-2 spike protein​18​. Several viruses employ mucin-like domains as glycan shields involved in immune evasion​18​. Although prediction of O-linked glycosylation is robust, experimental studies are required to determine if these sites are utilized in SARS-CoV-2. Theories of SARS-CoV-2 origins It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-like coronavirus. As noted above, the RBD of SARS-CoV-2 is optimized for human ACE2 binding with an efficient solution different from those previously predicted​7,11​. Further, had genetic manipulation had been performed, one of the several reverse genetic systems available for betacoronaviruses would likely have been used​19​. However, the genetic data irrefutably show that SARS-CoV-2 is not derived from any previously used virus backbone​20​. Instead, we propose two scenarios that can plausibly explain the origin of SARS-CoV-2: (​i​) natural selection in an animal host prior to zoonotic transfer, and (​ii​) natural selection in humans following zoonotic transfer. We also discuss whether selection during passage could have given rise to SARS-CoV-2. 1. Natural selection in an animal host prior to zoonotic transfer As many early cases of COVID-19 were linked to the Huanan market in Wuhan​1,2​, it is possible that an animal source was present at this location. Given the similarity of SARS-CoV-2 to bat SARS-like coronaviruses​2​, it is likely that bats serve as reservoir hosts for its progenitor. Although RaTG13, sampled from a ​Rhinolophus affinis bat​1​, is ~96% identical overall to SARS-CoV-2, its spike diverges in the RBD suggesting that it may not bind efficiently to the human ACE2 receptor (​Fig. 1a​)​7​. Malayan pangolins (​Manis javanica​) illegally imported into Guangdong province contain coronaviruses similar to SARS-CoV-2​21​. Although the RaTG13 bat virus remains the closest relative to SARS-CoV-2 across the genome​1​, some pangolin coronaviruses exhibit strong similarity to SARS-CoV-2 in the RBD, including all six key RBD residues (​Fig. 1​)​21​. This clearly shows that the SARS-CoV-2 spike protein optimized for binding to human-like ACE2 is the result of natural selection. Neither the bat nor pangolin betacoronaviruses sampled to date have polybasic cleavage sites. Although no animal coronavirus has been identified that is sufficiently similar to have served as the direct SARS-CoV-2 progenitor, the diversity of coronaviruses in bats and other species is massively undersampled. Mutations, insertions and deletions, can occur near the S1/S2 junction of coronaviruses​22 showing that the polybasic cleavage site can arise by a natural evolutionary process. For a precursor virus to acquire both the polybasic cleavage site and mutations in the spike protein suitable for human ACE2 receptor binding, an animal host would likely have to have a high population density - to allow natural selection to proceed efficiently - and an ACE2 gene that is similar to the human orthologue. 2. Natural selection in humans following zoonotic transfer It is possible that a progenitor to SARS-CoV-2 jumped into humans, acquiring the genomic features described above through adaptation during undetected human-to-human transmission. Once acquired,