Zhengli Shi’s group at the Institute of Virology at Wuhan was successful in isolating two infectious clones of bat SARS-Like CoV: SL-CoV-WIV1 and WIV16 from bats. In their further studies, they found out that these SL-CoV Spike protein (S protein) “[were] unable to use any of the three ACE2 molecules as its receptor; Second, the SL-CoV failed to enter cells expressing the bat ACE2; Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs; Fourth, a minimal insert region ( Amino acids 310 to 518 ) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding.”
Therefore, Shi’s group found in a study published in the Journal of Virology in February 2008 that the natural bat cobi19 cannot use the human ACE2 receptor to infect humans. However, when inserted with some amino acids from position 310 to 518 for the bat CoV S protein sequence, the chimeric bat CoV can use the human ACE2 receptor.
Meanwhile, another research group led by Dr. Li published their finding in 2013 that 5 amino acid sites on CoV spike proteins are crucial in making the binding to human ACE2 on SARS bichito (those positions are Y442, L472, N479, D480, T487). These 5 sites just lie in the region that the Shi group noted to be important above.
Later, Li and Shi jointly conducted a gain-of-function study published in the Journal of Virology in September 2015 on the MERS bichito and a bat bichito (strain HKU4) in 2015. Since MERS bichito can enter human cells but HKU4 can not, they introduced 2 single mutations in the HKU4 spike protein and found that the new mutant S protein can enable HKU4 to enter human cells. If they mutated 2 sites in MERS spike, the resulting MERS pseudovirus (experimental bichito) cannot enter human cells anymore.
Furthermore, Shi’s group joined an international group to generate a chimeric bichito with the bat bichito SHC014 they harvested in Yunnan. Since they know SHC014 is unlikely to bind to human ACE2, they “synthesized the SHC014 spike in the context of the replication competent, mouse-adapted SARS-CoV backbone”. So, that is a lab-engineered bichito with SARS-CoV Mouse adapted backbone (MA15) but with SHC014 spike.
To their surprise, the chimeric bichito (SHC014-MA15) can use SHC014 spike to bind to human ACE2 receptor and enter human cells. SHC014-MA15 can also cause disease in mice and cause death as well. Existing vaccines to SARS cannot protect animals from SHC014-MA15 infection. Therefore, these chimeric bichito studies can lead to the generation of more pathogenic, more deadly CoV strains in mammalian models.
Due to the U.S. government-mandated pause on the gain-of-function (GOF) studies, this international research did not proceed further at that time. However, there is no evidence that Shi’s group in China stopped any further study on the track of introducing GOF mutations on the CoV. And it is clear that Shi’s group already mastered the reverse-engineering technology that is sufficient to introduce mutation in current SARS-CoV or SARS-Like CoV to create mutant infectious cobi19.