Host Range And Structural Analysis Of Bat-origin RshSTT182/200 Coronavirus Binding To Human ACE2 And Its Animal Orthologs

Emerging and re-emerging infectious diseases pose significant challenges to global public health,economy and security.Over the past two decades,epidemics caused by three coronaviruses,sever acute respiratory syndrome coronavirus(SARSCoV),Middle East respiratory syndrome coronavirus(MERS-CoV),and sever acute respiratory syndrome coronavirus 2(SARS-CoV-2),have posed significant threats to global public health.As of February 2023,the onging SARS-CoV-2 pandemic has resulted in more than 700 million infections and 6.87 million deaths worldwide(https://www.who.int/emergencies/diseases/novel-coronavirus-2019).Bats are considered natural hosts for coronaviruses,and several studies suggest that coronaviruses are vulnerable to mutation and spillover events[1-4].Therefore,the surveillance of coronaviruses in bats is critical for understanding the potential risk of cross-species and zoonotic transmission,as well as for public health prevention and control.The RshSTT182 and RshSTT200 coronaviruses were identified in bat samples previously collected in Cambodia,expanding the geographic distribution of SARSCoV-2-related coronaviruses of Sarbecovirus subgenus[4].These viruses share 92.6%genome-wide identity and 84.3%amino acid identity of the receptor binding domain(RBD)of spike to SARS-CoV-2(both viruses have identical RBD sequence).Whether RshSTT182 and RshSTT 200 pose a potential risk of cross-species transmission to humans or coexisting animal hosts becomes an important scientific question.In this study,we found that the binding affinity to human angiotensin-converting enzyme 2(hACE2)of RshSTT182/200 RBD is at a low-affinity level(KD=18 μM),which was-3 orders of magnitude lower than that of the SARS-CoV-2 RBD(KD=18.9 nM).Next,we determined the structure of the RshSTT182/200 RBD in a complex with hACE2 and identified the key residues that influence receptor binding.We further found that the D487N mutation significantly enhances the binding affinity of the RshSTT182/200 RBD to hACE2;the amino acid deletion in the β4β5 loop of RshSTT182/200 RBD,also observed in many Sarbecoviruses,is an important factor affecting the interaction between RBD and hACE2.Furthermore,the pseudovirus infection experiments revealed that RshSTT182 and its mutant pseudoviruses displayed weak transduction efficiency into HeLa-hACE2 cells.These findings suggest that RshSTT182/200 still requires further evolution to break the interspecific barrier and spillover to humans.RshSTT182 and RshSTT200 were identified from 2010 bat samples by retrospective sequencing and are not representative of the current evolutionary status of coronavirus in Cambodia,which suggests that we should pay more attention to the evolution and prevalence of RshSTT182 and RshSTT200 with other coronaviruses in Cambodia.Invasion is a key step of viral infection and the interaction between virus and host cell receptors is a prerequisite for viral invasion.Evaluation of the binding between virus and host cell receptors can effectively assess the potential host range.To better understand the receptor binding spectrum of RshSTT182/200,we evaluated the interactions of RshSTT182/200 RBD with ACE2 of different species by flow cytometry and surface plasmon resonance(SPR),respectively.We evaluated the interactions of the RshSTT182/200 RBD with ACE2 orthologs from 39 animal species(including 18 bat species ACE2 orthologs)and found that the RshSTT182/200 RBD extensively recognized 21 out of the 39 species ACE2 orthologs,but affinities were generally weak,with most of at the micromolar level.And RshSTT182/200,as a bat-derived coronavirus,its RBD recognized only 5 out of 18 bat ACE2 orthologs,its bat receptor binding spectrum was narrower than that of SARS-CoV-2.In addition,RshSTT182 pseudovirus could effectively enter HeLa-fox ACE2 cells and HeLa-R.affinis ACE2 cells,suggesting that RshSTT182 and RshSTT200 can potentially infect susceptible animals with high-affinity ACE2 receptors.These results indicate the potential host range of RshSTT182/200 and the importance of surveillance of susceptible animal hosts.We also evaluated the cross-reactive immune response to RshSTT182/200 of serum samples from recovered COVID-19 convalescent and ZF2001? vaccinees as well as eight therapeutic monoclonal antibodies(MAb).We found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182/200 with degrees of reduction,indicating that SARS-CoV-2 induced immune responses have basic immune responses to bat-derived Sarbecoviruses RshSTT182 and RshSTT200.But RshSTT182 and RshSTT200 still had immune escape potential against the SARS-CoV-2 vaccine and MAb.These results indicate the significance of universal vaccine design and the development of prevention strategies for coronaviruses with pandemic potential.