Genetic diversity of pangolin coronaviruses reveals a key immuno-evasive substitution at spike residue 519.

Malayan pangolins are unusual hosts for several SARS-CoV-2-related coronaviruses, which have previously been known to only infect Rhinolophus bats. Much debate has hence surrounded their possible role as intermediate hosts in the emergence of SARS-CoV-2, but the virological phenotypes of most pangolin coronaviruses (pCoVs) remain unclear. Here, we comprehensively analyze all pCoVs to date identified from trafficked pangolins seized in the Guangdong province of China, which are remarkably similar to SARS-CoV-2 in the spike (S) protein. We explore the genetic diversity within these viruses and uncover how this diversity translates to different virological phenotypes. Strikingly, several Guangdong pCoVs harbor a lysine substitution at residue 519 of the S protein, which contributes to marked immune evasion by modulating the conformational state of the S protein. Furthermore, we highlight that a divergent immuno-evasive mutation at residue 519 of the S protein was acquired by SARS-CoV-2. These findings support that pangolin- and human-infecting coronaviruses likely represent independent spillover events from natural bat reservoirs, and that immuno-evasive mutations at residue 519 may be a common direction of viral evolution in coronaviruses that infect non-bat hosts.IMPORTANCEPangolins are frequently moved through illegal wildlife trade, creating opportunities for animal viruses to cross borders and encounter people. Guangdong pangolin coronaviruses are genetically close to SARS-CoV-2, particularly in spike, but their biological properties have been poorly defined. By analyzing all the available spike sequences of Guangdong pangolin coronaviruses and testing representative spikes in functional assays, we show that closely related pangolin coronaviruses can differ substantially in susceptibility to antibody neutralization. Notably, a single substitution at spike residue 519 can shift this phenotype by altering spike conformational dynamics, supporting the idea that residue 519 has been repeatedly targeted during adaptation outside bat reservoirs. These findings highlight spike residue 519 as a practical molecular marker to help flag immune-evasive, spillover-prone sarbecoviruses and to prioritize surveillance at wildlife-trade interfaces.