SARS-CoV-2 Evolution in Humans Enables Its Transmission to Nonhuman Primates

Zoonotic viruses frequently cross-species barriers, but the molecular processes enabling reverse zoonosis remain poorly defined. The COVID-19 pandemic provided an unprecedented opportunity to track SARS-CoV-2 evolution in humans and its capacity to infect nonhuman primates. Our earlier analyses of primate ACE2 sequences identified key substitutions that confer resistance to wild-type (WT) SARS-CoV-2 and are conserved in New World monkeys (NWMs), establishing a strong species barrier. Using pseudovirus assays with HeLa cells expressing New World monkey ACE2 (nwmACE2), we show that while WT and Alpha strains could not infect, later variants such as Delta, Omicron, and especially the XBB lineage (XBB.1.5, EG.5.1) acquired robust infectivity toward nwmACE2. More recent lineages (JN.1, LB.1, KP.3.1.1) displayed reduced but persistent cross-species infectivity. Molecular dynamics simulations and a combined mutation prioritization framework converged key receptor-binding domain residues (including N405, N417, R452, N477, K478, A484, P486, S490, R498, and Y501) as critical contributors of adaptation to NWM receptors. These mutations act synergistically to overcome ACE2 barriers in NWMs, correlating with documented natural infections in the field. Collectively, our findings demonstrate that human-driven viral evolution has progressively enhanced the potential of SARS-CoV-2 for cross-species transmission to nonhuman primates, underscoring the need for ongoing surveillance to mitigate future reverse zoonosis crises.