Abstract: bioRxiv preprint doi: https://doi.org/10.1101/2021.12.31.474653; this version posted January 3, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry. Thomas P. Peacock1#, Jonathan C. Brown1#, Jie Zhou1#, Nazia Thakur2, Joseph Newman2, Ruthiran Kugathasan1, Ksenia Sukhova1, Myrsini Kaforou1, Dalan Bailey2, and Wendy S. Barclay1* # These authors contributed equally to this work Department of Infectious Disease, Imperial College London, UK, W2 1PG 1 The Pirbright Institute, Woking, Surrey, UK, GU24 0NF 2 *Corresponding author: w.barclay@imperial.ac.uk Abstract At the end of 2021 a new SARS-CoV-2 variant, Omicron, emerged and quickly spread across the world. It has been demonstrated that Omicron’s high number of Spike mutations lead to partial immune evasion from even polyclonal antibody responses, allowing frequent re-infection and vaccine breakthroughs. However, it seems unlikely these antigenic differences alone explain its rapid growth; here we show Omicron replicates rapidly in human primary airway cultures, more so even than the previously dominant variant of concern, Delta. Omicron Spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than other variants. Omicron Spike mediates enhanced entry into cells expressing several different animal ACE2s, including various domestic avian species, horseshoe bats and mice suggesting it has an increased propensity for reverse zoonosis and is more likely than previous variants to establish an animal reservoir of SARS-CoV-2. Unlike other SARSCoV-2 variants, however, Omicron Spike has a diminished ability to induce syncytia formation. Furthermore, Omicron is capable of efficiently entering cells in a TMPRSS2-independent manner, via the endosomal route. We posit this enables Omicron to infect a greater number of cells in the respiratory epithelium, allowing it to be more infectious at lower exposure doses, and resulting in enhanced intrinsic transmissibility. bioRxiv preprint doi: https://doi.org/10.1101/2021.12.31.474653; this version posted January 3, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.