Evolution of ACE2-independent SARS-CoV-2 infection and mouse adaption after passage in cells expressing human and mouse ACE2

Abstract

Human ACE2 (hACE2) is the key cell attachment and entry receptor for SARS-CoV-2, with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe the emergence of a SARS-CoV-2 strain capable of ACE2-independent infection, and evolution of mouse-adapted SARS-CoV-2 by in vitro serial passaging of virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses evolved with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused characteristic lung histopathology. One mouse-adapted virus also evolved to replicate efficiently in several ACE2-negative cell lines across several species, including CRISPR/Cas9 ACE2 knockout cells. An E484D substitution is likely involved in ACE2-independent entry and has appeared in only ≈0.003% of human isolates globally, suggesting it provided no significant selection advantage in humans. ACE2-independent entry reveals a SARS-CoV-2 infection mechanism that has potential implications for disease pathogenesis, evolution, tropism and perhaps also intervention development.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression

“…An alternative to using hACE2 transgenic mice is the generation of mouse-adapted viruses that can utilize mACE2, allowing infection of wild-type mice [ 62 – 68 ]. To compare the gene expression profile induced by mouse-adapted SARS-CoV-2 to that observed in humans, we analyzed previously published data derived from C57BL/6J mice infected with the mouse-adapted SARS-CoV-2 strain, MA1 [ 68 ] ( Table 1 ). Using an identical analysis pipeline to that used in the current study, Yan et al reported 1027 DEGs due to MA1 infection at 4 dpi, of which 825 were scoDEGs.…”

“…An alternative to using hACE2 transgenic mice is the generation of mouse-adapted viruses that can utilize mACE2, allowing infection of wild-type mice [62][63][64][65][66][67][68]. To compare the gene expression profile induced by mouse-adapted SARS-CoV-2 to that observed in humans, we…”

“…analyzed previously published data derived from C57BL/6J mice infected with the mouseadapted SARS-CoV-2 strain, MA1 [68] (Table 1). Using an identical analysis pipeline to that used in the current study, Yan et al reported 1027 DEGs due to MA1 infection at 4 dpi, of which 825 were scoDEGs.…”

“…These mice develop a robust respiratory disease that histologically resembles severe COVID-19 [19][20][21]. These mice have been widely used for evaluation of new interventions [22][23][24][25][26][27][28][29], and for virology and immunopathology studies [30][31][32]. However, SARS-CoV-2 infected K18-hACE2 mice show a number of differences [19], perhaps the most important difference is a fulminant brain infection that is associated with the generally lethal outcome in this model after infection with original strains of SARS-CoV-2 [33].…”

“…This mouse model of COVID-19 is generally less severe, with infections usually self-limiting and non-lethal [ 38 , 39 ]. A third model used a mouse-adapted strain of an original SARS-CoV-2 isolate, MA1, which is able to utilize mACE2 and is able to infect wild-type mice efficiently [ 32 ]. Herein we elucidate bioinformatic methods for validating mouse models by comparing the transcriptional responses of mouse lungs and human lungs/lung tissues after SARS-CoV-2 infection.…”