Search

Angiotensin-converting enzyme 2 (ACE2) is the primary entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but ACE2-independent entry has been observed in vitro for strains with the spike-E484D substitution. Here, we conduct a whole-genome CRISPR-Cas9 knockout screen using SARS-CoV-2 mouse adapted 1 (SARS-CoV-2 MA1 ), which carries spike-E484D, to identify the ACE2-independent entry mechanisms. SARS-CoV-2 MA1 infection in HEK293T cells relies on heparan sulfate and endocytic pathways, with TMEM106B, a transmembrane lysosomal protein, the most significant contributor. While SARS-CoV-2 MA1 productively infects human brain organoids and K18-hACE2 mouse brains, it does not infect C57BL/6J or Ifnar -/- mouse brains. This suggests that ACE2-independent entry via TMEM106B, which is predominantly expressed in the brain, does not overtly increase the risk of SARS-CoV-2 neuroinvasiveness in mice with endogenous Ace2 expression. Importantly, SARS-CoV-2 MA1 does not replicate in the Ace2 -/- mouse respiratory tract. Overall, this suggests that robust ACE2-independent infection by SARS-CoV-2 MA1 is likely an in vitro phenomenon with no apparent implications for infection in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1 -/- and Ifnar -/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients., Competing Interests: Declaration of interests The user committee for the RepliSAFE project included members from MSD Animal Health. MSD Animal Health provided TC-83 replicons and antibodies but otherwise provided no other resources or funding, had no input into the manuscript nor the decision to publish., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)