Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection (preprint)

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kinetics in vivo using reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualization in vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualized in vivo infection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.

Virological characteristics of the SARS-CoV-2 XBB.1.5 variant (preprint)

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants (preprint)

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

Decomposing the sources of SARS-CoV-2 fitness variation in the United States (preprint)

The fitness of a pathogen is composite phenotype determined by many different factors influencing growth rates both within and between hosts. Determining what factors shape fitness at the host population-level is especially challenging because both intrinsic factors like pathogen genetics and extrinsic factors such as host behaviour influence between-host transmission potential. These challenges have been highlighted by controversy surrounding the population-level fitness effects of mutations in the SARS-CoV-2 genome and their relative importance when compared against non-genetic factors shaping transmission dynamics. Building upon phylodynamic birth-death models, we develop a new framework to learn how hundreds of genetic and non-genetic factors have shaped the fitness of SARS-CoV-2. We estimate the fitness effects of all amino acid variants and several structural variants that have circulated in the United States between February and September 2020 from viral phylogenies. We also estimate how much fitness variation among pathogen lineages is attributable to genetic versus non-genetic factors such as spatial heterogeneity in transmission rates. Up to September 2020, most fitness variation between lineages can be explained by background spatial heterogeneity in transmission rates across geographic regions. Furthermore, no genetic variant including the Spike D614G mutation has had a significant effect on population-level fitness. Instead, the rapid increase in the frequency of the Spike D614G can be explained by the variant having a spatial transmission advantage due to first establishing in regions with higher transmission rates during the earliest stages of the pandemic.

SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation (preprint)

Summary Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ∼6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses. Highlights Cell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19 Drug screen reveals critical role for membrane lipid composition in fusion Spike’s unusual membrane-proximal cysteines and aromatics are essential for fusion Cholesterol tunes relative infectivity of SARS-CoV-2 viral particles

The Potential for SARS-CoV-2 to Evade Both Natural and Vaccine-induced Immunity (preprint)

SARS-CoV-2 attaches to the surface of susceptible cells through extensive interactions between the receptor binding domain (RBD) of its spike protein and angiotensin converting enzyme type 2 (ACE2) anchored in cell membranes. To investigate whether naturally occurring mutations in the spike protein are able to prevent antibody binding, yet while maintaining the ability to bind ACE2 and viral infectivity, mutations in the spike protein identified in cases of human infection were mapped to the crystallographically-determined interfaces between the spike protein and ACE2 (PDB entry 6M0J), antibody CC12.1 (PDB entry 6XC2), and antibody P2B-2F6 (PDB entry 7BWJ). Both antibody binding interfaces partially overlap with the ACE2 binding interface. Among 16 mutations that map to the RBD:CC12.1 interface, 11 are likely to disrupt CC12.1 binding but not ACE2 binding. Among 12 mutations that map to the RBD:P2B-2F6 interface, 8 are likely to disrupt P2B-2F6 binding but not ACE2 binding. As expected, none of the mutations observed to date appear likely to disrupt the RBD:ACE2 interface. We conclude that SARS-CoV-2 with mutated forms of the spike protein may retain the ability to bind ACE2 while evading recognition by antibodies that arise in response to the original wild-type form of the spike protein. It seems likely that immune evasion will be possible regardless of whether the spike protein was encountered in the form of infectious virus, or as the immunogen in a vaccine. Therefore, it also seems likely that reinfection with a variant strain of SARS-CoV-2 may occur among people who recover from Covid-19, and that vaccines with the ability to generate antibodies against multiple variant forms of the spike protein will be necessary to protect against variant forms of SARS-CoV-2 that are already circulating in the human population.