Clade-3 Bat Sarbecovirus PRD0038 Reveals Constraints on Coronavirus Emergence and Immune Sensitivity

Technical Summary: Clade-3 Bat Sarbecovirus PRD0038 Reveals Constraints on Coronavirus Emergence and Immune Sensitivity

Problem Statement
Zoonotic Sarbecoviruses, including SARS-CoV and SARS-CoV-2, pose a documented threat to global health. While global surveillance has identified bats (Rhinolophus spp.) as principal reservoirs, African Sarbecoviruses remain poorly characterized compared to Asian strains, creating a significant gap in understanding the diversity and emergence potential of this high-risk clade. Specifically, the biological properties of the recently identified African clade-3 Sarbecovirus, PRD0038, were largely unknown. Critical questions remained regarding its live virus growth kinetics, receptor tropism in non-bat mammals, in vivo pathogenesis, and susceptibility to existing countermeasures. Furthermore, the structural dynamics of its spike protein and how specific mutations might influence receptor accessibility and conformational states (e.g., RBD-up vs. RBD-down) were unexplored.

Methodology
The authors employed a comprehensive multidisciplinary approach to characterize a recombinant PRD0038 (rPRD0038):

• Reverse Genetics: A cDNA clone of the full-length PRD0038 genome was generated and recovered in Vero cells expressing R. affinis ACE2. The team identified naturally occurring quasispecies variants during recovery and incorporated specific nonsynonymous substitutions (D603G, Q617K, etc.) into a stable recombinant backbone. Derivative strains encoding GFP or nano-luciferase (nLuc) reporters were created for quantitative assays.
• Structural Biology: Single-particle cryo-electron microscopy (cryo-EM) was used to determine the structures of the wild-type (WT) and mutant (D603G, Q617K, and D603G-Q617K) spike protein ectodomains. 3D variability analysis (3DVA) was performed to quantify conformational heterogeneity and dynamics.
• Host Range and Tropism: The virus was tested in non-permissive DBT cells and Vero cells expressing ACE2 orthologs from diverse species (bats, civets, camels, rabbits, cows, humans, mice). Replication was also assessed in primary human bronchial epithelial (HBE) cells and immortalized epithelial cells from other bat species.
• Antiviral and Immunological Susceptibility: Susceptibility to FDA-approved antivirals (remdesivir, molnupiravir, nirmatrelvir) was measured using nLuc reporter assays. Neutralization was assessed using sera from vaccinated non-human primates (NHPs), hospitalized COVID-19 patients, and mice infected with various SARS-CoV-2 variants (ancestral and Omicron). A panel of 142 monoclonal antibodies (mAbs) from the Coronavirus Immunotherapeutic Consortium (CoVIC) and other sources was tested for cross-neutralization.
• In Vivo Modeling: To overcome the inability of PRD0038 to use mouse ACE2, the authors generated a novel transgenic mouse model (K18-RaACE2) expressing the R. affinis ACE2 receptor. These mice were challenged with rPRD0038 to assess replication, pathogenesis, and the efficacy of prophylactic molnupiravir treatment.

Key Results

• Recovery and Evolution: The authors successfully recovered rPRD0038. Sequencing revealed that naturally occurring substitutions, specifically D603G and Q617K in the spike protein, were selected during cell culture adaptation.
• Structural Dynamics: Cryo-EM analysis showed that the WT PRD0038 spike exists predominantly in a closed (3-RBD-down) conformation. In contrast, the D603G-Q617K double mutant shifted the equilibrium toward open states, with ~40% of particles in a 1-RBD-up conformation and ~37% in a 2-RBD-up conformation. Structural analysis indicated that D603G disrupts a stabilizing interaction between the SD2 subunit and the S2 helix, while Q617K introduces electrostatic repulsion at the SD2/SD1 interface. Together, these mutations destabilize inter-protomer contacts, facilitating RBD-up transitions.
• Host Tropism: rPRD0038 efficiently replicated in cells expressing ACE2 from R. affinis, R. alcyone, R. sinicus, civets, camels, rabbits, and cows. However, it failed to replicate in cells expressing human ACE2 or primary human airway epithelial cells. Replication was also undetectable in cells from geographically distinct bat species (Perimyotis subflavus, Lasiurus borealis, Pteropus alecto).
• Therapeutic Susceptibility: Despite antigenic divergence, rPRD0038 remained highly sensitive to FDA-approved antivirals (remdesivir, nirmatrelvir, molnupiravir).
• Immune Recognition: Pre-existing SARS-CoV-2 immunity conferred reduced cross-neutralizing activity against PRD0038. Sera from mice infected with Omicron variants showed significantly lower neutralization titers compared to those infected with ancestral SARS-CoV-2. However, specific broadly neutralizing mAbs targeting conserved cryptic epitopes (particularly RBD-7/class-4 and select S2 regions) retained potent neutralizing activity.
• In Vivo Pathogenesis: The K18-RaACE2 mouse model supported high-titer viral replication in the lungs but did not result in weight loss or gross lung discoloration, indicating limited pathogenicity in this model. Prophylactic treatment with molnupiravir significantly reduced lung viral titers, validating the model for countermeasure testing.

Significance and Claims
The paper claims that these findings illustrate functional and immunological constraints that regulate the emergence potential of clade-3 bat Sarbecoviruses. Specifically:

1. Emergence Constraints: The requirement for specific adaptive mutations (like D603G/Q617K) to shift spike conformation and the current inability to use human ACE2 suggest that PRD0038 is currently constrained from immediate human emergence, though intermediate hosts (civets, camels, rabbits, cows) present potential spillover risks.
2. Therapeutic Readiness: The susceptibility to existing small-molecule antivirals and specific broadly neutralizing antibodies suggests that current countermeasures may be effective against a PRD0038-like emergence, provided the virus retains these conserved structural features.
3. Model Utility: The study provides a validated R. affinis ACE2-expressing mouse model, addressing a critical gap in the ability to evaluate in vivo pathogenesis and therapeutics for Sarbecoviruses that do not utilize mouse ACE2.
4. Surveillance Implications: The identification of specific mammalian species capable of supporting PRD0038 entry highlights the need for integrated surveillance at the human-animal interface in Africa to monitor potential intermediate hosts.

The authors conclude that while PRD0038 demonstrates substantial divergence from SARS-CoV-2, it remains a viable target for existing therapeutics, and its study informs broader strategies for pandemic preparedness against emerging Sarbecoviruses.