Rahal Guaragna Machado, R. | bioRxiv Science

bioRxiv2025

Characterization and evolutionary history of novel SARS-CoV-2-related viruses in bats from Cambodia

Circulating bat coronaviruses present a significant pandemic threat, yet our understanding of their genetic diversity and evolutionary dynamics remains limited. Over 3 years, we sampled 1,462 bats in Cambodias Steung Treng province, identifying extensive and diverse coronaviruses co-circulation. Using metatranscriptomic and amplicon sequencing, we generated 33 complete sarbecovirus genomes, revealing novel lineages that cluster into four distinct groups, each associated with different Rhinolophus bat species. Our analysis highlights rapid migration and recombination of sarbecovirus lineages over short distances and timescales. Of note, the receptor-binding domains of two novel viral groups exhibit high similarity to SARS-CoV-2, and pseudovirus assays confirmed the ability of this spike protein to mediate entry into cells expressing human ACE2, suggesting a potential zoonotic risk. The observed genetic diversity underscores the urgent need for continuous surveillance to identify high-risk animal-to-human interfaces and inform pandemic preparedness.

microbiology↗

bioRxiv2021

SARS-CoV-2 papain-like protease PLpro in complex with natural compounds reveal allosteric sites for antiviral drug design

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

biochemistry↗