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Bats are recognized as natural reservoirs for an array of diverse viruses, particularly coronaviruses, which have been linked to major human diseases like SARS-CoV and MERS-CoV. These viruses are believed to have originated in bats, highlighting their role in virus ecology and evolution. Our study focuses on the molecular characterization of bat-derived coronaviruses (CoVs) in Canada. Tissue samples from 500 bat specimens collected in Canada were analyzed using pan-coronavirus RT-PCR assays to detect the presence of CoVs from four genera: Alpha-CoVs, Beta-CoV, Gamma-CoV, and Delta-CoV. Phylogenetic analysis was performed targeting the RNA-dependent RNA polymerase (RdRP) gene. Our results showed an overall 1.4% CoV positivity rate in our bat sample size. Phylogenetic analysis based on the ~600 bp sequences led to the identification of an unclassified subgenus of Alpha-CoV, provisionally named Eptacovirus. The findings contribute to a better understanding of the diversity and evolution of CoVs found in the bat species of Canada. The current study underscores the significance of bats in the epidemiology of CoVs and enhances the knowledge of their genetic diversity and potential impact on global public health. [ABSTRACT FROM AUTHOR]

Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.