Post-transcriptional processing and modification of viral RNA, including alternative splicing, polyadenylation, and methylation, play crucial roles in regulating viral gene expression, enhancing genomic stability, and increasing replication efficiency. These processes have significant implications for viral biology and antiviral therapies. In this study, using Oxford Nanopore Technology (ONT) direct RNA sequencing (DRS), we provided a comprehensive analysis of the transcriptome and epitranscriptome features of the HIV-1 B (NL4-3) subtype strain and, for the first time, characterized these features in the CRF01_AE (GX2005002) subtype strain. We identified 11 novel splicing sites among the 61 RNA isoforms in NL4-3 and defined the splicing sites for GX2005002 based on its 63 RNA isoforms. Furthermore, we identified 74 and 79 chemically modified sites in the transcripts of NL4-3 and GX2005002, respectively. Although differences in poly(A) tail length were observed between the two HIV-1 strains, no specific correlation was detected between poly(A) tail length and the number of modification sites. Additionally, three distinct N6-methyladenosine (m⁶A) modification sites were identified in both NL4-3 and GX2005002 transcripts. This study provides a detailed analysis of post-transcriptional processing modifications in HIV-1 and suggests promising avenues for future research that could potentially be applied as new therapeutic targets in HIV treatment.

RNA molecules are inherently dynamic, forming complex structural ensembles of alternative conformations that direct their cellular functions. Current methods for resolving RNA structural heterogeneity and dynamics have been developed for short-read sequencing–based structure probing, limiting their sensitivity and resolution. Here, we introduce DeCoRE (deconvolving complex RNA structural ensembles), a computational method that leverages direct RNA sequencing to resolve RNA structural heterogeneity and dynamics at the individual transcript level. DeCoRE combines enhanced structural signal detection with direct clustering of sequencing reads to sensitively deconvolve RNA structural ensembles at single-nucleotide resolution. We demonstrate DeCoRE’s capabilities by resolving the known alternative conformations of the lysine and TPP riboswitches and uncover a previously uncharacterized cotranscriptional folding pathway of bacterial ribosomal RNA, where two conformations coexist during early elongation and progressively converge into a single mature conformation. DeCoRE represents a powerful tool for dissecting RNA structural heterogeneity and dynamics, advancing our understanding of RNA function.

Plague persistence relies on complex host–vector–environment interactions, yet the ecological contribution of soil microbiomes to natural foci remains poorly understood. We performed shotgun metagenomic sequencing of 143 soil samples across four distinct plague foci in Inner Mongolia, China, to characterize microbiome features associated with contrasting epidemiological states. Taxonomic, functional, and network analyses showed that the three active plague foci exhibited lower microbial diversity, simplified co-occurrence networks, and enrichment of specific taxa, particularly Streptomyces. Conversely, the quiescent Marmota sibirica focus harbored a more complex and interconnected microbial network with greater richness and exclusive detection of Yersinia bacteriophages, showing significant concordance with bacterial community structure. These patterns indicate distinct microbiome configurations associated with specific host and geographic settings, highlighting the need to disentangle host- and site-specific effects from broader epidemiological status. Compared with surface and deep soil, burrow-associated soil exhibited greater microbial differentiation and higher richness of bacteriophages, antimicrobial resistance genes, and virulence-associated genes, suggesting its potential as a sensitive environmental matrix for ecological monitoring. Overall, our findings identify distinct soil microbiome signatures—specifically taxonomic shifts, altered network complexity, exclusive Yersinia phage detection, and functional gene enrichment—associated with epidemiological heterogeneity across natural plague foci, providing a baseline for understanding ecological variation among endemic habitats.