Comparative transcriptome analysis unveils unique antiviral immune signatures of Rhinolophus pusillus

Rhinolophus pusillus is currently the only known horseshoe bat that simultaneously harbors SARS-CoV-related coronaviruses (SARSr-CoVs), SARS-CoV-2-related coronaviruses (SC2r-CoVs), and a sarbecovirus recombinant lineage with a mosaic genome combining features of both SARS-CoV and SARS-CoV-2. However, the molecular basis underlying multi-virus coexistence and viral tolerance in this species remains incompletely defined. Here, we focus on basal gene expression patterns to investigate the immune landscape of this species.

Multi-tissue transcriptomes were generated from six individuals of R. pusillus and Rhinolophus ferrumequinum, and integrated with publicly available RNA-seq datasets from multiple mammalian species relevant to zoonotic disease. Cross-species comparative transcriptomic analyses were performed to assess basal immune activity. Weighted gene co-expression network analysis (WGCNA) was applied to identify tissue-specific regulatory modules, and differential expression analyses were conducted to characterize immune-related transcriptional differences. In addition, long non-coding RNA (lncRNA)-mRNA regulatory networks were constructed to explore coordinated immune regulation.

R. pusillus exhibited elevated basal transcriptional activity of innate antiviral immune processes compared with other mammals, with significant enrichment of genes involved in innate immune responses and antiviral defense. In contrast, transcriptional programs associated with inflammatory regulation were relatively attenuated. WGCNA identified distinct tissue-specific functional modules with coordinated regulatory architectures. R. pusillus displayed transcriptional features consistent with an enhanced capacity for viral infection tolerance relative to R. ferrumequinum. LncRNA analyses indicated coordinated regulation of inflammatory responses and DNA damage repair through lncRNA-mRNA regulatory networks.

By integrating comprehensive transcriptomic analyses of protein-coding RNAs with the prediction of lncRNA, this study provides a systematic molecular framework based on constitutive gene expression for understanding the mechanisms of viral tolerance in R. pusillus under conditions of multi-virus coexistence. These findings advance our understanding of bat immunobiology and offer insights into infection-related immune adaptations in R. pusillus as a key viral reservoir species.