Transcriptomics analysis of in vivo Opisthorchis viverrini: Stage-specific gene expression and novel genes with stable expression in mammalian stages for opisthorchiasis intervention

Opisthorchis viverrini, Southeast Asian liver fluke, is a parasitic flatworm that has been widely spread in Asia and is a major risk factor for bile duct cancer - cholangiocarcinoma (CCA). Over 12 million people are at risk of opisthorchiasis, and the associated CCA is known to have claimed around 20,000 deaths per year in Thailand alone, with the number projected to be underestimated elsewhere. There is an opportunity to develop more efficient drug targets and diagnostic biomarkers, and these are urgently needed. Despite previous studies on gene expression analysis and reference genome, the knowledge on the biological processes of O. viverrini during its development remains largely unclear. Our study employed RNA-sequencing transcriptomics of O. viverrini developmental stages within hamsters from the juvenile stage (14-day post-infection), the adult stage of acute infection (42 days), and the adult stage of chronic infection (180 days). Differential gene expression and functional analyses were performed, and genes with stable expression (GSEs) were identified based on the transcript per million (TPM) normalization method and coefficient of variation. Our results show that key genes in juveniles were mostly associated with proteolysis, energy metabolism, signal transduction, and development. Significantly up-regulated genes in adult O. viverrini were associated with parasitic reproductive systems and parasitism. A total of 2,011 GSEs were identified, with 27 genes being highly expressed, and 628 genes showing no orthologues in the human reference genome. From this novel dataset, we illustrated insight into parasite biology, which revealed key molecular processes during intra-mammalian infection, and provided the candidate targets for biomarkers, drugs, and vaccine development. This valuable information will contribute to opisthorchiasis diagnostics and prevention in endemic countries and provide leads for future characterization of essential genes across liver fluke species.