Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle.

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity. Author summary Plasmodium vivax is the most prevalent malaria-causing parasite species outside of Sub-Saharan Africa and has many unique and poorly understood biological characteristics that make it particularly challenging to study and combat. Transcriptomic profiling of P. vivax under various conditions has the potential to unlock new experimental abilities and aid in elucidating biology and the development of clinical interventions. However, a lack of a robust in vitro culture system for this parasite has restricted transcriptomic studies to researchers with timely access to fresh human isolates from clinics, which often are in resource-poor settings, as well as nearby, well-equipped laboratories for sample processing. This study aimed to gain insight into the differential survival of P. vivax in various culture media from the parasites transcriptional signature in each media. By implementing low-input RNA library preparation strategies, this study obtains robust transcriptomic data at various parasite development stages and in different culture conditions from just 1000 FACS-purified, P. vivax-infected erythrocytes from viable cryopreserved patient isolates. With these data, we find culture media has little effect on transcriptional profile, we characterize invasion ligand expression across intraerythrocytic development and between clinical isolates, and we define the transcriptome of sexual, transmissible stages of the P. vivax parasite. These results highlight the establishment and utility of a powerful platform for studying the transcriptomic biology of this particularly challenging parasite.