“Russian Doll Virus”: Using RNAscope in-situ hybridization to reveal host-virus interactions of Matryoshka RNA viruses within Avian Haemosporidian parasites
Lilly Raphaelian
Department of Biology
Faculty Supervisor: Ravinder Sehgal
“Russian doll” viruses describe a host-pathogen interaction in which parasites themselves are infected by viruses. Eukaryotic parasites of the order Haemosporidia, which cause diseases such as malaria, haemoproteosis, and leucocytozoonosis, have recently been shown to harbor novel Matryoshka RNA viruses (MaRNAVs). Although the presence of MaRNAVs in wild avian populations has been confirmed by PCR, the cellular localization and site of infection within haemosporidian parasites remain unknown, largely due to the lack of suitable in situ detection methods for blood smears. Here, we establish a novel fixation and RNAscope in situ hybridization protocol optimized for RNA preservation and detection on blood smear slides. The protocol was first developed and validated using mouse blood smears, then adapted and applied to avian blood smears to detect MaRNAV-5 RNA within haemosporidian parasites. This approach enables direct visualization of viral RNA at the cellular level while preserving parasite morphology, without the need for host sacrifice. By enabling localization of MaRNAV-5 within parasitic protozoans, this study provides new insight into virus-parasite interactions in avian haemosporidia. More broadly, the standardized RNAscope blood smear protocol presented here offers a powerful and minimally invasive tool for studying host-pathogen and parasite-virus dynamics in wild and domestic animals, with potential applications in ecological, clinical, and epidemiological research on vector-borne diseases.