Name: Jessica Ray

Email: jessicalouiseray@gmail.com

Author: Jessica Ray, Jens Larsen, Runar Thyrhaug, Gunnar Bratbak, Aud Larsen and Ruth-Anne Sandaa

Author affiliation: Department of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway

Abstract title: Live and let die: Molecular methods for studying host-virus interactions using Chrysochromulina ericina and CeV-10B as a model system

Absstract:

The unicellular alga Chrysochromulina ericina (Haptophyta: Prymnesiophyceae) is a common species in Norwegian coastal waters. CeV-01B is a large dsDNA virus assigned to the Phycodnaviridae family that infects this species. Here we report the development of molecular methods a) to study the physiological response of C. ericina to CeV infection and b) to quantify C. ericina and CeV in both culture and seawater samples. CeV infection induces DNA fragmentation in C. ericina cells within 30-120 min post-infection, suggesting activation of a programmed cell death (PCD)-like pathway in C. ericina. Taking advantage of this, we have developed a method where we use fluorescently-labeled DNA strand breaks that occur during virus infection as a proxy for CeV-infected C. ericina cells. Employing an in situ nick translation (ISNT) assay, which allows incorporation of fluorescently-labeled nucleotides at nicks in DNA of fixed cells, we could enumerate infected cells and track the progression of viral infection by fluorescence microscopy. We are currently investigating the efficacy of a commercial TdT-mediated dUTP nick-end labelling (TUNEL) kit for flow cytometric detection DNA fragmentation. This assay utilizes the same principles as the ISNT assay to fluorescently label DNA fragmentation, but has the advantage that it is compatible with flow cytometry for detection and enumeration of labeled cells. Optimization of such flow cytometric methods for enumeration of infected versus uninfected cells would greatly improve our understanding of the dynamics of viral infection in this alga. We have in addition developed a real-time PCR assay targeting the CeV mcp gene sequences. This has made it possible to detect and quantify both free CeV particles and host cells harbouring CeV. Optimization of these methods may allow closer investigation of C. ericina host-virus dynamics in natural ecosystems.