QUANTIFICATION AND CHARACTERIZATION OF VIRAL INFECTION CYCLE USING IMAGING FLOW CYTOMETRY

Imaging Flow Cytometry is an emerging technology that combines the high speed, high throughput abilities of Flow Cytometry with the high content imagery of microscopy. Using the unique imaging and analysis abilities of the ImageStreamX, a high-throughput imaging flow cytometer, we show here for the first time a method to track and characterize the progression of the Mimivirus infection cycle in a quantitative manner. The recently discovered Acanthamoeba polyphaga Mimivirus (APMV) is a giant virus that infects Acanthamoeba species. It belongs to the NucleoCytomplasmic Large DNA Viruses (NCLDVs) clade, large dsDNA viruses sharing the unique feature of replicating (entirely or partially) at their host’s cytoplasm. These viruses share a unique feature – the formation of a viral factory (VF) at their host cytosol during the infection cycle. The VF is a large compartment within which central infection events occur, including DNA replication, transcription, DNA packaging and capsid assembly. We designed a series of features describing the morphological changes of the infected cell and the formation and maturation of the VF throughout the infection cycle. This allows us to monitor changes in the infected amoeba under different experimental conditions and to unravel the involvement of key elements in the infection cycle. Among the features used to track the progression of the infection cycle are the identification of the early formation of the VF, its localization in the infected cell, VF development and progression, as well as quantifying changes in the cells’ morphology and granularity. In addition, we developed a method for directly counting viral particles. The characterization and quantification methods presented here for the first time may serve as a valuable tool in viral studies.