New Insights from the High-Resolution Monitoring of Microalgae-Virus Infection Dynamics.

Gino Schiano Di Visconte,Andrew Spicer,Michael J Allen

doi:10.3390/v14030466

Abstract

Investigation of virus-induced microalgal host lysis and the associated infection dynamics typically requires sampling of infected cultures at multiple timepoints, visually monitoring the state of infected cells, or determining virus titration within the culture media. Such approaches require intensive effort and are prone to low sensitivity and high error rates. Furthermore, natural physiological variations can become magnified by poor environmental control, which is often compounded by variability in virus stock efficacy and relatively long infection cycles. We introduce a new method that closely monitors host health and integrity to learn about the infection strategy of Chloroviruses. Our approach combines aspects of spectrometry, plaque assays, and infection dose assessment to monitor algal cells under conditions more representative of the natural environment. Our automated method exploits the continuous monitoring of infected microalgae cultures in highly controlled lab-scale photobioreactors that provide the opportunity for environmental control, technical replication, and intensive culture monitoring without external intervention or culture disruption. This approach has enabled the development of a protocol to investigate molecular signalling impacting the virus life cycle and particle release, accurate determination of virus lysis time under multiple environmental conditions, and assessment of the functional diversity of multiple virus isolates.

Highlights

There are a variety of tools, techniques, and methodologies for characterising viruses, their life cycles, and their properties
Three main approaches are taken: direct observations derived from their impact on their host (such as viral plaque assay [1], infection dose (ID50 ) [2], and immunofluorescence foci assays [3]); observations based upon specific components of the virus or infected hosts; and, lastly, direct physical assessment/observation of whole viral particles
Through inclusion/exclusion experiments, we subsequently identified that the presence of vitamins B1 and B12 are essential for the growth of this microalgal strain

Summary

Introduction

There are a variety of tools, techniques, and methodologies for characterising viruses, their life cycles, and their properties. The most commonly used techniques are viral plaque assay [14], transmission electron microscopy [15], and flow cytometry [16], together with mass spectrometry for metabolite analysis and next-generation sequencing/metagenomics for genomic/transcriptomic analysis. In order to determine Phycodnavirus host lysis and/or release time, the only possible way was to sample infected cultures at multiple timepoints and monitor visually the state of the individual infected cells or determine a virus particle count in the media. Such approaches require intensive effort and are prone to low sensitivities. They are further exacerbated by poor environmental control, variation in virus stock quality, and relatively long infection life cycles

Methods

Results

Conclusion