Abstract
The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.
Highlights
The discovery of Mimivirus opened a new era in virology (La Scola et al, 2003)
We observed a significant decrease (90%) in the percentage of amoebae and an increase in the number of debris following mechanical lysis and infection with Mimivirus at 24 and 48 h compared to the percentage of control amoebae
The detection limit of the FACS was of 102 amoebae/ml, which is sufficient for detecting the arbitrary threshold of an amoebal loss greater than 50% after 24 h
Summary
The discovery of Mimivirus opened a new era in virology (La Scola et al, 2003). It was proposed that the virus be classified under a new order Megavirales (Colson et al, 2012). Other members of this order are pathogens of animals and several unicellular eukaryotes. Mimivirus deserved some special attention, because it was larger than the largest virus known before and because it was the first virus having dimensions (particle size and genome complexity) that significantly overlap with those typical of parasitic cellular microorganisms. The recent discovery of Pandoraviruses and Pithovirus revealed the extremely large particle size and genetic contents
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