A subscription to JoVE is required to view this content. Sign in or start your free trial.
Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.
During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.
Nucleocytoplasmic large DNA viruses (NCLDV) are extremely diverse, defined by four families that infect eukaryotes1. The first described viruses with genomes above 300 kbp were Phydcodnaviridae, including Paramecium bursaria Chlorella virus 1 PBCV12. The isolation and the first description of Mimivirus, showed that the size of viruses doubled in terms of both the size of the particle (450 nm) and the length of the genome (1.2 Mb)3. Since then, many giant viruses have been described, usually isolated using an amoeba co-culture procedure. Several giant viruses with different morpho....
1. Amoeba Culture
Single cell micro-aspiration is a micromanipulation process optimized in this manuscript (Figure 1). This technique enables capture of a rounded, infected amoeba (Figure 2A) and its release in a novel plate containing uninfected amoebae (Figure 2). It is a functional prototype that applies to the co-culture system and has successfully isolated non-lytic giant viruses. This approach was used for the first time in the.......
The duration of the single cell micro-aspiration handling and its good functioning is operator-dependent. The different steps of the experiment require precision. The use of the micromanipulation components of the workstation must be under constant control by observing the process of micro-aspiration and the release of the cell. The follow-up by microscopic observation is necessary for capture and transfer of a cell. An experienced operator can take 1 to 2 h to isolate 10 cells and retransfer them one by one depending on.......
The authors would like to thank both Jean-Pierre Baudoin and Olivier Mbarek for their advice and Claire Andréani for her help in English corrections and modifications. This work was supported by a grant from the French State managed by the National Research Agency under the "Investissements d’avenir (Investments for the Future)" program with the reference ANR-10-IAHU-03 (Méditerranée Infection) and by Région Provence Alpes Côte d’Azur and European funding FEDER PRIMI.
....Name | Company | Catalog Number | Comments |
Agarose Standard | Euromedex | Unkown | Standard PCR |
AmpliTaq Gold 360 Master Mix | Applied Biosystems | 4398876 | Standard PCR |
CellTram 4r Oil | Eppendorf | 5196000030 | Control the cells during the microaspiration process |
Corning cell culture flasks 150 cm2 | Sigma-aldrich | CLS430825 | Culture |
Corning cell culture flasks 25 cm2 | Sigma-aldrich | CLS430639 | Culture |
Corning cell culture flasks 75 cm2 | Sigma-aldrich | CLS430641 | Culture |
DFC 425C camera | LEICA | Unkown | Observation/Monitoring |
Eclipse TE2000-S Inverted Microscope | Nikon | Unkown | Observation/Monitoring |
EZ1 advanced XL | Quiagen | 9001874 | DNA extraction |
Glasstic Slide 10 With Counting Grids | Kova International | 87144E | Cell count |
Mastercycler nexus | Eppendorf | 6331000017 | Standard PCR |
Microcapillary 20 µm | Eppendorf | 5175 107.004 | Microaspiration and release of cells |
Micromanipulator InjectMan NI2 | Eppendorf | 631-0210 | Microcapillary positioning |
Nuclease-Free Water | ThermoFischer | AM9920 | Standard PCR |
Optima XPN Ultracentrifuge | BECKMAN COULTER | A94469 | Virus purification |
Petri dish 35 mm | Ibidi | 81158 | Culture/observation |
Sterile syringe filters 5 µm | Sigma-aldrich | SLSV025LS | Filtration |
SYBR green Type I | Invitrogen | unknown | Fluorescent molecular probes/ flow cytometry |
SYBR Safe | Invitrogen | S33102 | Standard PCR; DNA gel stain |
Tecnai G20 | FEI | Unkown | Electron microscopy |
Type 70 Ti Fixed-Angle Titanium Rotor | BECKMAN COULTER | 337922 | Virus purification |
Ultra-Clear Tube, 25 x 89 mm | BECKMAN COULTER | 344058 | Virus purification |
Explore More Articles
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved