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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Amoeba Culture

  1. Use Vermamoeba vermiformis (strain CDC19) as a cell support.
  2. Add 30 mL of protease-peptone-yeast extract-glucose medium (PYG) (Table 1) and 3 mL of the amoebae at a concentration of 1 x 106 cells/mL in a 75 cm2 cell culture flask.
  3. Maintain the culture at 28 °C.
  4. After 48 h, quantify the amoebae using counting slides.
  5. To rinse, harvest the cells at a concentration of 1 x 106 cells/mL and pel.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Agarose StandardEuromedexUnkownStandard PCR
AmpliTaq Gold 360 Master MixApplied Biosystems4398876Standard PCR
CellTram 4r OilEppendorf5196000030Control the cells during the microaspiration process
Corning cell culture flasks 150 cm2Sigma-aldrichCLS430825Culture
Corning cell culture flasks 25 cm2Sigma-aldrichCLS430639Culture
Corning cell culture flasks 75 cm2Sigma-aldrichCLS430641Culture
DFC 425C cameraLEICAUnkownObservation/Monitoring
Eclipse TE2000-S Inverted MicroscopeNikonUnkownObservation/Monitoring
EZ1 advanced XLQuiagen9001874DNA extraction
Glasstic Slide 10 With Counting GridsKova International87144ECell count
Mastercycler nexusEppendorf6331000017Standard PCR
Microcapillary 20 µmEppendorf5175 107.004Microaspiration and release of cells
Micromanipulator InjectMan NI2Eppendorf631-0210Microcapillary positioning
Nuclease-Free WaterThermoFischerAM9920Standard PCR
Optima XPN UltracentrifugeBECKMAN COULTERA94469Virus purification
Petri dish 35 mmIbidi81158Culture/observation
Sterile syringe filters 5 µmSigma-aldrichSLSV025LSFiltration
SYBR green Type IInvitrogenunknownFluorescent molecular probes/ flow cytometry
SYBR SafeInvitrogenS33102Standard PCR; DNA gel stain
Tecnai G20FEIUnkownElectron microscopy
Type 70 Ti Fixed-Angle Titanium RotorBECKMAN COULTER337922Virus purification
Ultra-Clear Tube, 25 x 89 mmBECKMAN COULTER344058Virus purification

References

  1. Iyer, L. M., Aravind, L., Koonin, E. V. Common Origin of Four Diverse Families of Large Eukaryotic DNA Viruses. Journal of Virology. 75 (23), 11720-11734 (2001).
  2. Li, Y., et al. Analysis of....

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