Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

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 pellet the amoebae by centrifugation at 720 x g for 10 min. Remove the supernatant and resuspend the pellet in the appropriate volume of starvat....

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.......

Materials

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