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 morphologies and genetic contents can be isolated from Acanthamoeba sp. cells, including Marseilleviruses, Pandoraviruses, Pithoviruses, Mollivirus, Cedratviruses, Pacmanvirus, Tupanvirus, and recently Medusavirus^{4,5,6,7,8,9,10,11,12,13,14,15,16,17}. In parallel, the isolation of Vermamoeba vermiformis allowed the isolation and description of the giant viruses Faustovirus, Kaumoebavirus, and Orpheovirus^18,19,20. Other giant viruses were isolated with their host protists, such as Cafeteria roenbergensis²¹, Aureococcus anophagefferens²², Chrysochromulina ericina²³, and Bodo saltans²⁴. All of these isolations were the result of an increasing number of teams working on isolation and the introduction of high throughput strategy updates^25,26,27,28, such as the improvement of the co-culture system with the use of flow cytometry.

In 2016, we used a strategy associating co-culture and flow cytometry to isolate giant viruses²⁷. This strategy was developed to increase the number of samples inoculated, to diversify protists used as cell supports, and to quickly detect the lysis of the cell support. The system was updated by adding a supplemental step to avoid preliminary molecular biology identification and quick detection of an unknown viral population as in the case of Pacmanvirus²⁹. Coupling flow cytometry to cell sorting allowed for separation of a mixture of Mimivirus and Cedratvirus A11³⁰. However, we later encountered the limitations of the separation and detection of these viral subpopulations by flow cytometry. After sequencing, when we assembled the genomes of Faustovirus ST1²⁵ and Faustovirus LCD7 (unpublished data), we surprisingly found in each assembly two supplemental genomes of two novel viruses not identified in public genome databases. However, neither flow cytometry nor transmission electronic microscopy (TEM) showed that the amoebaes were infected by two different viruses, Clandestinovirus ST1 and Usurpativirus LCD7. We designed specific PCR systems to amplify Faustovirus, Usurpativirus, and Clandestinovirus markers respectively based on their genomes; our purpose was to have PCR-based systems that enable verification of the purity of the viruses being separated. However, end-point dilution and flow cytometry failed to separate them. The isolation of this single viral population was difficult because neither the morphology nor replicative elements of Clandestinovirus and Usurpativirus populations have been characterized. We detected only one viral population by flow cytometry due to the overlapping of the two populations (tested after the effective separation). We tried to separate them using single particle sorting on 96-well plates, but we did not observe any cytopathic effects, and we detected neither Clandestinovirus nor Usurpativirus by PCR amplification. Finally, it was only the combination of end point dilution followed by single amoeba micro-aspiration that enabled separation of these two low-abundance giant viruses from Faustoviruses. This method of separation is the object of this article.

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 starvation medium to obtain 1 x 10⁶ cells/mL (Table 1).

2. Propagation of Stock Virus in Amoebae

NOTE: Before dilution, it is important to culture the stock sample to obtain enough fresh culture, then proceed to filtration.

1. Use 1 x 10⁶ of amoeba culture in starvation medium.
2. Inoculate the mixture of viruses issued from the stock solution (Faustovirus/Usurpativirus LCD7 or Faustovirus/Clandestinovirus ST1) after the co-culture process on the cell support at a multiplicity of infection (MOI) of 0.01.
   NOTE: The MOI is important to reduce the abundance of the major viral population and the number of infected cells.
3. Incubate at 30 °C until cytopathic effects (CPE) are induced, such as amoebal rounding or lysis, approximately 10 to 14 h after infection.
4. Collect the media and filtrate through a 5 µm filter to remove cellular debris.

3. End-point dilution

1. Perform a serial dilution (10^-1 to 10^-11) of the viral sample in starvation medium (Table 1).
2. Inoculate 2 mL of 1 x 10⁶ Vermamoeba vermiformis contained in each Petri dish with 100 µL of the mixture inoculum.
3. Place the Petri dishes into a sealable plastic bag at 30 °C.
4. Begin observing the Petri dishes with inverted optical microscopy at 6 h postinfection and check cell morphology every 4 to 8 h.
5. At the appearance of the cytopathic effect characterized by rounding cells, begin the single cell micro-aspiration process.

4. Single Cell Micro-aspiration

1. Prepare the host.
   NOTE: This preparation is made for the release of infected single cells to a fresh cell support.
   1. Treat the amoebae in the culture with an antimicrobial agent containing 10 µg/mL of vancomycin, 10 µg/mL of imipenem, 20 µg/mL of ciprofloxacin, 20 µg/mL of doxycycline, and 20 µg/mL of voriconazole. This mixture is used to avoid bacterial and fungal contamination.
      NOTE: The procedure takes place on a bench outside the microbiological safety station. Add 2 mL of amoebae concentrated at 1 x 10⁶ cells/mL each into 15 Petri dishes. For amoeba adherence, incubate the culture at 30 °C for 30 min.
2. Select the Petri dish used for the micro-aspiration from the limit dilution according to the following criteria: 1) absence of any visible contamination by fungal and bacterial agents, 2) evidence of cytopathic effect of amoebae due to the viruses, and 3) prelysis and rounding phase of the amoebae (to avoid aspiration of viral particles).
3. Set up a workstation with the following materials (see Figure 1A,B):
   Micromanipulator, which allows microcapillary positioning;
   Manual control pressure device, used to aspirate and release the cells into the microcapillary;
   Inverted microscope;
   Plug and play motor modules;
   Camera;
   Computer module to visualize manipulation and take pictures.
4. Choose a microcapillary (see Figure 1C).
   NOTE: The size of the cells, the deformation and adhesion of their membranes to the surfaces, and the cellular motility can impact the smooth progress of the micro-aspiration. The microcapillary diameter can be precisely chosen and adapted to specific cell types depending on their sizes and methods of aspiration. A microcapillary of 20 µm inner diameter was used to aspirate a rounding amoeba (diameter ~10 µm). This allows the upkeep of an internal position and an easy release of the cell.
5. Mount the system.
   1. Fix the operating angle of the gripping system on the motorized module at 45°.
   2. Perform a double installation, first on the gripping system, and then on the microcapillary.
   3. Focus on the cells after running a few drops of oil through the microcapillary.
      NOTE: The mineral oil with biological compatibility is supplied by the device.
   4. Complete mounting following manufacturer's recommendations.
6. Clone cells (see Figure 2A,B).
   NOTE: This procedure is similar to the one described by Fröhlich and König³¹.
   1. Place the Petri dish containing 2 mL of infected amoebae under the microscope.
   2. Focus first on the cells, and then on the microcapillary immersed in the culture.
   3. Pick a rounded single cell and bring the microcapillary closer to the micromanipulator.
   4. Exert soft aspiration with manual pressure control on the cell, taking it inside the microcapillary. Remove the single cell from the first sample and release in the cellular support, then incubate it at 30 °C.
   5. Conduct daily observations with an inverted optical microscope to observe the appearance of the cells and to monitor the emergence of the cytopathic effect.

5. PCR Screening

NOTE: Following step 4, a systematic screening by PCR is crucial to confirm the separation. In both Usurpativirus/Faustovirus and Clandestinovirus/Faustovirus, the design and application of the specific primer and probe systems were done using Primer-BLAST online³² (Table 2).

1. Extract DNA from a part of the positive culture samples (i.e., where a cytopathic effect is observed), using an automated extraction system according to the manufacturer's protocol.
2. Use appropriately designed primers.
   NOTE: Here we designed primers to amplify core genes annotated as RpB2 (Faustovirus), LCD7 major capsid protein (Usurpatvirus) and minor capsid protein (Clandestinovirus)
3. Perform standard PCR using a thermocycler.
   1. Carry out 20 µL PCR reactions with 50 µM of each primer (Table 2), 1x Master Mix, and RNase free water.
   2. Activate the Taq DNA polymerase for 5 min at 95 °C, then follow with 45 cycles of 10 s denaturation at 95 °C, annealing of the primers for 30 s at 58 °C, and extension for 30 s at 72 °C.
4. Run the PCR products on a 1.5% agarose gel, stain with DNA gel stain (Table of Materials), and visualize with UV.

6. Virus Production and Purification

1. Put the rest of the Petri dish culture back in a small flask.
2. For the virus production, prepare 15 flasks of 145 cm², containing 40 mL of Vermamoeba vermiformis in starvation medium and 5 mL of the isolated virus already transferred from the Petri dish to small flasks.
3. Treat with the same antibiotic and antifungal mixture used in step 4.1.
4. Incubate at 30 °C. Observe every day with inverted optical microscopy.
5. After the complete infection, pool all flasks. Use a 0.45 µm filter to eliminate debris.
6. Ultracentrifuge all supernatants at 50,000 x g for 45 min.
7. After centrifugation, remove the supernatant from each tube by aspiration and resuspend the pellet in 1 mL of phosphate buffered saline (PBS).
8. Purify the virus produced using 25% sucrose (27.5 g sucrose in 100 mL of PBS, sterilized by filtration).
9. Centrifuge 8 mL of sucrose and 2 mL of the viral suspension at 80,000 x g for 30 min. Resuspend the viral pellet in 1 mL of PBS. Store it at -80 °C.

7. Negative Staining and Transmission Electron Microscopy

NOTE: Bou Khalil et al. previously published this protocol²⁷.

1. Deposit 5 μL of the lysis supernatant onto the glow-discharged grid. Leave for approximately 20 min at room temperature.
   NOTE: The glow-discharge allows us to obtain a hydrophilic grid by plasma application.
2. Dry the grid carefully and deposit a small drop of 1% ammonium molybdate on it for 10 s. Leave the grid to dry for 5 min.
3. Proceed to electron microscopy observations at 200 keV.

8. Characterization of Clandestinovirus ST1 and Usurpativirus LCD7

1. Characterize pure populations of Clandestinovirus ST1 and Usurpativirus LCD7 using genome sequencing, genome assembly, bioinformatics analyses, and study of their replicative cycle as we have done for other viruses^10,20,29.

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