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

Single Cell Micro-Aspiration is a method to complement the different techniques used in the field of microbiology in general and in virology in particular. It could answer key question and resolve problems encountered during giant viruses isolation by separating a virus from a viral mixture presenting a low abundance Virus as it was the case for turnover viruses, clandisinal virus, and uzibati virus from another one, which is Faustovirus, present in high abundance anyway. The main advantage of this approach is the use of an indirect strategy.

It consists of separating and cloning the infected host to obtain a viral separation. So, this method monitors the different steps from capture to the release of cells and confirm the sorting process by microscopic observation. Finally, molecular biology is used to confirm the sorting and electronic microscopy to observe the separated viral particles.

This approach will solve the major problem of virus separation. It is a prospective for cloning ameba or cloning protists in general. The person who is trying to use this technique for the first time must be rigorous and precise when controlling pressure.

The visual control is essential to confirm the capture and the release of only one cell. Use Vermamoeba Vermiformis, strain CDC 19, as a cell support. Add 30 milliliters of PYG medium and three milliliters of the amoeba at a concentration of one times ten to the sixth cells per milliliter in a 75 square centimeter cell culture flask.

Maintain the culture at 28 degrees Celsius in an incubator. After 48 hours, transfer 10 milliliters of the amoeba cell culture on to a counting slide and place it under a microscope to quantify the amoebae. To rinse, harvest 30 milliliters of the cell culture in a tube and pellet the amoebae by centrifugation at 720 times G for 10 minutes.

Remove the supernatant and resuspend the pellet in the appropriate volume of starvation medium to obtain the concentration of one times 10 to the sixth cells per milliliter. Prepare the cellular support with the addition of antimicrobial agent. In a biosafety cabinet, add one times 10 to the sixth of amoeba culture into two milliliters of starvation medium.

Then inoculate 100 milliliters of the stock solution containing the mixture of viruses into the amoebae cell culture support at a multiplicity of infection of 0.01. Incubate the infected amoebae cell culture support. Add 30 degrees Celsius for 10 to 14 hours or until cytopathic effects are induced such as amoebal rounding or lysis.

Next, use a syringe to collect the media and filtrate through a five micron filter to remove cellular debris. Perform a cereal dilution of the viral sample in starvation medium in different wells. Inoculate two milliliters of one times 10 to the sixth Vermamoba Vermaformis contained in each Petri dish with 100 milliliters of the mixture inoculum.

Then, place the Petri dishes into a sealable plastic bag at 30 degrees Celsius. At six hours post infection, observe the Petri dishes with inverted optical microscopy and check cell morphology every four to eight hours. The anti-microbial treatment is for the cellular support.

Select among the Petri dishes the ones absent of any visual contamination by fungal and bacterial agents and with evidence of cytopathic effect of amoebae due to the viruses and with pre-lysis and rounding phase of the amoebae to avoid aspiration of viral particles. Set up a work station with micro manipulator, manual control pressure device, inverted microscope, Plug and Play Motor modules, camera, and computer module. Choose a micro capillary of 20 micron inner diameter to allow the upkeep of an internal position and an easy release of the cell.

Fix the operating angle of the gripping system on the motorized module at 45 degrees. Perform a double installation, first on the gripping system and then on the micro capillary. Focus on the cells.

To clone cells, place the Petri dish containing two milliliters of infected amoebae under the microscope. Focus first on the cells and then on the micro capillary immersed in the culture. Picking rounded single cell and bring the micro capillary closer to the micromanipulator.

Exert soft aspiration with manual pressure control on the cell taking it inside the micro capillary Remove the single cell from the first sample and release it in the amoebae's cell culture support. Then incubate it at 30 degrees Celsius. Carefully control the pressure to be able to aspire a single cell and control your aspiration by the observation of the release of a single cell.

Conduct daily observations with an inverted optical microscope to observe the appearance of the cells and to monitor the emergence of the cytopathic effect. Now operate an automated extraction system to extract DNA from part of the positive culture samples where a cytopathic effect is observed. Design primers to amplify poor genes annotated as RBP2 for Faustovirus, major capsid protein for Usurpatvirus, and minor capsid protein for Clandestinovirus.

Perform standard PCR using a thermocycler according to the manuscript. Carry out 20 microliliters of PCR reactions with 50 micromolar of each primer, 1X Master Mix, and ribonuclease free water. Run the PCR products with DNA gel stain on a 1.5%agarose gel at the voltage of 135 volts and visualize with UV.This protocol optimizes a micromanipulation process with single cell micro aspiration.

This technique enables the capture of a rounded and infected amoeba and its release in a novel plate containing uninfected amoebae. This approach successfully isolated a new low abundance giant virus named Userpatvirus LCD7. Your Userpatvirus was only observed in clone seven with presence of Userpatvirus LCD7 DNA and absence of Faustovirus DNA.

Electron microscopy revealed the appearance of Clandestinovirus ST1, which has a typical icosahedral morphology without fibrils as well as Usurpatvirus LCD7 with an icosahedral capsid of about 250 nanometers. Low cytometry confirm the overlapping of populations of Faustovirus and Nauvoirus. Once mastered, this procedure can be done easily.

The most important thing to remember when attempting this procedure is the necessity of a minimum observations ritual concerning the size of the entities with the good handling of the competent of the work station and the check of any contamination problem. This approach can be used as a sorting technique on the basis of cell morphology. Currently it is used in our laboratory for the cloning of specific amoeba.

One critical point of this technique is to maintain the cell integrity. Indeed, we can have some problems with the non observation of the cell or release. This is due mostly to the disintegration of the cell into the micro capillary.

This method could be an efficient alternative when flaw telemetry and fact sorting are not available and when specific technique limitations are reached. Don't forget that in some cases the cell is not released from the micro capillary. This is due in part to the amoebae characteristics and their capacity to adapt and then from the cell membrane.