Isolation of Mosquito-Associated Virus from Mosquito Homogenate

Abstract

Source: Huang, D., et al. Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes. J. Vis. Exp. (2022).

This video demonstrates the step-by-step procedure for the in vitro isolation of mosquito-associated viruses from mosquito homogenates using fibroblast-like mammalian cells.

Protocollo

1. Virus isolation
   NOTE: All steps were carried out in a biosafety level 2 (BSL-2) laboratory. The safety level requirement of the biosafety laboratory was determined by the biosafety risk assessment based on the regulations of different countries and regions. The process must be performed in a biosafety cabinet.
   1. Remove the cell culture medium from the 24-well plates and add 100 µL of the cell culture maintenance medium and 100 µL of the supernatant of the mosquito homogenate (P0) to each well.
   2. Incubate the plates at 28 °C or 37 °C for 60 min, gently shaking them every 15 min to prevent cell drying.
   3. Remove the supernatant and rinse each well gently with 600 µL of cell culture maintenance medium to remove the debris completely.
   4. Add 800 µL of cell culture maintenance medium to each well and keep the plates in a 5% CO₂ humidified incubator at 28 °C or 37 °C for 7 days.
   5. Monitor the cell state of each well under the microscope daily.
   6. Collect the supernatants of cells (P1 supernatants) on the 7^th day and store the supernatants at -80 °C.
   7. Repeat steps 1.1-1.6 twice to get P2 and P3 supernatants.
      NOTE: In step 1.3, rinsing each well gently with 600 µL of cell culture maintenance medium was optional for the P2 and P3.
   8. Depending on which well shows the cytopathogenic effect (CPE) cells die, pyknosis, and detach from the surface; fusion with adjacent cells to form syncytia; or the appearance of nuclear or cytoplasmic inclusion bodies add 300-400 µL of the supernatant of this CPE well into each well of the new 6-well plates containing cells (the cell confluency was 80%-90%) to greatly amplify the viruses.
   9. Collect the supernatants and aliquot them into 2 mL screw cap storage tubes (500 µL per tube). Store them at -80 °C.
      NOTE: After three generations of successive passaging in cells, the samples that did not induce CPE were discarded. If necessary, the generations of successive passing in cells can be increased.

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
24-well plates	CORNING	3524	Containers for cell
Screw cap storage tubes (2 mL)	biofil	FCT010005
Refrigerator (-80 °C)	sanyo	MDF-U54V
Incubator (37 °C)	Panasonic	MCO-18AC	Instrument for cell culture
Dulbecco’s minimal essential medium (DMEM)	Gibco	C11995500BT	medium for vertebrate cell lines