Intranasal Infection of a Mouse with a Pathogenic Influenza Virus

Resumen

Source: Wang, M., et al, Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice. J. Vis. Exp. (2020).

This video demonstrates intranasal infection of a mouse with a pathogenic influenza virus to establish a respiratory tract infection. The procedure outlines the administration of the virus into the nasal cavity. The virus enters host cells, replicates, and activates the immune system.

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

NOTE: Virus infection of mice and isolation of organs should be performed under Animal Biosafety Level 2 (ABSL2) conditions.

1. Inoculation of PR8 influenza virus and recording of mice weight

1. Prepare 8-week-old male C57BL/6 mice for infection in the ABSL2 room.
   NOTE: This protocol is also suitable for experiments with female mice.
2. Dilution of PR8 virus: take out the virus from the -80 °C freezer and incubate on ice until it melts into liquid. Vortex the stock virus thoroughly and dilute the virus to 2 plaque-forming unit (PFU)/µL with sterile phosphate-buffered saline (PBS), 135 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM disodium hydrogen phosphate (Na₂HPO₄), 1.8 mM potassium dihydrogen phosphate (KH₂PO₄) in a pre-chilled 1.5 mL tube.
3. Mice anesthetization: weigh each mouse and calculate the volume (4-fold (µL) the mouse weight (g)) of sodium pentobarbital (2 mg/mL) to be used. Inject the calculated volume of sodium pentobarbital intraperitoneally.
   NOTE: This step is to make mice breathe steadily and peacefully, so that an accurate titer of virus could be inoculated intranasally. Too fast or too slow heartbeats indicate inappropriate anesthetization. In addition, the use of vet ointment is recommended to avoid eye dryness.
4. Intranasal Inoculation: vortex the diluted PR8 virus thoroughly. Pipette 10 µL and carefully perform intranasal inoculation on one side drop by drop. After finishing the inoculation of all the mice in one cage (a maximum of 5 mice) on this side, repeat inoculation on the other side (keep the breathing of the mouse peaceful and steady throughout the inoculation). Each mouse is infected with 40 PFU of the PR8 virus in total.
5. Place the mice in sternal recumbency in warm cages for better revival.
6. Monitor the mouse weight daily for 10 days. (The infection day is recorded as Day 0).