Schughart4 project protocol

Susceptibility to influenza A (H3N2) viral infection: body weight, viral loads and leukocyte profiles in females of the 8 inbred founder strains of the Collaborative Cross   (2016)

Leist SR, Kollmus H, Schughart K

See also: Schughart4 animal documentation


Project protocol - Contents

Workflow and sampling

Step
Procedure
Data collected
1
Mouse-adapted H3N2 produced and prepared for intra-nasal delivery
-
2
Mice weighed to determine subsequent weight changes Body weight
3
Mice anesthetized
-
4
Mice infected with H3N2 intra-nasally or with PBS alone
-
5
Body weight determined daily post-infection Body weight
6
Mice euthanized and blood samples taken for hematology analysis Leukocyte profiles
7
Lungs harvested and tested for viral load using FFU assay Viral load

Equipment and supplies

  • Incubator
  • Balance
  • Syringes and needles
  • Centrifuge
  • Micro tubes containing EDTA (Sarstedt)
  • VetScan HM5 (Abaxis)

Reagents and solutions

  • Ketamine/xylazine (100 mg/mL and 20 mg/mL, respectively)
  • H3N2: mouse-adapted virus strain influenza A/HK/01/68 obtained from Otto Haller, University of Freiburg GERMANY
  • Natrium chloride solution
  • Phosphate-buffered saline
  • Isofluroran (cp-pharma, Burgdorf GERMANY)

Procedure: H3N2 preparation/delivery, daily body weights and terminal leukocyte profiles and viral load in the lungs

    1. H3N2 is produced in the allantoic cavity of 10-d-old embryonated hen eggs for 48h at 37°C.
    2. Mice are weighed to determine dosing.
    3. Mice are anesthetized by intra-peritoneal injection with a mixture of Ketamine/Xylazine in sterile natrium chloride solution.
    4. Mice are intra-nasally infected with 20 µL virus solution of sterile phospate-buffered saline (PBS) using a concentration of 10 focus-forming units (FFU), or with PBS alone.
    5. Body weight is measured daily and determined as percentage of initial weight at day 0 (pre-infection).
    6. Mice are euthanized with Isofluoran (160 µL) and exsanguinated via puncture of the retro-bulbar vein plexus and lungs are collected.
    7. Blood is immediately measured in a hematologic system (VetScan).
    8. Viral load is determined on MDCK II (Madin-Carby Canine Kidney II) cells using the FFU assay. Briefly, lungs are homogenized and debris removed by centrigufation; samples are stored at -70 °C until analysis. Serial dilutions of lung homogenates are prepared and viral titers determined.

Data collected by investigator

  • Body weight pre-infection
  • Body weight post-infection (expressed as percentage of pre-infection weight)
  • Leukocyte profiles
  • Viral load