Graham1 project protocol

West Nile virus infection in males of F1 hybrids (RIX) of Collaborative Cross strains of mice   (2015)

Graham JB, Thomas S, Gale Jr. M, Lund JM
With: Swarts J, McMillan AA, Ferris MT, Suthar MS, Treuting PM, Ireton R

Project protocol - Contents

Workflow and sampling

Data collected
WNV preparation and inoculation Syringes and needles
2 Health assessment for 26 days
- Body weights and clinical scores
3 Harvest spleen and brain and prepare cells for flow cytometry and analysis Dissection kit; flow cytometry machine Lymphocyte populations
4 Serum preparation and WNV-specific IgM ELISA Spectrophotometer WNV-specific IgM levels
5 Harvest kidney (in addition to spleen and brain) for RNA extraction and analysis Dissection kit; PCR machine WNV levels and Ifnb1 and Ifit1 RNA expression

Equipment and supplies

  • Freezer (-80°C)
  • Refrigerated centrifuge
  • 70 µm nylon mesh
  • Hemacytometer
  • 96-well plates
  • Flow cytometry machine (Becton-Dickinson LSRII with BD FACSDiva software)
  • FlowJo software
  • Spectrophotometer
  • Precellys 24 machine (for homogenization of tissues; Bertin Technologies, FRANCE)

Reagents and solutions

  • West Nile virus (WNV) TX-2002-HC (WN-TX)
  • Vero cell lines
  • Phosphate buffered saline (PBS)
  • Ammonium-chloride-potassium (ACK) lysis buffer
  • Fluorescence-activated cell sorting (FACS) buffer (1X PBS, 0.05% fetal bovine serum)
  • RPMI medium
  • Hypertonic Percoll
  • Trypan blue
  • WNV NS4b-H2Db tetramer (Immune Monitoring Lab, Fred Hutchinson Cancer Research Center)
  • Foxp3 fixation/permeabilization concentrate and diluent (eBioscience)
  • AmCyan Live/Dead stain (Invitrogen)
  • Directly conjugated monoclonal antibodies:
    • CD3-ECD (143-2C11)
    • CD4-BV605 (RM4-5)
    • CD8-BV650 (53-6.7)
    • Foxp3-Alexa700 (FJK-16S)
    • NS4b class I tetramer-allophycocyanin
  • Goat-anti-mouse IgM-horseradish peroxidase (HRP; eBioscience)
  • 3,3',5,5'-Tetramethylbenzidine (TMB)
  • RNA-later
  • RNeasy kit (Qiagen)
  • DNase (Ambion)
  • SYBR green
  • PCR primers for WNV, interferon beta 1 (Ifnb1), and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) (see Graham et al., 2015)

Procedure: West Nile virus preparation and inoculation

  1. West Nile virus is propagated as previously described (Suthar et al., 2010).
  2. Working stocks are generated from supernatants collected from infected Vero cell lines and stored at -80°C.
  3. Mice are subcutaneously inoculated in the rear footpad with 100 PFU WNV.

Procedure: Daily health assessment

  1. Mice are monitored daily for morbidity (weight loss) and clinical disease scores. Scoring:
    • 0=healthy mouse
    • 1=ruffled fur, lethargy, hunched posture, no paresis, normal gait
    • 2=altered gait, limited movement in one hind limb
    • 3=lack of movement, paresis in one or both hind limbs
    • 4=moribund
  2. Categories:
    • Asymptomatic: RIX:CC(041x012), RIX:CC(017x004)
    • Symptomatic: RIX:CC(005x001), RIX:CC(024x023)
    • Asymptomatic with CNS involvement: RIX:CC(004x011)

Procedure: Leukocyte preparation and analysis

  1. Mice are euthanized and perfused with 10 mL PBS to remove residual intravascular leukocytes.
  2. Spleens are homogenized and treated with ACK lysis buffer to remove red blood cells, washed, and resuspended in FACS buffer. Brains are harvested into RPMI and a suspension created through mechanical disruption.
  3. The suspensions are added to hypertonic Percoll to create a 30% Percoll solution, vortexed, and centrifuged at 1250 rpm for 30 min at 4°C.
  4. After aspirating supernatant, any remaining red blood cells in the cell pellet are lysed with ACK, washed, passed through a nylon mesh, and resuspended in FACS buffer.
  5. Cells are counted using a hemacytometer and trypan blue exclusion.
  6. Cells are plated at 1 x 106 cells/well and stained for surface markers for 15 min on ice.
  7. For tetramer staining, cells are stained with the WNV NS4b-H2Db tetramer.
  8. Cells are subsequently fixed, permeabilized, and stained intracellularly with antibodies for 30 min on ice.
  9. AmCyan Live/Dead stain is used in all panels for identification of live cells.
  10. Flow cytometry is performed on each sample.

Procedure: Serum preparation and IgM ELISA

  1. Serum is prepared at designated endpoints and stored at -80°C until ready for analysis.
  2. Sample wells are coated with 106 PFU WNV/well and incubated at 4°C overnight.
  3. The next day, plates are washed 4 times, and standards and samples (in duplicate) are incubated for 2h at room temperature, followed by incubation with goat anti-mouse IgM-HRP.
  4. TMB substrate is used and the plate read via a spectrophotometer at 450 nm.

Procedure: RNA extraction and analysis

  1. Kidneys are harvested (in addition to spleen and brain).
  2. Organs are suspended in RNA-later and stored.
  3. Organs are suspended in PBS and homogenized at 1500 rpm for 20 s.
  4. Total RNA is extracted and analyzed via RT-qPCR for WNV (E gene) and interferon beta 1 (Ifnb1) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1). Primer sequences are available in Graham et al., 2015.

Data collected by investigator

  • Body weights
  • Clinical scores
  • Lymphocyte populations
  • WNV-specific IgM
  • WNV levels
  • Ifnb1 levels
  • Ifit1 levels


Suthar MS, Ma DY, Thomas S, Lund JM, Zhang N, Daffis S, Rudensky AY, Beven MJ, Clark EA, Kaja MK, Diamond MS, Gale M, Jr. (2010) IPS-1 is essential for the control of West Nile virus infection and immunity. PLoS Pathog 6:e1000757.