| WNV preparation and inoculation
||Syringes and needles
||Health assessment for 26 days
||Body weights and clinical scores
||Harvest spleen and brain and prepare cells for flow cytometry and analysis
||Dissection kit; flow cytometry machine
||Serum preparation and WNV-specific IgM ELISA
||WNV-specific IgM levels
||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
- Freezer (-80°C)
- Refrigerated centrifuge
- 70 µm nylon mesh
- 96-well plates
- Flow cytometry machine (Becton-Dickinson LSRII with BD FACSDiva software)
- FlowJo software
- Precellys 24 machine (for homogenization of tissues; Bertin Technologies, FRANCE)
- 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)
- 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
- West Nile virus is propagated as previously described (Suthar et al., 2010).
- Working stocks are generated from supernatants collected from infected Vero cell lines and stored at -80°C.
- Mice are subcutaneously inoculated in the rear footpad with 100 PFU WNV.
Procedure: Daily health assessment
- 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
- 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
- Mice are euthanized and perfused with 10 mL PBS to remove residual intravascular leukocytes.
- 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.
- 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.
- 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.
- Cells are counted using a hemacytometer and trypan blue exclusion.
- Cells are plated at 1 x 106 cells/well and stained for surface markers for 15 min on ice.
- For tetramer staining, cells are stained with the WNV NS4b-H2Db tetramer.
- Cells are subsequently fixed, permeabilized, and stained intracellularly with antibodies for 30 min on ice.
- AmCyan Live/Dead stain is used in all panels for identification of live cells.
- Flow cytometry is performed on each sample.
Procedure: Serum preparation and IgM ELISA
- Serum is prepared at designated endpoints and stored at -80°C until ready for analysis.
- Sample wells are coated with 106 PFU WNV/well and incubated at 4°C overnight.
- 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.
- TMB substrate is used and the plate read via a spectrophotometer at 450 nm.
Procedure: RNA extraction and analysis
- Kidneys are harvested (in addition to spleen and brain).
- Organs are suspended in RNA-later and stored.
- Organs are suspended in PBS and homogenized at 1500 rpm for 20 s.
- 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.
- 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.