Materials and Reagents

The neurovirulent (RP-9) of Japanese encephalitis virus (JEV)
Aedes albopictus mosquito cell line C6/36
BHK21 cell line
20% sucrose
Phosphate buffered saline (PBS)
FreeStyle 293-F cells (Life Technologies, Invitrogen™, catalog number: R790-07 )
pcDNA3/CLEC5A_ECD.Fc plasmid (or plasmids carrying extracellular domain of other C-type lectin receptor)
293 fectin Transfection Reagent (Life Technologies, Invitrogen™, catalog number: 12347-019 )
Gibco FreeStyle 293 Expression Medium (Life Technologies, Invitrogen™, catalog number: 12338018 )
Protein A sepharose beads (GE Healthcare, catalog number: 17-0780-01 )
HRP-conjugated anti-human IgG (Fc) (Jackson ImmunoResearch Laboratories, 109-035-003 )
3,3,5,5-tetramethylbenzidine (TMB) (BD Pharmingen, catalog number: 555214 )
1x RPMI-1640 (Life Technologies, Gibco^®, catalog number: 31800-022 )
Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 11091-148 )
BSA (Sigma-Aldrich, catalog number: A2153 )
KH₂PO₄
Na₂HPO₄
Blocking buffer (see Recipes)
1 L PBS buffer (see Recipes)

Equipment

TECAN ELISA Reader
Beckman Ultracentrifugation
Pasteur pipette
Centrifuge tubes (Corning Incorporated)
Microtiter plate (Corning, Costar^®, catalog number: 9018 )

Procedure

Infected C6/36 cells (1 x 10⁷) with JEV (M. O. I. = 0.1) for 2 h followed by removing virus and replenishing fresh RPMI 1640 medium containing 5% fetal bovine serum.
After three days, supernatants were harvested and stocked; cells were further refilled with new fresh medium for another three days.
Collect viral supernatant (10 ml/tube) and centrifuging supernatant at 8,000 rpm for 30 min at 4 °C.
Add 10 ml of supernatant into centrifuge tube followed by 1 ml of adding 20% sucrose cushion at the bottom of centrifuge tube by using Pasteur pipette adding sucrose solution into bottom of the centrifuge tube.
Centrifuge supernatant at 28,000 rpm (SW28) for 3.5 h at 4 °C.
Remove supernatant and suspending the JEV particles in 0.5 ml PBS, and viral titer was determined by typical plaque assay using BHK21 cell line.
30 μg of pcDNA3/CLEC5A_ECD.Fc plasmid or extracellular domain of other C-type lectin receptor (CLR) plasmid were transfected into suspended FreeStyle 293-F cells (3 × 10⁷/30 ml) by using 30 μl of 293 fectin? Transfection Reagent.
Supernatants were harvested at 48 h of post transfection and subjected to protein purification by protein A sepharose beads.
For Fc.funsion prtoen purification, supernatants were passed through a protein A column [pre-equilibrated with 0.05 M Tris-HCl (0.1 M, pH 7.0 )], after binding, washed column with 20 ml of Tris-HCl (0.1 M, pH 7.0) and eluted with 4 ml of Tris-Glycine (0.1 M, pH 3.0) buffer.
50 μl of sucrose-cushion-purified JEV particles (5 × 10⁶ pfu) in PBS were coated on microtiter plates for overnight at 4 °C, and washed with 250 μl of PBS for 3 times before adding CLR fusion proteins and control protein.
Plate was filled with blocking buffer containing 1% BSA and 1% fetal bovine serum in PBS at room temperature for 1 h to reduce the detection background.
Both human and murine CLEC5A.Fc fusion proteins (0.05 mg/ml in PBS; 100 μl/well) and hu-IgG1 control fusion proteins were added at the amount of 0.05 mg/ml; 100 μl /well in room temperature for 2 h to interact with coated JEV particle.
Remove unbound JEV particle by gentle washing with 250 μl of PBS twice.
HRP-conjugated anti-human IgG (Fc) (1:5,000) were used to detect fusion proteins for 1 h at room temperature followed by washing with 250 μl of PBS twice and using 100 μl of 3,3,5,5-tetramethylbenzidine (TMB) as substrate and detecting the absorbance at 450 nm in ELISA reader.

Recipes

Blocking buffer
1% BSA
1% FBS
1 L PBS buffer (pH to 7.2-7.4)
900 ml H₂O
8 g NaCl or 27.4 ml 5 M
0.2 g KCl
0.19 g KH₂PO₄ or 1.4 ml 1 M
0.61 g Na₂HPO₄ or 4.3 ml 1 M

Acknowledgments

This protocol is adapted from Chen et al. (2012). We thank Drs. Caroline Milner and Carl Brown for critical comments. We are grateful for Dr. Tzyy-Wen Chiou, Dr. J-J Liang, Y-L. Lee, Chih-Ya Yang, Yu-Chiuan Wang and C-H, Chao for technical assistance. We also thank the Taiwan Mouse Clinic which is funded by the National Research Program for Genomic Medicine (NRPGM) at the National Science Council (NSC-99-3112-B-001-021) of Taiwan for technical support in histopathology experiments. We also acknowledge the technical services provided by the Transgenic Mouse Model Core Facility of the National Research Program for Genomic Medicine, NSC, and the technical services provided by Flow cytometry Core Facility of National Yang Ming University.

References

Chen, S. T., Liu, R. S., Wu, M. F., Lin, Y. L., Chen, S. Y., Tan, D. T., Chou, T. Y., Tsai, I. S., Li, L. and Hsieh, S. L. (2012). CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. PLoS Pathog 8(4): e1002655.
Chen, S. T., Lin, Y. L., Huang, M. T., Wu, M. F., Cheng, S. C., Lei, H. Y., Lee, C. K., Chiou, T. W., Wong, C. H. and Hsieh, S. L. (2008). CLEC5A is critical for dengue-virus-induced lethal disease. Nature 453(7195): 672-676.