TITLE: Guidelines for microneutralization testing of human antibodies to West Nile virus
AUTHORS: Anna  Nagy, Nikolett Csonka, Mária Takács

[DESCRIPTION]
West Nile virus (WNV), a mosquito-borne member of the family Flaviviridae, genus Flavivirus is an emerging pathogen, which is endemic in most part of Europe, especially in the central and southern regions of the continent. The vast majority of human infections remains asymptomatic, though approximately 20% of infected individuals develops milder, influenza-like disease and less than 1% has severe neurological manifestation. The laboratory diagnosis of human flavivirus infections could be challenging due to the serological cross-reactivity and the overlapping endemic areas with other members of the flavivirus genus. Microneutralization assay considered to be a gold standard confirmatory method in the arbovirus serological diagnostic. The detection of neutralizing antibodies provides a highly specific and sensitive tool for evaluation and better interpretation of other serological test results. Furthermore, microneutralization is a species independent method and the neutralization antibody titer correlates with the immune protection. 

[STEPS]
SECTION: Preparation of samples
1. Tested specimens: human serum, plasma
SECTION: Preparation of samples
3. Heat inactivation: inactivate all sera to be assayed at 56°C for 30 minutes to limit the effects that complement, or adventitious virus may have on the final results.
SECTION: Preparation of samples
4. Optional: Filtrate the serum specimens through 0.22-micron filters to remove particulates is not necessary.
SECTION: Dilution of specimens and controls
5. Dilution of specimens and controls:  For endpoint titration, 2-fold (50%) serum dilutions should be used. These dilution series lead to a more precise estimate of the endpoint titer than higher dilutions factors. All samples should be tested in parallel titration.
SECTION: Dilution of specimens and controls
6. Prepare serial two-fold dilutions of test sera and positive and negative control sera: add 50 ul cell culture media without fetal bovine serum (FBS) to each well of a sterile 96-well cell culture microplate. (1- 1 row of the microplate for each sample.)
SECTION: Dilution of specimens and controls
7. Add 50 µl of heat-inactivated serum to the first well of the 96-well microplate containing 50 µl of diluent, thus making an initial serum dilution of 1:2.
SECTION: Dilution of specimens and controls
8. Perform serial two-fold dilutions of each of the test sera and the positive and negative control sera by transferring 50 µl of the 1:2 serum dilution to the next well and so on. After each transfer, mix by gentle suspending; change pipette tips and continue the dilution series to the end of the required range.
SECTION: Dilution of specimens and controls
9. After the final transfer, discard the remaining 50 µl.
SECTION: Dilution of specimens and controls
10. To the last well of each row of the microplate, prepare the ‘serum only’ control by adding 50 µl diluent + 50 µl specimen or control, resulting 1:2 dilution of the tested specimen or control sera. Finally, add 50 µl diluent again. This well will not contain virus suspension and will serve as a control to exclude any cytotoxic activity of the tested specimen.
SECTION: Test procedure
11. Rapidly thaw a vial of virus, that was previously stored at -80°C.
SECTION: Test procedure
12. Dilute the virus stock in cell culture medium (without FBS) to the dilution previously determined: the fixed dose of WNV is 100 TCID50 (tissue culture infective dose 50%).
SECTION: Test procedure
13. The working dilution of the virus stock = 100 TCID50 per well.
SECTION: Test procedure
14. Add equal volumes (50 µl) of the diluted virus to each well, except to the ‘serum only’ controls. Avoid contamination of the pipette tips by serum. (The final dilution of the tested specimens will be: 1:4)
SECTION: Test procedure
15. Besides the serum control and the positive + negative controls, it is necessary to use a virus control. It is recommended to prepare a ‘virus titration’ control in each assay. Virus titration control can be prepared in a separate 96-well microplate. Add 50 µl of each dilution of the serially diluted virus stock, in at least three parallel wells of the microplate. Then, add 50 µl cell culture medium without FBS (diluent) to each well. Diluent represents the ‘volume of the tested specimen’.
SECTION: Test procedure
16. Cover the microplates by sealing foil.
SECTION: Test procedure
17. Mix the microplates by gentle shaking.
SECTION: Test procedure
18. Place the microplates into a 37°C; 5% CO2 incubator for 90 minutes.
SECTION: Test procedure
19. Add 100 µl Vero or Vero E6 cell suspension (600000 cells/ml) in cell culture medium with 10% FBS to each well of the microplate.
SECTION: Test procedure
20. Prepare ‘cell control’: add 100 µl cell suspension + 100 µl cell culture medium without FBS (diluent). (You can use a separate microplate for this step.)
SECTION: Test procedure
21. Place the microplate into a 37°C; 5% CO2 incubator for 5 – 7 days.
SECTION: Test procedure
22. Evaluation: read the results by using an inverted microscope.     
SECTION: Preparation of samples
2. Used controls in this assay: Positive serum control, Negative serum control, Serum only control, Virus titration control, Cell control