By Radhakrishnan Padmanabhan, Subhash G. Vasudevan
Infection via flaviviruses comparable to dengue virus serotypes (DENV 1-4), jap encephalitis virus (JEV), tick-borne encephalitis virus (TBE), yellow fever virus (YFV) and West Nile virus (WNV) effect hundreds of thousands of lives and reason tens of hundreds of thousands of mortalities every year. Dengue is a world public wellbeing and fitness emergency in particular considering there's no preventative vaccine or antiviral remedy for dengue sickness. Dengue: equipment and Protocols deals the expanding variety of dengue researchers a one-stop protocol e-book with options compiled from the best laboratories engaged on dengue. Chapters disguise themes akin to dengue virus isolation from scientific samples, quantification of human antibodies opposed to the virus, assays to quantify the virus debris, the generally used mouse version to check dengue pathogenesis, vaccine and antiviral efficacies. Written within the winning Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, with no trouble reproducible protocols and notes on troubleshooting and warding off identified pitfalls.
Authoritative and simply obtainable, Dengue: tools and Protocols seeks to serve either execs and newbies with its well-honed methodologies on dengue research.
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Extra info for Dengue: Methods and Protocols
Neutralization assay using Vero cells: Dilute a prior titrated virus stock to 50 μL/well using 2 h infection medium. 1, transfer 50 μL of diluted virus to each appropriate well in a U-bottom 96-well plate. Transfer 50 μL of diluted monoclonal antibody or human serum into the appropriate wells in the plate, mix by pipetting and incubate plate at 37 °C in an incubator containing 5 % CO2 for 1 h. After the 1 h incubation, aspirate medium from Vero cell-coated wells, transfer the antibody-virus mixture to the aspirated wells, and incubate for 2 h at 37 °C in an incubator containing 5 % CO2.
Gently add 5 mL of 24 % (w/v) sucrose to the bottom of the suspension using a Pasteur pipette. Do not mix the two solutions. 5 h. Invert the tube to remove the sucrose and wick off the excess solution. Add 500 μL NTE buffer to cover the pellet. This is kept overnight at 4 °C to allow the pellet to soften (see Note 8). 5 mL Eppendorf tube. Centrifuge on a benchtop centrifuge at 16,000 × g for 2 min. Transfer the supernatant into a new Eppendorf tube and repeat the centrifugation process. 7. Prepare a potassium tartrate-glycerol gradient ranging from 10 to 30 % in an ultra-clear tube suitable for the specific ultracentrifuge rotor.
16. Let slides air-dry completely. 17. Place a drop of mounting fluid onto the slide and gently lay down the coverslip. 18. View slides using a fluorescent microscope (see Note 12). 19. Record the number of infected mosquitoes for each dilution. 20. Calculate virus titers using the method of Reed and Muench and express the titers in terms of the dose that infects 50 % of the mosquitoes inoculated (MID50 per ml). Mosquito Inoculation Technique 4 23 Notes 1. The needles can be pulled using a simple alcohol lamp if a needle puller is not available.