来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

The overall goal of this procedure is to recover infectious virus from a two part Zika virus Z-DNA clone. This method can answer key questions in the field of flavivirology, such as vaccine strategies, pathogenesis, transmission, and virus evolution. The main advantage of this technique is that the two-part system is more stable than full length approaches.

Though this method can provide insight into Zika virus, it can also be applied to other viral systems that have proven difficult to clone due to plasmid instability. Demonstrating the procedure will be James Weger, a post-doc from my laboratory. Begin by setting up digestion reactions for both plasmids encoding the Zika virus genome, P1 and P2.Digest 3.3 micrograms of P1 with ApaL1 and BamH1 High Fidelity in the presence of a competent phosphatase in a final volume of 100 microliters.

For P2, digest 13.3 micrograms of P2 DNA with ApaL1 and EcoR1 High Fidelity in a final volume of 100 microliters. Incubate both reactions at 37 degrees celsius for 1.2 hours. After purifying the digested plasmids using a commercial gel and PCR cleanup kit, add 30 microliters of elution buffer preheated to 70 degrees celsius to the column and incubate for five minutes.

After spinning down the purified digested plasmids, prepare the ligation reaction by combining 29 microliters of purified P1 and 29 microliters of purified P2 with T4 DNA ligation reaction buffer and T4 DNA ligase in double distilled water to a final volume of 100 microliters. Incubate the ligation reaction at room temperature for two hours. After incubation, purify the ligated plasmid as before.

This time use 10 microliters of preheated elution buffer. Next, run the undigested plasmids, digested plasmids, and ligation on an agarose gel. A successful ligation should give rise to a ligation product of around 11Kb.

After confirming that the plasmids run at the correct molecular weight, set up a T7 in vitro transcription reaction using eight microliters of purified ligation reaction and incubate for four hours at 37 degrees celsius. After the incubation, measure the RNA concentration via a UV spectrophotometer. One or two days prior to electroporation, prepare one T182 flask of Vero cells per construct to be recovered.

Include a T182 flask to be used as a mock electroporation control. When cells are at 70 to 80%confluency, warm DMEM containing 15%FBS and 10 millimolar heaps in a water bath heated to 37 degrees celsius. Detach the cells by trypsinization according to standard methods and collect the cells and medium containing 10%FBS to inactivate the trypsin.

Then centrifuge the cells at 150 times G for five minutes at four degrees celsius. After washing the cells three times with PBS, we suspend each cell pellet in RPMI 1640 plus 10 millimolar heaps to a concentration of around 0.5 times 10 to the seven cells per milliliter. Transfer 200 microliters of the cell suspension to a sterile tube.

Next, add 20 microliters of water as the mock control or 20 microliters of RNA to each set of cells. Mix the cells gently by flicking the tube, and then transfer the contents of the tube to a two millimeter gap electroporation cuvette. Set the electroporator to 170 volts, zero Omega resistance, and 950 microfarads.

Pulse the cells once and immediately add 600 microliters of the pre-warmed medium. Add the cells to a T75 tissue culture flask containing 10 milliliters of the pre-warmed medium. Remove any remaining medium from the cuvette by using the dropper that is included with the cuvette.

Swirl the flask to mix the medium and cells, and then place it in the 37 degree celsius incubator. The next day and each day thereafter, check the cells for viability until 50 to 75%cell death is observed relative to the mock electroporated control. When 50 to 75%cell death is seen, harvest the supernatant in a conical tube, and then centrifuge at greater than 3, 000 times G for 10 minutes at four degrees celsius to remove cellular debris.

Following centrifugation, transfer the clarified supernatant to a new tube and add FBS to a final concentration of 20%Additionally, add sterile heaps to a final concentration of 10 millimolar as a buffering agent to preserve virus infectivity while frozen. Finally, mix the virus by vortexing and aliquot into screw cap files. Store the virus at minus 80 degrees celsius for future use.

This image shows the similar effect of wild-type PRVABC59 and infectious clone-derived Zika virus on the growth kinetics of the non-human primate Vero cell line. This image shows the effect of both viruses on human placental cells. Similar growth kinetics are seen in a mosquito-derived C6/36 line.

This survival curve shows data obtained from four week old interferon alpha, beta, and gamma receptor knockout mice that were inoculated with 1, 000 PFU of PRVABC59 or the infectious clone-derived virus. Aedes aegypti were dissected 14 days after a blood meal containing Zika virus. Plaque assay were used to assess infection in mosquito bodies in legs to measure dissemination, or in salivary secretions to measure transmission of infectious virus.

Rates are presented as the percent of the total mosquitoes that were tested. While attempting this procedure, it's important to remember to sequence the entire virus. It's also important to remember to harvest the virus at 50 to 75%CPE to minimize viral replication cycles.

This technique is important for researches in the field of flavivirology to explore vaccines, pathogenesis, and virus evolution in Zika virus.