This procedure generates recombinant rift valley fever virus MP 12 strain encoding mutated NSS by reverse genetics and aims to characterize the phenotypes for vaccine development. First, using a transfection system in Bhk T seven nine cells, which stably express T seven RNA polymerase recover recombinant MP 12 strain mutants then amplify the recovered recombinant MP 12 strain mutant in Vero E six cells and titrate the amplified MP 12 mutants by plaque assay. The final step of the procedure is to screen for the lack of interferon, alpha or beta suppression by mutant NS proteins.
Ultimately, results can facilitate the preparation of candidate live attenuated vaccine for Rift Valley Fever through a reverse genetics system. The main advantage of this technique over existing method, like randomly introducing mutation into viral genome by using chemical nitrogen or serial passage, is the ability to introduce designation mutation into viral genome by using genic system. This method can help address key questions in the field of valley fever vaccines such as the significance of the major ence factor NSS in the immunogenicity of vaccine candidates.
In generally individuals new to this method will struggle because swift water fever virus, like in form target blocks and visualization of block required an optimized technique. Visual demonstration of this method is critical as the viral recovery and hydration steps are difficult to learn because step-by-step protocol for this method has not been reported. Using six centimeter dishes, place the T seven nine baby hamster kidney cells, which stably express T seven RNA polymerase and place an incubator at 70 to 80%Cell co fluency change media to complete MEM alpha without hygromycin B.Within an hour begin the transfection process for recovery of rift valley fever virus.
Create one set of plasmids encoding viral genomic RNAs for full length viral RNA expression and a second set encoding viral gene Open reading frames for viral protein expression. Now add 30 microliters of transit LT one to 385 microliters of Optum in a 1.5 milliliter tube. Vortex briefly and incubate for five minutes.
At room temperature, slowly add resulting liposomes to the 10 microgram plasmid sets mixed gently by pipetting and incubate for 15 minutes at room temperature in a drop by drop fashion, add the transfection mixture to bhk T seven nine cells and incubate for 24 hours. Replace the culture supernatant with complete MEM alpha and culture for four additional days. Harvest the culture supernatants into a 50 milliliter tube and remove cell debris by centrifugation.
Now Eloqua the supernatants into screw cap five milliliter cryo tubes and store this passage. Zero virus stock at minus 80 degrees Celsius to facilitate downstream experiments. Amplify P zero virus in cells lacking interferon alpha and beta genes grow 80%confluent Vero E six cultures in complete DMEM, replace the medium with the diluted P zero virus and incubate for an hour.
Remove the inocular and culture in 10 milliliters of complete DMEM for three to four days until the cytopathic effect of Vero E six cells becomes apparent. Harvest the supernatant as described earlier and designate the samples as E six P one using six well plates grow Vero E six cells to 80%co fluency in complete DMEM. Aspirate the medium from the Vero E six cells and add 400 microliters of tenfold serial dilutions of the E six P one virus preparation incubate for an hour.
Meanwhile, prepare two 15 milliliter tubes for the agar overlay tube. A containing seven milliliters of 1.2%noble agar in water and two B containing seven milliliters of two x complete MEM and 10%trip dose phosphate broth after 15 minutes in the water baths decant tube B into tube A and use a pipette to mix them together. Replace the viral inocular with two milliliters per well of a one-to-one mixture of tubes A and B.Continue to culture cells for three to four days.
Prepare the auger overlay by adding 1.2%noble agar two x complete MEM and neutral red to tube A to B and a third tube labeled neutral red put tube A into a 42 degree Celsius water bath and the other two tubes into a 37 degree Celsius water bath for 15 minutes. Then mix together seven milliliters of tube A seven milliliters of tube B and 0.5 milliliters of neutral red aliquot two milliliters of the mixture into each well of a six well plate and incubate the plate overnight. Now determine the number of platforming units per milliliter C 57 wild type mouse embryonic fibroblasts encode a secreted embryonic alkaline phosphatase gene inducible by NF kappa B and IRF three or seven.
Here we assay for seep activity induced by innate immune responses infect sub cofluent cells in 12 well plates with either recombinant MP 12 encoding NS mutations or MP 12 control and incubate for one hour. Remove the inocular add one milliliter per well of complete DMEM and culture for 14 hours. Harvest culture supernatants for a seep assay to each well of a 96 well plate add 200 microliters of quantum blue and add 50 microliters of each sample seal and incubate the plate at 37 degrees Celsius for one hour.
Measure the OD at 650 nanometers using a plate reader. The Rift Valley fever virus has a tripartite negative sense RNA genome named SM and L segment compared to NSS of MP 12 strain. The open reading frame of C 13 type is truncated by 69%Typically, the reverse genetic system generates viable recombinant MP 12 viruses with titers higher than one times 10 to the six PFU per milliliter C 13 type virus.
Lacking N'S functions form large turid plaques while MP 12 formed clear plaques of various sizes compared to mock infected cells at 14 hours. Post infection. C 57 wild type meth cells infected with MP 12 do not increase the level of seep in culture.
Supernatant by contrast, C 13 type infection induces an increased level of seep by 14 hours post infection northern blots using an RNA probe specific to mouse ISG 56 mRNA confirm the upregulation of ISG 56 mRNA in the absence of N'S expression. Following this procedure as a method like mouse immunization and testing of neutralizing the antibody can interrogate questions such as immunogenicity of NSS mutant. This technique paved the way for researchers in the field of with Valley Fever to explore the non-structural gene functions in infected cells and animals.
Don't forget that working with infectious virus can be extremely dangerous, so always take precautions such to avoid. Inhale AOLs. After watching this video, you should have a good understanding of how to recover and titrate recombinant MP 12 strains and how to screen NSS mutants lacking interferon alpha beta suppression function.
