The overall goal of this procedure is to recover infectious mu eye neurovirus using two reverse genetic systems. Hello, I'm Armando Arias from section of Virology in Imperial College of London. In our group, we are interested in dissecting the molecular mechanisms under underlying neurovirus replication.
Noroviruses are a major cause of gastroenteritis. Worldwide jet molecular techniques for their characterization are still relatively new. The identification of neo antiviral approaches and insights into the neurovirus lifecycle have been limited due to their inability to complete a productive infection in cell culture.
The recent isolation of an antivirals capable of infecting mice and propagating cell culture, namely mirror neurovirus, has opened new avenues for the investigation of these important pathogens. This report, we will demonstrate two strategies that allow the generation of genetically defined neuro neurovirus isolates in cell culture. Both strategies are based on the generation of viral transcripts capped at the five prime end.
The first involves the synthesis in capping of viral RNA in vitro prior to transfection into cells. The second approach entails the transcription of MI neuro vial, CDNA inside of cells expressing T seven RNA polymerase Procedure overview. The protocol for the recovery of neuro norovirus from capped RNA transcripts is initiated by linear rising.
The plasmid containing MV CD NA for mmv one. This is done with the restriction enzyme NHE one. The DNA is next transcribed in vitro into RNA using T seven RNA polymerase.
Subsequently, the M-M-V-R-N-A is capped in vitro and is transfected into cells to allow the recovery of infectious MMV direct recovery of infectious MNV using FAL PX virus expressing T seven RNA polymerase for MMV recovery from directly transfected cDNA clones, the cells are first infected with a recombinant FAL PX virus expressing T seven RNA polymerase FAL PX infection will result in the expression of T seven RNA polymerase as well as viral RNA capping enzymes that may cap some of the synthesized RNA. The M-M-V-C-D-N-A is then transfected into the FPV infected cells by lipid mediated transfection. The T seven RNA polymerase then produces the M-M-V-R-N-A, some of which may then be capped by the F-P-V-R-N-A capping machinery.
A ribo zyme sequence guarantees that each RNA transcript contains a defined three prime end. The capped MMV transcripts will then be translated into protein prior to replication, leading to the production of new infectious particles. Protocol for RNA transcription and capping for the recovery of infectious MMV synthesis of infectious capped MMV transcripts.
Firstly, mix the following reagents, transcription, buffer nucleotides, water and linearized plasmid containing the M-M-V-C-D-N-A. Then add RNA in and T seven RNA polymerase to the reaction mixture. Many commercial kits are available for this purpose and provide a reproducible method for RNA synthesis.
The transcription reaction is carried out by incubating the mix at 37 degrees Celsius for at least two hours. Analyze a small alcott of this transcription reaction on a non denaturing agros gel. First, clean the gel equipment with the detergent and wash with RNAs free water prior to use.
Ensure that agros gels prepared using RNAs free reagents. M-V-R-N-A will run at approximately three killer bases on a non denaturing agro rose gel. In addition, an Agilent bioanalyzer can be used to provide a rapid method of analyzing RNA integrity.
RNA should then be purified to remove unincorporated nucleotides. Many methods are available, however, in this protocol, we purify the RNA by lithium chloride precipitation as a cost-effective alternative pellets the RNA by centrifugation at maximum speed for 15 minutes. Remove the s supernatant taking care not to disturb the translucent pellet and wash using 70%ethanol.
Resuspend the MMV transcript in RNA storage solution and ensure that it is entirely dissolved. Heating the RNA to 65 degrees C may aid this process later. Quantify the RNA by spectrometry.
Analyze the RNA integrity by running a small aliquot on a 1%non denaturing agros gel. As you can see, the RNA integrity after lithium chloride precipitation should remain the same to improve the capping efficiency. Heat an aliquot of the RNA at 65 degrees for 10 minutes.
Place the tube immediately on ice. To avoid degradation or refolding, prepare a capping reaction mixture as suggested by the manufacturer. We typically add about 60 micrograms of M-N-V-R-N-A to a final reaction of 100 microliters.
Although the reaction can be scaled down mixed thoroughly and incubate at 37 degrees for one hour, purify the RNA by lithium chloride precipitation as before. It is essential to check the integrity of the RNA again before proceeding with the transfection. Step protocol for MMV recovery by RNA electroporation into raw cells.Resus.
Suspend the flask of raw cells into DMEM. Ensure you form a single cell suspension. Determine the concentration of viable cells in a hemo cytometer using trian blue exclusion.
Pellet the cells and carefully resus. Suspend them in fresh antibiotic-free media aliquots 8 million cells per transfection and pellet them in a micro fuge. Remove the media and wash the cells in 500 microliters of PBS centrifuge again and remove the PBS resuspend the cells in 130 microliters of room temperature.
Neon resus suspension solution. Add the appropriate amount of capped RNA, typically 1.3 micrograms per 130 microliters of cells equivalent to one microgram of RNA per 6 million cells. Cells mixed with transcripts are collected in the 100 microliter neon transfection tip.
Great care should be taken not to introduce bubbles at this stage. Then electro operate using a single pulse at 1700 volts for 25 milliseconds. Release the cells into an eend orph tube containing one mil of antibiotic free media.
Distribute the cells from the tube into independent wells containing antibiotic free DMEM with 10%FCS incubate the cells at 37 degrees for 24 to 72 hours. MMV recovery by RNA lip perfection into BSR T seven cells as before CAP MMV transcripts are generated by MMV CD nna plasmid LINEARIZATION T seven polymerase in vitro transcription and in vitro capping transcripts are then lip affected into BS RT seven cells. Trypsin is a monolayer of bsrt seven cells and seed 7.5 times 10 to the five viable cells into six well plate dishes.
Incubate the cells at 37 degrees overnight. Remove the media from the cells and replace it with three mils of fresh media lacking antibiotics. To ensure the maximum efficiency of transfection, mix one to two micrograms of capped M-M-V-R-N-A with 100 microliters of optimum.
Also mix four microliters of lipectomy 2000 with 100 microliters of optimum. Combine the samples containing RNA and lipectomy and mix them thoroughly by pipetting up and down 15 times. Leave the mixture at room temperature for 20 minutes.
Then add the transfection complexes containing capped MMV transcripts in a drop price fashion to the cell monolayer. Shake the plate in perpendicular directions. Incubate the cells for 24 to seven two hours.
Protocol for direct recovery of infectious MMV from CDNA in cells expressing T seven RNA polymerase as described earlier. This protocol is based on the expression of T seven RNA polymerase and oxx viral capping enzymes in cells infected by a help of oxx virus. M-N-V-C-D-N-A under control of a T seven promoter is then transfected into the cells.
Firstly, remove cell culture media and add to each well 700 microliters of F pox virus diluted in antibiotic free media. A multiplicity of infection of around 0.5 PFU per cell is generally used, shape the plates perpendicularly and incubate for one hour at 37 degrees.Afterwards. Add two meals of antibiotic free media and incubate the cells for an additional hour at 37 degrees to allow T seven RNA polymerase expression MNV CG NNA transfection, remove the media from the infected cells and add three mills of fresh media without antibiotics.
Prepare a mixture of one microgram of M-M-V-C-D-N-A and lipectomy as explained in the previous section. Mix thoroughly by preparing up and down. Keep the tube at room temperature for 20 minutes, then mix again and add drop pies to the cell monolayer.
Shake the plate gently in perpendicular directions. Incubate the cells at 37 degrees for 24 to 72 hours to confirm the recovery of infectious MMV by the different reverse genetics approaches shown in the course of this video release infectious varion from cells by repeated freeze thawing. Determine the in each sample by standard procedures such as TCID 50 or plaque assay representative results.
The availability of these reverse genetic systems to study mi Neurovirus has provided an unprecedented ability to dissect the role of viral sequences in neurovirus replication and pathogenesis in the natural host. Both reverse genetics approaches explained in this video are highly efficient for the recovery of infectious neuro and neurovirus and cell culture. As shown in this figure.
Infectious MMV, the title is exceeding 10 to the five TCID 50 per mil are recovered after transfection of capped MM V transcripts into either raw cells or BS RT seven cells. The transfection of MM V-C-D-N-A into BSRT seven cells previously infected with help of foul pops virus results in viral titers exceeding 10 to the four TCID 50 per mil. Viral titers obtained by reverse genetics approaches are similar to Titus rescued in transfect with RNA, isolated from infectious virus, highlighting the high efficiency of reverse genetic systems described here.
