The overall goal of this procedure is to investigate various stages of the Hepatitis C virus replication cycle. This is accomplished by first generating HCV genomic RNA, transcripts from Linearized HCV plasmid constructs. The second step is to transfect the cells with H-C-V-R-N-A.
Next, the cells are plated for various time points and assays. The final step is to collect the cell culture supernatant for measuring viral titer and harvest transfected cells for protein and RNA. Ultimately reverse transcription, PCR Western blotting immunofluorescence assay and HCV titer are performed and demonstrate a robust HCV infectious cell culture system.
The main advantage of this infectious hepatitis C virus cell culture system over the HCV replicant system is that viral entry vion assembly and egress steps can be investigated. This protocol can help answer the key questions in the hepatitis C virology field, such as Vion, morphogenesis, and host a pathogen interaction. The implications of this technique extend toward vaccine development as it allows characterization of attenuated viral strains that can be evaluated as potential vaccine candidates.
Though this method can provide insight into hepatitis C virus, it can also be applied to other RNA viruses in the family of lab verde. Generally, individuals new to this method will be faced with the challenge of generating high quality HCV genomic RNA studying. The complete replication cycle of HCV became feasible in cell culture following the discovery of a genotype two A HCV Japanese fulminant Hepatitis one isolate.
Visual demonstration of virological assay is critical because the assay will require expertise in both molecular and cellular techniques Using an intra genotype two A chimeric virus, F-N-X-H-C-V and F-N-X-H-C-V poll null HCV for evaluation of viral replication. Linearize previously generated viral plasmids by digestion with XBA one restriction enzyme in a two milliliter tube. Then treat the plasmids with mung bean nuclease to generate blunt ends.
Purify the digested plasmids by anion exchange chromatography. Verify the integrity of the linearized plasmid by subjecting DNA to AROS gel electrophoresis. Next, add the linearized plasmid DNA template into a 0.2 milliliter tube containing T seven RNA polymerase reaction components to synthesize HCV genomic RNA transcripts.
Purify the newly synthesized DNAs treated RNA using an RNA purification kit. Then verify the RNA production by Agros gel electrophoresis. Following this, quantify the RNA by spectral photometry.
Detach HU seven based adherent cells using trypsin enzyme treatment and collect the cells into a 50 milliliter conical. Two following centrifugation resus. Suspend the cell pellet with cold low serum media After repeating the centrifugation, resuspend the cells in low serum media at one times 10 to the seven cells per milliliter.
Next, mix a total of 10 micrograms of transcribed viral RNA with 400 microliters of resuspended cells into a pre chilled 0.4 centimeter electroporation vet deliver the viral RNA into the cells using an electroporation at 270 volts, 100 ohms and 950 micro ferres. When finished, resuspend the electroporated cells in 10 milliliters of complete growth media with 15%FBS at this point, plate the cells in both T 25 flasks at about 1.2 times 10 to the six cells per flask and 48 well plates at one times 10 to the fourth cells per well for 4 48 and 96 hour time points. Replace the media at four to eight hours post transfection with fresh supplemented growth media with 10%FBS to remove dead cell debris from the cultured flasks and plates.
Using a serological pipette harvest the cell culture supernatants at the 48 N 96 hour time points into a 15 milliliter conical tube. Then remove cellular debris from the collected samples by centrifugation at 15, 000 RPM for 10 minutes at four degrees Celsius following centrifugation. Store the cell-free supernatants at negative 80 degrees Celsius.
Lyce the cells for protein and RNA analysis by western blot and reverse transcription quantitative PCR or R-T-Q-P-C-R at the indicated time points. Reverse transcribe one microgram of total cellular RNA using reverse transcriptase enzyme and a specific primer for the HCV sense strand that binds to the five prime untranslated region in a 0.2 milliliter tube. Also reverse transcribe F-N-X-H-C-V-R-N-A of known genome copies using an HCV sense strand primer using a realtime PCR system.
Carry out QPCR by using 50 nanograms of the resulting transcribed CD NA using specific HCV primers and DNA binding green dye containing QPCR super mix. Use the following conditions when running QPCR to determine the H-C-V-R-N-A copy number following QPCR. Resolve the cell lysate from the viral RNA transfected at 96 hours.
Post transfection using SDS page. Then transfer the resolved proteins in the gel to a poly vine di fluoride membrane by trans plot turbo method. Block the membrane using a blocking solution containing 5%skim milk and 0.2%tween 20 in PBS and placed in a container.
Incubate the membrane with primary mouse monoclonal antibody and S3 at a one in 1000 dilution and beta actin at a one in 5, 000 dilution in a four degrees Celsius cold room. After incubation, add goat anti mouse immunoglobulin G conjugated to horse radish peroxidase at a one in 5, 000 dilution and detect by chemiluminescence. At this point, fix the H-C-V-R-N-A transfected cells using methanol for 30 minutes at negative 20 degrees Celsius for the immunofluorescence assay.
When finished, wash the cells with PB S3 times after blocking with immunofluorescence assay blocking buffer use rabbit polyclonal anti NS five a primary antibody and mouse monoclonal anti DS RNA antibody J two at a dilution of one to 200 and incubate for five hours to overnight in a four degrees Celsius cold room. Following incubation, wash the cells with PB S3 times after the primary antibody. Then add goat anti rabbit immunoglobulin G 4 8 8 polyclonal secondary antibody and goat anti muse immunoglobulin 5 9 4 polyclonal secondary antibody at a one in 1000 dilution and incubate for one hour at room temperature on a tabletop rocker.
After washing the cells with PB S3 times stain the nuclei using herx die and view using a fluorescent microscope. Next plate naive hue, 7.5 0.1 cells at approximately three times 10 to the third cells per well. Using a 96 well plate the next day, perform tenfold serial dilution of cell-free culture supernatant harvested from H-C-V-R-N-A transfected cells using growth media and inoculate in triplicate onto hue.
7.5 0.1 cells Fix the cells at 72 hours post-infection using methanol for 30 minutes at negative 20 degrees Celsius after removing the cells from the freezer amino stain for H-C-V-N-S five A protein using the previously described conditions using a fluorescent microscope. Count the NS five, A positive cell foci in the well with the highest viral dilution and calculate the average number of focus forming units per milliliter. The linearized HCV plasmid quality was assessed by gel electrophoresis.
The H-C-V-D-N-A was subjected to T seven RNA polymerase mediated in vitro transcription, which yielded a single RNA product at 9.6 Kilobases. R-T-Q-P-C-R Results indicated that the wild type virus replicated the genome efficiently. The wild type exhibited a one to three log higher level of genome replication compared to poll null virus.
The wild type virus produced the NS three protein involved in viral protein cleavage and genome replication. The wild type virus also expressed NS five a protein and the NS five a protein and double stranded RNA co localized in the cellular cytoplasm of wild type transfected cells suggesting active viral replication virus titer measurements indicated that the wild type virus is infectious and produces over 10, 000 foci forming units per milliliter of infectious particle at six hours post transfection. Both wild type and pole null viruses had similar luciferase activities indicating a similar input level of transfected.
RNA had been translated, however, at 48 hours and 96 hours post transfection. The wild type virus exhibited increased genome replication levels compared to poll null virus. The wild type virus also produced viral NS three protein.
The poll null virus had base level luciferase activity, whereas the wild type virus had a two to three log higher level of replication compared to poll null reporter virus. Once mastered Hepatitis TC virological assays can be done in two weeks if they're performed properly While attempting this procedure, it's important to have robust cells and high quality HCV genomic RNA transcripts. Following this procedure, characterize HCV strains can be tested in animal model systems to investigate in vivo fitness and host pathogen interactions.
The development of an infectious HCV cell culture system paved the way for researchers to explore the virion, morphogenesis and viral egress steps of the HCV replication cycle. After watching this video, you should have a good understanding of how to perform various virological assays for characterizing different stages of the Hepatitis C virus replication cycle. Don't forget that working with Hepatitis C virus can be extremely hazardous and by safety precautions such as wearing appropriate personal protective equipment should always be taken while performing this procedure.
