In this video, acute and latent herpes virus infections are evaluated following intranasal infection in mice by plaque and infectious center assays. First to mimic physiological exposure, mice are infected by intranasal administration of neuron gamma herpes virus. 68, 5 to seven days later, lung tissue is collected and homogenized.
Then a plaque assay is performed to determine the acute viral load To determine the latent viral load, the spleen is harvested from the mouse. 12 to 14 days after infection, a single cell suspension of infected SPOC sights is generated and an infectious center assay is performed. Results from these assays reveal the spatiotemporal profiles of viral replication and infectivity in vivo.
So the main advantage of this technique over existing methods like using the tissue culture system for the study of viral infection, is that it takes advantage of marine gamma herpes virus infection of mice for the in vivo characterization of the lifecycle and or pathogenesis of gamma herpes viruses. Begin this protocol by infecting six week old mice with gamma herpes virus 68 or gamma HV 68, 5 to 10 minutes after administering anesthesia perform a toe pinch. It is important to ensure that the mice are fully anesthetized.
If they are not intranasal, administration of the virus will cause them to sneeze and they will not receive the correct dose. Next place the tip of a pipette in the opening of the mouse's nose and slowly deliver 15 microliters of PBS containing 500 to 2, 500 plaque forming units of gamma HV 68 drop by drop. The mouse should inhale the drops and bubbles should not form repeating the other nostril.
Place the mouse on a heating pad and allow it to recover for 10 to 15 minutes. Once the mouse has regained consciousness, it can be returned to its cage to determine the virus titer in acutely infected lungs. Plaque assays performed five to seven days after intranasal infection.
Wash the lungs harvested from sacrificed mice by placing the mink cold PBS stir briefly then remove the PBS and repeat this wash once more. To remove any remaining blood cells, place one of the lungs in a five milliliter round bottom tube containing one milliliter of ice cold complete DMEM. Place the tube on ice, then place the other side of the lungs in another tube and submerge it in a slurry of dry ice and ethanol.
To rapidly freeze it. Store the tube at minus 80 degrees Celsius for examining viral DNA loads in the lungs by QPCR. Next, take the tissue that was placed on ice and homogenize it at maximum speed for one second.
Then freeze and thaw the sample by placing the tube on dry ice for 10 minutes. Then transferring it to a 37 degree Celsius water bath for two minutes. Wash the homogenizer with 70%ethanol and PBS.
Then repeat the process of homogenization and freeze thaw with the next sample. Cleaning the homogenizer between each sample. Centrifuge the homogenized tissue at 3000 RPM for 10 minutes of four degrees Celsius.
Transfer the supernatant to a new micro centrifuge tube. To prepare serial dilutions. Label the S supernatant tube as 10 to the zero.
Then label eight additional 1.5 milliliter micro centrifuge tubes containing 540 microliters of plain DMEM as 10 to the minus one to 10 to the minus eight. Transfer 60 microliters of the undiluted homogenate from the tube labeled 10 to the zero to the tube labeled 10 to the minus one cap vortex. Then withdraw 60 microliters and transfer it to the tube labeled 10 to the minus two.
Continue with the serial dilutions through 10 to the minus eight. Next, remove the vari cell monolayers prepared a day earlier from the incubator. The cell should be evenly distributed and the cell density should reach 30 to 40%on the day of the plaque assay.
Labeling the plate for assaying. Each dilution in duplicate aspirate the medium then to each of the 12 wells. Add 200 microliters per well of the serially diluted homogenate in reverse order from 10 to the minus, eight to 10 to the zero.
Place the vari cells in a humidified incubator at 37 degrees Celsius, 5%carbon dioxide for one hour every 15 minutes during the incubation, gently swell the plates to evenly distribute the virus over the monolayer following the incubation, aspirate the inoculums and replace them with two milliliters per well of overlay medium. Then place the plates back in the incubator for one week after a week has passed. Aspirate overlay medium and replace it with two milliliters of fixed stain medium for the plaque assay.
Incubate the plate for at least one hour of room temperature. Pour off the fixed stain medium and wash with water twice. Then allow the plates to air dry with the lids off when the plates are dry.
Count the number of plaques at each dilution to minimize error. Exclude any wells that have fewer than 10 or more than 100 plaques. Calculate the virus tighter using the following formula in which the titer in plaque warming units per milliliter is determined by dividing the number of plaques per well by the volume of the inoculum per well and multiplying that by the dilution factor to determine latent viral load.
An infectious center assay is performed 12 to 14 days after the intranasal infection. Begin by pooling spleens harvested from sacrificed mice in one milliliter of chilled DMEM at 10 milliliters of DMEM with 2%FBS to a 10 centimeter plate and transfer the spleens into the plate. Next, use ice cold medium to wet.
Two frosted end glass microscope slides. Place the spleens on the frosted side of one slide Mechanically dissociate the spleen by pressing and rubbing the two slides together until all of the red clumps are crushed. This will also remove fat particles if they are present, holding the slide over a 10 centimeter plate.
Pipette DMEM over it to rinse the contents into the plate, flip and wash the other side. Then repeat this process with the other slide. Gently pour the entire tissue suspension through a cell strainer into a 50 milliliter conical screw cap tube.
Then wash the 10 centimeter dish containing the spleen homogenates with 10 milliliters of PBS and pour it through the cell strainer centrifuge the single cell suspensions for 10 minutes at 1, 300 RPM and discard the snat after the spin. Discard the snat and add five milliliters of a CK buffer. Delays the red blood cells.
Flick the cell pellets to break up any clumps. Incubate for five minutes at room temperature and then centrifuge your gain a 1, 300 RPM following the spin. Discard the senna and re suspend the pellets in PBS with two FBS by flicking and gentle pipetting.
Then place the cells back on ice count viable cells using an automatic cell counter. Once the cell numbers have been determined, spin down the PBS resuspended SP cytes at 375 times G for four minutes. Discard the supinate and resuspend the cells in four milliliters of complete DMEM label.
Five 15 milliliter conical tubes containing 2.4 milliliters of DMEM for fivefold serial dilutions, one to five, one to 25, 1 to 125, 1 to 250 and freeze and thaw. Then dilute 600 microliters of PLE cyte suspension into the first tube and mix well. Do not vortex.
Continue with the serial dilution for each tube. Next, aspirate the medium from the prepared six well monolayer of various cells prepared one day earlier seed one milliliter of the serial diluted cytes onto various cells in duplicate 10 wells will be used. Incubate the plates for eight to 12 hours, aspirate the seeded cyte suspension and add four milliliters of overlay medium to each well incubate for an additional six days after six days have passed.
Determine the levels of spleen infectious centers by plaque assay as before to assess the role of the viral anti apoptotic protein BCL two in gamma herpes virus 68 LITIC and latent infection bowel C mice were infected with either gamma HV 68 or a mutant strain of gamma HV 68 containing non-functional viral BCL two and acute and latent infection rates were assessed as shown in this video as shown here. Viral BCL two replicated at levels comparable to wild type gamma HV 68 in the lungs after seven days of intranasal infection in BC mice. No statistically significant differences in the lung Titus of the virus were detected between the two groups.
These data indicate that viral BCL two is not a crucial factor for the acute infection of Gamma H HV 68 in mice. However, by 28 days post infection, the titer of viral BCL two mutant virus in the spleens dropped six to tenfold compared to the wild type as measured by infectious center assay. This suggests that the viral BCL two mutant gamma H 68 virus is defective in the maintenance of splenic latency after infection to verify the results obtained by infectious assay or to determine whether infection rates were reduced due to a reduction in viral latent loads versus a failure of the latent virus to reactivate X vivo viral genome levels were assessed by QPCR as shown here.
The viral genome load of the viral BCL two mutant virus was severely reduced compared to that of wild type viruses at day 28 in repeated experiments. The close correlation between viral genome loads and the frequency of latent gamma HV 68 viral BCL two mutant virus reactivation, ex vivo at latent stage of infection highlights a latency defect of this mutant virus infection. Following this procedure, other methods like limiting dilution PCR can be performed in order to answer additional questions like to evaluate the frequency of viral genome bearing cells.
