The overall goal of the following experiment is to use various virological and biochemical approaches to dissect the role of a host signaling pathway in the lytic replication of a model herpes virus. Here as an example, we examine the role of the mitochondrial antiviral signaling protein Mavs during infection with herpes virus gamma HV 68. First to examine the role of Mavs during acute infection, wild type and Mavs knockout mice are infected with gamma HV 68 following an acute viral infection.
The mouse lung is collected and viral loads are determined by a plaque assay in which the samples are serially diluted and plated. On monolayers the number of plaques is then counted. Increased viral loads in the knockout mice suggests that the gene of interest plays an antiviral role.
By contrast, reduced viral loads in knockout mice indicate that the gene is required for viral infection To validate these findings. In vitro viral multi-step growth kinetics are examined in mouse embryonic fibroblasts, wild type, and mavs knockout mouse embryonic fibroblasts are infected with gamma HV 68 and incubated for various times. The cells are then collected and plaque assays are performed to compare multi-step growth kinetics between wild type cells and mavs deficient cells.
To further validate these findings, wild type and mavs knockout mouse embryonic fibroblast cells are infected with gamma HV 68. Then DNA and RNA are separately extracted from the infected cells and the viral genomic transcripts are analyzed by real-time PCR. We first had the idea for this protocol when we studied the role of a host immunity pathway during a virus litic infection.
This protocol can be potentially applied to investigate the rules of host signaling pathways during the infection by other pathogens. Begin this procedure by allowing six to eight week old gender matched litter made mice to acclimate for a period of four days after shipment. The following experiment will be performed in a biohazard facility.
In preparation, put on personal protective equipment including a lab coat, gloves, a mask and booties working in a cabinet of biosafety. Level two. Prepare the viral suspension with 40 to 10, 000 PFU of gamma HV 68 and 30 microliters of sterile PBS for each mouse that will be infected.
Keep the viral suspension on ice following an intraperitoneal injection of ketamine Xylazine. Ensure that the mice have been sedated by performing a toe pinch. Then using a pipette deliver 30 microliters of the viral suspension dropwise into either the left or the right nostril.
Lay the mouse on its side for five to 10 minutes to facilitate the airway delivery of the virus into the lung. Then place the mice back into their cage and monitor them until they have fully recovered from sedation. Next lung tissue is collected from the injected mice to prepare a cell lysate for assessing viral titer at various days post-infection.
Place the lungs harvested from sacrificed mice into sterile 1.5 milliliter screw cap tubes containing 500 microliters of 1.0 millimeter zirconia silica beads on ice. If not proceeding to the next step on the same day. Store the samples at minus 80 degrees Celsius.
Otherwise, add one milliliter of cold serum free DMEM. Place the tube in a bead beater and press start to homogenize the lungs for 30 seconds. To avoid overheating the sample which may inactivate the virus.
Chill the tube on ice for at least one minute following 30 seconds of homogenization. Then repeat this process. Next, centrifuge the homogenized tissue at 16, 000 RCF at four degrees Celsius for one minute.
Following the spin, the cell debris will be at the bottom of the tube and the lipids will be at the surface. Immediately Take the snat as shown here to perform a plaque assay using a NIH three T three or BHK 21 monolayer. Next, a plaque assay is performed To determine the viral titer present in the sample, begin the plaque assay by making serial dilution of the viral supernatant.
First using a multichannel pipette add 270 microliters of media to a 96 well plate. Leave the first row blank. Then add 200 microliters of supernatant to the first row of the 96 well plate plate each sample in triplicate.
Next, transfer 30 microliters of the sample from the first row to the next row and mix. Then transfer 30 microliters of the mixture to the next row and mix. Repeat this step multiple times to make tenfold serial dilutions.
Next, add the diluted viral supernatant onto the cell monolayer. In a 24 well plate. Place the plate in an incubator and shake the plate every 30 minutes during a two hour incubation following the incubation to remove cell debris, aspirate the supernatant from the plate.
Then wash the cells once with fresh medium. After the wash, add medium containing methylcellulose to the cells. Incubate the cells for four to six days.
After four to six days have passed. Use a microscope to read the plaque number for each sample. Record the number of plaques for each dilution.
Then calculate the mean and standard deviation. Wells containing too many or too few plaques cannot be used to accurately calculate titer. So for each sample, use a dilution in which there are seven to 70 plaques.
This sample diluted 1000 fold has an average of 23 plaques per well. To calculate the viral titer in the undiluted solution, multiply the dilution factor by the number of plaques by the number of microliters in a milliliter. Then divide that by the volume of diluted supernatant added per well.
So in this example, 1000 times 23 plaques times 1000 microliters per milliliter divided by 100 microliters equals 2.3 times 10 to the fifth forming units per milliliter. Next, a viral stock solution with a known titer is used to determine the growth kinetics of gamma HV 68 in mouse embryonic fibroblasts ORMs begin by splitting wild type myths and those deficient in a host gene into a 24 well plate at 10, 000 cells per well. The next day, replace the medium in each well with 0.5 milliliters of a low MOI gamma HV 68 suspension.
Place the plate in the incubator at 37 degrees Celsius and allow the incubation to proceed for two hours throughout the incubation. Rock the plate every 30 minutes after the incubation. Use a pipette to remove the viral suspension and add 0.5 milliliters of fresh complete DM EM containing 8%fetal bovine serum to virus infected cells.
Place the cells back in the incubator at various days post-infection. Harvest the medium end cells by pipetting up and down multiple times. Transfer them to sterile 1.5 milliliter centrifuge tubes immediately freeze the tubes at minus 80 degrees Celsius to release gamma HV 68 from the Mets.
Perform three cycles of freezing and thawing. Thaw the cells by placing them at 37 degrees Celsius. Then vortex and place them back in the minus 80 degrees Celsius freezer.
Determine viral titer by a plaque assay using a NIH three T three or BHK 21 monolayer as shown earlier to examine whether DNA or RNA replication is affected by deficiency in host genes or viral genes. Begin with infected mes at various days post-infection, discard the supernatant, rinse the cells with cold PBS and trypsin ice cells. Then pellet the cells by centrification at 1000 RCF at room temperature for one minute following the spin, discard the supernatant and store the cells at minus 80 degrees Celsius.
Extract total DNA and RNA, which is of host and viral origin. Then centrifuge the spin columns. Perform real-time PCR using total DNA and primer specific for viral lytic transcripts.
Determine the relative quantity of intracellular gamma HV 68 genome in reference to a host housekeeping gene such as beta actin. Prepare CDNA with total RNA and oligo DT primer. Perform real-time PCR using CD NA and primers as above to determine the relative quantity of viral transcripts.
In reference to that of the host housekeeping gene Mavs is short for mitochondrial antiviral signaling as shown here. The Mavs adapter molecule relays signaling from cytosolic rig eye like receptors to activate NF kappa B and interferon regulatory factors IRFs that in turn upregulate the gene expression of pro-inflammatory cytokines and interferons. The viral loads in the lung of gamma HV infected wild type mice and the litter mate mice deficient in Mavs are shown here.
Deficiency in Mavs significantly reduced viral loads in the lung at 10 days post infection indicating that Mavs is required for efficient viral infection in vivo. This figure shows a viral multi-step growth curve on wild type mouse embryonic fibroblasts, and those deficient in Mavs deficiency in Mavs. Significantly delayed viral growth on mouse embryonic fibroblasts indicating that Mavs is required for efficient viral replication in vitro mRNA levels of viral lytic genes in gamma HV 68 infected mouse embryonic fibroblasts were analyzed by real-time PCR deficiency in Mavs, significantly reduced the mRNA levels of three essential lytic genes.
All these results collectively support the conclusion that Mavs is required for gamma HV 68 transcription activation, and lytic replication. After watching this video, you should have a good understanding of how to interrogate the host pathogen interaction at a multiple levels, both in VO and Vevo. Don't forget that working with pathogens could be extremely hesitant.
Precautions such as wearing personal protection equipment should be taken while performances proceeding.
