The overall goal of this experimental procedure is to evaluate the pathogenesis, humoral responses, and protection efficacy induced by Influenza A wild-type viruses or Influenza vaccines using a mouse model of infection. This method can help answer the questions about the biology of Influenza A viruses such as, viral pathogenesis, immuno responses and efficacy of Influenza vaccines as well as, anti-virus. The main advantage of this technique is that the mouse has an immune system similar to humans and multiple strains are available that can be exploited will evaluate the genetic basis of Influenza A virus infections.
For this method can provide insight into the efficacy of new Influenza vaccines, it can be applied also to older systems such as, evaluating potential antiviral treatments including addressing antibodies or drugs. After raising and marking six to eight week old C57B6 mice under pathogen free conditions and preparing Influenza A virus or IAV dilutions under aseptic conditions. Use a scale to weigh the mice and record the weight.
Once a mouse is anesthetized and the depth of anesthesia has been verified according to the text protocol, place the mouse in dorsal recumbency. Insert a pipette tip containing 30 microliters of the virus inoculum in the nostril and slowly, but constantly eject the solution. To evaluate for morbidity and mortality, infect three to four groups of female mice with 10 full dozes of PR8 wild type as described in the text protocol.
Monitor and weigh the mice over the next 14 days, at approximately the same time to minimize weight variations due to food ingestion. After euthanizing the animals that survived the viral infection and calculating the viral 50%mouse lethal dose or MLD50 according to the text protocol. Infect female six to eight week old mice with viral doses from 10 to 10 to the fourth fluorescent forming units or FFU of PR8 wild type.
On days two and four, to collect the mouse lungs and nasal mucosa for evaluation. After euthanizing the animals, place an animal in dorsal recumbency and use 70%ethanol to disinfect the fur over the thorax. Use scissors to cut the skin from the base of the abdomen to the sternum.
Then, with scissors, make one centimeter cuts from the base of the incision to the lateral parts of the mouse. Remove the skin from the entire upper part of the mouse to the base of the abdomen where the first incision was made. Then use the scissors to cut off the head and place it in a dry sterile petri dish.
Use scissors to cut the junctions between the maxilla and the mandible and discard the mandible. Then, with the scissors, make two cuts at the ends of the zygomatic arcs and remove them. Use dissecting forceps to remove the eyes.
Next, with the dissection forceps, hold the cranium and use a scalpel to cut the cranium along the sagittal suture. Lift the two halves of the cranium with the brain to see the nasal mucosa. Using the scalpel, cut the part of the cranium with the brain and discard it.
Place the remaining of the cranium with the nasal mucosa in a sterile tube. Store the tube on ice if the samples are processed the same day or freeze quickly on dry ice if the samples will be processed later. To avoid contamination of samples, use chlorine dioxide disinfectant, to clean and disinfect the dissecting tools between each tissue recovered and between each animal dissection.
Then, to collect the lungs, place the animal in dorsal recumbency and use scissors to cut the plura. Starting at the base of the ribcage, use scissors to cut the ribs one centimeter on both sides of the sternum to the superior part of the ribs. Make a cross section with the scissors between the two incisions in the superior part of the ribcage and remove the chest place.
Hold the lungs with the forceps, make a cut at the end of the trachea just before the lungs and put the lungs into a sterile tube. Place the lungs or nasal mucosa into a sterile dounce homogenizer and add one milliliter of cold infection medium. Homogenize the sample by moving the pestle up and down for approximately one minute at room temperature until the lungs are completely disrupted.
Put the homogenized sample in a sterile tube. Centrifuge the samples at 300 x G for five to 10 minutes at room temperature. Collect the supernatant in a new sterile tube and discard the pellet.
Store the supernatant at four degree celsius if viral titration is performed on the same day. The day before titration, seed 96 well plates with MDCK epithelial cells in tissue culture medium so that they reach approximately 80 to 90%confluence. Incubate the cells in a 37 degree celsius incubator with 5%carbon dioxide overnight.
To prepare one to 10 dilutions of the homogenized lung or nasal mucosa samples, add 90 microliters of infection medium to each of the wells of a 96 well plate. Add 10 microliters of the supernatant from one of the homogenized samples to wells A1, A2 and A3, to evaluate the viral title of each sample and triplicate. Then, add 10 microliters of another supernatant of the homogenized samples to wells, A4, A5 and A6.After adding the sample supernatant to row A, use a multi channel pipette to mix by pipetting up and down.
Transfer 10 microliters from row A to row B and mix well. Change the tips between dilutions and repeat the transfer from row B to row C and continue in a similar manner until reaching the last row, H.Using a vacuum aspirator multi channel adapter, remove the tissue culture medium from the MDCK cells in the 96 well plates and use 100 microliters per well of 1x PBS to wash the cells twice. Beginning with the higher dilutions and moving to the lower dilutions, transfer 50 microliters per well of the serially diluted supernatant of the homogenized samples to the 96 well plate of MDCK cells, transferring the higher dilutions from row H to row A of cells.
Put the 96 well plate on a rocking platform for one hour at room temperature to allow viral absorption. Then, using a vacuum aspirator multi channel adapter, remove the inoculum and add 100 microliters per well of post infection medium. Incubate the infected cells for eight hours in a 33 degree celsius or a 37 degree celsius incubator with 5%carbon dioxide.
Proceed to fixing, staining, imaging and viral title calculation as described in the text protocol. In this experiment, mice were infected with different doses of PR8 wild type. The body weight and survival of five mice per group were measured through 14 days post infection.
Mice immunized with higher FFU of PR8 wild type, lost weight more quickly and severely and all of them died as a result. The MLD50 of PR8 wild type in this experiment, calculated with the reed and muench method was 1.5 x 10 to the second FFU. To analyze the humoral responses induced against IV infection, 14 days after immunization, mice were bled to analyze the presence of antibodies against total viral proteins and the presence of neutralizing antibodies targeting the viral HA protein, using a hemoagglutination inhibition or HAI and a virus micro neutralization or VN assay.
As seen here, results from the assay indicate that the sera for mice immunized with a higher dose of PR8 live attenuated Influenza virus or LAIV had higher antibody titles against total PR8 proteins than sera of mice immunized with a lower dose. Using the HAI and VN assays, sera from mice with the greatest amount of PR8 LAIV had a higher title of neutralizing antibodies while the serum from a mouse immunized with the lower dose had the lowest title of neutralizing antibodies against PR8. After watching this video, you should have a good understanding of how to infect mice in with Influenza A viruses, how to evaluate by their pathogenesis, humoral and immune responses and bio replication in the upper and lower respiratory tract.
After its development, this technique paved the way for researchers in the field of Influenza A virus to explore pathogenicity, immunogenicity and protection efficacy of live antibody vaccines, using a mouse model of infection.
