The overall goal of this procedure is to efficiently quantify influenza viral titers in the airways of infected mice. Mice are first inoculated intranasally with influenza virus at selected time. Points after infection.
The M are sacrificed and bronchoalveolar lavage or BAL fluid is collected. Next, the BAL fluid is used to infect MDCK cells. Infected cells can then be stained for the presence of viral proteins using specific primary antibodies, followed by infrared conjugated secondary antibodies.
Infrared signals are read using the Lycor Odyssey scanner and the Odyssey software can be used to determine viral titers. Using a standard curve, quantitative determination of viral titers can be obtained. The major advantages of this technique over the existing methods is the precise quantification of the viral titers, the reproducibility, and the small volume that is required to perform this assay.
The implications of this technique can be extended toward diagnosis and therapy of various respiratory viral illnesses because it can be used to quantify viral titers in clinical samples Following this procedure. Other method like QPCR can be performed in order to answer additional questions such as viral copy number. While attempting this technique, it is important to remember to check the titer standard control virus after this development.
This technique path the way for the researcher in the field of viral logic to explore virus titer or twist stain viral protein in clinical samples. Once master this technique can be performed in 17 hours. It performed precisely Infect mice intranasally with 5, 000 platforming units or PFU of PR eight influenza virus in 30 microliters of sterile PBS or with PBS alone as a negative control for BAL fluid collection from euthanized animals first, cut that the thoracic cavity open and make a one centimeter incision parallel to the trachea to expose it with fine sterile scissors.
Make a small incision in the trachea, infuse the trachea with 0.5 milliliters of 1%BSA containing PBS and gently aspirate the lavage fluid out. Store the BAL fluids at negative 20 degrees centigrade to quantify viral titers in lavage fluids. First culture 5 million MDCK cells in 20 milliliters of complete RPMI in a T 75 flask overnight at 37 degrees centigrade the next day, harvest the cells and plate them in an optical flat bottom black 96 well plate at 10, 000 cells per well.
Then incubate the plate overnight at 37 degrees centigrade following the incubation. Gently aspirate off the supernatant and wash the cells twice in complete but serum free DMEM containing 0.2%BSA instead of FBS. Once the cells are washed, add 50 microliters of BAL fluid or 50 microliters of a suspension containing a known concentration of virus per well.
Then add 50 microliters per well of DMEM, medium containing TPCK treated trypsin at 0.2 micrograms per milliliter to enhance viral attachment, entry and replication. Incubate the cells for one hour at 37 degrees centigrade to allow the virus to absorb. Then add 100 microliters of FBS containing complete RPMI to each well culture the cells overnight to allow the infection to propagate following the overnight incubation.
Wash the cells with 100 microliters of 1%B-S-A-P-B-S. Once the cells are washed, add 100 microliters per well of 1%para formaldehyde and incubate the plates for five minutes at room temperature to fix the cells Next wash with 100 microliters per well of 1%B-S-A-P-B-S and incubate the cells for a further 30 minutes for blocking. Once the cells have been blocked, add 50 microliters per well of primary antiviral M two antibody diluted at one to 1000, and incubate the plates for one hour at room temperature to stain any viral protein present after the incubation, wash the cells three times in 1%B-S-A-P-B-S and incubate them with 100 microliters per well of infrared dye conjugated secondary antibody at a one to 200 dilution for one hour at room temperature in the dark once the cells are stained, wash them three times as previously.
Then position the plates on the reading glass platform of the Licor Odyssey infrared scanner. Select the 96 well plate setting on the reader using the Licor Odyssey software. Identify the blank negative control wells, then quantify the fluorescence from the whole plate.
Use the auto shape tool to draw the region of interest or ROI in the middle of a test. Well use the ROI from a negative control. Well, to set the baseline fluorescence value for the assay, then introduce the two opposing cross hairs provided by the Odyssey software within the ROI to measure the intensity of the fluorescence across the well.
Next, scan the plate using the 780 nanometer channel for detection and 680 nanometer as the reference wavelength. Once the read command has been activated, fluorescent intensities at uniform intervals of the ROI will be measured. And then the collected data points integrated.
A standard deviation multiplier will determine the level of signal over the baseline that is included in the ROI be sure to select the integrated intensity option for calculations since it represents the net pixel volume for defined areas independently of size. Background fluorescence will be quantified from the mock infected controls and will be used to estimate the integrated intensity in test wells. The infrared fluorescence from duplicate wells containing serial dilutions of titrated virus are used to construct a standard curve.
The standard curve can then be used to determine the titers of BAL fluid from infected mice. In this experiment, a significant increase in viral load was observed at day two and four. Post infection virus was cleared by day 10 as shown here.
Viral titers measured using this assay correlate well with mouse weight loss following infection. This technique can also be applied to human samples. Shown here is a quantification of H one N one virus from a human nasal swab.
The limit of detection here is 10 to the third TCID or tissue culture infective dose. After watching this video, you also have good understanding of how to estimate influenza virus titer using infrared die based assay. Visual demonstration of this technique is critical because using the corp imager can be difficult and this will help simplify and demonstrate our methods for quantifying viral titers in a 96 or 384 well plate.
And finally, don't forget that working with virus can be extremely hazardous and precursors such as personal protective equipment should always be taken while performing this procedure.
