代替体外ウイルスのカプシド構造健全性の定量方法

The overall goal of these methods is to gain further insight into the effects of a treatment on the infectivity or integrity of the human norovirus capsid. This method can help answer key questions in the field of non-enveloped viral inactivation. Such as what is happening to the viral capsid's ability to infect a cell after a given treatment.

The main advantage of these methods is that they can provide additional mechanistic insight into the action of any given treatment on the virus. Though this method can provide the insight into human norovirus inactivation strategies, it also has potential to be applied to other non-enveloped viruses such as rhinovirus or hepatitis A.To begin, dilute purified human norovirus major capsid protein, assembled in capsids to 50 micrograms per milliliter in 1X phosphate buffered saline. Use a maximum volume of 15 microliters in the individual capped PCR tubes.

Ensure that the tube contains at least 600 nanograms of capsid and that there is 600 nanograms of capsid per treatment ligand combination. Next, preheat the thermocycler to the desired heat treatment. Then, preheat an additional thermocycler to four degrees celsius for immediate cooling.

Add the tubes with capsid solution to the thermocycler for the desired time. After heating, immediately transfer the tubes to the precooled four degrees celsius cycler for five minutes. Briefly spin down the tubes in the centrifuge to get all the droplets to the bottom of the tube.

Dilute the treated capsid solutions and PBS to three micrograms per milliliter. Ensure that there is at least 200 microliters of diluted treated capsid. Next, apply 100 microliters of capsid solution to each well ensure that there are at least two wells per treatment.

Additionally, include two control wells with 100 microliters of PBS for each ligand. This provides the background absorbance of the assay. Cover the plate with a lid and seal the edges with paraffin film to reduce evaporation.

Leave the plate overnight on a shaking incubator at four degrees celsius or for two hours at room temperature. Following incubation, remove the treated capsid solutions from the plate. Then, add 200 microliters per well of blocking buffer.

Incubate for two hours at room temperature or sealed at four degrees celsius overnight. Meanwhile, dilute the biotinylated aptamer to one micromolar in nuclease-free water. Ensure that there is enough solution for 200 microliters total for each treatment.

Also, dilute the chosen biotinylated HPGA to 30 microliters per milliliter in the blocking buffer. Again, ensure that there is enough solution for 200 microliters total volume for each treatment. Following incubation, remove the blocking buffer and wash the plate three times with 200 microliters per well of PBST.

Pat the plate upside down on sterile paper towels to dry the residual moisture. Next, at 100 microliters per well of the ligand binding solutions ensuring that there are two wells per heat treatment ligand combination, incubate the covered plate on an orbital shaker at about 60 RPM for one hour at room temperature. After washing the wells as described in the text protocol add 100 microliters per well of 0.2 micrograms per milliliter of streptavidin horseradish peroxidase and PBS.

Incubate for 15 minutes at room temperature with gentle shaking on an orbital shaker before washing the wells again. Then, after another wash, add 100 microliters per well of TMB substrate and allow the color to develop for five to 15 minutes. Observe the wells for color.

Be sure to consistently develop for the same time between replicate plates and be aware of the absorbance range of the plate reader used. Stop the development of the reaction with 100 microliters per well of one molar phosphoric acid. Immediately read the plate at 450 nanometers.

Create a new size standard operating procedure in the DLS instrument software as detailed in the text protocol. Then, select the green run arrow within the software and name the sample. Dilute the heat-treated virus-like particles or VLPs one to 10 in 1X PBS for final concentration of five micrograms per milliliter.

Then, filter the sample with a one micron pore size to remove dust particles. DLS is very sensitive to dust, as large particles scattered much more light than small particles. Transfer 50 microliters of the diluted VLPs into a small volume disposable cuvette.

Insert the cuvette into the sample chamber of the instrument. Start the run and use the correlogram cumulant fit and expert advice tabs to evaluate the data quality. During the run, check that the poly disperse of the index value is less than 0.3 representing a quality measurement.

In the multiview tab, make sure the correlation coefficient is constant and close to but not more than one at a short time and drops off sharply at zero at a later time, characteristic of particle size. Use the expert advice tab for feedback on the data quality during the measurements. After the measurement is complete, check again at the poly disperse of the index value is less than 0.3.

Make sure that the cumulant fit line follows the data points closely in the cumulants fit tab. Create a new size standard operating procedure in the DLS instrument software as detailed in the text protocol. Select the run arrow within the software to open and name a new sample and allow the instrument to reach the temperature equilibrium.

Transfer the VLP suspension to a small volume quartz cuvette avoid bubbles and pipette slowly against the wall of the cuvette. After the instrument has reached temperature equilibrium, insert the cuvette into the sample chamber. After waiting 10 seconds for the sample temperature to equilibrate, start the run and simultaneously start a timer.

Stop the timer at the end of the first measurement. Take this time as the first time point. Obtain subsequent time points from the measurement times recorded by the software.

Perform heat treatment as before except substitute PBS for 10 millimolar heaps, pH 7.4 and do not further dilute capsids after treatment. Next, pipette the entire 15 microliter drop of treated capsid solution onto paraffin film. Grab the grid edge with tweezers allowing them to close on the edge to hold the grid and place grid carbon side down on top of the drop of treated solution.

Let the grid incubated for 10 minutes to allow the capsid to attach to the grid. Depending on the purity of the capsid preparation, add washes of the heap solution in the form of 10 to 15 microliter droplets placed in line after the treated capsid droplet. After 10 minutes, pick up the grid with the droplet.

Place the grid nearly perpendicular to a piece of filter paper to wick away the droplet. Then, use the grid to pick up the droplet of 10 to 15 microliter heaps buffer and hold it for 30 seconds. After 30 seconds of wash time wick away with another piece of filter paper.

Next, place a 15 microliter droplet of two percent uranyl acetate solution on the paraffin film in an empty area. Pick up the droplet of uranyl acetate with the grid and hold it for 45 seconds. Then, wick away the uranyl acetate being sure to remove most of the solution.

Place the grid carbon side up on a piece of filter paper being careful that the grid does not stick to the moisture on the tweezers. Then, place the filter paper with the grid on it in an open glass petri dish. After repeating this process for all treatments, place the petri dish halves with the grids in a desiccator overnight to dry prior to observing with TEM.

The following graph displays reduction of the binding ability of human norovirus capsids over time when treated at 68 degrees celsius. As can be seen, HPGA binding is the first signal to completely disappear. At this point, it is assumed that the large majority of capsids in the solution have lost the ability to bind host cell HPGAs and thus cannot be internalized into the cell.

Aptamer binding mirrors that of HBGA, though slightly more heat treatment is required to remove the signal. Shown here is a DLS correlation coefficient curve representative of good quality data. At time close to zero, the correlogram is constant at a value close to, but below one.

It then drops off sharply to zero at a time characteristic of the particle size. Size results from this experiment can likely be trusted. Conversely, this correlation coefficient curve is representative of poor quality data.

The correlogram starts at a value above one and gradually drops off to zero. Results from this experiment should not be used. Here, particle size data shows a clear aggregation profile for capsids treated at 68 degrees celsius with super aggregates forming after about 20 minutes.

The time at which the super aggregation occurs is characteristic of the susceptibility of the VLPs to that treatment. This figure visualizes the TEM results for heat treatment of human norovirus capsids. Here, the capsids were being treated at 60 to 80 degrees celsius from left to right.

As can be seen, capsid morphology clearly changes with treatment as capsids aggregate and denature. A similar phenomenon is noted for capsids treated at 68 degrees celsius for zero to 25 minutes. Once mastered, these techniques can be done in one to 24 hours if they are performed properly.

After its development, this technique provided an additional avenue for researchers in the field of food virology to explore the mechanisms and effects of inactivation on the capsid of human noroviruses. Following this procedure, other methods like a real-time RT-PCR with and without RNase pretreatment, STS page of viral capsids, and plaque assays with cultivable surrogate viruses can be performed to get a more complete overall view of viral reduction after a given treatment. After watching this video, you should have a good understanding of how to utilize ligand binding, dynamic light scattering, and transmission electron microscopy to gain further mechanistic insights into the effects of inactivation treatment on the human norovirus capsid.

Don't forget that working with some of the reagents used can be hazardous and precautions such as wearing personal protective equipment and following laboratory safety best practices should always be taken while performing this procedure.