Metodo EPA 1615. Misura di Enterovirus e Norovirus Presenza in Water di cultura e RT-qPCR. II. Totale coltivabili Virus Assay

The overall goal of the following protocol is to measure the level of infectious virus particles in a water sample. This is achieved by using an alkaline-based extract solution to elute negatively-charged virus particles from an electro-positive filter that has been concentrated with virus from drinking or environmental water. The collection of virus onto an electro-positive cartridge filter, is covered in Part One of this series.

As the second step, the pH of the eluate is slowly reduced, which results in the formation of a flock of these extract proteins and virus present in the sample. Then the flock is collected by centrifugation, dissolved in a sodium phosphate buffer, and filter sterilized to remove bacteria and mold. Next, the final concentrated eluate is inoculated onto Buffalo green monkey kidney cells, and the cells are observed for cytopathic effect, or CPE, for 14 days, in order to detect virus replication.

To release any cell-associated virus particles, all flasks of cells are frozen at the end of 14 days, or when the CPE in any flask is 75%or greater. Following a second and third passage to confirm that the CPE is due to virus replication, a Most Probable Number, or MPN calculator program, is used to determine the most probably number per liter of each sample analyzed. Ultimately, the total culturable virus acid component of EPA Method 1615, provides a measure of the level of infectious virus particles in the water sample.

This total virus assay is just one component of EPA Method 1615. The complete method measures enterovirus, and norovirus occurrence, in drinking and environmental waters. Any virus findings, can then be used as input data to measure risk from exposure to these viruses in drinking and in recreational water.

The main advantage of this protocol, is that it provides a single standardized approach for measuring viruses in water. The approach is actually based upon the Information Collection Rule method that was published in 1996. The main problem that new people have with this method is keeping the BGM cell line free of contamination and deciphering CPE, or cytopathic effects.

Shay Fout and I will be demonstrating the illusion and organic flocculation procedures, followed by the cell culture technique. To perform the first elution, place 500 milliliters of room-temperature buffered 1.5%beef extract, pH 9.0, in a gradated cylinder. Then, open the filter cartridge housing, and add a sufficient amount of beef extract to cover the electropositive filter completely.

Place the rest of the beef extract into a beaker. Replace the filter housing lid. After one minute of contact time, use a peristaltic pump and sterile tubing to pass the beef extract solution in the housing, along with that remaining in the beaker, slowly through the filter.

Collect the eluate into a sterile 2-liter glass beaker. Repeat these steps with an additional 500 milliliters of buffered 1.5%beef extract, except using a contact time of 15 minutes. Add the beef extract from the second elution to the 2-liter beaker containing that from the first elution and add a sterile stir bar to the beaker.

Place the beaker containing the eluate on a stir plate. Turn on the plate, and increase the stirring speed until a vortex is formed. Slowly adjust the pH of the eluate to 3.5 plus or minus 0.1 by drop-wise addition of 1.2 molar hydrochloric acid, or HCL.

Add the acid drop-wise, because a rapid addition will inactivate the virus. During this time, the eluate will become cloudy, as a precipitate begins to form. Reduce the mixing speed to a slow stir, and continue to monitor and maintain the pH of the eluate at 3.5 plus or minus 0.1 at room temperature for 30 minutes.

Then, pour the precipitated beef extract suspension into one or more centrifuge bottles, and centrifuge for 15 minutes at 2, 500 times g at four degrees Celsius. Remove the bottles from the centrifuge, and slowly decant the supernatant to prevent the loss of the pelleted precipitate. The supernatant can be discarded.

For beef extract lots that dissolve within 5 minutes, add 30 milliliters of 0.15 molar sodium phosphate, pH 9.0, to the centrifuge bottle containing the precipitate. See the text protocol for instructions on dissolving precipitates that do not dissolve within five minutes. Stir for 10 minutes after the precipitate is completely dissolved.

Next, remove the stir bar, and centrifuge the dissolved precipitant for 10 minutes, at 4, 000 to 10, 000 times g, and four degrees Celsius. Carefully pour the supernatant into a glass beaker without disturbing the pellet. Add a stir bar to the beaker, and discard the pellet.

Place the beaker onto a magnetic stirrer, and stir the solution. Add 1.2 molar HCL drop-wise do adjust the pH to between 7.0 and 7.5. Filter sterilize the supernatant by passage through a sterilizing filter containing a prefilter that has been pre-treated with 15 milliliters of buffered 1.5%beef extract, pH 7.0 to 7.5.

See the text protocol to calculate the Assay Sample Volume, or S, for all test samples. Then, calculate the Inocolum Volume by dividing the S by 10. Using these calculations, divide the final concentrated sample volume, which is the sample obtained following filter sterilization, into three sub-samples, as described in the text protocol.

Innoculate 10 test vessels containing a monolayer of BGM cells for each test sample, with the volume of sub-sample one, equal to the Inoculum Volume, along with the total culturable virus quantal assay controls. For the Lab Fortified Blank, and the Lab Fortified Sample Matrix, prepre five, 25, and 125-fold dilutions using sub-sample three and 0.15 molar sodium phosphate, pH 7.0 to 7.5, as a diluent. In addition to the vessels inoculated with undiluted sub-sample one, inoculate 10 washed cell culture test vessels for each dilution series, using an Inoculum Volume on each test vessel.

Distribute the inoculum over the surface of the cell monolayers, by tilting the vessels back-and-forth. Incubate the test vessels at room temperature for 80-120 minutes on a mechanical rocking platform at one to five oscillations per minute, or with rocking of the vessels every 15 to 20 minutes to allow any virus present to absorb to the cells. Then, add pre-warmed maintenance medium to the test vessels, before incubating them at 36.5 plus or minus one degree Celsius.

Look for the appearance of cytopathic effects in each test vessel using a microscope for the first three days, and then examine them every two to three days up to day 14. Transfer any test vessels that show greater than 75%cytopathic effects to a freezer set at or below minus 70 degrees Celsius. After examining the vessels on the last day, freeze all remaining cultures, and the total culturable virus quantal assay controls at or below minus 70 degrees Celsius.

Then, thaw all the cultures, and filter greater than 15%of the medium from every CPE-positive test vessel, through a 0.2 micron sterilizing filter. The next step is to perform a second passage of all first passage test vessels using washed BGM kidney cell test vessels. To do this, innoculate the new test vessels with an inoculation volume that represents 10%of the thawed medium from all negative test vessels, as well as from the filtered medium of positive vessels.

Repeat the steps done for the first passage, freezing any test vessel that was negative on the first passage, and positive on the second. Then, perform a third passage as described for the second passage, using only the negative assay controls, and cell cultures that were negative during the first passage and positive in the second passage. Identify individual test vessels as virus positive when they show cytopathic effects in both the first and second passages, or, in the case where cytopathic effect does not occur until the second passage, in both the second and third passages.

Use the U.S.Environmental Protection Agency's Most Probable Number Calculator with the default program settings set as in the text protocol, to calculate the virus titers of all test samples. Then, use the values obtained for the Most Probable Number of infectious units per milliliter, in addition to the upper and lower confidence limits per milliliter, to calculate the Most Probable Number of infectious units per liter, and corresponding confidence limits, as described in the text protocol. The mean percent recovery is shown for Poliovirus from ground and reagent-grade water samples.

Mean Poliovirus recovery from ground water samples averaged 58%with a coefficient of variation, of 79%No culturable virus was detected in any of the duplicate un-seeded groundwater field samples. Method performance also was measured, using Lab Fortified Blank samples, modified by using two different seed levels. The twelve reagent-grade water samples included six seeded with 300 MPN of infectious units, and six seeded with 1, 000 MPN of infectious units of Poliovirus.

These controls performed similarly, with a mean recovery of 111%and a coefficient of variation of 100%While performing this procedure, it's important to remember to use aseptic technique, and good laboratory practices to prevent the contamination of samples. It is also important to remember that when working with viral pathogens, you should take precautions such as the use of gloves, whenever performing this procedure. After watching this video, you should have a good understanding of how dilute viruses and electropositive filters and perform organic flocculation and cell culture assays.

In addition to this procedure, you can also perform molecular assays to identify and detect additional virus types.