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Adsorption Experiments

Virus adsorption was determined by passing approximately 35 L of unseeded seawater through the filter, followed by 500 ml of seawater seeded with adenovirus and norovirus, and then 5.5 L of unseeded water to push any remaining unadsorbed viruses through the filter. Samples of the 500 ml of seeded influent and the 6 L of effluent (corresponding to the 500 ml of seeded seawater filtrate and the subsequent 5.5 L of unseeded seawater filtrate) were collected for real-time PCR or RT-PCR analysis. The results of these experiments are displayed in Table 3 for adenovirus 41. The average percent adsorption for adenovirus 41 in seawater was 97.6% (± 0.64%). In all three experiments, the concentration of norovirus in the seawater filtrate was below the detection limit of the real- time RT-PCR assay. The minimum RT-PCR units needed for detection was calculated to be 1.5 x 106. Using this detection limit and comparing it to the number of RT-PCR units detected in the spiked seawater, the percent adsorption of norovirus was calculated to be greater than or equal to 43%.

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Table 3. Percent adsorption of adenovirus 41 in seawater to Argonide Nanoceram cartridge filters

Elution Experiments

The results of the elution experiments are shown in Table 4 and Table 5 for

adenovirus 41, and in Table 6 for norovirus GII.4. The concentration of each virus in PCR or RT-PCR units in the seeded seawater was estimated using the Ct value of the viral stock solution, and the eluent was sampled for quantitative analysis by real-time PCR or RT-PCR. The concentration of viruses in the 20-40 L of spiked seawater was below the detection limit of the PCR assay. Average percent recoveries for adenovirus with immediate elution were 3.2% (± 2.3%), 1.0% (± 0.30%) and 0.69% (± 0.40%) using beef extract, beef extract with 0.1% Tween 80 and beef extract with 0.01% Tween 80, respectively. The average percent recovery for adenovirus with a 3-day soak in beef extract prior to elution was 0.69% (± 0.17%). Average norovirus percent recoveries with immediate elution were 111% (± 29%), 119% (± 26%) and 88% (± 24%) using beef extract, beef extract with 0.1% Tween 80 and beef extract with 0.01% Tween 80, respectively. Norovirus recovery could not be

determined for the 3-day soak experiments because the virus stock used in previous

experiments was exhausted and the titer of the new norovirus stock solution was too low for detection in the eluate by real-time RT-PCR. The addition of Tween 80 did not improve elution of either virus from the filter.

Trial Virus in Influent (500 ml) Virus in Filtrate (6 L) Percent Adsorption

1 2.3x109 3.8x107 98.3%

2 2.2x109 6.5x107 97.0%

3 2.6x109 6.5x107 97.5%

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Table 4. Elution of adenovirus 41 with 3% beef extract using a peristaltic pump

a

3% BE = 3% beef extract – 0.1M glycine eluent at pH 9.5 with and without 0.1% - 0.01% Tween 80

Table 5. Percent recovery of adenovirus 41 with a 3-day soak in 3% beef extract followed by elution with a peristaltic pump

Trial Virus in Influent (40 L) Virus in Eluatea (500 ml) Percent Recovery

1 8.3x108 5.3x106 0.64%

2 1.3x109 7.4x106 0.55%

3 1.0x109 8.8x106 0.88%

Average 0.69% (± 0.17%)

a

Eluate = 3% beef extract with 0.1M glycine

Table 6. Elution of norovirus GII.4 with 3% beef extract using a peristaltic pump

a

3% BE = 3% beef extract – 0.1M glycine eluent at pH 9.5 with and without 0.1% - 0.01% Tween 80

PEG Precipitation Experiments

The results of the PEG precipitation experiments are shown in Table 7. For

adenovirus, average percent recoveries were 10% (± 3.6%), 36% (± 14%), 42% (± 13%), and 89% (± 20%) using 6% PEG - 0.1 M NaCl, 6% PEG - 0.3 M NaCl, 9% PEG - 0.1 M NaCl,

Elution replicates - % recovered Eluent a Estimated Adenovirus input (PCR units) 1 2 3 4 Average adenovirus % recovered (± st dev) 3% BE 1.0x109 4.6% 1.9% 5.7% 0.65% 3.2% (± 2.3%) 3% BE, 0.1% Tween 80 1.0x109 1.0% 0.61% 1.2% 1.3% 1.0% (± 0.30%) 3% BE, 0.01% Tween 80 1.0x109 1.3% 0.46% 0.58% 0.43% 0.69% (± 0.40%)

Elution replicates - % recovered Eluent a Estimated Norovirus input (PCR units) 1 2 3 4 Average norovirus % recovered (± st dev) 3% BE 3.5x106 86% 88% 133% 139% 111% (± 29%) 3% BE, 0.1% Tween 80 3.5x106 95% 140% 99% 141% 119% (± 26%) 3% BE, 0.01% Tween 80 3.5x106 99% 53% 103% 98% 88% (± 24%)

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and 9% PEG - 0.3 M NaCl, respectively. For norovirus, average percent recoveries were 38% (± 3.7%), 43% (± 7.3%), 51%, and 69% (± 4.0%) using 6% PEG - 0.1 M NaCl, 6% PEG - 0.3 M NaCl, 9% PEG - 0.1 M NaCl, and 9% PEG - 0.3 M NaCl, respectively. The standard deviation is not given for one experiment because a bottle broke in the centrifuge, resulting in only two experimental replicates. The highest recoveries for both viruses were observed using 9% PEG and 0.3 M NaCl (unpaired t-test, p < 0.05).

Table 7. Percent recovery of adenovirus 41 and norovirus GII.4 from seeded beef extract using PEG precipitation

6% PEG 9% PEG

0.1 M NaCl 0.3 M NaCl 0.1 M NaCl 0.3 M NaCl

Adenovirus 41 (n=4) 10% (± 3.6%)* 36% (± 14%) 42% (± 13%)** 89% (± 20%)

Norovirus GII.4 (n=3) 38% (± 3.7%) 43% (± 7.3%) 51%*** 69% (± 4.0%) * percent recovery (± st dev)

** n=3 *** n=2

Next, the efficiency of PEG precipitation was determined for actual seawater eluents. This experiment was performed in order to determine what effects the salts and anything else concentrated from seawater might have on PEG precipitation. The results of this experiment are shown in Table 8. Adenovirus average percent recoveries were 1.9% (± 0.48%), 3.7% (± 1.7%), 29% (± 14%), and 34% (± 12%) using 6% PEG - 0.1 M NaCl, 6% PEG - 0.3 M NaCl, 9% PEG - 0.1 M NaCl, and 9% PEG - 0.3 M NaCl, respectively. Norovirus average percent recoveries were 5.6% (± 1.1%), 5.4% (± 0.46%), 52% (± 7.8%), and 59% (± 4.8%) using 6% PEG - 0.1 M NaCl, 6% PEG - 0.3 M NaCl, 9% PEG - 0.1 M NaCl, and 9% PEG - 0.3 M NaCl, respectively. Overall, percent recoveries were considerably lower using actual seawater eluents. Higher average percent recoveries were observed for both viruses using

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9% PEG rather than 6% PEG (unpaired t-test, p < 0.05). However, no significant difference in average percent recoveries was observed between 0.1 M NaCl and 0.3 M NaCl when combined with 9% PEG for either adenovirus (unpaired t-test, p = 0.325) or norovirus (unpaired t-test, p = 0.122).

Table 8. Percent recovery of adenovirus 41 and norovirus GII.4 from actual eluates using PEG precipitation

6% PEG 9% PEG

0.1 M NaCl 0.3 M NaCl 0.1 M NaCl 0.3 M NaCl

Adenovirus 41 (n=4) 1.9% (± 0.48%)* 3.7% (± 1.7%) 29% (± 14%) 34% (± 12%)

Norovirus GII.4 (n=3) 5.6% (± 1.1%) 5.4% (± 0.46%) 52% (± 7.8%) 59% (± 4.8%) * percent recovery (± st dev)