key: cord-0881712-47xgtixh authors: Oh, C.; Kim, K.; Araud, E.; Wang, L.; Shisler, J. L.; Nguyen, T. H. title: A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads date: 2021-12-08 journal: nan DOI: 10.1101/2021.12.07.21267392 sha: 36677eca5f89ce17a51f58b384d9c03b3d7681a4 doc_id: 881712 cord_uid: 47xgtixh Viruses are present at low concentrations in wastewater, and therefore an effective concentration of virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the concentrated over that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency became significantly higher when calculated based on virus titers than genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk due to wastewater contaminated with infectious enteric viruses. PCR inhibitors were not concentrated by PGM-MBs, suggesting this tool will be successful for use with environmental samples. The PGM-MBs method is cost-effective (0.43 USD/sample) and fast turnaround (3 hours from virus concentration to genome quantification), and thus this method can be implemented for high throughput facilities. Based on good performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, we are confident that the PGM-MBs method can be applied for successful WBE and ultimately provides valuable public health information. Enteric viruses are the leading cause of gastroenteritis worldwide. Many enteric viruses, such as 51 rotavirus, norovirus, and adenoviruses, are shed in fecal material (Kang, 2017; Kapikian, 1996) . 52 Other airborne viruses, like SARS-CoV-2, are also shed in the human stool (Hu et al., 2020) . 53 Thus, wastewater-based epidemiology (WBE) can be a powerful tool to detect viral transmission 54 in communities even before the appearance of clinical cases (Ahmed et influent wastewater was used for sensitivity and specificity experiments (Section 3.1, 3.2, 3.3, 172 and 3.6). The filtrate was stored at -80 until used. The filtrate was thawed and stored at 4 for 173 less than two weeks, a time period where we conducted all experiments in this study. 174 Three-day composite sewage samples were collected from January to July 2020 from 10 175 different neighborhood-level sewersheds across Champaign County, IL, USA (Table S3) . These 176 sewage samples were stored at 4 for less than two weeks before being used to test the impact of 177 the filtration process on performances of the PGM-MBs method (Section 3.4) and the tolerance 178 of the PGM-MBs method to PCR inhibitors (Section 3.5). In addition, the SARS-CoV-2 179 concentration in these sewages were measured for a smaller aliquot of these samples kept at -80 180 for less than a week and analyzed as soon as they were defrosted (Section 3.7). Information 181 including sampling locations and dates was summarized in Table S3 . 182 183 In an experiment conducted to evaluate performances of the PGM-MBs method with different 185 viral genome concentrations, we spiked 100 μ L of either TV, RV, AdV, NL63 or TGEV into 10 186 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint mL of filtered influent wastewater ( Table S3). The initial genome copies spiked to wastewater 187 were about 10 7 gc for TV and NL63 and about 10 8 gc for RV, AdV, and TGEV. The wastewater 188 was serially diluted in 10-fold increments (four serial dilutions for TV and NL63 and five serial 189 dilutions for RV, AdV, and TGEV) and the three biological replicates were prepared. The results 190 from this experiment can be found in Section 3.2. We conducted a set of experiments to understand the impact of solid particles on 206 performances (RE and tolerance to PCR inhibitors) of the PGM-MBs method. We chose 6 207 sewage samples collected from different sampling locations or times. These sewage samples 208 were left in a refrigerator at 4 for two hours without disturbance to mimic sedimentation. The 209 liquid near the surface of these samples was used for two experiments. In the first experiment, 210 this liquid was used as it is. In the second experiment, this liquid was filtered by 0.22 μ m pore 211 size filters. Each of five viral species (about 10 6 gc) was added to 10 mL of either the filtered or 212 the unfiltered samples before these samples were subjected to the PGM-MBs. The concentrations 213 of viral genomes in samples before and after the PGM-MBs method were quantified and the 214 recovery efficiencies were calculated. In addition, we tested the impact of solid particles as PCR 215 inhibitors. In these experiments, we used the same pairs of unfiltered and filtered samples 216 described above. Then, we applied the PGM-MBs method to the sewage samples, to which we 217 did not spike any viruses. After we obtained the final extracts from the PGM-MBs sample, we 218 spiked 1 μ L of TV genomes to 10 μ L of the final extracts. Because TV is a Rhesus monkey virus, 219 they are not expected in the sewage samples. The same number of TV genomes was added to 220 PCR inhibitor-free water (molecular biology grade water) as control samples. The results about 221 the impact of solid particles on the performances of the PGM-MBs method are presented in 222 Section 3.4. 223 We also tested tolerance of the PGM-MBs method to PCR inhibitors in environmental 224 samples. We collected 19 different sewage samples and 1 sample from lagoon (Table S3) . We 225 also dissolved humic acid (41747, Alfa Aesar, USA), which was extracted by alkaline extraction 226 method from brown coal, to molecular biology grade water at a final concentration of 20 mg C/L. MBs method, we put 10 μ L of the PGM-MBs to 10 mL of the samples adjusted to 50 mM of 232 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. recovery efficiencies with filtered and unfiltered sewage samples (Fig. 6) . There were no outliers 267 (i.e., all data for the paired t-test exist within a range from Q1-1.5IQR and Q3+1.5IQR). Also, 268 normalities of the differences between two data sets were checked by Shapiro-Wilk test. Two-269 sample proportion test was conducted to compare two binomial proportions of Ct values less 270 than 40 from control wastewater and virus-spiked wastewater in Table 2 . We added notes to 271 Table 2 showing if sample size (n) times proportion (p) is greater than 5, which is an assumption 272 for the two-sample proportion test. All statistical analysis was conducted by OriginPro 2019b. 273 274 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint We tested MgCl 2 concentration and genome extraction method to optimize the PGM-MBs Whitney test, p<0.05). Therefore, 50 mM was determined as the optimal concentration of MgCl 2 290 for the PGM-MBs method. 291 Second, we established a heat denaturation method to release genomes from viruses 292 bound to PGM-MBs as an alternative to using nucleic acid extraction kits. This method only 293 requires the addition of proteinase K followed by heating up at 95 for 10 minutes. Mini Kit and with the heat denaturation method. The y-axis represents the recovered viral 306 genomes divided by average number of viral genomes by extraction kit. Statistical analysis of 307 two groups of results were conducted by a non-parametric test (Mann-Whitney Test; *: p<0.05 308 and ns: no significant difference). 309 310 Genome concentrations of the spiked viruses in the initial wastewater samples are plotted on the 312 X axis of (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Eq. 5 shows that LOQ PGM-MBs are inversely proportional to Volume Initial solution , so the LOQ PGM-MBs 362 will be lowered as the volume of the initial solution increases if the other variables such as 363 Volume Final solution , LOQ F , and RE remain constant as Volume Initial solution changes. Therefore, we 364 can reasonably assume that Volume Final solution and LOQ F will be maintained if the same virus 365 concentration method and quantification instruments are used. We also assumed RE is 366 maintained when the volume ratio of the PGM-MBs to the initial solution is fixed to 10 µL to 10 367 mL. Therefore, we can derive Eqs. 6-7. 368 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and MF values that were experimentally determined with the 10 mL of the initial solution (Table 375 S4). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint viruses. We serially diluted the lab-grown viruses to the filtered influent wastewater and 406 determined the genome concentrations and infectious virus titers. The slopes in Fig. 4 indicate 407 the ratio of genome concentrations to infectious virus titers in the virus-spiked wastewater. As 408 shown in Fig. 4 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Rusiñol et al., 2020) . Therefore, the recovered virus genomes by the PGM-MBs better 432 represents the risk stemming from the enteric viruses in the environment than those determined 433 by conventional counterparts. Genome concentration Virus titer TV NL63 TGEV AdV RV * ns * * * All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. sewage samples (Fig. 6A) . The paired sample t-test results showed that the differences between 446 the Filtered and the Unfiltered samples were not significant different except for RV (p>0.05). 447 This finding support that the presence of solid particles in the sewage samples did not 448 significantly affected performances of the PGM-MBs method. We also tested the impact of solid 449 particles as PCR inhibitors. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint samples 469 We designed experiments to test if the PGM-MBs method can reduce the PCR inhibitors from 470 the environmental samples. PCR inhibition was evaluated by comparing the Δ Ct values for 21 471 different samples (Fig. 7) . Out of the 21 samples, 19 samples have (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. LG, 1-fold Not detected All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint influent wastewater showed a few positive samples for TV, AdV, NL63, and TGEV, but 20 506 cases out of 76 (33%) for RV ( Table 2) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 2) The number of positive samples/total samples was presented with positivity rate in 525 parentheses. At least five molecular replicates and six technical replicates (n>30) were 526 measured by the qPCR. 527 3) Not applicable. Group of data does not satisfy the assumptions for the two-sample 528 proportion test (i.e., np<5). 529 4) Positivity rates of wastewater and virus spiked wastewater were analyzed by the two-530 sample proportion test. 531 5) All the positive Ct values regardless of the viral species were pooled, and an average was 532 presented with a standard deviation. of NL63 recovery efficiency. We found that NL63 recovery efficiencies for the filtration and the 545 PGM-MBs method were normally distributed (Shapiro-Wilk test, p>0.05) in wide ranges from 546 1.4 to 18.6% (Fig. S4) . The wide range of recovery efficiencies were also reported elsewhere 547 (Randazzo et al., 2020) , and this is probably because the two methods basically collect viral 548 genomes through virus adsorption to their media (i.e., electronegative membrane filter and the 549 PGM-MBs), which depends on the water characteristics (pH, ionic strength, or competing 550 substances) (Gutierrez and Nguyen, 2012 were not significantly different between the two methods (paired sample t-test p>0.05). 556 Therefore, we concluded the PGM-MBs method can be applied for monitoring SARS-CoV-2 in 557 wastewater. 558 559 560 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The adaptability to high throughput instruments, low price, and a short operational time should 571 be considered to evaluate virus concentration methods. Using magnetic beads and heat 572 denaturation for collecting viruses and extracting viral genomes, respectively, allow the PGM-573 MBs method to satisfy those three requirements. Karthikeyan et al. (2021) demonstrated that a 574 magnetic-bead-based approach could be implemented for an automated nucleic acid purification 575 system (Thermo Fisher Scientific, USA), which enabled high throughput analysis (96 samples 576 per run). In addition, using the PGM-MBs will further lower the cost for WBE. For example, 577 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint consumables for the production of the 10 µL PGM-MBs (10 mL wastewater sample analysis) 578 cost 0.413 USD (Table S6) . Essential materials for this method, including magnetic beads, 579 mucin, magnets, and proteinase K are not proprietary. Also, the entire process for the PGM-MBs 580 method only takes less than 3 hours, including concentrating viruses (30 minutes), extracting 581 genomes (10 minutes), and quantifying viral genomes (90 minutes), which is much shorter than 582 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267392 doi: medRxiv preprint the PGM-MBs method with various experiments using different virus concentrations (Fig. 3) (Table 3) . Given the findings from Fig. 3 and Table 3 , we concluded 606 that the PGM-MBs method is comparable to or better than conventional methods to concentrate 607 various enteric viruses, including SARS-CoV-2 from environmental samples. 608 609 This study first introduced a novel approach to concentrate enteric viruses from the 611 environment using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). This novel 612 method is simple, fast, affordable, and comparable with conventional methods. The optimized 613 PGM-MBs method takes less than 3 hours from virus concentration to genome quantification 614 and costs less than 0.5 USD for 10 mL volume of sample without expensive instruments such as 615 an ultracentrifuge. We systematically demonstrated that the performance of PGM-MBs method 616 is comparable to or better than conventional methods to concentrate various enteric viruses, 617 including SARS-CoV-2, from environmental samples. We also discovered that the PGM-MBs 618 method is robust to environmental samples, which features the existence of different viral species 619 and PCR inhibitors. Taken all together, we concluded that the PGM-MBs method can readily be 620 used for urgent SARS-CoV-2 surveillance to cope with the current COVID-19 pandemic or 621 monitoring other enteric viruses for better public health management. 622 623 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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