key: cord-0314078-t5papovw
authors: Roldan-Hernandez, L.; Graham, K.; Duong, D.; Boehm, A.
title: Persistence of endogenous SARS-CoV-2 and pepper mild mottle virus RNA in wastewater settled solids
date: 2022-01-07
journal: nan
DOI: 10.1101/2022.01.06.22268855
sha: ac70b2d2ffbef89eef0dfd20794424b7ad61c10a
doc_id: 314078
cord_uid: t5papovw

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4{degrees}C, 22{degrees}C, and 37{degrees}C for 10 days. The concentration of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA was measured using an RT-ddPCR. Limited decay (< 1 log10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 - 0.098 day-1 for SARS-CoV-2 RNA and 0.010 - 0.091 day-1 for PMMoV RNA, depending on temperature conditions. Slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets.

Introduction coefficient of SARS-CoV-2 RNA, and the average residence time of wastewater prior to sample 48 collection. In some models, PMMoV RNA is used as an endogenous viral process control and as 49 a fecal strength control. 5 The accuracy of these models is still uncertain and further research is 50 still needed to improve them. Understanding the decay rate of SARS-CoV-2 and PMMoV RNA is 51 critical in their implementation.

Only a few studies [13] [14] [15] [16] [17] have documented the persistence of SARS-CoV-2 RNA and infectious 54 SARS-CoV-2 viruses in wastewater (Table 1) , all those studies have been conducted using 55 wastewater influent as the experimental matrix. Additionally, most of these studies do not 56 measure the decay of endogenous SARS-CoV-2 RNA, rather they seed the wastewater with an 57 exogenous virus. A recent study by Chik et al. 18 found that surrogate spikes of SARS-CoV-2 (in 58 this case gamma-irradiated inactivated SARS-CoV-2) may exhibit different solid-liquid 59 partitioning behaviors compared to SARS-CoV-2 naturally found in wastewater. Virus 60 persistence can be influenced by the degree of sorption to solids. 19 To date, no study has 61 documented the decay of SARS-CoV-2 RNA in wastewater primary settled solids, based on a 62 systematic search of the literature on 11 November 2021 (Table S1) . contributing to a given wastewater sample; however, it may also correct for variation in RNA 68 recoveries between samples 5 , and for losses of RNA during storage. 20 PMMoV is a non-69 enveloped single-strand RNA virus highly abundant in human feces and wastewater. 21, 22 Studies 70 have shown that PMMoV RNA is remarkably stable in wastewater and exhibits almost no 71 . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 7, 2022. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 7, 2022. CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint POTW using the average concentration of SARS-CoV-2 and PMMoV RNA at each time point 185 (day 0, 2, 4, 6, 8, and 10). k and the time needed to achieve a 90% reduction in concentration 186 (T90) were calculated using the following equations: 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 January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint where β0 is the intercept, βi is the regression coefficient for each factor xi (temperature, RNA 208 target, and POTW), and ! is the residual standard error of the model. log10k values of SARS-CoV- 209 2 RNA from this study were also compared to previously reported log10k values of SARS-CoV-2 210 RNA in wastewater using a linear regression model in R. log10k values were used in that analysis 211 because previous work has shown an exponential relationship between k and temperature for 212 viruses in environmental waters. 28,29 P<0.05 was used to assess statistical significance. Table 2 shows the first-order decay rate constants and 226 time needed to achieve a 90% reduction in concentration for SARS-CoV-2 (N1 and N2 targets) 227 and PMMoV RNA. k was significantly different from 0 for all conditions (p<0.05) and varied 228 from 0.010 to 0.091 day -1 depending on the RNA target and temperature conditions. The linear 229 model was a good fit for most decay curves (median r 2 = 0.60, median RMSE = 0.21; Table 2 ); 230 . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. Figure 1 shows the decay curves of all RNA targets in primary settled solids and Table 2 239 summarizes the first-order decay constants and T90 for all RNA targets and temperature Table 1 ). The slope of log10k 276 . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. organic matter, dissolved oxygen, solid content, and microbial activity. 19, 29, 32 In this study, the 288 decay rate constants of all RNA targets increased with temperature, which is consistent with 289 previous studies of viral decay in the environment. [13] [14] [15] [16] [17] 24 However, limited information is 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 January 7, 2022. It should be noted that SARS-CoV-2 has not been cultured from raw wastewater 34 therefore the 323 scientific evidence to date indicates that detection of RNA in these samples is not associated with 324 infectious viral particles. Moreover, the decay rate constants reported herein are not to be 325 misinterpreted as decay rate constants for infectious SARS-CoV-2. Bivins et al. 14 CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint Table 1 (results from previous studies) and Table 2 (results from this study).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 7, 2022. ; https://doi.org/10.1101/2022.01.06.22268855 doi: medRxiv preprint

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