key: cord-0701531-nnwtmaqz
authors: Wurtzer, S.; Waldman, P.; Ferrier-Rembert, A.; Frenois-Veyrat, G.; Mouchel, J.-M.; Boni, M.; Maday, Y.; Obepine, C.; Marechal, V.; Moulin, L.
title: Several forms of SARS-CoV-2 RNA can be detected in wastewaters : implication for wastewater-based epidemiology and risk assessment.
date: 2020-12-22
journal: nan
DOI: 10.1101/2020.12.19.20248508
sha: fe4cca90500ed56e450ba90314f3f391bb2a2e4e
doc_id: 701531
cord_uid: nnwtmaqz

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health emergency of international concern. Although SARS-CoV-2 is considered to be mainly transmitted by inhalation of contaminated droplets and aerosols, SARS-CoV-2 is also detected in human feces and in raw wastewaters suggesting that other routes of infection may exist. Monitoring SARS-CoV-2 genomes in wastewaters has been proposed as a complementary approach for tracing the dynamics of virus transmission within human population connected to wastewater network. The understanding on SARS-CoV-2 transmission through wastewater surveillance, the development of epidemic modeling and the evaluation of SARS-CoV-2 transmission from contaminated wastewater are largely limited by our knowledge on viral RNA genome persistence and virus infectivity preservation in such an environment. Using an integrity based RT-qPCR assay this study led to the discovery that SARS-CoV-2 RNA can persist under several forms in wastewaters, which provides important information on the presence of SARS-CoV-2 in raw wastewaters and associated risk assessment.

stools from asymptomatic carriers with a largely unknown prevalence . This likely 55 reflects SARS-CoV-2 replication in the gut (Luz et al., 2020) . Accordingly high level of viral RNA have 56 been detected in wastewaters in different countries and potential cases of transmission via 57 wastewater have been reported (Yeo et al., 2020a; Yuan et al., 2020) . In addition to the risk of exposure 58 for sewage workers, wastewaters containing potentially infectious SARS-CoV-2 may enter the aquatic 59 environment via wastewater discharge thus potentially resulting in pollution of surface waters (Kumar 60 et al., 2020; Naddeo and Liu, 2020; Rimoldi et al., 2020; Wurtzer et al., 2020a) and to a lesser extent 61 groundwaters. Such a pollution could locally affect the quality of water ressources used for the 62 production of water intended to human consumption. Moreover, the persistence of infectious virus in 63 treated effluents of wastewater treatment plant could cause problems for agricultural activities 64 the contamination of wastewater by SARS-CoV-2 raises the same concerns as human seasonal enteric 66 viruses (Okoh et al., 2010) . 67

The monitoring of SARS-CoV-2 genomes in raw wastewater was successfully used for estimating the 68 dynamics of viral pandemic in population linked to a wastewater network (Medema et al., 2020; 69 Nemudryi, n.d.; Randazzo et al., 2020; Wurtzer et al., 2020b) . However many questions remain to be 70 answered to better assess the risk of transmission of SARS-CoV-2 through wastewaters(Elsamadony et 71 al., 2021; Lodder and de Roda Husman, 2020). Indeed RT-qPCR protocoles that are currently used can 72 not distinguish between partial or full-length, virion associated or free viral genomes (Prevost et al., 73 2016 (Prevost et al., 2016) . We propose that SARS-CoV-2 genomes can exist under three different 96 states at least: genomic RNA protected within an infectious particle, genomic RNA protected in a non-97 infectious structure, free total or partial genomic RNA. SARS-CoV-2 persistence and integrity were 98 compared to an enteric virus -Coxsackievirus B5 -that is commonly found in feces and wastewater. 99

The analysis of 87 raw wastewater samples collected from April to July 2020 in Paris area confirmed 100 supplemented with penicillin (50 U/mL) and streptomycin (50µg/mL), TPCK trypisin (1µg/mL) without 119 fetal bovin serum. The supernatant, collected after cytopathic effect observation, was clarified by 120 centrifugation at 2,000 x g for 15 min and stored at -80 °C before using. 121 122

Raw wastewater samples were homogenized, then 11 ml were centrifugated at 200 000 x g for 1 hour 124 at +4°C using a XPN80 Coulter Beckman ultracentrifuge equipped with a swing rotor (SW41Ti). Viral 125 pellets were resuspended in 200 μL of PBS 1X buffer as previsouly described by Wurtzer & al. 126 127

Five raw wastewater samples were collected in july 2020 in different WWTP and scored negative for 129 SARS-CoV-2 and enterovirus genome. These <24h old samples were centrifugated at 4,000 xg for 15 130 min for removing the largest particles and supernatants were filtred on membrane with 0,45µm 131 porosity. The filtrates were stored at +4°C and used within the following 24h. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint

The quantification of SARS-CoV-2 genome in wastewater has been proposed as an alternative strategy 186 to monitor the dynamics of pandemic SARS-CoV-2 virus. However, this approach is highly dependent 187 on the persistence of SARS-CoV-2 RNA in wastewaters. In addition, it is of upmost importance to 188 provide convenient tools to distinguish free viral RNA and virion-associated RNA as a first approach to 189 evaluate the concentration of infectious virus particle in matrix from which SARS-CoV-2 is technically 190 difficult to isolate, such as stools or wastewaters. Since viral genomes are protected by viral proteins 191 and surrounded by a cell-derived enveloped in infectious particles, we assumed that we could 192 distinguish between free and protected viral genomes using an integrity RT-qPCR based assay. 193

Briefly, two 1L-raw wastewater samples were collected by the 3 rd (sample S1) and the 7 th (sample S2) 194 of april 2020 in Greater Paris area, a period when SARS-CoV-2 genomes were easily detected (Wurtzer 195 et al., 2020b). Samples were analyzed less than 24h after the time of the sampling (day 0). The rest of 196 each sample was split into 2 parts and stored at +4°C or +20°C for 10 days and 12 days respectively. 197

Total SARS-Cov-2 viral RNA (vRNA) and protected viral RNA (pRNA) were quantified by RT-qPCR. As 198 shown on figure 1, less than 10 % of the total viral RNA was under a protected form. SARS-CoV-2 vRNA 199 and pRNA concentrations were relatively stable for 7 (S1) and 12 (S2) days respectively at +4°C while 200 they were slighly less stable when stored at +20°C. 201 202

Infectious enteric virus such as coxsackievirus B5 are commonly found in wastewaters, but the ability 204 of enveloped virus, like SARS-CoV-2, to persist under an infectious form is still debated. To address this 205 question the persistence of SARS-CoV-2 in raw wastewater was compared to that of coxsackievirus B5 206 (CV-B5) using three different indicators namely the quantification of total RNA (vRNA), protected viral 207 RNA (pRNA) and infectious particles (TCID50). Five raw wastewater samples, that were negative for 208 SARS-CoV-2 and enterovirus genome by RT-qPCR (data not shown) were used. The detection of 209 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in intended to evaluate more precisely the effect of temperature on SARS-CoV-2 using CV-B5 as a control. 233

For this purpose, we first exposed samples spiked with infectious SARS-CoV-2 and CV-B5 to increasing 234 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint temperature for 10 minutes. Then we evaluated the effect of the treatment on infectious particles or 235 total RNA stability (vRNA). Viral genome protection was evaluated as before by an integrity RT-qPCR 236 based assay (pRNA). 237 CV-B5 infectiosity was preserved up to 42°C and then dramatically decreased up to 70°C, as previously 238 Total and protected viral RNA were quantified in 87 raw wastewater samples that were collected from 253 April to July 2020 in Greater Paris area. vRNA and pRNA concentrations ranged from 1.4x10 3 to 5.2x10 6 254 GU/L and from 0.7x10 3 to 1.8x10 6 genome units/L respectively (figure 4A). Total viral RNA were 255 significantly higer than protected RNA in each sample (p<0.0001). The pRNA/vRNA ratio was comprised 256 between 0 and 100%, with a median value of 20.1%. In wastewater samples with vRNA concentrations 257 <100,000 (n=39) and >100,000 GU/L (n=22), the median ratio was 29,6% (max=99,3%) and 28.1% 258 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint (max=100%) respectively. This ratio was significantly lower in samples with low genome concentration 259 (n=26; <10,000 GU/L; median ratio = 0%; max = 18.8%) compared to <100,000 GU/L and >100,000 260 GU/L samples (p=0.015 and p=0.006 respectively) ( figure 4B) . but also on the half-life of total viral RNA in raw wastewater. In this study, we showed that total viral 274 RNA (vRNA) concentration in raw wastewater was stable for at least 7 days provided that the samples 275 were stored at +4°C until analysis, which is in agreement with previous work(Bivins et al., 2020). 276 Importantly, freezing water samples had a negative impact on the relevance of the measurment (data 277 not shown), at least in our protocol. Such a delay is important to be taken into consideration to 278 organize campains from the sampling to the analysis, including transportation to specialized 279 laboratories. Although our study was performed on a limited number of samples, the results suggested 280 that vRNA concentration was not dramatically affected by the composition of wastewater samples 281 over 24h-incubation time, a period of time that is compatible with the travel of the viral genomes from 282 emission of human faeces to raw wastewater sampling at the inlet of WWTP. As importantly SARS-283 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in simply non-infectious, or that cell culture system was not adapted for such highly chemically or 305 microbiologically contaminated samples(Cashdollar and Wymer, 2013). Efforts for concentrating and 306 isolating infectious viruses from hydric environment are usually successful for naked virus that are less 307 sensitive to chemicals. In this study, infectious SARS-CoV-2 was spiked in negative wastewater samples 308 and viable viruses were quantified up to 24 hours, without pretreatment of sample before cultivation. 309 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint Whereas such an exposure only midly affected coxsackievirus B5 viability, SARS-CoV-2 infectivity was 310 clearly affected at 20°C depending on the nature of the sample. These results are in agreement with 311 previous work (Bivins et al., 2020) . We brought here additional evidences that sample temperature had 312 a strong impact on virus viability since the SARS-CoV-2 infectivity was not significantly modified at +4°C 313 for 24h whereas it is slightly affected at 20°C. Both viruses infectivity was fully preserved up to 42°C 314 for shorter incubation times (10 min The present study confirmed that evaluating total vRNA widely overestimated the number of 323 infectious particles within wastewaters. Nevertheless, the relatively long persistence of SARS-CoV-2 324 genomes was surprising with regards to its supposed fragility compared to surrogates. Whether the 325 regions that are amplified by RT-qPCR came from total or partial genomes cannot be assessed by such 326 assays. A tool for assessing the integrity of naked virus particles already showed that genome of naked 327 RNA viruses can be protected from degradation by the capsid, a structure that remains non-permeable 328 to intercalating dye. Our comparative study on SARS-CoV-2 and CV-B5 demonstrated that viral 329 genomes can be found in multiple states i.e. infectious protected, non-infectious protected and 330 unprotected forms. Unpublished data showed that such dyes (Ethidium monoazide or propidium 331 monoazide) targeted secondary structures within single stranded RNA (hairpins or IRES for 332 picornaviruses) (Wurtzer et al., 2018) . In addition previous study showed that capsid integrity is lost at 333 42°C for CV-B5, with a maximum access of SyBR green II to viral RNA at 50°C (Waldman et al., 2017) . In 334 the case of coronaviruses, a lipid layer protects the RNA genome that is closely associated to 335 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint nucleoproteins. The lipid layer is probably very labile in wastewater, which may contain detergent 336 residues for example, and unstable at high temperature. Nonetheless integrity measurments showed 337 that vRNA remained protected from intercalating dye up to 70°C-incubation. These results suggested 338 other structures such as viral nucleoproteins may limit access of the dye to SARS-CoV-2 RNA, in 339 addition to the viral envelop. It is to note that SARS-CoV-2 and CV-B5 shared a similar profil of 340 sensitivity to temperature, although SARS-CoV-2 genome appeared to be better protected than CV-B5 341 genomes. This assay was used on a large panel of samples, confirming that less than 30% of the viral 342 perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted December 22, 2020. ; perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint 394 395 B All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.19.20248508 doi: medRxiv preprint

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