key: cord-1038225-5qrkkuxx
authors: Le Guernic, Antoine; Palos Ladeiro, Mélissa; Boudaud, Nicolas; Do Nascimento, Julie; Gantzer, Christophe; Inglard, Jean-Christophe; Mouchel, Jean-Marie; Pochet, Cécile; Moulin, Laurent; Rocher, Vincent; Waldman, Prunelle; Wurtzer, Sébastien; Geffard, Alain
title: First evidence of SARS-CoV-2 genome detection in zebra mussel (Dreissena polymorpha)
date: 2021-06-01
journal: bioRxiv
DOI: 10.1101/2021.05.28.446136
sha: 2ba2ed0198cc8b5431fc2a6a20342ca30465e9ff
doc_id: 1038225
cord_uid: 5qrkkuxx

The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater at laboratory scale in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters. Graphical abstract

Zebra mussels (2.98 ± 0.38 g; 2.51 ± 0.19 cm) were collected during October and November 145 of 2019 from Der lake (51290 Giffaumont-Champaubert, France, N 48°33'35"; E 4°45'11") and 146 brought back to the laboratory, where they were maintained in 1,000 L aerated tanks with 750 147 L of municipal drinking water (13.46 ± 1.77°C; pH 8.15 ± 0.17; 597 ± 27 mS/cm; 0.21 ± 0.05 148 mg/L nitrites; 58.05 ± 13.54 mg/L nitrates; 0.14 ± 0.42 mg/L ammoniac). Mussels were kept 149 several months before the experiments, under these acclimation conditions. Throughout this 150 acclimation step, mussels were fed ad libitum, twice per week, with Nannochloropsis (Nanno 151 3600, Planktovie, Marseille, France). 152 153

The Reims WWTP is located at 16 chemin des Temples, 51370 Saint-Thierry (49°16'49.566" 155 N, 3°59'32.625" E) and is managed by Grand Reims. The center Seine WWTP is located 5 156

Boulevard Louis Seguin, 92700 Colombes (48°55'57.936" N, 2°14'38.58" E) and is managed 157 by the SIAAP. Their characteristics are summarized in Annex 1. Briefly, the two treatment 158 plants have common characteristics, namely physical and chemical treatment of wastewater 159 and sludge, as well as biological treatment of wastewater. The biological treatment of the two 160

WWTPs is mechanical (anaerobic and aeration tanks, biofilters, etc.) and does not include a 161 step with chlorine. At the end of the water treatment process, this water is discharged into the 162 Vesle for the Reims WWTP, and into the Seine for that of the SIAAP. Two exposures to effluent were performed on dates corresponding to the two epidemiological 167 waves observed in France. The first one was performed from 07 th April 2020 to 07 th May 2020, 168 while the second one was performed from 25 th September 2020 to 27 th November 2020. These 169 cages were deposited into the 1,000 L acclimation tanks. Polyethylene cages, having a volume of 931 cm 3 , and exhibiting 5 x 5 mm mesh, contained 150 mussels, and were then deposited 171 by two at the study sites. For the site of Reims, cages were placed on the sediment with a 172 water column height of at least 40 cm above them and were connected to the bank with a 173 cable, while for the center Seine site, cages were placed inside the WWTP in a tank receiving 174 treated wastewaters. 175

For the earlier experiment (April and May 2020), mussels were caged at the exit of the Reims 176 WWTP (49°16'39.5" N, 3°59'06.5" E) and inside that of center Seine (48°55'57.936" N, 177 2°14'38.58" E). Caging and sampling kinetics are described in Table 1 . The digestive glands 178 of three zebra mussels were grouped together to have enough biological material for the 179 analyses. At each sampling time, ten pools of three digestive glands are recovered for Reims 180 WWTP, and five pools of three digestive glands for SIAAP WWTP. The samples were then 181 directly frozen by liquid nitrogen vapours and then stored at -80°C before the analyses. 182

Concerning the experiment conducted in October and November 2020, mussels were only 183 exposed to the exit of Reims WWTP. As previously described, dissections were performed at 184 laboratory and pools of digestive glands were then stocked at -80°C until RT-qPCR. 185

For each different caging periods, less than 10% mortality was reported. 186 187 The experimental procedure is identical for all four experiments, as described below. Before 196 the experiments, mussels were placed in 10 L aerated glass tanks in the dark with control of 197 the temperature at 13°C. Four tanks containing each 30 D. polymorpha, were implemented: i) 198

with 100% (4 L) of Cristaline Aurele drinking water (spring Jandun, France); ii) 10% of raw 199 wastewater coming from the WWTP of Reims and collected the day before (drinking water q.s. 200 4 L); ii) 33% of raw wastewater (drinking water q.s. 4 L); and iv) 100% of raw wastewater. 201

These waters were changed every day, and the input of raw wastewater came, each 202 experiment day, from a sample the day before. Concerning the first exposure (August 2020), 203 samples were collected on D1, D2, D3 and D4. For both September exposures, samples were 204 collected only on D3, and mussels were not fed during the experimentation step, while 205 concerning the last exposure (November 2020), that lasted longer (sampling time on D1 and 206 D7), mussels were fed every day with Nanno 3600 algae (Planktovie, Marseille, France) before 207 the water change. For this last exposure, two tanks containing 30 zebra mussels were placed 208 for the 100% raw wastewater condition. As previously described, dissections were performed 209 at laboratory and pools of digestive glands were then stocked at -80°C before RT-qPCR 210 analysis. During the exposures carried out at the end of September and in November, mussels 211 in 100% and 33% raw sewage conditions could be dissected respectively before D3 and D7 212 according to their general condition (in particular the time required to close the valves). For 213 these experiments, 15 pools of 3 mussels were dissected before D3 (September) or D7 214 (November) because of the toxicity of untreated wastewater, undiluted or two-thirds diluted. 215 216

Analyses of SARS-CoV-2 genome in raw wastewater were realised by the Obepine group 218 Health and Human Services, 2020) respectively (Table 2) . Then these data were synthesized 224 into an indicator obtained by data assimilation with a digital model of the Kalman filter type 225 (Forward-Backward). This graph was constructed only with envelop protein gene. Data for the 226 Reims and center Seine (SIAAP) WWTPs were collected from April 2020 to January 2021, and 227 compared to periods of confinement and curfew observed in France (Réseau Obepine, 2021) . 228

This information is available on the Obepine network site (Réseau Obepine, 2021) . (2020), were provided by Eurogentec (Liege, Belgium) and are described below (Table 2Table  250 2). Unlike water samples, the viral load within the digestive gland mash cannot be 251 Diagnostics, TX, USA) before RT-qPCR, and extraction controls were performed by adding 10 256 µL of this positive standard to digestive gland pools from mussels not exposed (between 257 dissection and freezing). This positive extraction control allowed the obtention of an extraction 258 yield between initial and final concentration of 70% for the E and N genes, and of 28% for the 259 RdRp gene. The positive detections of the SARS-CoV-2 genome in the digestive tissues of 260 zebra mussels were validated by a second passage of these samples in reverse transcription 261 polymerase chain reaction. 262 263 

Obepine group has perfomed the wastewater analyses on the two WWTPs studied, and 271

summarized Figure 1A (Réseau Obepine, 2021) . Table 3 (Table 3) . 277

During the first exposures of zebra mussels to treated wastewater (spring), water 278 contamination by the SARS-CoV-2 genome was very high (almost 500,000 copies/L for E 279 gene), but dropped considerably until it reached its lowest values at the end of these exposures 280 ( Figure 1A or <DL, Table 3 ). On the other hand, the exposures to treated wastewater carried 281 out at the end of 2020 corresponded to a period when the index was quite high (between 50 282 and 150, Figure 1 ). During this second caging exposure, genome concentrations of SARS-283

CoV-2 in raw water remained stable (between 38,000 and 91,000 copies/L for E gene, Table  284 3). This range of values was also found within exposures carried out in the laboratory after half 285 of September 2020. Indeed, a notable increase in concentrations between the two experiments 286 carried out in September 2020 was observed (Table 3) . 287 288 294 295 3.2. Detection of SARS-CoV-2 genome in digestive tissues of zebra mussels exposed to 296 treated wastewaters 297

The number of pools of digestive tissues from mussels caged in potentially contaminated 298 wastewater as well as the number of pools with detection of the SARS-CoV-2 genome (at least 299 one of the three genes tested) are shown on Figure 1B and on Figure 2A . Table 4 described 300 the detections of the SARS-CoV-2 genome in zebra mussel samples. 301

The first objective of our study was whether the SARS-CoV-2 genome could be detected by 302 the zebra mussel caged at the exit of the WWTP. RNA of SARS-CoV-2 was found in digestive 303 glands of mussels caged at the exit of both center Seine and Reims WWTPs (Table 4, Figure  304 2A May 07, 2020 during spring caging, and from September 18 to November 20, 2020 during 306 autumn caging, Table 4 ). Since the concentration values of the various SARS-CoV-2 genes 307 were obtained in raw wastewater, the connection with their detection in caged mussels 308 exposed to treated wastewater must be considered with caution. During the first caging 309

campaign, corresponding to a decreasing phase of the raw wastewater index ( Figure 1A) , 10% 310 of the exposed mussel pools showed positivity to the SARS-CoV-2 genomes in their digestive 311 tissues ( Figure 2A) . Surprisingly, when only the results from the center Seine WWTP were 312 considered, this percentage raised to 21%, compared to 5% at the outlet of Reims WWTP. In 313

Reims, the two positive samples were reported in week 16, corresponding to very high 314

concentrations of viral genomes in raw wastewater (464,844 copies of E gene per liter), but 315 also in week 18, during which however the concentrations in the wastewater were below the 316 detection limit (Table 3) . For the center Seine WWTP, even with less data, the same 317 observation was made, namely that the detection of SARS-CoV-2 genome in mussels mainly 318 occurred during weeks 18 and 19 when the concentrations found in the raw wastewater were 319 much lower (44,178 and 2,045 copies of E gene per liter respectively, Table 3 ). The detection 320 of the SARS-CoV-2 genomes in digestive tissues of zebra mussels was therefore possible 321 even with small amount present in the aquatic environment, and this detection lasted several 322

The second experiment was performed when the concentration of the SARS-CoV-2 genome 324 in raw wastewater increased until a plateau ( Figure 1A) , and showed 18% of positivity to the 325 virus genome in mussels (only for the Reims WWTP, Figure 2A) . Looking more closely, 326 genome positivity for SARS-CoV-2 in mussels was mainly observed during weeks when the 327 concentration in the water was quite high (about 70,000 copies of E gene per liter, weeks 40, 328 45, 47, especially for the E gene, Table 3) . There are therefore still gray areas as to the fate of this virus within hydrosystems. 338

Among the three genes used to detect SARS-CoV-2 genome, only the envelope (E) and of the 339 nucleocapsid (N) genes were detected in mussels (Table 4 ). Even with maximum 340 concentration of the samples, no detection of the RNA-dependent RNA polymerase (RdRp) 341 gene was reported, contrary to N gene detected only with the maximum concentration (Table  342 4). The same conclusion can be made with regard to the concentrations of the three SARS-343

CoV-2 genes in untreated wastewater. In fact, the concentrations for the E gene were 344 approximately 6 times higher than those of the RdRp gene and 2 times higher than those of 345 the N gene (Table 3 ). These differences may be due to the various PCR efficiencies for these 346 genes but also to the non-homogeneous fragmentation of viral genomes inside our biological and these two genes were expressed in tissues lysates of Crassostrea gigas after controlled 364 exposure to heat-inactivated SARS-CoV-2. In our study, the viral genome positivity of the 365 digestive gland samples was mainly linked to the E gene, and a few of these samples also had 366 positivity via the N gene (Table 4 ). Despite the lack of harmonization on the methods used, it 367 would have been interesting to use other specific genes of SARS-CoV-2 (Orf1ab, RdRp IP4 368 set, other regions of N or E genes, etc.) to potentially improve its detection within digestive 369 glands of zebra mussels. 370

Few studies had reported detection of SARS-CoV-2 genome in treated wastewaters. In our 371 study, detection of the SARS-CoV-2 genome in the digestive glands of zebra mussels exposed 372 at the WWTP outlet was observed. The use of a filter feeder and sessile species could explain 373 this difference. The detection of SARS-CoV-2 genomes directly in wastewater was often 374 represented by a value at a time point as well as on a volume of water which is not fully 375 representative of the water mass. Correlatively, zebra mussels, because of their sessility and Also in the marine environment, the Ruditapes genus had shown its efficiency in accumulating 384 the SARS-CoV-2 genome in their digestive tissues (Polo et al., 2021) . The authors of this study 385 concluded that mollusc bivalves can be used as biomonitoring tools for various anthropogenic 386 contaminants, including the SARS-CoV-2 virus. During our study, zebra mussels were useful 387 to detect SARS-CoV-2 genome in both untreated and treated wastewaters, even if the 388 concentrations in wastewater was under the detection limit (1,000 copies/L). All these characteristics make such bivalve, and particularly zebra mussels, good indicators for the 390 detection of SARS-CoV-2 genomes in such environments. These organisms can potentially 391 support or even improve the sensitivity of the direct detection of the viral genome in water 392 samples. 

To further support the use of this sentinel species as an indicator of the presence of the SARS-407

CoV-2 genome in environmental waters, improvements on the extraction and detection of the 408 SARS-CoV-2 genome in the digestive tissues of zebra mussels are required. Indeed, to 409 improve detection of SARS-CoV-2 genome inside this complex biological matrix, the PCR 410 cycle number has been increased to 50. Of the total positive detection data on E gene (Table  411 4), 73% had Cq lower than 42.75, but a few were higher (all Cq were comprised between 35.64 412 to 46.32). These high values underlined the limits of detection or extraction of this viral genetic 413 material, and particularly in the biological matrix used here. Contrary to the quantification of 414 SARS-CoV-2 genome in water, a pre-concentration step to concentrate the viral genome 415 before analyses is not necessary (Kitajima et al., 2020) . Moreover, the number of digestive 416 glands per pool was not elevated (3). Several modifications can be considered to improve the 417 viral extraction, such an addition of a purification step to limit as much as possible the 418 enzymatic inhibitors which could be found in the biological matrix, preventing the good 419 progress of the detection. In parallel, new experiments could be performed to improve the 420 sensitivity of the detection of SARS-CoV-2 genome in mussels but also to characterize the 

Out of a total of 666 mussels exposed to water potentially contaminated by the SARS-CoV-2 495 genome, i.e. 222 pools of digestive glands, 33 pools showed positivity to the genome of this 496 virus, representing almost 7%. This detection was observed during the two major 497 Moreover, SARS-CoV-2 genomes was detected in D. polymorpha as well at the outlet of the 499

Reims wastewater treatment plant as that of center Seine one. This corroborated the results 500 in untreated wastewater but also brought a novelty with the resilience of the genetic material 501 of the virus after treatment of these waters. This detection is proportional to the contamination 502 in the wastewaters and can allow a temporal and spatial monitoring. The zebra mussel 503 therefore appears to be an attractive candidate for detecting the presence of the SARS-CoV-504 2 genome in raw and treated wastewaters, but also in other hydrosystems. The detection of 505 the genome of other enteric viruses could be relevant using this sentinel species. 506 507 Acknowledgments 508

The authors are deeply grateful to the MeSeine observatory from SIAAP (Ile-de-France public 509 sanitation service) and the Obepine group for their contribution and advice to this study. This 510 study was also supported by the ACTIA VIROcontrol Joint Technological Unit. 511 512

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genome in pools (orange) according to exposure, exposure condition and sampling times. A: Results obtained after 429 exposure to treated wastewaters (spring and autumn) on the zebra mussels caged after Reims The second aim of this study was to assess the interest of using zebra mussels as bioindicator 438 of water contamination by the SARS-CoV-2 genome. Controlled laboratory exposures were 439 therefore put in place to address the questions raised during exposure to treated wastewater. 440Also, to maintain the natural contamination by SARS-CoV-2 in the receiving aquatic 441 environment, mussels were exposed to raw wastewater from Reims WWTP. 442Regarding the experiments performed at a laboratory scale, the genome detection of SARS-443CoV-2 genome was higher when the mussels were directly submitted to raw wastewater 444 (100%) compared to the diluted wastewaters (33 or 10%, Figure 2C ). Indeed, the most 445 concentrated condition (100% raw wastewater) resulted in a positivity of 28% of the samples 446 (10/32), greater than 15% (4/27) and 13% (4/31), caused respectively by conditions 33% and 447 10% of raw wastewater ratios. Zebra mussels were therefore useful to detect the SARS-CoV-448 2 genome in accordance with its presence in wastewaters. Furthermore, there was a similarity 449 between the growing phase of the COVID-19 pandemic after summer 2020, confirmed by an 450 increase of the raw wastewater index between July and October ( Figure 1A) , and the detection 451 of the SARS-CoV-2 genome in mussels ( Figure 2B ). Indeed, when exposed to raw 452 wastewaters in August, none of the 15 pools of exposed mussel digestive gland had the 453 genome of this virus, and this number increased with time. During the first exposure in early 454September 2020, 7% (1/15) of the pools exhibited positivity for the SARS-CoV-2 genome, to 455 increase to 38% (5/13) at the end of September, almost identical to the values found in 456 November (33%, 12/36, Figure 2B ). This result was in accordance with the sudden increase in 457 the concentration of the SARS-CoV-2 genome in raw wastewater between early and late 458 September (Table 3) . This increase was all the more important for the E gene and also 459 continued after September. For illustration, the concentrations of these genes in the raw 460 wastewater were lower than those of the 33% diluted wastewater (Table 3) . This originally 461 suggested that the zebra mussel can be used as indicator of the SARS-CoV-2 genome 462 detection in proportion to the contamination load present in freshwater environment and 463 contributes to emphasis the uses of zebra mussels as sentinel species for SARS-CoV Nonetheless, even if the laboratory experiments allowed to expose zebra mussels to higher 469 SARS-CoV-2 genome contamination, the experimental plan used in our study (short exposure 470 due to the possible toxicity of raw wastewaters) only allowed the qualitative detection of the 471 presence of SARS-CoV-2 genome in organisms but did not allow the genome quantification. 472Indeed, the possible toxicity of raw wastewater, causing an advanced dissection of organisms 473 exposed to the most concentrated raw sewage conditions (18% of samples during the 474 exposure at the end of September and 26% of samples during the last exposure, in November), 475 did not allow mussels to be exposed any longer. To dispense with the toxicity of raw 476 wastewater, a longer exposure of the mussels (from 14 to 21 days) in the laboratory to a non-477 infectious SARS-CoV-2 or to low pathogenic CoV strains could improve characterization of 478 virus accumulation within mussels (Desdouits et al., 2021; Wurtzer et al., 2020b) . 479Thanks to our results, the use of this bivalve as a bioindicator and possible matrix to follow the 480 presence of SARS-CoV-2 genome in water is conceivable, whether at the level of treatment 481 plants, but also at the level of freshwater (rivers, etc.) or in countries whose water treatment 482 structures are still underdeveloped. As announced by several recent studies, the bivalve taxon 483 represents a complementarity, even a more than plausible alternative for the detection of