# Environmental tracking of viral pathogens in surface waters around Paris using high-throughput PCR

Prunelle Waldman, Audrey Fraisse, Mai-Lan Tran, Sylvie Perelle, Sandra

Martin-Latil

► To cite this version:

Prunelle Waldman, Audrey Fraisse, Mai-Lan Tran, Sylvie Perelle, Sandra Martin-Latil. Environmental tracking of viral pathogens in surface waters around Paris using high-throughput PCR. Science of the Total Environment, 2026, 1020, pp.181554. 10.1016/j.scitotenv.2026.181554. hal-05532508

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Submitted on 2 Mar 2026

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# Environmental tracking of viral pathogens in surface waters around Paris using high-throughput PCR

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Prunelle Waldman<sup>a, *,</sup>, Audrey Fraisse<sup>a</sup>, Mai-Lan Tran<sup>b</sup>, Sylvie Perelle<sup>a</sup>, Sandra Martin-Latil<sup>a, c, *</sup>

## HIGHLIGHTS

* Norovirus GI levels were significantly higher in the Marne than in the Seine.

* Higher river flow was associated with lower norovirus GI concentrations.

* A PCR array was applied for rapid detection of 30 viruses in river water.

* Seasonality had a greater impact on virus prevalence than river location.

* Aichivirus, adenovirus, and bocavirus were the most prevalent enteric viruses.

## GRAPHICAL ABSTRACT

### Environmental tracking of viral pathogens in surface waters around Paris using high-throughput PCR

<figure><img src="image_4.png" /></figure>

The PCR array allows fast, simultaneous detection of diverse viral pathogens, making it an effective tool for environmental monitoring.

## ARTICLE INFO

### Keywords:

Surface water

Viral contamination

RT-qPCR

High-throughput PCR

## ABSTRACT

Surface waters that receive discharges from wastewater treatment plants and other sources of pollution are therefore highly susceptible to microbiological contamination. They can be contaminated by a wide variety of viruses, some of which are known to persist in the environment for extended periods. In this study, we assessed the viral contamination of the Seine and Marne rivers in the Paris area by analyzing 42 water samples collected in both winter and summer 2023. Norovirus GI and GII were quantified by RT-qPCR, and a high-throughput PCR array was used for the qualitative detection of 30 viruses, including enteric viruses, respiratory viruses, and indicators of fecal contamination. Noroviruses were detected in 10–82% of the samples tested, depending on the river and season, at concentrations ranging between 5.8 × 10¹ and 1.1 × 10³ genome copies/L. Across the entire sampling campaign, the most prevalent human viruses detected using the PCR array were aichivirus, adenovirus, and bocavirus (74–83%), followed by enterovirus, astrovirus, norovirus, and cosavirus (12–26%). Some other viruses were sporadically detected (sapovirus, hepatitis A and E viruses, OC43, measles virus). The PCR array enables the simultaneous detection of diverse viruses in a single assay, making it a powerful tool for environmental surveillance of multiple pathogens. Its adaptability allows for the detection of a broad range of targets, delivering results within a short timeframe, typically within the same day.

<footnote><sup>a</sup> Université Paris-Est, Anses, Laboratory for Food Safety, Enteric Viruses Unit, 14 rue Pierre et Marie Curie, 94701, Maisons-Alfort Cedex, France</footnote>

<footnote><sup>b</sup> Université Paris-Est, Anses, Laboratory for Food Safety, IdentyPath, 14 rue Pierre et Marie Curie, 94701, Maisons-Alfort Cedex, France</footnote>

<footnote><sup>c</sup> Anses, Laboratory for Animal Health, Virology Joint Research Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France</footnote>

<footnote>* Corresponding authors at: Anses, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.</footnote>

<footnote>E-mail addresses: prunelle.waldmanvigne@anses.fr (P. Waldman), sandra.martin-latil@anses.fr (S. Martin-Latil).</footnote>
# 1. Introduction

Human enteric viruses are the leading cause of food- and waterborne illnesses worldwide (Cook and Richards, 2013), causing over a billion infections each year, several hundred thousand of which lead to death (Bányai et al., 2018; Du et al., 2022; Lucero et al., 2021). Enteric viruses refer to a wide variety of viruses, belonging to many viral genera, which infect and replicate in the gastrointestinal tract of the host. Some of the most common enteric viruses are noroviruses, rotaviruses, adenoviruses, astroviruses, and enteroviruses (Bishop and Kirkwood, 2008). These viruses are transmitted through the fecal-oral route. They are responsible for various diseases, including not only gastroenteritis but also hepatitis, neurological disorders, muscular syndromes, and chronic diseases (Boussettine et al., 2020). Human infections can occur directly by contact with infected individuals or indirectly after contact with contaminated surfaces or ingestion of contaminated food or water. They are excreted by infected individuals at high concentrations (up to $10^{13}$ genome units/g of stool) (Caballero et al., 2003; Teunis et al., 2015). Since wastewater treatments are partially effective in eliminating them, viruses can be discharged through wastewater treatment plant effluents in environmental waters (Iaconelli et al., 2017; Prevost et al., 2015; Xagoraraki et al., 2014). Due to their high resistance, they can persist for extended periods depending on physicochemical environmental conditions (Fong and Lipp, 2005; Rzezutka and Cook, 2004; Sanchez and Bosch, 2016). Furthermore, the virological safety of aquatic environments can also be compromised during rainfall events that cause combined sewer overflows, leading to the discharge of raw wastewater into the environment (Hata et al., 2021, 2014). As a result, the viral load in environmental water influenced by wastewater effluents reflects the level of their circulation in the local population (Prevost et al., 2015).

Progress has been made in detecting viruses in environmental water using qPCR-based methods. Concentrations ranging from $10^2$ to $10^5$ genome copies (gc)/L of norovirus and other enteric viruses like adenovirus, astrovirus, rotavirus, and hepatitis A and E viruses in river water samples were reported in several countries (Beyer et al., 2020; D'Ugo et al., 2016; Farkas et al., 2018; Prevost et al., 2015; Rutjes et al., 2009a). In addition, a recent meta-analysis of 61 studies estimated a global prevalence of human norovirus in river water at 43.5% (Ekundayo et al., 2021). Nevertheless, data on virus occurrence in environmental waters remains limited due to the relative complexity of setting up appropriate analytical methods and the associated cost. Virus quantification based on molecular tools does not provide information on the infectivity of the viruses detected. However, infectious enteric viruses such as adenovirus, rotavirus, and enterovirus have been isolated from surface water (Jiang et al., 2001; Rutjes et al., 2009b; Salvador et al., 2020; Sedji et al., 2018), showing that these viruses can persist in surface water for long enough to pose a health risk (Li et al., 2023).

Environmental waters are therefore highly susceptible to viral contamination, and a matrix associated with an important risk of virus transmission. Indeed, waterborne outbreaks can occur through multiple exposure pathways, including the consumption of tap and bottled drinking water sourced from contaminated water supplies (Blanco et al., 2017; Jacqueline et al., 2022; Kauppinen et al., 2019; Moreira and Bondelind, 2016) as well as through aquatic recreational activities (Schets et al., 2018; Sinclair et al., 2009). Water is also a primary vector of contamination in foodborne outbreaks, particularly through the consumption of horticultural produce irrigated with contaminated water (Barrimah et al., 1999), and filter-feeding shellfish that bio-accumulate viruses (Campos et al., 2016; Le Guyader et al., 2008; Yan et al., 2022). In Europe, regulatory frameworks for assessing the microbiological quality of drinking and environmental waters rely exclusively on bacterial indicators of fecal contamination. In particular, the EU Directive on the management of bathing water quality (Directive 2006/7/EC) mandates that member states monitor only *Escherichia coli* and intestinal enterococci to determine microbiological water quality (Directive 2006/7/EC, 2006). Nevertheless, there is no strict correlation

between the presence of bacterial indicators and the occurrence of viral pathogens (Richiardi et al., 2023), meaning that the detection of such indicators should not replace the detection of viral pathogens to assess risks posed by waterborne viruses.

In addition to waterborne enteric viruses, several non-enteric viral pathogens that had long been considered to pose no risk of water-mediated transmission were detected in wastewater or environmental waters. These include papillomaviruses, polyomaviruses (La Rosa et al., 2012), monkeypox virus, influenza viruses (Heijnen and Medema, 2011), coronaviruses (Blanco et al., 2019; La Rosa et al., 2023), and other respiratory viruses (Ahmed et al., 2023). For some of these viruses, their detection in water raised the question of a potential alternative route of transmission by water, either by ingestion or aerosolisation (Bofill-Mas et al., 2001; Maal-Bared et al., 2022; Wathore et al., 2020). Given the significant diversity of pathogens that can be transmitted through water, and the limitations of relying solely on microbial indicators for providing accurate risk assessment models, one of the challenges in managing water quality is the development of effective detection methods for monitoring pathogens.

In this study, we investigated the occurrence of human viral pathogens in the Seine and Marne rivers in the Paris area during the winter and summer seasons in 2023. We quantified noroviruses of genogroups I (NoV GI) and II (NoV GII), and monitored the occurrence of 30 viruses including enteric viruses, respiratory viruses, and viral indicators of fecal contamination, using a high-throughput PCR array.

# 2. Material and methods

## 2.1. Sampling

Two sampling campaigns were carried out in 2023, from 4 January to 31 March (winter period), and from 23 August to 22 September (summer period), to collect river water samples. Samples were collected upstream of Paris (Île-de-France, France) from both the Seine River and its tributary, the Marne River. Both rivers receive effluents from wastewater treatment plants (WWTP). In the Île-de-France region, the Seine and the Marne are important sources of drinking water. Half of the drinking water supplied to Paris is produced by two drinking water treatment plants (DWTP) that use water from these rivers. The Seine and the Marne are also used for recreational water activities, including designated bathing areas. Each river was sampled at two different sites, as shown in Fig. 1 which also shows the WWTP, DWTP and bathing areas. The GPS coordinates of the sampling sites were 48.85326, 2.54294 and 48.81599, 2.41329 (Marne) and 48.74756, 2.43525 and 48.80729, 2.40977 (Seine). Five liters of water were collected in polyethylene containers and stored at -20 °C until processing. A total of 23 water samples were collected in winter (10 in the Seine and 13 in the Marne) and 19 water samples were collected in summer (11 in the Seine and 8 in the Marne). Additional data regarding these samples (24-hour rainfall, pH, temperature, water flow rate) are provided in Table S1.

## 2.2. Virus concentration and nucleic acids extraction

Water samples (5 L) were artificially spiked with approximately $10^7$ gc (100 µL) of the process control, murine norovirus (MNV-1). The primary virus concentration was performed as described by Cuevas-Ferrando et al. (2019) with minor modifications by Dead End Hollow Fiber Filtration (DEUF) disposable Rexeed-25A dialysis filters (Asahi Kasei Medical, Co., Ltd.) with a 30 kDa molecular weight cut-off and a surface area of $2.5 \text{ m}^2$. An N6-3L high-flow industrial peristaltic pump (Dutscher) was used. Briefly, the filter was first pretreated with 6.25% calf serum by circulating this blocking solution for 5 min at 1800 mL/min. The filter filled with the blocking solution was placed on a rocking shaker for at least 2 h at 4 °C. The filter was mounted and flushed at 1800 mL/min with 1 L of ultrapure water and then with the river water sample. The volume of the filtered sample was recorded. Finally, the
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Map of the study area. Four sampling points (●) were considered in the Marne and Seine rivers upstream of Paris (France). In the study area, effluents from two wastewater treatment plants (WWTP) are discharged into the rivers, and water from these rivers is used by four drinking water treatment plants (DWTPs) to produce drinking water. Two authorized swimming areas (■) are also indicated. The grey arrow indicates the direction of flow of the Seine.</figcaption></figure>

filter was mounted in reverse and the backflush elution was performed by flushing 300 mL of PBS supplemented with 0.001% Antifoam A (Sigma), 0.01% sodium polyphosphate (Sigma), and 0.01% Tween 80 (Sigma) solution at 600 mL/min. The volume of the concentrate was recorded for the calculation of virus concentrations.

The secondary concentration was performed by polyethylene glycol (PEG) precipitation. Three hundred milliliteres of concentrate were divided by transferring 150 mL into two centrifuge bottles. Two grams of beef extract (Sigma) were added to each tube and mixed thoroughly until dissolution. Then 50 mL of 5× PEG/NaCl were added to each tube and placed on a rocking shaker at 4 °C overnight. Finally, the samples were centrifuged at 4200 ×g for 1 h, and the pellets were resuspended in 1 mL PBS and stored at -80 °C until nucleic acids extraction.

Nucleic acids extraction was performed on a NucliSENS® easyMAG® instrument (bioMérieux). The lysis was performed off-board by thoroughly mixing the concentrates to a final volume of 3 mL NucliSENS® Lysis Buffer. After 15 min of incubation at room temperature, the nucleic acids extraction was performed on the instrument using the “Specific A” protocol with 50 µL of magnetic silica beads. The nucleic acids were eluted in 100 µL of elution buffer, aliquoted and stored at -80 °C.

## 2.3. Virus quantification by RT-qPCR

NoV GI, NoV GII and MNV-1 were quantified by TaqMan real-time RT-PCR with the CFX96™ real-time PCR detection system (Bio-Rad). Reactions were performed by using the RNA UltraSense™ One-Step Quantitative RT-PCR System (Life Technologies) in 25 µL final volume containing 1× of RNA UltraSense™ reaction mix, 1.25 µL of RNA UltraSense™ enzyme mix, 2 U RNase inhibitor, 62.5 ng of bovine serum albumin, 500 nM of forward primer, 900 nM of reverse primer, 250 nM of probe (see Table S2 for primer and probe sequences) and 5 µL of RNA extract. The cycling conditions included a reverse transcription at 55 °C for 1 h followed by the activation of the polymerase at 95 °C for 5 min,

45 cycles of denaturation at 95 °C for 15 s, and elongation at 60 °C for 1 min and 65 °C for 1 min. The fluorescence was recorded at the end of the elongation. Standard curves were generated from tenfold dilutions of RNA transcripts (5 × 10⁶ to 5 × 10¹ gc/PCR reaction) in ultrapure water. The slopes (S) of the regression lines were used to calculate the amplification efficiency (E) of the RT-qPCR reactions according to the formula E = 10|-1/S|-1 (Fig. S1). A negative control containing all the reagents except the RNA template was included in each run. RNA samples were analyzed in duplicate undiluted or five-fold diluted. The limits of detection (LOD₈₀) were established at 1 × 10⁰ cg/µL for the NoV GI and MNV-1 RT-qPCR assays and 1 × 10¹ cg/µL for the NoV GII RT-qPCR assay (Table S3.A).

## 2.4. Reverse transcription and pre-amplification

Reverse transcription (RT) reactions were performed using the SuperScript™ IV First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol with 11 µL of viral RNA and 2.5 µM random hexamers, in 20 µL reaction mixtures. Immediately after the RT, cDNAs were pre-amplified. Specific forward and reverse primers of each assay were combined to prepare a single primer pool containing each primer at a 200 nM final concentration (see Table S2 for primer and probe sequences). Pre-amplification reactions were performed in a 5 µL reaction mixture containing 1 µL of PreAmp Master Mix (Standard BioTools), 1.25 µL of the 200 nM primers pool (final concentration of each primer: 60 nM), 1.25 µL of cDNA and 1.25 µL of PCR-grade water. The thermal conditions were as follows: 10 min of initial activation at 95 °C and 14 cycles including a denaturation at 95 °C for 15 s and an elongation at 60 °C for 4 min. Amplified products were immediately diluted (1/5) with PCR-grade water and stored at -80 °C until the high-throughput qPCR analysis one week later.

## 2.5. High-throughput PCR

The specific detection of 30 viral genomes was performed by high-throughput qPCR (HT-qPCR) with a BioMark HD system using 48.48 dynamic array IFC plates (Standard BioTools). This system allows analyzing up to 48 samples with up to 48 specific assays. First, control line fluid was injected into the 48.48 IFC plate which was then primed in an IFC controller MX. For each sample, 5 µL of a reaction mixture containing 1× of RNA UltraSense™ reaction mix (Life Technologies), 0.25 µL of RNA UltraSense™ enzyme mix (Life Technologies), 1× ROX reference dye (Life Technologies), 1× GE sample loading reagent (Standard BioTools) and 3.3 µL of DNA sample were deposited on the right part of the plate. For each assay, 5 µL of a mix containing 2.5 µL of 2× Assay loading (Standard BioTools), and 2.5 µL of 20× oligonucleotides stock (10 µM of forward primer, 18 µM of reverse primer, and 5 µM of probe; sequences listed in Table S2) were deposited on the left part of the plate. The reaction mix were loaded in each of the 2.304 chambers with an IFC controller MX and the loaded plate was then transferred to the BioMark HD system. The qPCR thermal cycling conditions were as follows: 15 min of activation at 95 °C followed by 40 cycles of 15 s at 95 °C for denaturation, 1 min at 60 °C and 1 min at 65 °C for elongation. The fluorescence was recorded at the end of the elongation steps. The limits of detection (LOD₈₀), expressed as RNA input concentrations prior to the reverse transcription step, were established at <1 × 10⁰ cg/µL for the MNV-1 high-throughput PCR assay and 1 × 10¹ cg/µL for the NoV GI and NoV GII high-throughput PCR assays (Table S3.B).

## 2.6. Statistical analyses

Statistical analysis and graphic construction were performed with GraphPad Prism (version 8.0.1; GraphPad Software Inc., La Jolla, USA). NoV concentrations in the Seine and Marne and during the two sampling periods were compared with one-way ANOVA and Tukey's post-hoc test. In all cases, *p*-value <0.05 was considered significant.
# 3. Results and discussion

## 3.1. Performance of the concentration method

The performance of the method for the viral concentration in surface water was evaluated based on recovery rates of MNV-1, used as a process control, spiked with a known amount prior to sample processing. The recovery rate of MNV-1 was determined from undiluted RNA and five-fold diluted RNA to account for possible PCR inhibition (Fig. 2). MNV-1 was detected in all samples with a mean recovery rate of 1.44 ± 0.30% from undiluted RNA and 6.29 ± 0.98% from five-fold diluted RNA. This difference indicates the co-concentration of PCR inhibitors from surface water samples. For 38/42 samples, the recovery rates from diluted samples were greater than 1%, which is the quality assurance level specified in the ISO 15216-1:2017 method (ISO 15216-1, 2017) for the detection of norovirus and HAV in bottled water. These results are in agreement with a previous study, which reported a median recovery of the control process (Mengovirus) < 0.5% in undiluted RNA samples and between 2.89% and 5% in 10-fold diluted samples, using the same concentration method from surface water samples (Cuevas-Ferrando et al., 2021).

In the literature, various recovery rates of viruses from surface water have been reported depending on the concentration method, sample volume, and the virus considered. For example, recovery rates of 0.053–1.2% for mengovirus, AdV, or NoV (Petterson et al., 2015), and 4–33% for mengovirus, NoV and HAV (Borgmästars et al., 2017) were reported using a concentration in two steps (filtration and PEG precipitation). Additionally, recovery rates of 18–83% for AdV, EV, RV or NoV were obtained using a three-step concentration method (filtration, organic flocculation, ultracentrifugation) (Prevost et al., 2015), but another three-step concentration method (filtration, organic flocculation, ultrafiltration) led to mengovirus recovery <1% (Salvador et al., 2020).

## 3.2. Concentration of norovirus in the Seine and Marne rivers

A total of 42 river water samples were collected from 4 different sample points (Fig. 1) and noroviruses were analyzed by RT-qPCR (Fig. 3).

NoV GI was detected with frequencies of only 10% (winter) and 37% (summer) in water samples from the Seine, and 54% (winter) and 82% (summer) in water samples from the Marne. In the Marne, the median concentration was about $5.8 \times 10^1$ gc/L in winter and $2.4 \times 10^2$ gc/L in summer. The maximum concentration measured was $1.1 \times 10^3$ gc/L in the Marne River. No significant differences were observed between the two seasons in either river. However, during summer, the concentration of NoV GI was significantly higher in the Marne compared to the Seine.

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Recovery of MNV-1 in surface water samples. MNV-1 was quantified in spiked river water samples (n = 42) by RT-qPCR from undiluted or 5-fold diluted RNA extracts (mean ± SEM).</figcaption></figure>

Regarding NoV GII, detection frequencies in the two rivers ranged between 50% and 75% across both seasons. Median concentrations in winter and summer were $2.3 \times 10^1$ and $4.4 \times 10^1$ gc/L, respectively, in the Seine River, and $9.1 \times 10^1$ and $2.0 \times 10^2$ gc/L in the Marne River. No significant differences in NoV GII concentrations were observed between seasons or between the two rivers.

Correlation analysis between NoV concentrations and environmental parameters (Table S4) revealed a significant negative association between NoV GI levels and water flow rate ($r = -0.340$). This indicates that higher flow rates are associated with lower viral levels. This relationship can be explained by a dilution effect, whereby increased water flow disperses viral particles more effectively, reducing their measured concentrations. From a public health perspective, this finding indicates that periods of low flow may pose a greater risk of contamination, as reduced dilution favors higher concentrations of NoV in surface waters. Both the Seine and Marne rivers are impacted by multiple treated wastewater discharges along their courses, which constitute major sources of microbial contamination (Prevost et al., 2015). On sampling days, flow rates ranged from 34 to 264 m³/s in the Marne and from 78 to 458 m³/s in the Seine (Table S1). The average flow rate of the Seine was higher than that of the Marne. This disparity may have contributed to the lower detection frequency of norovirus in the Seine, as higher flow rates can promote greater dilution and dispersion of microbial pollutants (Patil et al., 2022). In this study, 24-hour rainfall was not correlated with viral contamination, although rainfall events are generally recognized as a major driver of water quality degradation (Hata et al., 2014). This lack of correlation may be explained by the absence of intense rainfall events during the sampling period. Using the threshold proposed by Tornevi et al. (2014), intense rainfall events (>15 mm over 24 h) occurred during a single sampling day (Table S1).

Correlation analysis also revealed that flow rate is negatively correlated with temperature ($r = -0.748$) and positively correlated with pH ($r = 0.373$), while temperature and pH exhibit a strong negative correlation ($r = -0.714$). These relationships reflect typical seasonal variations, with higher flow rates and higher pH values in winter due to increased precipitation and runoff. Overall, these patterns highlight the interconnected dynamics of the rivers' physical and chemical parameters.

Once released into surface waters, enteric viruses may persist for extended periods depending on environmental conditions such as temperature, solar irradiation, and organic matter content, while being subject to dilution, transport, and partial inactivation (Li et al., 2023; Pinon and Vialette, 2018). Quantitative data on human enteric viruses in river waters are still scarce in the literature. Nevertheless, a study conducted about ten years before the present one in the same geographical area reported median NoV GI and NoV GII loads ranging from approximately $10^2$ to $10^3$ gc/L in the Seine and Marne rivers, with detection frequencies exceeding 70% (Prevost et al., 2015). Norovirus occurrence measured in the present study is slightly lower than previously reported. Although this difference can be attributed to the use of different methodologies in the two studies, it may also suggest a possible improvement in water quality. Indeed, over the last decade, wastewater treatment systems in the Île-de-France region underwent upgrades aimed at improving effluent quality. Treatment capacities were increased to accommodate population growth and to improve resilience to wet-weather conditions, thereby reducing the discharge of untreated or partially treated wastewater into receiving waters. Additional measures were implemented to make the Seine swimmable for the 2024 Olympic Games. Stormwater retention basins were constructed to capture rain-water and reduce untreated wastewater discharges during heavy rainfall events (Préfecture de la région d'Île-de-France, 2024). The sewer network was modernized and expanded, faulty domestic connections were corrected and boats were connected to the sewer system to prevent direct wastewater inputs into the rivers. Some WWTP, including the plant located near our Seine sampling sites, have also been upgraded to improve the removal of microbiological pollutants through the
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Concentrations of NoV GI and NoV GII in the Seine and Marne Rivers. River water samples were collected in winter (n = 13 in the Marne River, n = 10 in the Seine River) and summer (n = 11 in the Marne River, n = 8 in the Seine River) 2023. NoV GI and NoV GII were quantified by RT-qPCR. The percentages of positive samples are also indicated. The dotted line represents the limit of detection (LOD); medians are indicated by horizontal lines. n.s.: not significant, *: p < 0.05.</figcaption></figure>

implementation of tertiary treatments (performic acid) (Rocher and Azimi, 2021).

Other authors reported NoV GI and NoV GII mean concentrations of 6.1 × 10² and 3.7 × 10³ gc/L in a river located in northeastern France (Sedji et al., 2018), NoV GI and NoV GII concentrations of about 10² gc/L in a North Walian River in UK (Farkas et al., 2018), and NoV GII concentrations over 10⁴ gc/L in the Ruhr River in Germany but for only 2/24 positive samples (Mackowiak et al., 2018). The detection of noroviruses in watercourses indicates that these viruses are circulating in the population. However, the detection of viral genomes does not provide information on the risk linked to water use. Very few data are available on the infectivity of noroviruses detected in naturally contaminated environmental waters (Carmona-Vicente et al., 2024). Indeed, a replication system for noroviruses remained inaccessible until the recent development of human intestinal enteroids (HIE) capable of amplifying infectious noroviruses (Ettayebi et al., 2016). In addition, there are still limitations in assessing the infectivity of noroviruses in environmental matrices, such as the selective HIE permissiveness to certain viral strains and the sensitivity of this approach. Nevertheless, this culture system has enabled laboratory-scale studies under controlled conditions to demonstrate that some norovirus strains can remain infectious for several weeks in various water matrices such as seawater, lake water, and tap water (Desdouits et al., 2022; Esseili et al., 2025; Kennedy et al., 2023; Rexin et al., 2024; Shaffer et al., 2022).

### 3.3. Prevalence of multiple pathogenic viruses in the Seine and Marne rivers

To extend the screening to a larger number of viruses, all 42 river water samples were analyzed using a high-throughput PCR array. The prevalence of each virus according the river and season is shown in Fig. 4. Over the entire monitoring period, the most prevalent pathogenic viruses were aichivirus (35/42, 83%), adenovirus (32/42, 76%), and bocavirus (31/42, 74%) whatever the river. The other enteric viruses detected were enterovirus (11/42, 26%), astrovirus (9/42, 21%), NoV GII (9/42, 21%), cosavirus (6/42, 14%), NoV GI (5/42, 12%), HEV (2/42, 5%), sapovirus (1/42, 2%) and HAV (1/42, 2%). Bocavirus, astrovirus, and adenovirus were more prevalent in winter samples, while enterovirus and aichivirus were more frequently detected in summer samples, suggesting a seasonal effect. Among the respiratory viruses included in the detection panel, only OC43 and measles virus were detected once, in two different samples, during the winter campaign. The two viral indicators of fecal contamination, CrAssphage and PMMoV, were detected in all samples except one in which PMMoV was

<table><caption>**Fig. 4.** Prevalence (%) of river water samples positive for the detection of different viruses using high-throughput PCR. Water samples were collected in winter (Marne river: 13; Seine river: 10) and summer (Marne river: 11; Seine river: 8) 2023.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Winter</th><th colspan="2">Summer</th><th rowspan="2">Overall</th></tr><tr><th>Marne River</th><th>Seine River</th><th>Marne River</th><th>Seine River</th></tr></thead><tbody><tr><td>Hepatitis A virus</td><td>0</td><td>0</td><td>0</td><td>13</td><td>2</td></tr><tr><td>Hepatitis E virus</td><td>0</td><td>0</td><td>9</td><td>13</td><td>5</td></tr><tr><td>Rotavirus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Norovirus GI</td><td>15</td><td>10</td><td>18</td><td>0</td><td>12</td></tr><tr><td>Norovirus GII</td><td>31</td><td>10</td><td>18</td><td>25</td><td>21</td></tr><tr><td>Norovirus GIV</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Sapovirus</td><td>0</td><td>0</td><td>9</td><td>0</td><td>2</td></tr><tr><td>Adenovirus</td><td>85</td><td>80</td><td>64</td><td>75</td><td>76</td></tr><tr><td>Enterovirus</td><td>15</td><td>10</td><td>36</td><td>50</td><td>26</td></tr><tr><td>Cosavirus</td><td>15</td><td>20</td><td>18</td><td>0</td><td>14</td></tr><tr><td>Bocavirus</td><td>100</td><td>100</td><td>27</td><td>63</td><td>74</td></tr><tr><td>Aichivirus</td><td>77</td><td>70</td><td>91</td><td>100</td><td>83</td></tr><tr><td>Astrovirus</td><td>31</td><td>40</td><td>0</td><td>13</td><td>21</td></tr><tr><td>Tick-borne encephalitis virus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Poliovirus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Influenza A virus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Influenza B virus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>CoV 229E</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>CoV HKU1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>CoV NL63</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>CoV OC43</td><td>0</td><td>10</td><td>0</td><td>0</td><td>2</td></tr><tr><td>SARS-CoV-2</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>SARS-CoV-2 Omicron</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Respiratory syncitial virus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Measles virus</td><td>0</td><td>10</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Monkeypox virus</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>CrAssphage</td><td>100</td><td>100</td><td>100</td><td>100</td><td>100</td></tr><tr><td>PMMoV</td><td>100</td><td>100</td><td>91</td><td>100</td><td>98</td></tr></tbody></table>

not detected.

These prevalence values were used to calculate Pearson correlation coefficients to quantify the strength of the association by river or sampling season (Table S5). The association was stronger for samples collected from different rivers during the same season (0.987 and 0.960)
than for samples collected from the same rivers during the two seasons (0.883 and 0.909). This indicates that, in the context of the present study, the seasonal effect had a greater impact on virus prevalence than local effects.

In a review describing the occurrence of human pathogenic viruses in drinking water and the water sources contributing to its production, adenovirus, aichivirus, rotavirus, astrovirus, NoV GI, and NoV GII were the most frequently detected viruses (prevalence >30%) in surface water (river, lake) samples (Panizzolo et al., 2023). In addition, a one-year survey on the occurrence of pathogenic viruses in the Seine and Marne (175 samples) conducted in 2013–2014 identified NoV GII, adenovirus, NoV GI, rotavirus, astrovirus, and aichivirus as the most prevalent viruses with detection frequencies between 15% and 84% (Prevost et al., 2015). Bocavirus is less frequently investigated, but its presence in surface water was reported for the first time in 2008 in 40.8% of the 120 river water samples tested in Germany (Hamza et al., 2009). Regarding HEV and HAV, meta-analyses on their occurrence in water environments reported detection frequencies in surface water (river, lake, sea) of 7.4% and 10%, respectively (Takuissu et al., 2023, 2022). However, higher prevalence data of HEV were found in other European countries such as Portugal (77.7%) (Salvador et al., 2020), Germany (30.0%) (Beyer et al., 2020), Italy (25%) (Idolo et al., 2013), and the Netherlands (17%) (Rutjes et al., 2009a).

Our findings are broadly consistent with these previous results, except for rotavirus, which we did not detect. However, we previously detected rotavirus in a significant proportion of treated wastewater samples from a wastewater treatment plant located in the study area and discharging effluent into the Marne River (WWTP shown in Fig. 1) (Waldman et al., 2025). A possible explanation for the lack of detection in river samples is that the concentration method used is not well adapted for rotavirus, or that rotavirus levels in the rivers are too low to be detected.

Detection rates for NoV GI and GII using the PCR array were 12% and 21%, respectively, lower than those obtained by RT-qPCR, which were 45% and 64%. These differences between the two methods were statistically significant ($\chi^2$ test, $\alpha = 0.05$), reflecting the reduced sensitivity of the PCR array compared to RT-qPCR. This slightly reduced sensitivity is likely due to the very small reaction volumes used on the array (9 nL). Although a pre-amplification step is employed to compensate for these low volumes, it appears that it does not fully restore the sensitivity to the level achieved by RT-qPCR. The experimental LOD₈₀ is higher for the PCR array ($1 \times 10^1$ cg/µL) than for RT-qPCR ($1 \times 10^0$ cg/µL) for NoV GI, and similar for NoV GII ($1 \times 10^1$ cg/µL) (Table S3.B). All samples testing positive for NoV GI or NoV GII were also positive by RT-qPCR, with the exception of one sample for each target. Moreover, PCR array-positive samples tended to have higher viral concentrations, indicating that the PCR array is less sensitive than standard RT-qPCR, although this trend did not reach statistical significance (Fig. S2).

The PCR array has previously been used for the detection of viruses in wastewater, which typically contains high concentrations of human viruses (Waldman et al., 2025). However, our findings show that it is also suitable for analyzing environmental matrices with lower viral loads, such as surface waters, and can successfully detect a broad diversity of viruses despite its slightly reduced sensitivity compared to RT-qPCR. The PCR array was used in this study to track viruses at sites close to Olympic venues where the triathlon and marathon swimming events took place during the summer of 2024.

It is important to note that the detection of viral genomes using molecular methods does not inform on viral infectivity and therefore does not allow for a direct assessment of infectious risk, which would necessitate cell culture-based approaches. Indeed, some viral genomes detected in environmental samples may originate from inactivated viruses and thus may not represent an actual health risk. However, evidence from the literature suggests that at least some of the viral pathogens detected in rivers may remain infectious. A survey conducted in Portugal in 2019 on 25 surface water samples tested for the presence

of HEV reported the detection of genomes and infectious particles in 77.8% and 23%, respectively (Salvador et al., 2020). Moreover, infectious human adenoviruses were detected in 14/15 of river water samples collected in France in 2016 (Sedji et al., 2018). Bathing in the Seine and Marne rivers is prohibited, except in a limited number of designated and managed areas subject to microbiological monitoring. Access to these areas by the general public has only been permitted since 2025. Despite this restriction, swimming in non-authorized areas occasionally occurs. In this study, measured levels of NoV genomes were relatively low; however, as demonstrated by PCR array analysis, it is important to consider the presence of a mixture of enteric viruses, which can have very low infectious doses in humans. Consequently, a residual risk to the local population associated with recreational water exposure cannot be completely ruled out. In contrast, the risk of exposure via drinking water derived from the Seine and Marne rivers is considered very low, due to the application of multi-barrier disinfection processes in DWTPs (coagulation/flocculation and sedimentation, filtrations, chemical and physical disinfentions).

## 4. Conclusion

This study investigated the presence of human viruses in the Seine and Marne rivers in the Paris area in 2023. Noroviruses GI and GII were quantified by RT-qPCR, and the presence of 28 pathogenic viruses was determined by a high-throughput PCR array, highlighting the constant contamination of the two rivers by different enteric viruses. Once released into surface waters, enteric viruses may persist for extended periods depending on environmental conditions, while undergoing dilution, transport, and partial inactivation. Such contamination highlights rivers as dynamic reservoirs and vectors for human viruses, with potential exposure of local populations primarily through recreational water use, particularly during warm periods. Although detected viral concentrations were generally low, the concomitant presence of multiple enteric viruses, known to be infectious at low doses, suggests that a residual public health risk cannot be completely excluded. However, as genome detection does not reflect viral infectivity, a direct assessment of infectious risk would require complementary cell culture-based approaches. Despite this limitation, the results provide valuable insight into the environmental circulation and potential fate of human viruses in urban rivers. Finally, the PCR array proved to be an efficient tool for the rapid and simultaneous monitoring of multiple pathogens in environmental samples. Its adaptability makes it particularly relevant for large-scale monitoring and early warning strategies, supporting integrated environmental surveillance frameworks.

### CRediT authorship contribution statement

**Prunelle Waldman:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. **Audrey Fraisse:** Writing – review & editing, Methodology. **Mai-Lan Tran:** Investigation. **Sylvie Perelle:** Writing – review & editing, Resources. **Sandra Martin-Latil:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

### Funding sources

This work was part of the FOREWARN project supported by the French National Research Agency and the ERA-Net Cofund AquaticPollutants (Joint Call 2020).

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prunelle Waldman reports financial support was provided by French
National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2026.181554.

## Data availability

Data will be made available on request.

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