Science of the Total Environment 863 (2023) 160902 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate ⁎ Albert Perlas a,b, , Kateri Bertran a,c, Francesc Xavier Abad a,c, Carles M. Borrego d,e, Miquel Nofrarías a,c, Rosa Valle a,c, Lola Pailler-García a,c, Antonio Ramis a,b, Martí Cortey b, Vicenç Acuña d,f, Natàlia Majó a,b a Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia, Spain b Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain c IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia. Spain d Catalan Institute for Water Research (ICRA), C. Emili Grahit 101, 17003 Girona, Spain e Grup d'Ecologia Microbiana Molecular, Institut d'Ecologia Aquàtica, Universitat de Girona (UdG), Plaça Sant Domènec 3, 17004 Girona, Spain f Universitat de Girona (UdG), Plaça Sant Domènec 3, 17004 Girona, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • We used artificial streams to assess avian influenza environmental persistence. • Artificial streams remained infectious be- tween one and two days. • Avian influenza viral RNA was detected until the end of the experiment (14 days). • Experiments simulating real environ- ments provide a more realistic approach. • Mediterranean wetlands conditions could facilitate transmission during short pe- riods. A R T I C L E I N F O A B S T R A C T Editor: Warish Ahmed Avian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to op- timize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV Keywords: persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimiz- Influenza ing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Birds Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and Water Persistence similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment Environment presence (i.e., 10–13 °C vs. 16–18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV Mediterranean (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points Sediments until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end ⁎ Corresponding author at: Unitat mixta d'Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia, Spain. E-mail addresses: albert.perlas@uab.cat (A. Perlas), kateri.bertran@irta.cat (K. Bertran), xavier.abad@irta.cat (F.X. Abad), cborrego@icra.cat (C.M. Borrego), miquel.nofrarias@irta.cat (M. Nofrarías), rosa.valle@irta.cat (R. Valle), lola.pailler@irta.cat (L. Pailler-García), Antonio.ramis@uab.cat (A. Ramis), marti.cortey@uab.cat (M. Cortey), vacuna@icra.cat (V. Acuña), natalia.majo@uab.cat (N. Majó). http://dx.doi.org/10.1016/j.scitotenv.2022.160902 Received 3 October 2022; Received in revised form 1 December 2022; Accepted 8 December 2022 Available online 13 December 2022 0048-9697/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A. Perlas et al. Science of the Total Environment 863 (2023) 160902 of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sed- iment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled stud- ies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more re- alistic approach for assessing environmental persistence of AIVs. 1. Introduction environmental conditions to validate results from laboratory investigations are rare (Reeves et al., 2020). Only recently, a combination of field- and Avian influenza viruses (AIVs) belong to the Orthomyxoviridae family laboratory-based approaches suggested that waterfowl AIVs could remain and have a segmented, single-stranded RNA genome. AIVs have an evolu- viable for months in surface water of northern wetlands in North tionary plasticity that allows them to replicate in different species like wa- America, supporting the tenet that surface waters may act as an important terfowl, poultry, and mammals, including humans. Based on the ability to vehicle in which AIV may be both transmitted and maintained, potentially cause disease and death in chickens, AIVs can be classified into two differ- serving as an environmental reservoir for infectious AIVs (Ramey et al., ent pathotypes: low pathogenic (LP) and highly pathogenic (HP). LPAIVs 2022b). Nevertheless, the effect of UV light and/or water movement were can be asymptomatic, but they typically cause mild to moderate respiratory not examined in that study. disease in chickens, often accompanied by a decrease in water or feed con- Most information about AIV persistence in water has been obtained sumption and drops in egg production. In contrast, HPAIVs cause severe from studies where AIV was diluted in water and maintained inside a systemic disease with very high mortality in chickens and other Galliformes tube (virus-in-a-tube experiments), under controlled conditions lacking (Reperant et al., 2012). Outbreaks of AIV currently represent a big chal- the realism of natural ecosystems. Here, we used artificial streams that lenge for the poultry sector, wildlife, and public health, as exemplified by can mimic biotic and abiotic natural conditions of waterfowl habitats, pro- the 2021–2022 HPAIV epizootic in Europe (European Food Safety Author- viding a more refined inference on the duration of AIV infectivity in wet- ity, 2022). Even if there is a low zoonotic risk of the current HPAIV strains land systems. More specifically, we assessed the effect of temperature circulating in Europe, the emergence of strains with zoonotic potential is an (10–13 °C vs. 16–18 °C), the effect of sediments (with the associated micro- obvious concern, as some HPAIVs have been able to replicate in humans biota), and the interaction of these two variables on LPAIV water persis- (European Food Safety Authority, 2022). tence in a more realistic model. Based on previous works, we Wild waterfowl are the main reservoir of AIVs (Pantin-Jackwook and hypothesized that: 1) colder water temperature (10–13 °C) might have a Swayne, 2009); they can disseminate these viruses globally through their greater protective effect on infectious viral particles than warmer water migration routes and occasionally spread them to poultry, sometimes temperature (16–18 °C); 2) the presence of sediments could have a sorption with devastating effects (Blagodatski et al., 2021; Fourment et al., 2017; effect on AIVs protecting them from abiotic effects; and 3) correspondingly, Lycett et al., 2016). Furthermore, migratory waterfowl can also suffer the interaction of colder water temperature with the presence of sediments from high mortality rates by HPAIV infections, threatening wildlife conser- could have the main protective effect on AIVs. Given recent HPAIV detec- vation (Ramey et al., 2022a). Therefore, the study of waterfowl habitats tions in wetlands from the Mediterranean coast of Catalonia (Ministerio that intersect different avian migration routes is key to understanding trans- de Agricultura, 2022) and throughout Southern Europe, we also used our mission dynamics and, ultimately, designing optimal management strate- results to speculate on AIV persistence in Mediterranean waterfowl habitats gies. Since transmission of AIV among wild birds occurs mainly via the during the winter-spring period. faecal-oral route through water containing viral particles (Hinshaw et al., 1979, 1980; Stallknecht et al., 2010), environmental transmission through 2. Materials and methods this vehicle may facilitate spill-overs to poultry. Decades of laboratory-based investigations provide evidence that AIV 2.1. Virus stock may remain infectious in water and sediments for extended periods (weeks or months) under experimentally controlled conditions. The role The LPAIV isolate A/Duck/Italy/281904/06 H1N1 LPAIV was used of some abiotic factors is well studied: low water temperatures, neutral (kindly provided by Dr. Ana Moreno from the Istituto Zooprofilattico pH, and low-salinity water conditions substantially increase AIV persis- Sperimentale della Lombardia e dell'Emilia Romagna, Italy). The working tence (Dalziel et al., 2016; Morin et al., 2018; Pepin et al., 2019). The effect stock was prepared in embryonated chicken eggs (ECE) (Spackman and of biotic factors has also been studied, showing that in filtered and sterilized Killian, 2014), filtered (0.2 μm), and titrated in Madin–Darby Canine Kid- water AIV can persist for longer periods than in untreated water collected in ney (MDCK, ATCC CCL-34) cells using the TCID50, calculated following natural sources with the presence of microorganisms (Guan et al., 2009; the Reed and Muench method (Hierholzer and Killington, 1996). Keeler et al., 2013; Nazir et al., 2010; Nielsen et al., 2013; Zarkov, 2006; Zhang et al., 2014). Furthermore, wild bird-origin AIVs have repeatedly 2.2. Experimental design been isolated (Hinshaw et al., 1979; Ito et al., 1995; Lebarbenchon et al., 2011; Markwell and Shortridge, 1982; Okuya et al., 2015) or detected by The experiment was performed at the artificial streams facility of the RT-PCR (Hénaux et al., 2012; Lickfett et al., 2018) from surface water, Catalan Institute for Water Research (ICRA, Girona, Catalonia, Spain). and isolated (Poulson et al., 2017) or detected by RT-PCR (Himsworth This indoor experimental system simulates the natural water environment et al., 2020; Lang et al., 2008) from sediments collected from freshwater under controlled conditions (Romero et al., 2019). Here, four different and estuarine wetlands. Regarding the amount of AIV needed to initiate treatments were evaluated (three replicates per treatment), with two sedi- an infection in waterfowl via the faecal-oral route, a recent study suggests ment conditions (presence [S1] vs. absence [S0]) and two temperature con- that even very low titers, around 2 log10 50 % tissue culture infection ditions (10–13 °C [T0] vs. 16–18 °C [T1]) akin to those found in a dose (TCID50) may be sufficient (Ahrens et al., 2022). These findings are Mediterranean river during winter-spring (Fig. 1). Water temperature var- in line with the hypothesis that environmental transmission plays a crucial ied over time following day/night cycles of light, a constant parameter role in AIV epidemiology. However, field experiments carried out under from the experimental set-up, as explained below. Each artificial stream 2 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 Fig. 1. A. Schematic representation of the artificial streams. B. Artificial streams at ICRA, showing six of the 12 streams used in the experiment. was 2 m long, had a rectangular cross-section of 50 cm2 and was set up as an a necessary step for the sediment microbiota to colonize the water. The ac- open system with constant slope and steady, uniform flow. Water and sed- climation process was verified as explained below (Section 2.3). Following iments were obtained from a non-polluted lower section of the Ter River acclimation, water and sediment samples from all streams were collected (Jafre, Catalonia, Spain) close to a wetland area where many species of wa- and used as negative controls. Subsequently, all streams were spiked with terfowl are present (Aiguamolls del Baix Ter, Catalonia, Spain). Sediments 53.5 ml of H1N1 LPAIV at a titer of 7.3 log10 TCID50 directly into each were mostly sandy and median sediment grain size was 0.02 mm. Con- 5.5 l stream, resulting in a spike dose of approximately 5.3 log10 TCID50/ trolled parameters constant to all treatments were daily cycles of photosyn- ml per stream. The dilution was carried out by gently spiking the virus lon- thetic active radiation (PAR) (12 h daylight + 12 h darkness) that emulated gitudinally on each stream water surface. Following the virus spike, all the solar radiation, reproducing all the wavelengths at the same intensity streams were sampled and monitored for 14 days as detailed below. modifying water temperature, and 5.5 l of water in a recirculating flow of Water evaporation was compensated for by adding 400 ml of Ter river 0.01 l/s. These cycles of photosynthetic active radiation were chosen to water per stream daily. As a positive control, viral persistence was also mimic autumn day/night cycles in the Mediterranean region, the season assessed under virus-in-a-tube conditions at Centre de Recerca en Sanitat when some waterfowl species arrive from their summer breeding areas. Animal (IRTA-CReSA, Bellaterra, Catalonia, Spain). Briefly, 24 Eppendorf The recirculating flow of 0.01 l/s was the lowest flow possible in the artifi- tubes (12 tubes at 10 °C and 12 tubes at 16 °C) containing each 1.5 ml of cial streams facility and was chosen to mimic very low flow in wetland water from Ter River without sediment were spiked with H1N1 LPAIV at areas and to avoid a fast eutrophication process due to still water in the a final titer of 5.3 log10 TCID50 per ml and maintained at their correspond- streams. All streams were in acclimation for 3 weeks prior to virus spike, ing temperature for 14 days. 3 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 2.3. Characterization of water and sediments and detected by one-step Taqman real-time RT-PCR (rRT-PCR) in Fast7500 equipment (Applied Biosystems, USA) (Spackman et al., 2002). Water and sediment samples were collected throughout the experiment A standard curve from the same region of the AIV M1 gene was obtained for characterization purposes. Dissolved oxygen, pH, and specific conduc- as previously described by our group (Sánchez-González et al., 2020) and tivity were measured every 48 h by noon at each artificial stream using used to obtain the gene equivalent copies from the rRT-PCR results. Fur- Wissenschaftlich-Technische Werkstätten (WTW) hand-held probes thermore, virus isolation from water and sediment samples was conducted (Weilheim, Germany). Water temperature in each artificial stream was re- in ECEs by standard methods (Spackman and Killian, 2014). Briefly, 3 ECEs corded every 10 min during all the experiment by means of VEMCO were inoculated with 0.2 ml of undiluted water and sediment samples by minilog temperature data loggers (TR model; AMIRIX Systems Inc., Halifax the chorioallantoic sac route. Standard hemagglutination assay was used Canada) (5–35 °C, ±0.2 °C). Photosynthetically active radiation was also to test the allantoic fluid from each ECE for virus replication (Killian, recorded every 10 min using 4 quantum sensors (LI-192SA; LiCOR Inc., Lin- 2014). Hemagglutinating allantoic fluids were assumed to represent the coln, NE, USA) located across the whole array of streams. presence of infectious AIV in water and sediment samples. Biofilm colonization was allowed in the artificial streams for 3 weeks before virus spike. Briefly, superficial sediments (1–10 cm depth) were 2.6. Characterization of the sediment microbiota brought from a nearby lowland river (Ter River near the mouth, Girona), then spread, with no further treatment, in the artificial streams forming a DNA was extracted from biofilms developed on sediments using the layer of 3–4 cm depth. A colonization period was then allowed so that the FastDNA Spin for Soil kit (MP Biomedical) according to the manufacturer's biofilms could adapt and grow on the artificial streams. During this accli- instructions. High-throughput multiplexed 16S rRNA gene sequencing with mation period, biofilm status was monitored twice per week for their max- the Illumina MiSeq System (2 × 250 PE) was carried out using primer pair imum photosynthetic efficiency. These measurements provided 515f/806r (Caporaso et al., 2011) targeting the V4 region of the 16S rRNA information on the physiological status of the biofilm (Sabater et al., gene complemented with Illumina adapters and sample specific barcodes. 2007) in the artificial streams and were made to assess the physiological Details on the analysis of sequence datasets including comparison of similarity of the biofilms in all the artificial streams before exposure to alpha and beta diversity of samples across treatments are described in Sup- the different temperatures. Biofilm samples were collected in streams plementary Material. with sediments after the acclimation period (pre-spike) and at 14 days post-spike at both temperature ranges (see Section 2.6). 2.7. Statistical analysis 2.4. Sampling of water and sediment Results obtained from AIV rRT-PCR were used to evaluate the effects of the temperature and the presence or absence of sediments by two methods. Water and sediment samples were collected from all streams at 12 dif- First, a Kruskal-Wallis Test (Kruskal and Wallis, 1952) was used to assess ferent time-points: pre-spike (as a negative control), 0 (10 min post- differences between groups. If any differences were found, a Dunn's Test spike), 1, 2, 3, 4, 5, 6, 7, 9, 11, and 14 days post-spike. Briefly, 9 ml of (Dunn, 1961) for multiple comparisons was applied to test which treat- water per stream and time-point were collected with a micropipette and ments were significantly different. Second, to compare the treatments for stored in 1.5-ml aliquots (Suppl. Photo1) at −75 °C. Approximately 2 ml each time point, a mixed-effects model with the stream as a fixed factor of sediments per stream and time-point were collected following the and treatment and time point as random factors was adjusted, along with “core” method (Suppl. Video 1), with a 15-ml falcon tube and stored at a contrast of the estimated marginal means. Results obtained from virus iso- −75 °C. Similarly, one tube per temperature condition and time-point lation were used to determine differences between treatments in their sur- from the virus-in-a-tube experiment was collected and stored at −75 °C. vival curves with a Gehan-Breslow-Wilcoxon test. All the statistical analyses were performed with the R software (R Core Team, 2020) and the subse- 2.5. Viral detection and quantification in water and sediment samples quent packages: car, FSA, lmerTest, emmeans and tidyverse. Water and sediment samples were used for virus isolation and RNA ex- 3. Results traction. While water samples were processed without any additional manip- ulation, the procedure for extracting virus from sediments was optimized for 3.1. Physical and chemical characterization of water and sediment samples virus recovery prior to processing samples. To that end, two different methods were tested and compared using sediment samples collected as negative con- The temperature of the streams under colder conditions was maintained trols from our experiment that were subsequently spiked with 5.3 log10 between 10 °C and 13 °C (mean of 11.6 °C and a standard deviation of 0.95), TCID50/ml of sediment with H1N1 LPAIV. For the first method, a sterile while the temperature of the streams under warmer conditions was main- gauze was wet with 5 ml of brain heart infusion broth supplemented with 2 tained between 16 °C and 18 °C (mean of 17.5 °C and a standard deviation % Penicillin/Streptomycin (Invitrogen, Spain). Then the sediment pebbles of 0.89) throughout the experiment, temperature variation was due to day/ were manually rubbed with the wet gauze, and the gauze was placed in a night PAR cycles. The pH in all the streams and time points was around 8, sealable plastic bag. A masticator homogenizer (IULmicro, Spain) was used and the specific conductivity was moderate (≈500 μs/cm), without signif- for 1.5 min to punch and squeeze the supernatant out of the gauze and inside icant differences among streams at any time. The physical and chemical the plastic bag. Finally, the supernatant was collected into sterile 50-ml falcon characterization of the water did not show significant differences among tubes using a micropipette and squeezing the remaining supernatant out of treatments throughout the experiment (Suppl. File 1). the gauze with a 20-ml syringe. This sediment supernatant was used for virus isolation and RNA extraction alongside water samples. For the second 3.2. Characterization of the sediment microbiota method, the same steps were followed, but a pestle instead of a masticator was used, as previously described (Stephens and Spackman, 2017). The first Characterization of microbial communities in the streams containing method using the masticator was able to recover 4.75 log10 TCID50, in con- sediments was performed to assess potential variations across treatments trast with the method using the pestle which only recovered 4.2 log10 (i.e., water temperature) that could affect viral persistence during the ex- TCID50, validating the use of the method using the masticator. perimental period. Bacterial communities in sediments did not show signif- Water and processed sediment samples were used for quantification of icant differences in richness and diversity among streams differing in water M gene copies. Viral RNA was extracted using Nucleospin RNA virus kit temperature (10–13 °C vs. 16–18 °C) (Suppl. Fig. S2). However, a Principal (Macherey–Nagel, Düren, Germany) following manufacturer's instructions. Coordinates Analysis (PCoA) ordination of samples using the Bray-Curtis A highly conserved region of 99 bp present in AIV M1 gene was amplified dissimilarity distance segregated samples according to both the water 4 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 Fig. 2. M gene copies in water samples in the four analysed treatments in the artificial streams experiment. Results are expressed as mean and standard deviation (three replicates per treatment) expressed in log10 values. temperature (horizontal axis, 26 % sample variance, Suppl. Fig. S3) and the However, the interaction between 10 and 13 °C temperatures and the ab- experimental time (time 0 vs. time 14 days, vertical axis, Suppl. Fig. S3) sence of sediment compared with the interaction of 16–18 °C temperatures (PERMANOVA test, F-value = 2.4207; p < 0.016). and the presence of sediment showed the most significant differences in LPAIV RNA persistence (Suppl. Table S1). In sediment samples, no signifi- 3.3. Viral RNA quantification in water and sediment samples cant differences in viral RNA decay were observed between streams at dif- ferent temperatures (Kruskal-Wallis test) (Suppl. Table S2). Significant Water and sediment samples from the artificial streams experiment lower numbers of genomic copies were detected in sediment samples com- were used for RNA extraction and quantification of H1N1 LPAIV M gene pared to water samples on the first days (taking together both temperature copies by rRT-PCR at 12 different time points. All negative control samples conditions), but similar numbers were found in both types of samples on (samples collected pre-spike) were negative, confirming the absence of AIV subsequent days (Fig. 4 and Suppl. Table S2). from water and sediments from Ter River. Overall, water and sediment rRT- PCR results showed presence of viral RNA throughout the 14-day experi- 3.4. Viral isolation from water and sediment samples ment, although a steady decline over time was observed (Figs. 2 and 3). In water samples, significant differences in viral RNA decay were only To detect infectious H1N1 LPAIV, viral isolation in ECE from water and observed between streams at different temperatures (10–13 °C vs. 16–18 sediment samples was carried out. Viral isolation from water samples was °C) regardless of sediment condition (multiple comparison Dunn's test). successful from all the streams at 0 days post-spike, from six of 12 streams Fig. 3. M gene copies in sediment samples in the two analysed treatments in the artificial streams experiment. Results are expressed as mean and standard deviation (three replicates per treatment) expressed in log10 values. 5 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 Fig. 4. M gene copies in sediment samples and water samples from streams with sediments. Results are expressed as mean and standard deviation (three replicates per treatment) expressed in log10 values. at 1 day post-spike, from one stream at 2 days post-spike, and no infectious environmental conditions (Horm et al., 2012; Ramey et al., 2022b), the per- viruses were detected beyond that (Table 1). No significant differences in sistence of AIV in water has not been yet fully characterized in simulated viral survival among treatments were detected (Table 1). No infectious environments. In this study, we mimicked biotic and abiotic natural condi- virus was recovered from any of the sediment samples. In contrast to the ar- tions of waterfowl habitats using artificial streams to analyse the effect of tificial streams, viral persistence in water under virus-in-a-tube conditions temperature and sediments on the persistence of LPAIV. Even if we still yielded infectious viruses from all samples at all time points, both at 10 didn't fully characterize the simulated environment, we tried to go one °C and 16 °C temperature conditions (Table 1). step further mimicking factors such as UV light cycles and streamflow. First, we characterized the streams after the acclimation period. Similar 4. Discussion physical and chemical properties were found among the streams at the same temperature and sediment conditions. Water had a neutral-to-basic Most of the data regarding environmental AIV persistence comes from pH and moderate specific conductivity throughout all the experiment; virus-in-a-tube experiments, which do not completely reproduce real envi- these characteristics were previously associated with the longest AIV persis- ronmental conditions (Dalziel et al., 2016; Morin et al., 2018; Pepin et al., tence periods (Dalziel et al., 2016; Keeler et al., 2014; Spackman and 2019). Even if some studies have attempted to reproduce more realistic Killian, 2014). Furthermore, the characterization of bacterial communities Table 1 Results of virus isolation from water samples for each replicate by treatment in each of the different experiment setups (i.e., artificial streams and virus-in-a-tube conditions). Results are shown as the number of positive replicates/total replicates. NA = not apply. Treatment Sediment Temperature Experiment Day 0 Day 1 Day 2 Day 3 setup to14 S1-T0 Present 10–13 ºC Artificial 3/3 3/3 0/3 0/3 streams S0-T0 Absent 10–13 ºC Artificial 3/3 1/3 1/3 0/3 streams S1-T1 Present 16–18 ºC Artificial 3/3 1/3 0/3 0/3 streams S0-T1 Absent 16–18 ºC Artificial 3/3 1/3 0/3 0/3 streams NA NA 10 ºC Virus-in-a- 3/3 3/3 3/3 3/3 tube NA NA 16 ºC Virus-in-a- 3/3 3/3 3/3 3/3 tube 6 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 in the sediments was also analysed to assess the potential effect of the sed- short viral persistence in our artificial streams compared to our virus-in-a- iment microbiota on viral persistency. No great differences in richness and tube experiment, performed as a positive control. Our artificial streams re- diversity of sediment microbial communities were observed across treat- sults are in line with a simulated environment study that reproduced real ments, although beta diversity analyses revealed differences between conditions of Cambodian lakes in experimental aquatic biotopes, where streams according to water temperature and incubation time. This observa- LPAIV isolation from water was possible during a maximum of 4 days tion suggests that maturation of biofilm communities was clearly influ- post-spike (Horm et al., 2012). Nonetheless, another realistic study per- enced by ambient conditions (i.e., temperature). Recently, Romero and formed in Alaska showed longer persistence results, with evidence for inter- co-workers reported a 92 % similarity between bacterial communities annual LPAIV persistence (Ramey et al., 2022b). The differences observed from the artificial streams and those from the source location (Romero between these simulated environments studies are probably related to differ- et al., 2019). Here, since the conditions of the artificial streams were very ences in water temperature, since higher temperatures were used to repro- similar to those of the source river (non-polluted reach of the river Ter, see duce Cambodia's temperatures (25 °C) than Alaska's (0 °C–16 °C), and to the Materials and Methods), we can assume that the studied sediment microbiota different methodologies used, even if initial spiking doses were like ours. was analogous to that in the real, streambed biofilms. Overall, our results in- We suggest that abiotic factors such as UV light with the associated variation dicate that the conditions, both abiotic and biotic, in the artificial streams in water temperature and/or the water movement, which were not incorpo- were comparable to those existing in surface water from the area of Spain rated in previous studies (Horm et al., 2012; Ramey et al., 2022b) but were from which water and sediment was collected and where AIV may persist. analysed in ours, could be related to our short viral persistence. In fact, UV Viral RNA genomic copies throughout the experiment were determined light, at a specific wavelength, is used as a biosafety technique to inactivate in water and sediment samples by rRT-PCR. It is worth mentioning that we AIV in routine laboratory work (Nishisaka-Nonaka et al., 2018). Furthermore, did not concentrate water samples since our goal was to obtain the most re- infectious viruses were not protected inside a tube as in virus-in-a-tube exper- alistic results. Genomic copies of the spiked LPAIV were detected both in iments. For these reasons, PAR cycles could have greatly influenced the re- water and sediment samples until the end of the experiment (14 days), sug- sults. We suggest that northern breeding areas, with very few hours of light gesting that the use of environmental samples for viral RNA detection could during winter and an incidence angle that can make the radiation less pene- be a good surveillance method for the early detection of viral RNA from trating, could reduce the effect of this factor. We also hypothesize that faeces AIVs, as was previously suggested but not to reveal actual infectious AIV or feathers could protect viral particles against UV light. In any case, further virus (Coombe et al., 2021; Hood et al., 2021; Pepin et al., 2019). However, studies need to be done to confirm these hypotheses. fewer genomic copies were found at day 0 than expected, a phenomenon No infectious LPAIV was recovered from any of the sediment samples, probably related to viral aggregation after the addition of LPAIV to water suggesting that sediments did not play a major role in viral persistence in at different pH and conductance levels (Floyd and Sharp, 1977, 1978; our study. However, several studies have described that sediments can act Kahler et al., 2016; Pradhan et al., 2022). Significant differences in genomic as AIV reservoirs, although viral infectivity was not evaluated (Densmore copies in water were observed between streams at different temperature et al., 2017; Himsworth et al., 2020; Lang et al., 2008) or a gem carrier conditions regardless of the sediment condition, but not between streams was used (Nazir et al., 2011). In fact, one study tried to isolate infectious with or without sediments at the same temperature condition. This observa- virus from sediment samples and was successful only at high infectious tion suggests that, in our study, the presence of sediments —and therefore doses (≥100 plaque-forming units per 200 μl), linking such low recovery of associated biotic factors— played a less important role in the degradation rate from sediment samples to inhibition, disruption of viral particles, or or retention of viral RNA than the temperature. These results could be asso- viral attachment to the sediment (Numberger et al., 2019). ciated with the absence of organisms capable of filtering or feeding LPAIV Our study was performed in artificial streams mimicking the environ- in our artificial streams (Ma et al., 2021; Meixell et al., 2013; Root et al., mental conditions of the waterfowl habitat in a Southwestern European re- 2020). Also, water movement (even if minimal) could be the reason for gion with a Mediterranean climate (Catalonia, Spain) during the winter- the low sediment retention. However, a significant combined effect of tem- spring period. In this specific region and conditions, we can find AIV sus- perature and sediments in viral decay was observed, with higher viral mo- ceptible species, such as the grey heron (Ardea cinerea), the yellow-legged lecular signal in colder streams without sediments. Furthermore, there was gull (Larus michahellis), and the mallard duck (Anas platyrhynchos) among a reduction in the difference of RNA amplification signal between water others (Museu del Ter, 2022). Indeed, outbreaks of HPAIV in waterfowl samples and sediment samples over time, with almost no differences in have already been detected in recent years. For example, in a wetland the last days. These results suggest some degree of viral sorption in sediments. area from Catalonia (Parc Natural dels Aiguamolls de l'Empordà, Girona, In contrast, and as expected, the temperature had a clear significant effect on Spain), which is close to the Ter River, HPAIV has been detected in a viral detection in water samples, with significantly higher amounts of gen am- white stork (Ciconia ciconia) during the 2016–2017 season, and in a white plification signal detected in the colder streams. The favouring effect of cold- stork and a greylag goose (Anser anser) during the 2020–2021 season. water temperature in LPAIV persistence is widely known (Brown et al., 2009; More recently, seven positive wild birds were detected in wetlands in Cata- Dalziel et al., 2016; Stallknecht et al., 1990a, 1990b). The lack of significant lonia and 38 wild birds all around Spain during the 2021–2022 winter mi- differences in viral molecular signals in sediment samples between streams gration period (Ministerio de Agricultura, 2022). Our experimental results at different temperatures could be related to a protective effect of the sedi- corroborate that waterfowl habitats in these mild-temperature latitudes ments against abiotic factors, such as temperature. can still provide the right conditions for bird-to-bird (faecal-oral) transmis- The ultimate objective of our study was to analyse viral infectivity over sion of AIVs among waterfowl, both migratory and resident, that normally time to determine the infectious capacity of LPAIV in Mediterranean water- aggregate in a specific area for a short period, as seen in the recent out- fowl habitats. For that purpose, water and sediment samples were inocu- breaks with numerous AIV-positive wild birds from the same location. lated in ECE to determine their infectivity. The detection of viral RNA by Even if more experiments need to be done, with different water and sedi- rRT-PCR did not associate with the recovery of infectious particles in ments sources or with different AIV strains, our results seem to suggest water samples. Even though viral isolation was successful from all water that interannual persistence of LPAIV in water ecosystems is unlikely in samples at 0 days post-spike (10 min after spike) and from six streams on such Mediterranean climate. This underscores direct or almost direct day 1, isolation in ECE was only successful in one stream at 2 days post- faecal-oral transmission in contrast to environmental transmission spike. No significant differences in viral isolation results were observed be- (i.e., without the presence of infected birds), due to the low persistence tween streams differing in water temperature and presence of sediment, times found. However, these results could differ significantly in other lati- probably due to the rapid decrease in viral infectivity observed in all tudes with environmental conditions more favourable for AIV persistence. streams. Surprisingly, we found relatively short viral persistence compared In fact, studies reproducing Alaska's conditions suggest that its water eco- to virus-in-a-tube studies, even though our initial spiking dose was close to systems play a crucial role in LPAIV transmission among migratory water- other studies (Brown et al., 2009). Consistently, we also found relatively fowl, as they are a breeding place for these birds (Ramey et al., 2020, 7 A. Perlas et al. Science of the Total Environment 863 (2023) 160902 2022b). This highlights a paradox of our study, while it better approximates Blagodatski, A., Trutneva, K., Glazova, O., Mityaeva, O., Shevkova, L., Kegeles, E., Onyanov, N., Fede, K., Maznina, A., Khavina, E., Yeo, S.-J., Park, H., Volchkov, P., 2021. Avian in- real conditions in the field, it also narrows the parameters to which the re- fluenza in wild birds and poultry: dissemination pathways, monitoring methods, and sults can be applied, here restricted to some relevant conditions mimicking virus ecology. Pathogens 10, 630. https://doi.org/10.3390/pathogens10050630. a specific region and season. Furthermore, our study did not consider the Brown, J.D., Goekjian, G., Poulson, R., Valeika, S., Stallknecht, D.E., 2009. Avian influenza virus in water: infectivity is dependent on pH, salinity and temperature. Vet. 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TCID50 50 % tissue culture infection dose Dunn, O.J., 1961. Multiple comparisons among means. J. Am. Stat. Assoc. 56, 52–64. https:// ECE embryonated chicken eggs doi.org/10.1080/01621459.1961.10482090. PAR photosynthetic active radiation European Food Safety Authority, 2022. Avian influenza overview March – June 2022. EFSA J. 20, 7415. https://doi.org/10.2903/J.EFSA.2022.7415. PCoA Principal Coordinates Analysis Floyd, R., Sharp, D.G., 1977. Aggregation of poliovirus and reovirus by dilution in water. rRT-PCR real-time RT-PCR Appl. Environ. Microbiol. 33, 159–167. https://doi.org/10.1128/aem.33.1.159-167. 1977. Floyd, R., Sharp, D.G., 1978. Viral aggregation: effects of salts on the aggregation of poliovirus Supplementary data to this article can be found online at https://doi. and reovirus at low pH. Appl. Environ. Microbiol. 35, 1084–1094. https://doi.org/10. org/10.1016/j.scitotenv.2022.160902. 1128/aem.35.6.1084-1094.1978. Fourment, M., Darling, A.E., Holmes, E.C., 2017. The impact of migratory flyways on the spread of avian influenza virus in North America. BMC Evol. Biol. 17, 118. https://doi. Funding org/10.1186/s12862-017-0965-4. Guan, J., Chan, M., Grenier, C., Wilkie, D.C., Brooks, B.W., Spencer, J.L., 2009. Survival of This work was supported by the coordinated project RTA2015-00088- avian influenza and Newcastle disease viruses in compost and at ambient temperatures based on virus isolation and real-time reverse transcriptase PCR. Avian Dis. 53, 26–33. C03-03 of the Instituto Nacional de Investigación y Tecnologı́a Agraria y https://doi.org/10.1637/8381-062008-Reg.1. Alimentaria (INIA), by the coordinated project PID2020-114060RR-C33- Hénaux, V., Samuel, M.D., Dusek, R.J., Fleskes, J.P., Ip, H.S., 2012. Presence of avian influ- INFLUOMA, entitled “Unravelling the molecular mechanisms of avian in- enza viruses in waterfowl and wetlands during summer 2010 in California: are resident birds a potential Reservoir? PLoS One 7, e31471. https://doi.org/10.1371/journal. fluenza virus infection outcome in the avian host by using a multi-omics ap- pone.0031471. proach” of the funder Spanish Ministerio de Ciencia e Innovación, and by Hierholzer, J.C., Killington, R.A., 1996. Virus isolation and quantitation. Virology Methods the 2019 grant for research and conservation from the Fundació Barcelona Manual. Elsevier, pp. 25–46 https://doi.org/10.1016/B978-012465330-6/50003-8. Zoo. KB was funded by the Generalitat de Catalunya, Spain, Agència de Himsworth, C.G., Duan, J., Prystajecky, N., Coombe, M., Baticados, W., Jassem, A.N., Tang, P., Sanders, E., Hsiao, W., 2020. Targeted resequencing of wetland sediment as a tool for Gestió d'Ajuts Universitaris i de Recerca, Beatriu de Pinós postdoctoral fel- avian influenza virus surveillance. J. Wildl. Dis. 56, 397. https://doi.org/10.7589/ lowship (2017 BP 00105). MC was funded by the Ministry of Economy and 2019-05-135. Competitiveness, Spain, program Ramón y Cajal (grant RyC-2015-17154). Hinshaw, V.S., Webster, R.G., Turner, B., 1979. Water-bone transmission of influenza A vi- ruses? Intervirology 11, 66–68. https://doi.org/10.1159/000149014. Hinshaw, V.S., Webster, R.G., Turner, B., 1980. The perpetuation of orthomyxoviruses and CRediT authorship contribution statement paramyxoviruses in Canadian waterfowl. Can. J. Microbiol. 26, 622–629. https://doi. org/10.1139/m80-108. Hood, G., Roche, X., Brioudes, A., von Dobschuetz, S., Fasina, F.O., Kalpravidh, W., The project was conceived, and funding was obtained by MN, AR, and Makonnen, Y., Lubroth, J., Sims, L., 2021. A literature review of the use of environmental NM. The experiment was designed by AP, KB, VA, MC, FXA, and NM. The sampling in the surveillance of avian influenza viruses. Transbound. Emerg. Dis. 68, experiment was performed by AP, VA, and KB. Samples were collected 110–126. https://doi.org/10.1111/tbed.13633. Horm, V.S., Gutiérrez, R.A., Nicholls, J.M., Buchy, P., 2012. Highly pathogenic influenza A and processed by AP, KB, VA, and RV. LPG has done the statistical analysis. (H5N1) virus survival in complex artificial aquatic biotopes. PLoS One 7, e34160. CMB has done the microbiome analysis of sediments. Figures were pre- https://doi.org/10.1371/journal.pone.0034160. pared by LPG, AP, and KB. The manuscript was drafted by AP with correc- Ito, T., Okazaki, K., Kawaoka, Y., Takada, A., Webster, R.G., Kida, H., 1995. Perpetuation of influenza A viruses in Alaskan waterfowl reservoirs. Arch. Virol. 140, 1163–1172. tions from MC, KB, VA, FXA, MN, AR, LPG, and NM. All authors contributed https://doi.org/10.1007/BF01322743. to the article and approved the submitted version. Kahler, A.M., Cromeans, T.L., Metcalfe, M.G., Humphrey, C.D., Hill, V.R., 2016. Aggregation of adenovirus 2 in source water and impacts on disinfection by chlorine. Food Environ. Virol. 8, 148–155. https://doi.org/10.1007/s12560-016-9232-x. Data availability Karunakaran, A.C., Murugkar, H.v., Kumar, M., Nagarajan, S., Tosh, C., Pathak, A., Mekhemadhom Rajendrakumar, A., Agarwal, R.K., 2019. Survivability of highly patho- Data will be made available on request. genic avian influenza virus (H5N1) in naturally preened duck feathers at different tem- peratures. Transbound Emerg. Dis. 66, 1306–1313. https://doi.org/10.1111/tbed. 13148. Declaration of competing interest Keeler, S.P., Lebarbenchon, C., Stallknecht, D.E., 2013. Strain-related variation in the persis- tence of influenza A virus in three types of water: distilled water, filtered surface water, and intact surface water. Virol. J. 10, 13. https://doi.org/10.1186/1743-422X-10-13. The authors declare that they have no known competing financial interests Keeler, S.P., Dalton, M.S., Cressler, A.M., Berghaus, R.D., Stallknecht, D.E., 2014. 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