Harnessing Alpine Lake Bacteria for the Development of Synthetic Communities with Broad-Spectrum Antiviral Activity

Harnessing Alpine Lake Bacteria for the Development of Synthetic Communities with Broad-Spectrum Antiviral Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Harnessing Alpine Lake Bacteria for the Development of Synthetic Communities with Broad-Spectrum Antiviral Activity Anna Carratala, Laura Daniela Morales, Jessica Aubouy, Shannon David, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8367630/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In aquatic ecosystems, phagotrophic protists and bacteriophages are major biological factors shaping bacterial populations. However, the extreme environmental conditions and low nutrient concentrations in alpine lakes limit the abundance of protists. Thus, bacteriophages may represent the main biological factor shaping bacterial communities in these ecosystems. We hypothesized that alpine lake bacteria populations harbor diverse antiviral strategies against bacteriophages that could be used to fight against other viral pathogens. To evaluate the presence of bacteria with broad-spectrum antiviral activity in high altitude Swiss lakes, we developed a sampling campaign in 24 alpine lakes in Switzerland. We assessed the decay of two human viruses (echovirus 11 and influenza A virus) in lakewater and characterized the microbial abundance and metabolic diversity in the collected samples. In addition, we obtained a collection of 223 bacterial isolates and selected bacteriophage resistant bacteria to build synthetic communities with enhanced antiviral activity against the two tested human viruses, and one plant virus of commercial significance (Potato Virus X). This confirmed our hypothesis and drew for the first time a relation between bacteriophage resistance and viral inactivation. Our findings emphasize the importance of alpine lakes as hotspots of microbial diversity with unexplored biotechnological applications. Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Antimicrobials/Antiviral agents Alpine lakes microbial communities viral inactivation bacteriophage resistance synthetic communities Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Alpine lakes are unique ecosystems with extreme environmental conditions: low nutrient concentrations, low temperatures, ice cover during the wintertime, high UV radiation in summertime, short summertime and drastic light changes between the ice-covered and ice-free season [ 1 ]. Despite these harsh conditions, alpine lakes host diverse bacterial communities that play central roles in ecosystem functioning [ 2 ]. In aquatic ecosystems, phagotrophic protists and bacteriophages (also known as phages) are major biological factors shaping bacterial populations [ 3 ]. However, in alpine lakes bacterial communities are less influenced by protist grazing compared to low-land lakes due to the limited richness and diversity of the latest compared to non-alpine regions [ 4 ]. Since bacteriophages are generally highly resistant to extreme environmental conditions, they likely represent the main biological factor shaping bacterial communities in alpine aquatic ecosystems [ 5 ]. Bacteriophages are the most abundant microorganisms in aquatic systems [ 6 ], [ 7 ]. They lead to 20–40% of bacteria mortality and exert a huge selective pressure on their bacteria hosts. Bacteria-viral interactions in this context, are a constant and dynamic back and forth battle [ 8 ]. Bacteria adapt their immune systems and develop mechanisms to resist bacteriophage infections, and bacteriophages frequently mutate to counteract bacteria adaptations [ 9 ], [ 10 ]. Bacteria have developed numerous mechanisms against bacteriophage infections and have clustered them in so-called genomic “defense islands” across their genomes [ 10 ], [ 11 ]. These defense mechanisms range from the well characterized bacterial CRISPR/Cas system shared by multiple bacterial species to the most recently discovered Bacteriophage Exclusion (BREX) system in Bacillus subtilis [ 12 ], [ 13 ]. They involve phage DNA silencing through small RNA molecules, DNA degradation by restriction enzymes, bacterial programmed cell dead to avoid viral proliferation, and the production of chemical antiviral molecules like viperins [ 14 ], [ 15 ], [ 16 ]. Noteworthy, bacterial and human viperins have structural and functional similarities. While bacteria use these molecules to fight bacteriophage infections, humans seem to also use them to fight virus infections [ 17 ]. Bacterial antiviral molecules and their mechanisms can be potentially exploited to our advantage against viral pathogens of multiple hosts. Climate change, urbanization and mobility increase the risk of infectious disease emergence [ 18 ]. Viral pathogens, in particular, have high mutation rates, large population numbers, short generation times and rapidly become resistant to antiviral drugs and disinfectants [ 19 ], [ 20 ], [ 21 ]. There is a permanent lack of antiviral molecules effective against rapidly evolving and emerging human, animal and plant pathogens [ 22 ]. Aquatic ecosystems and, in general, extreme environments are potentially a rich source of microbial secondary metabolites with antimicrobial and antiviral activity. In the early 70s and 80s, Ward et al. and Cliver et al . described the antiviral activity of fresh water microorganisms and their proteases against Poliovirus, Echovirus-12, Rotavirus SA-11 and Coxsackievirus B5 [ 23 ], [ 24 ]. More recently, our lab further characterized lakewater bacterial isolates and their proteolytic activity. We found a correlation between viral inactivation and extracellular metalloprotease production [ 25 ]. As well, bacterial intra- and extra- cellular polysaccharides have shown antiviral activity against diverse viruses [ 26 ]. However, the potential of high-altitude aquatic environments as hotspots for the discovery of new antiviral compounds of bacterial origin remains largely unexplored. Due to the extreme environmental conditions and the high abundance of bacteriophages in alpine lakes, we hypothesized that bacteria in these environments have developed complex and diverse antiviral mechanisms against bacteriophages that likely contribute to the inactivation of multiple viruses. Thus, we aimed to explore broad-spectrum viral inactivation by bacteria communities and isolates from Swiss Alpine lakes. In Switzerland, 60% of the land is covered by the Alps with over 1,500 lakes present in these high-altitude regions. We conducted sampling campaigns in 24 alpine lakes located above the tree line in the Swiss Alps and investigated the inactivation of two human viruses (Influenza A virus and Echovirus 11) in water from selected lakes. In addition, we identified and selected bacteriophage resistant isolates to build two synthetic communities with increased broad-spectrum antiviral activity against the two human viruses and two plant viruses (Potato Virus X and Pepino Mosaic Virus). Our findings highlight high-altitude lakes as unexplored reservoirs of microbial and metabolic diversity. They offer promising opportunities for the discovery of novel broad-spectrum antiviral mechanisms and bioactive compounds, and for the design of synthetic microbial consortia with potential applications in the biocontrol of viral pathogens. RESULTS Microbial abundances and metabolic diversity in Swiss alpine lakes. A total of 24 alpine lakes were sampled in the Swiss Alps from eight distinct regions in the canton of Valais and one region in the Bernese Oberland (Fig. 1A). The abundance of bacteria, eukaryotes and tailed bacteriophages was determined in each lake to identify those showing a higher ratio of bacteriophages to bacteria and eukaryotes. We found great variability of microbial abundances between samples (Fig. 1B). Bacteria concentrations (16S) ranged from 300 genome copies/liter (gc/l) in Lac Bleu (Arolla) to more than 10 8 gc/l in Spilsee (Bellwald). Eukaryotic DNA (18S) ranged from 100 gc/l in Lac Bleu to 10 9 gc/l in Stellisee and Leisee (Zermatt). The concentration of tailed bacteriophages (g23) varied between < 10 gc/l in Small Lac de Vaux (Verbier) to 10 7 gc/l in Lengsee (Bellwald). We calculated the ratio of tailed bacteriophages over the sum of bacteria and eukaryotes concentration (measured in gc/l) in each lake, shown in Fig. 1C. At the time of sampling, few of the lakes had particularly high ratios of tailed bacteriophages per bacteria and protists. Lakes Mossjesee, Guggisee, Marjelensee and Salanfe had ratios of 0.12, 0.15, 0.10 and 0.69, respectively, compared to an average ratio of 0.01 in the other 20 lakes. The ratio of tailed bacteriophages to bacteria and eukaryotes was 60-times higher in Lake Salanfe compared to the average ratio of the rest of the lakes. To characterize functional traits of the microbial communities in the sampled lakes that could provide insights into their antiviral activity, we assessed carbon metabolism using Biolog EcoPlates. These plates contain 31 compounds classified as carbohydrates, amino acids, carboxylic acids, amines or alcohols (Fig. 2 ). Based on the number of metabolized carbon sources, we calculated Shannon diversity indexes to determine the metabolic diversities per each lake (Table S3). Lakes Bleu, Petit Aletsch 1 and Schwarzsee had the highest metabolic diversity (Shannon indexes > 3). Microbial communities present in these three lakes metabolized substrates in all six categories (carbohydrates, amino acids, carboxylic acids, amines and alcohols). In contrast, lakes Stellisee and Leisee were the least metabolically diverse (Shannon indexes < 1), as microbial communities from these lakes were unable to metabolize most of the substrates present. Microbial communities in alpine lakes show antiviral activity against human viruses. We have previously reported that bacterial communities from Lake Geneva inactivate human enteric viruses [ 25 ]. To confirm whether bacteria in Swiss alpine lakes also show antiviral activity against human viruses, we conducted inactivation experiments using two structurally different viruses: echovirus 11 (E11) and influenza A virus (IAV). For these assays, we selected four lakes with high proportion of tailed bacteriophages per bacteria and eukaryotes (Guggisee, Mossjesee, Marjelensee and Salanfe), and four lakes with low proportion (Lac Bleu, Schwartzsee, Small Lac de Vaux and Mittelsee) to evaluate whether elevated phage-to-host ratios are indicative of increased antiviral activity. We focused on the antiviral activity of bacteria present in the water. Therefore, the water used for these assays was filtered through a 0.8 µm membrane to remove protists, also known to inactivate human viruses [ 27 ]. We observed higher E11 inactivation compared to IAV in all the tested lakes (Fig. 3 ). E11 was inactivated > 1 log10 in five out of eight of the lakes (Fig. 3 A) while IAV was inactivated around 1 log10 in all lakes (Fig. 3 B). Out of the eight lakes, lake Mossjesee, an artificial reservoir resulting from glacial meltwater, had the greatest inactivation against both viruses (2.17 average log10 decay for E11; 1.03 log10 decay for IAV) over 7 days of incubation. E11 inactivation in water from this lake was significantly higher compared to lakes Salanfe and Mittelsee (P = 0.0387 compared to Salanfe and P = 0.0199 compared to Mittelsee). We did not observe a correlation between viral inactivation and the proportions of bacteriophages per bacteria and eukaryotes. However, two out of four of the chosen lakes with increased ratios of tailed bacteriophages (Mossjeesee and Marjelensee) showed the greatest inactivation against E11. Similarly, we did not identify any correlation between antiviral activity and lake metabolic diversities (Figure S1 ). Alpine lakes host bacteria with low susceptibility to bacteriophage infections. To identify and characterize specific bacteria with potential antiviral activity, we isolated single bacteria species from each lake regardless of the phage-to-host ratios (Fig. 4 A). In total, we obtained 223 different bacteria isolates across the 24 alpine lakes (Fig. 4 B). The number of bacterial isolates obtained varied per lake, ranging from 4 to 19. Lakes Guggisee and Oeschinensee yielded the highest number of isolates (19 and 16 cultures respectively), together accounting for approximately 15% of the total isolates. Lakes Stellisee and Wirbulsee yielded the lowest numbers of bacterial isolates (only 4 isolates from each of both lakes). Interestingly, Lakes Guggisee and Oeschinensee showed the lowest bacterial abundance (Fig. 1B), while lakes Stellisee and Wirbulsee were within the lakes with the greatest bacterial abundance. We hypothesized that bacterial isolates with lower susceptibility to bacteriophage infections would show increased antiviral activity against human and plant viruses. To identify bacteria with decreased susceptibility to bacteriophage infections and test our hypothesis, we created a “Phage Mix” by pooling virus fractions (0.22 µm filtered lakewater) from the surface water samples collected in the 24 lakes. This generated a phage cocktail that included a large abundance of diverse bacteriophages potentially capable of infecting most of the bacteria isolates (Fig. 4 A). All 223 isolates were grown independently and spiked with the alpine “Phage Mix”. After co-incubating bacteria isolates and phage mix, we compared the final concentration of bacteriophages to the initial concentration in the inoculum (Fig. 4 C). Most of the isolates (148 specifically) showed susceptibility to the Phage Mix as indicated by an increase in the bacteriophages’ titer after the incubation period. Conversely, we identified 75 isolates with decreased susceptibility indicated by a decrease in the concentration of bacteriophages after co-incubations. Lake Oeschinsee (9 isolates), Lac de Vaux (8 isolates), Leisee (8 isolates) and Lac de Salanfe (8 isolates) had the greatest number of phage resistant isolates. Interestingly, these lakes had the lowest metabolic diversity (Shannon 0.98 to 1.69) compared to other lakes. In terms of bacteriophage to bacteria and eukaryotes ratios, we did not observe any relation between bacteriophage resistant isolates and the calculated ratios. However, we noticed that Lac de Salanfe had the highest calculated ratios amongst all sampled lakes (Fig. 1C). Phage-resistant synthetic communities inactivate human and plant viruses. Previous studies performed in our laboratory showed greater viral inactivation in lakewater samples compared to single bacteria cultures, and a correlation between increased microbial diversity and enhanced inactivation [ 25 ], [ 28 ]. Therefore, we aimed to design microbial synthetic communities using isolates classified as “bacteriophage-resistant” and assess their antiviral activity against viral pathogens with different structures and hosts. We created two synthetic communities of 25 members each (communities 1 and 3 – Fig. 5 ). In parallel, we created a control community using 25 bacteriophage-susceptible bacteria (Control community). The three communities were spiked with either E11 or IAV and incubated for 4 days. After co-incubation, the synthetic communities of bacteriophage-resistant bacteria (communities 1 and 3) had enhanced antiviral activity compared to the bacteriophage-susceptible (control) community (Fig. 5 A). In both cases, we observed significantly (P value < 0.01) more inactivation between the communities (1 and 3) and the control. In addition, we observed that E11 had greater inactivation compared to IAV (E11 ~ 3 log10; IAV ~ 1 log10, in four days). Noteworthy, the synthetic communities showed greater inactivation against E11 as compared to the inactivation we previously observed in lakewater (~ 2 log10 decay in seven days). We determined the bacteria composition of the three communities (1, 3 and control) before viral inoculation using 16S amplicon sequencing (Fig. 5 B). We also confirmed the identity of individual bacteria using Sanger sequencing. We found high relative abundance of genera Brevundimonas , Serratia, Pseudomonas and Stenotrophomonas species across the three communities (control and communities 1 and 3). On the other hand, genera Alcaligenes and Microbacterium species were shared between communities 1 and 3 but were not present in the control community. We also identified bacteria uniquely found in each community. Bacillus sp. and Staphylococcus sp. were uniquely present in community 1, and Allorrhizobium species were uniquely found and abundant in community 3. Lastly, to identify some mechanisms behind the observed antiviral activity, we assessed protease secretion by individual bacteria from each community. Bacteria isolates were individually grown in skim milk agar (Figure S2A) and proteolytic activity was determined by measuring degradation halos around individual colonies. We did not observe a difference between the proteolytic activity of the community members present on each of the phage resistant and susceptible communities (Figure S2B). Since our previous results indicated that the developed synthetic communities exhibit antiviral activity against bacteriophages and human viruses, we hypothesized that they may possess broad-spectrum antiviral properties. To test this hypothesis, we performed viral inactivation assays against Potato Virus X (PVX) and Pepino Mosaic Virus (PepMV) using synthetic community 3 (Fig. 6 ). For these assays, this synthetic community was spiked with either plant virus, and then incubated at room temperature for four days. In parallel, both viruses were also diluted in PBS and incubated under the same conditions. These suspensions were used to mechanically inoculate tobacco plants and the infectivity of both viruses was assessed 19 days post-inoculation (dpi). We observed a decrease of PVX infectivity from 79% of plants infected with PVX alone to just 1% following the incubation with community 3 (Fig. 6 A and B). In contrast, PepMV showed no infectivity reduction in the presence of community 3. (Fig. 6 D and E). These results suggest that community 3 exhibits broad-spectrum antiviral activity but also displays some specificity in the extent of viral inactivation across different viruses. As controls, we included 10 untreated plants (five plants per virus) to evaluate crossed contamination in the greenhouse (Plant controls - Fig. 6 C and F) and observed one plant control positive for PVX. We also inoculated plants with community 3 to assess bacteria pathogenesis against plants and observed no pathogenicity. These results confirmed our working hypothesis and provide for the first time a link between resistance against phage infections and viral inactivation. DISCUSSION In this study we characterized broad-spectrum viral inactivation by bacteria communities and isolates from 24 Swiss Alpine Lakes. Across these lakes, we found heterogenous microbial abundances, diversity of carbon utilization and antiviral activity against two human viruses (E11 and IAV). Given the increased abundance of bacteriophages in alpine aquatic ecosystems, we hypothesized that bacteria populations in alpine lakes may have evolved antiviral mechanisms against bacteriophages that may be potentially harmful to other viral pathogens. To test our hypothesis, we generated a collection of alpine lake bacteria and used those classified as “bacteriophage-resistant” to assemble two synthetic communities that significantly inactivated E11, IAV and one plant virus (PVX) compared to a community built from bacteriophage-susceptible isolates. Although our research drew a relation between bacteriophage resistance and broad-spectrum antiviral activity, further research is necessary to identify and characterize shared molecular mechanisms. Our results show that microbial abundances differed from one lake to another suggesting that abundances are influenced by environmental and/or biological factors acting at very local scale. Previous research showed that high altitude lakes have bacteria abundances and diversities greatly dependent on local characteristics of each lake (e.g. ionic concentrations, nutrients, temperature and pH) [ 29 ]. Although, identifying and characterizing the influence of these specific factors on microbial abundances was beyond the scope of this work, we found enhanced antiviral activity in bacterial fractions of waters collected from some lakes with high proportion of phages compared to other microorganisms. These findings are consistent with previous observations on the inactivation of human pathogens in freshwater samples were protists were also removed [ 27 ], [ 30 ]. To further investigate the relationship between bacteriophage resistance and antiviral activity, we created a collection of 223 bacteria isolates from the 24 high-altitude Swiss lakes and assessed their susceptibility to a bacteriophage cocktail. We identified potential bacteriophage-resistance in 75 out of 223 isolates. The highest percentage of phage resistant bacteria were identified Lake Oeschinsee, Lac de Vaux, Leisee and Lac de Salanfe. Interestingly, Oeschinensee is the lake at the lowest altitude (1580 m), potentially experiencing the least extreme environmental conditions, and Lac Salanfe had the greatest phage to bacteria and eukaryotes ratio. Beyond these observations, we did not find any correlation between the microbial abundances or metabolic profile of these lakes and bacteriophage resistance. Our findings suggest that limited nutrients and high bacteriophage concentrations are not the only characteristics associated to bacteriophage resistance in bacteria populations. It is important to acknowledge that some methodological choices –including media selection for bacterial isolation, possible absence of phages capable of infecting certain isolates in the Phage Mix [ 7 ], [ 31 ] and reliance on g23 qPCR as an indicator of total phage concentration –may have influenced our results. Nevertheless, the identified bacterial subset represented promising candidates for further characterization and was used to develop synthetic communities with enhanced viral inactivation activity. Noteworthy, using these candidates we built two synthetic bacterial communities with enhanced viral inactivation. We found that communities composed of bacteriophage-resistant isolates significantly inactivated E11 and IAV compared to a composed of bacteriophage-susceptible bacteria. This confirmed our working hypothesis and demonstrated for the first time a potential link between phage resistance and viral inactivation is drawn. We tested one of these communities against two plant viruses and found antiviral activity against only one virus (PVX), suggesting that bacterial viral inactivation is partially selective. Identification of taxa present in the synthetic communities revealed bacteria genera previously reported to possess antiviral activity. For example, a protease produced by Serratia marcescens protected tobacco plants against Tobacco Mosaic Virus (TMV) [ 32 ], and secondary metabolites from Bacillus subtilis exhibit antiviral activity against RNA viruses and a pseudorabies virus (PVR) [ 33 ], [ 34 ]. While these antiviral compounds have been described in single isolates, their production and specific contribution to virus inactivation in mixed microbial communities remain unknown. Viral inactivation in freshwater was first described by Cliver et al. (1972) and Ward et al. (1986), who showed bacterial antiviral activity against enteroviruses and concluded that viral capsid proteins can be cleaved by proteolytic bacterial enzymes [ 24 ], [ 35 ]. Other studies on the fish pathogen Koi-Herpesvirus have shown significantly faster inactivation with bacteria present compared to bacteria free samples [ 36 ], [ 37 ]. They also suggest that proteases were likely responsible for the observed inactivation [ 36 ]. Here, we were unable to find a correlation between viral inactivation and proteolytic activity of the community members. This suggests that either our assays did not capture the relevant proteases associated to viral inactivation or that additional antibacterial mechanisms—chemical, enzymatic, or structural—contribute to viral decay. Further work is needed to elucidate the specific mechanisms underlying viral inactivation by the enhanced synthetic communities described here and to characterize the contribution of each individual species in the community. In summary, we developed and applied an innovative strategy to construct two synthetic bacterial communities with enhanced broad-spectrum antiviral activity from environmental samples collected in 24 Swiss alpine lakes. We further demonstrated that alpine lakes exposed to extreme environmental conditions harbor bacterial populations with antiviral properties. This was consistent with our previous observations in lowland lakes such as Lake Geneva. Notably, our results provide the first evidence linking resistance to indigenous environmental bacteriophages with inactivation of viral pathogens infecting diverse hosts, highlighting potential avenues for future biotechnological applications. Future research will aim to elucidate the molecular mechanisms underlying the broad-spectrum viral inactivation reported here. MATERIALS AND METHODS Alpine Lake sampling and sample processing. The lakes chosen for this study were high altitude lakes located in the Swiss Alps above 2000 meters of altitude. All samples were collected between September and November 2021. The list of lakes sampled in this project as well as their sampling date and altitude are shown in Table S1 . During each sampling campaign, two liters of surface water were collected using plastic containers made of polycarbonate and a telescopic sampling pole. Samples were kept at 4°C until further processing. Once in the laboratory, one liter of lakewater was filtered through 0.45 µm filters (Millipore™) using a conventional vacuum pump to retain protists, bacteria and bacteriophages (associated with the bacteria and organic matter). Water samples with higher turbidity and rich in suspended particles were first pre-filtered through 8.0 µm filters. The 0.45 µm filters were transferred to Bead Tubes of the DNeasy PowerWater kit (QIAgen, Germany). These tubes were frozen at -20°C until nucleic acid extractions were performed using the DNeasy PowerWater kit (QIAgen, Germany) following the manufacturer’s recommendations. Microbial abundance quantification in alpine lakes. Quantitative polymerase chain reaction (qPCR) assays were used to determine the abundance of picoeukaryotes, bacteria and bacteriophages targeting 18S, 16S and g23 genes, respectively [ 38 ], [ 39 ]. The 16S gene was targeted to quantify the abundance of bacteria using a Femto Bacterial DNA quantification kit (Zymo Research Corporation) following the manufacturer’s recommendations. The g23 gene encodes for a capsid protein of tailed bacteriophages and was used as a proxy of bacteriophage abundance given that no universal gene is available for viruses[ 40 ]. Table S2, shows the sequences of all the primers used in this study. Metabolic diversity of alpine lake communities. Biolog EcoPlates™ were used to characterize the metabolic profile of bacteria communities found in the studied lakes. Briefly, 1x of M9 minimal media (Sigma-Aldrich) was supplemented with 2mM of MgSO4 (PanReac AppliChem), 0.1 mM of CaCl2 (Thermo Scientific) and 1X of trace metals (Teknova). The content of each well of the EcoPlate™ was resuspended in 100 ul of supplemented M9 minimal. Then, 40 ul of lakewater were added on top of each well. Plates were incubated for 5 days at room temperature in the dark. Optical density of each well was measured at a wavelength of 590 nm using a microplate reader (BioTek – Synergy Mx). Each carbon source is present in triplicate in the EcoPlate™. Average optical density was reported per carbon source for each lake. To calculate metabolic diversity, compounds that yielded an optical density > 0.2 at 590 nm were used to calculate a Shannon index per lake using the following formula: H = − Σ (p i * ln(p i )) Here, H is the Shannon diversity index, Σ is the sum, p i is the proportion of compounds with signal > 0.2 nm and ln is the natural logarithm. Propagation of human and plant viruses. Echovirus 11 (E11, Gregory strain, ATCC VR737) and Influenza A (IAV) virus were cultured in the laboratory using Buffalo Green Monkey Kidney (BGMK) and Madin-Darby Canine Kidney (MDCK) cells, respectively. Stocks were generated by infecting sub-confluent monolayers of cells, following previously established protocols [ 27 ]. For E11 purification, viral particles were harvested from the infected cells through a series of freeze-thaw cycles. Subsequently, to remove cellular debris, the suspensions underwent centrifugation at 3000 × g for 5 minutes. Buffer exchange was performed into PBS using Amicon® Ultra filters in cycles of 4000 × g for 10 minutes until viral suspension was clear. For IAV purification, culture supernatant from the infected cells was cleared by centrifugation (2,500 × g, 10 min). Virus was concentrated and purified by pelleting through a 30% sucrose cushion at 112’400 × g in a SW31Ti rotor (Beckman) for 90 min at 4◦C. The resulting pellets were soaked overnight at 4°C in PBS (ThermoFisher, 18912014) and then fully resuspended by pipetting up and down. Pepino Mosaic Virus (PepMV) and Potato Virus X (PVX) were obtained from tobacco plants ( Nicotiana benthamiana and Nicotiana tabacum Xanthi, respectively ) mechanically inoculated with virus isolates from Agroscope collection in Changins (Nyon, Switzerland). Virus particles were semi-purified as previously described from symptomatic leaves stored at -20°C [ 41 ]. The abundance of particles was evaluated using a Tecnai G2 transmission electron microscope. The particles were diluted in 0.018 M phosphate-citrate buffer at pH 7 when required. All virus suspensions were aliquoted and stored at − 80°C until further use. Enumeration and quantification of human viruses. The concentration of infectious viruses was determined using a most probable number (MPN) infectivity assay for E11 and plaque assays for IAV. For MPN, 5 replicates per sample were used as described previously [ 20 ]. Plaque assays were performed using MDCK cells as described previously [ 42 ]. For quantification of human viruses, qPCR targeting Influenza A Matrix or Echovirus 11 viral protein 1 were performed (Table S2). For both viruses, RNA extractions from samples were performed, using the QIAamp Viral RNA Mini extraction kit (52906, QIAGEN GmbH, Hilden, NW, Germany) according to manufacturer’s instructions. For E11 quantification, RT-qPCR was performed on a Mic qPCR Cycler (Bio Molecular Systems) using the One Step SYBR® PrimeScript™ RT-PCR Kit (Takara). Each reaction (20 µL) contained 3 µL of template, 10 µL of 2× One Step SYBR RT-PCR Buffer III, 0.4 µL of TaKaRa Ex Taq HS (5 U µL − 1), 0.4 µL of PrimeScript RT enzyme mix II, 250 nM of each primer and 5.2 µL of water. The thermocycling conditions were as follows: RT at 42°C for 5 min, 10 s at 95°C, followed by 40 cycles of 95°C for 5 s, 52°C for 20 s and 60°C for 30 s. Each sample was run once, and the Cq values were determined using the micPCR software (v2.10.0; Bio Molecular Systems)[ 43 ]. For IAV quantification, extracted nucleic acids were eluted in elution buffer, and stored at − 20◦C until analysis. Reverse-transcription and amplification were performed using the One Step PrimeScript ™ RT-PCR Kit (RR064A, Takara Bio Inc., Kusatsu, JP-25, Japan) using primers targeting the Influenza A Matrix gene (Table S2). The RT-qPCR mix was composed of 7.5 µL of 2x One-Step SYBR RT-PCR Buffer, 0.3 µL of Takara Ex Taq HS (5 U/µL stock), 0.3 µL PrimeScript RT enzyme mix, 0.3 µL forward and reverse primers (10 µM stocks), and 3.3 µL RNase-free water, to which 3 µL of extracted RNA sample was added. The RT-qPCR was performed in a Mic Real-Time PCR System (Bio MolecularSystems, Upper Coomera, QLD, Australia) with the following parameters: 2 min at 50 ◦ C, 10 min at 95°C, 15 seconds at 95°C followed by 60 sec at 60°C for 40 cycles. A final step from 55°C to 95°C at 0.3°C/s was performed for the melting curve analysis. A Gblock gene fragment (Gblock SA, Ath, WHT, Belgium), as described in Olive et al. (2022), was used to create a standard curve for quantification over a range of 10 to 10 7 genomic copies per microliter (GC/µl)[ 44 ]. Inactivation of human viruses in lakewater. Inactivation experiments were conducted in lakewater filtered through 0.8 µm (Millipore™) (to remove heterotrophic protists) as described above. Specifically, 50 ml of filtered water were spiked with 100 µl of IAV and E11 to the same initial concentration (10 5 genome copies per ml). Then, 5 ml of spiked water was placed in 6-well plates in three replicates. The plates were incubated for 7 days at room temperature and 500 µl aliquots were taken on days 0, 3 and 7. These aliquots were frozen at -20°C until analyzed. Viral nucleic acids were extracted using the QIAamp Viral RNA Mini extraction kit (52906, QIAGEN GmbH, Hilden, NW, Germany) and virus concentrations were determined by qPCR throughout the experiment or by cell culturing as described above. Development of synthetic bacteria communities with antiviral activity. To develop synthetic bacteria communities with enhanced antiviral activity, we first aimed to identify specific bacteria species with antiviral properties in the lake water samples. To this end, we built a culture collection of isolates from all the sampled lakes. One liter of water from each lake was 0.45 𝜇m filtered. Filters were transferred to LB agar plates and incubated at room temperature for 72 hours. Then, single colonies were transferred to new LB plates. Serial transfers of isolated colonies into sterile LB plates were conducted until pure cultures were obtained. A total number of 223 pure bacterial isolates were obtained using this methodology. LB media was purchased from Invitrogen. Bacteria isolates were identified performing 16S rRNA gene sequencing using the primers 27 F and 786 R (Table S2). Samples were sent for conventional Sanger sequencing to Fasteris (Genesupport SA, Geneva, Switzerland). To identify which of the obtained isolates showed antiviral activity, we conducted phage susceptibility tests using the bacteriophage fraction obtained from each studied lake. Specifically, phage enrichments were conducted for each bacteria isolate obtained in this study (n = 223) following state-of-the-art protocols [ 45 ], [ 46 ]. The phage cocktail used to infect each bacteria isolate was a 1:1 mixture of all the control waters obtained from each lake which is called “Phage Mix” hereafter. This process was done to increase the chances for at least one phage type in the mix to be capable of infecting each bacteria isolate. To assess bacterial susceptibility to the “Phage Mix”, fresh cultures of each bacteria isolate were grown overnight in 5 ml of LB. Once bacteria cultures had an optical density at 600 nm around 0.6–0.8 (exponential phase), they were spiked with 1 ml of the phage mix. The tubes were then incubated at room temperature for 15 min and then gently agitated (50 rpm) for 2 more hours. Then, chloroform was added to the bacteria-phage co-cultures (hereafter named “Phage enrichments”) which were centrifuged for 10 minutes at 5000 rpm and filtered at 0.2 µm to remove all bacteria. This process was done for each bacteria isolate. To compare phage susceptibility of bacteria isolates, the phage titer yield in the “Phage Mix” and in each enrichment was determined by qPCR targeting the g23 gene [ 45 ], [ 46 ]. Bacteria cultures with phage concentrations equal or higher compared to the phage mix were considered “susceptible” and retained to build the control communities, while those yielding inferior concentrations were considered “resistant” and retained for the design of two different synthetic communities with enhanced antiviral properties. This approach assumes that resistant bacteria limit phage replication, so lower phage titers in the enrichment experiments reflect the bacteria’s ability to inhibit phage proliferation. Alpine synthetic communities against human and plant viruses. To confirm the enhanced antiviral properties of the obtained synthetic communities, three groups of 25 bacterial isolates (2 synthetic communities with enhanced activity and one control group) were mixed and used to assess their inactivation activity against E11 and IAV. Before the inactivation assays, isolates were independently grown in LB overnight at room temperature to avoid competition and were mixed in equal parts in a final volume of 50 ml. Once the bacteria isolates were mixed together, inactivation experiments against E11 and IAV were conducted as previously described. Two bacterial pools were composed of 25 unique bacterial isolates with decreased susceptibility to the “Phage Mix” and one control was composed of 25 bacteria isolates with increased phage susceptibility. Specifically, 50 ml of mixed bacteria isolates were diluted 100x using sterile lakewater and spiked with 100 µl of IAV and E11 to the same initial concentration (10 5 genome copies per ml). Then, 5 ml of spiked media with the isolates was placed in 6-well plates in five replicates. The plates were incubated for 4 days at room temperature. For inactivation assays against plant viruses, the most inactivating community against human viruses was selected. All the community members were grown as previously described and mixed the day of the assay. Previously purified PepMV and PVX viruses were independently spiked into the bacteria solution and incubated at room temperature for four days. After incubation, the viral-bacterial solutions were filtered sterilized to remove bacteria and used to infect 75 tobacco plants as described [ 41 ]. A control group was simultaneously inoculated with a viral solution incubated for 4 days at room temperature in the absence of bacteria. Inoculated plants were maintained under controlled greenhouse conditions (approx. 20 days). A group of uninoculated plants were placed in the same green house as plant controls. In addition, a group of 16 plants were inoculated only with the bacterial pool as a control of bacteria pathogenesis. Virus detection was performed an ELISA test with antibodies from Bioreba AG (Reinach, Switzerland), with two leaves taken from each plant [ 47 ]. The infection threshold was determined as the mean value of the non-inoculated controls plus three standard deviations, following established methodology. For PVX and PepMV, this corresponds to 0.16 and 0.28 absorbance units, respectively, after 18 hours of incubation. The infected plants were further divided into two groups: one consisting of highly infected plants, whose absorbance signal exceeded the upper detection limit of the ELISA plate reader (4 units), and another consisting of moderately infected plants, whose absorbance signal fell between the infection threshold and the upper detection limit. 16S amplicon sequencing of alpine synthetic communities. Once bacterial isolates were mixed, a sample from each community were taken for DNA extraction and 16S amplicon sequencing. Sample was 0.22 µm filtered and DNA from filters was extracted using DNeasy PowerWater Kit (Qiagen, Hilden, Germany) as previously described. DNA extracts and blanks were then used for 16S rRNA gene amplification using the 27F and 1492R (Table S2). The PCR reaction was carried out in a 50-µL volume containing 20 ng of input DNA and 25 µL of LongAmp Taq 2× master mix (New England Biolabs, Ipswich, MS, USA). Each PCR product was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using Qubit™ (dsDNA quantification, Broad Range). The sequencing library was prepared using the Native Barcoding Kit SQK-NBD114.24 (Oxford Nanopore Technologies, Oxford, UK) following the manufacturers’ instructions. The library was loaded into a FLO-MIN106 flow cell (Oxford Nanopore Technologies) and sequenced using a MinION Mk1C sequencing device for 72 hours. Sequencing files were basecalled using Dorado (v0.9.1) basecaller. FASTQ files were input into the MetONTIIME workflow [ 48 ] to estimate bacterial relative abundance and assign taxonomy. Feature tables and count matrix were imported into R and analysed using the microeco package [ 49 ]. Data analysis and visualization. Analysis of all the data obtained during the project was done with the dyplr and tidyverse packages RStudio [ 50 ]. All the plots were obtained in Rstudio using the ggplot package [ 51 ]. Correlation matrices and statistical analysis were performed using the Pearson correlation coefficient function in RStudio. Declarations FUNDING AND ACKNOWLEDGEMENTS This work was supported by the project ANTIVIRALPS (“Screening for novel antiviral compounds in alpine and polar lakes”) funded by the EPFL Science Seed Fund 2021, and the project HYDROVIR (“Optimizing the use of alpine lake water bacteria with antiviral properties to combat viral diseases in hydroponic cultures”) funded by the EPFL ENABLE Grant, 2022. The authors gratefully acknowledge Nathalie Dubuis for her assistance with plant virus preparation and sample analysis in Agroscope. References N. P. Adhikari, S. Adhikari, X. Liu, L. Shen, and Z. Gu, “Bacterial Diversity in Alpine Lakes: A Review from the Third Pole Region,” J. Earth Sci. , vol. 30, no. 2, pp. 387–396, Apr. 2019, doi: 10.1007/s12583-018-1206-5. H. Peter and R. Sommaruga, “Shifts in diversity and function of lake bacterial communities upon glacier retreat,” ISME J. , vol. 10, no. 7, pp. 1545–1554, July 2016, doi: 10.1038/ismej.2015.245. H. Brüssow, “Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity,” Mol. Microbiol. , vol. 65, no. 3, pp. 583–589, Aug. 2007, doi: 10.1111/j.1365-2958.2007.05826.x. J. C. Vogt, J. L. Olefeld, C. Bock, J. Boenigk, and D. C. Albach, “Patterns of protist distribution and diversification in alpine lakes across Europe,” MicrobiologyOpen , vol. 10, no. 4, p. e1216, Aug. 2021, doi: 10.1002/mbo3.1216. E. Jończyk, M. Kłak, R. Międzybrodzki, and A. Górski, “The influence of external factors on bacteriophages—review,” Folia Microbiol. (Praha) , vol. 56, no. 3, pp. 191–200, May 2011, doi: 10.1007/s12223-011-0039-8. T. Sime-Ngando, “Environmental bacteriophages: viruses of microbes in aquatic ecosystems,” Front. Microbiol. , vol. 5, July 2014, doi: 10.3389/fmicb.2014.00355. K. E. Wommack and R. R. Colwell, “Virioplankton: Viruses in Aquatic Ecosystems,” Microbiol. Mol. Biol. Rev. , vol. 64, no. 1, pp. 69–114, Mar. 2000, doi: 10.1128/MMBR.64.1.69-114.2000. C. A. Suttle, “Marine viruses — major players in the global ecosystem,” Nat. Rev. Microbiol. , vol. 5, no. 10, pp. 801–812, Oct. 2007, doi: 10.1038/nrmicro1750. J. E. Samson, A. H. Magadán, M. Sabri, and S. Moineau, “Revenge of the phages: defeating bacterial defences,” Nat. Rev. Microbiol. , vol. 11, no. 10, pp. 675–687, Oct. 2013, doi: 10.1038/nrmicro3096. A. Stern and R. Sorek, “The phage‐host arms race: Shaping the evolution of microbes,” BioEssays , vol. 33, no. 1, pp. 43–51, Jan. 2011, doi: 10.1002/bies.201000071. F. Tesson et al. , “Systematic and quantitative view of the antiviral arsenal of prokaryotes,” Nat. Commun. , vol. 13, no. 1, p. 2561, May 2022, doi: 10.1038/s41467-022-30269-9. S. Doron et al. , “Systematic discovery of antiphage defense systems in the microbial pangenome,” Science , vol. 359, no. 6379, p. eaar4120, Mar. 2018, doi: 10.1126/science.aar4120. T. Goldfarb et al. , “BREX is a novel phage resistance system widespread in microbial genomes,” EMBO J. , vol. 34, no. 2, pp. 169–183, Jan. 2015, doi: 10.15252/embj.201489455. A. Bernheim et al. , “Prokaryotic viperins produce diverse antiviral molecules,” Nature , vol. 589, no. 7840, pp. 120–124, Jan. 2021, doi: 10.1038/s41586-020-2762-2. S. Kronheim et al. , “A chemical defence against phage infection,” Nature , vol. 564, no. 7735, pp. 283–286, Dec. 2018, doi: 10.1038/s41586-018-0767-x. B. J. O’Hara, Z. K. Barth, A. C. McKitterick, and K. D. Seed, “A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome,” PLOS Genet. , vol. 13, no. 6, p. e1006838, June 2017, doi: 10.1371/journal.pgen.1006838. K. J. Helbig and M. R. Beard, “The Role of Viperin in the Innate Antiviral Response,” J. Mol. Biol. , vol. 426, no. 6, pp. 1210–1219, Mar. 2014, doi: 10.1016/j.jmb.2013.10.019. R. E. Baker et al. , “Infectious disease in an era of global change,” Nat. Rev. Microbiol. , vol. 20, no. 4, pp. 193–205, Apr. 2022, doi: 10.1038/s41579-021-00639-z. T. H. Bacon, M. J. Levin, J. J. Leary, R. T. Sarisky, and D. Sutton, “Herpes Simplex Virus Resistance to Acyclovir and Penciclovir after Two Decades of Antiviral Therapy,” Clin. Microbiol. Rev. , vol. 16, no. 1, pp. 114–128, Jan. 2003, doi: 10.1128/CMR.16.1.114-128.2003. A. Carratalà, H. Shim, Q. Zhong, V. Bachmann, J. D. Jensen, and T. Kohn, “Experimental adaptation of human echovirus 11 to ultraviolet radiation leads to resistance to disinfection and ribavirin,” Virus Evol. , vol. 3, no. 2, July 2017, doi: 10.1093/ve/vex035. Q. Zhong, A. Carratalà, H. Shim, V. Bachmann, J. D. Jensen, and T. Kohn, “Resistance of Echovirus 11 to ClO 2 Is Associated with Enhanced Host Receptor Use, Altered Entry Routes, and High Fitness,” Environ. Sci. Technol. , vol. 51, no. 18, pp. 10746–10755, Sept. 2017, doi: 10.1021/acs.est.7b03288. C. S. Adamson, K. Chibale, R. J. M. Goss, M. Jaspars, D. J. Newman, and R. A. Dorrington, “Antiviral drug discovery: preparing for the next pandemic,” Chem. Soc. Rev. , vol. 50, no. 6, pp. 3647–3655, 2021, doi: 10.1039/D0CS01118E. D. Cliver and J. Herrmann, “Proteolytic and microbial inactivation of enteroviruses,” Water Res. , vol. 6, no. 7, pp. 797–805, July 1972, doi: 10.1016/0043-1354(72)90032-2. R. L. Ward, D. R. Knowlton, and P. E. Winston, “Mechanism of inactivation of enteric viruses in fresh water,” Appl. Environ. Microbiol. , vol. 52, no. 3, pp. 450–459, Sept. 1986, doi: 10.1128/aem.52.3.450-459.1986. M.-H. Corre, V. Bachmann, and T. Kohn, “Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water,” ISME J , vol. 16, no. 8, pp. 1970–1979, Aug. 2022, doi: 10.1038/s41396-022-01246-3. W. Chaisuwan et al. , “The Antiviral Activity of Bacterial, Fungal, and Algal Polysaccharides as Bioactive Ingredients: Potential Uses for Enhancing Immune Systems and Preventing Viruses,” Front. Nutr. , vol. 8, p. 772033, Nov. 2021, doi: 10.3389/fnut.2021.772033. M. Olive, C. Gan, A. Carratalà, and T. Kohn, “Control of Waterborne Human Viruses by Indigenous Bacteria and Protists Is Influenced by Temperature, Virus Type, and Microbial Species,” Appl Env. Microbiol , vol. 86, no. 3, pp. e01992-19, Jan. 2020, doi: 10.1128/AEM.01992-19. A. Romanenko, H. Peter, J. Meibom, M. A. Borchardt, and T. Kohn, “Diversity of lake bacteria promotes human echovirus inactivation,” Appl. Environ. Microbiol. , vol. 91, no. 2, pp. e02366-24, Feb. 2025, doi: 10.1128/aem.02366-24. R. Wang et al. , “Bacterial and Archaeal Communities within an Ultraoligotrophic, High-altitude Lake in the Pre-Himalayas of the Qinghai-Tibet Plateau,” Indian J. Microbiol. , vol. 60, no. 3, pp. 363–373, Sept. 2020, doi: 10.1007/s12088-020-00881-8. R. S. Fujioka, P. C. Loh, and L. S. Lau, “Survival of Human Enteroviruses in the Hawaiian Ocean Environment: Evidence for Virus-Inactivating Microorganismst,” APPL Env. MICROBIOL , vol. 39, 1980. S. A. Gatea Kaabi and H. K. Musafer, “New Phage cocktail against infantile Sepsis bacteria,” Microb. Pathog. , vol. 148, p. 104447, Nov. 2020, doi: 10.1016/j.micpath.2020.104447. Y. Qin et al. , “Purification and Characterization of a Secretory Alkaline Metalloprotease with Highly Potent Antiviral Activity from Serratia marcescens Strain S3,” J. Agric. Food Chem. , vol. 67, no. 11, pp. 3168–3178, Mar. 2019, doi: 10.1021/acs.jafc.8b06909. X. Wang, G. Hao, M. Zhou, M. Chen, H. Ling, and Y. Shang, “Secondary metabolites of Bacillus subtilis L2 show antiviral activity against pseudorabies virus,” Front. Microbiol. , vol. 14, p. 1277782, Oct. 2023, doi: 10.3389/fmicb.2023.1277782. L. Yuan, S. Zhang, J. Peng, Y. Li, and Q. Yang, “Synthetic surfactin analogues have improved anti-PEDV properties,” PLOS ONE , vol. 14, no. 4, p. e0215227, Apr. 2019, doi: 10.1371/journal.pone.0215227. D. O. Cliver, “Experimental Infection by Waterborne Enteroviruses,” J. Food Prot. , vol. 44, no. 11, pp. 861–865, Nov. 1981, doi: 10.4315/0362-028X-44.11.861. A. Amtmann et al. , “Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus,” J. Fish Dis. , vol. 43, no. 2, pp. 185–195, Feb. 2020, doi: 10.1111/jfd.13106. T. Shimizu, N. Yoshida, H. Kasai, and M. Yoshimizu, “Survival of Koi Herpesvirus (KHV) in Environmental Water,” Fish Pathol. , vol. 41, no. 4, pp. 153–157, 2006, doi: 10.3147/jsfp.41.153. L. A. Amaral-Zettler, E. A. McCliment, H. W. Ducklow, and S. M. Huse, “A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes,” PLoS ONE , vol. 4, no. 7, p. e6372, July 2009, doi: 10.1371/journal.pone.0006372. T. Stoeck et al. , “Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water,” Mol. Ecol. , vol. 19, no. s1, pp. 21–31, Mar. 2010, doi: 10.1111/j.1365-294X.2009.04480.x. F. Zhu, R. Massana, F. Not, D. Marie, and D. Vaulot, “Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene,” FEMS Microbiol. Ecol. , vol. 52, no. 1, pp. 79–92, Mar. 2005, doi: 10.1016/j.femsec.2004.10.006. M. Mahillon, J. Brodard, I. Kellenberger, A. G. Blouin, and O. Schumpp, “A novel weevil-transmitted tymovirus found in mixed infection on hollyhock,” Virol. J. , vol. 20, no. 1, p. 17, Jan. 2023, doi: 10.1186/s12985-023-01976-6. S. C. David et al. , “Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS,” mSphere , vol. 8, no. 5, pp. e00226-23, Oct. 2023, doi: 10.1128/msphere.00226-23. O. Larivé, J. Brandani, M. Dubey, and T. Kohn, “An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes,” J. Virol. Methods , vol. 296, p. 114225, Oct. 2021, doi: 10.1016/j.jviromet.2021.114225. G. Motos et al. , “Dependence of aerosol-borne influenza A virus infectivity on relative humidity and aerosol composition,” Front. Microbiol. , vol. 15, p. 1484992, Oct. 2024, doi: 10.3389/fmicb.2024.1484992. N. Ács, M. Gambino, and L. Brøndsted, “Bacteriophage Enumeration and Detection Methods,” Front. Microbiol. , vol. 11, p. 594868, Oct. 2020, doi: 10.3389/fmicb.2020.594868. M. R. J. Clokie and A. M. Kropinski, Eds., Bacteriophages , vol. 501. in Methods in Molecular Biology, vol. 501. Totowa, NJ: Humana Press, 2009. doi: 10.1007/978-1-60327-164-6. A. N. Adams and M. F. Clark, “Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses,” J. Gen. Virol. , vol. 34, no. 3, pp. 475–483, Mar. 1977, doi: 10.1099/0022-1317-34-3-475. A. Matoute et al. , “Meat-Borne-Parasite: A Nanopore-Based Meta-Barcoding Work-Flow for Parasitic Microbiodiversity Assessment in the Wild Fauna of French Guiana,” Curr. Issues Mol. Biol. , vol. 46, no. 5, pp. 3810–3821, Apr. 2024, doi: 10.3390/cimb46050237. C. Liu, Y. Cui, X. Li, and M. Yao, “ microeco : an R package for data mining in microbial community ecology,” FEMS Microbiol. Ecol. , vol. 97, no. 2, p. fiaa255, Jan. 2021, doi: 10.1093/femsec/fiaa255. H. Wickham et al. , “Welcome to the Tidyverse,” J. Open Source Softw. , vol. 4, no. 43, p. 1686, Nov. 2019, doi: 10.21105/joss.01686. Hadley Wickham, ggplot2: Elegant Graphics for Data Analysis . Springer-Verlag New York, 2016. Additional Declarations There is NO Competing Interest. Supplementary Files DM2025AntiviralsSupplem151225.docx Supplementary information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8367630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":564796663,"identity":"58f62336-6c87-4b88-9e9e-b0abbf4454fb","order_by":0,"name":"Anna Carratala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3PMQrCMBTG8RcCWV3fpFeICFZBPEtCwKngASwYlzrVC+ghHHWzFOoiugYUFHqBTqKLGNwUbHUTzB+6vR/5CuBy/Xwn6ADQRAPRH91TAAE9ACa/I0k5qUyjOIeg2/fGuzgXwa7qMXt/XbwneNgqhFS1ZxtFUaT7RjskmkSbgjnG50g05QgKULC9nB9HmpLwvagZv3EhesixktGLuG2H85QUE278pn0l4YiKoQxXgpeRuiUtka4tyZotOVH1pf2XOCogVTvM5MHADpOZyc/dmsdofLoWkEfiaar9ViXgJf7ducvlcv1Bd9boUGzocYu4AAAAAElFTkSuQmCC","orcid":"","institution":"Swiss Federal Institute of Technology in Lausanne (EPFL)","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"","lastName":"Carratala","suffix":""},{"id":564796664,"identity":"5b25b91a-c048-44aa-8ccc-51fa97d85e90","order_by":1,"name":"Laura Daniela Morales","email":"","orcid":"","institution":"Swiss Federal Institute of Technology in Lausanne (EPFL)","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"Daniela","lastName":"Morales","suffix":""},{"id":564796665,"identity":"00aa36b7-be1e-40d0-86a9-58ac679d8ce5","order_by":2,"name":"Jessica Aubouy","email":"","orcid":"","institution":"Swiss Federal Institute of Technology in Lausanne (EPFL)","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Aubouy","suffix":""},{"id":564796666,"identity":"205f3591-1a9c-467b-9733-2d554fc041cd","order_by":3,"name":"Shannon David","email":"","orcid":"","institution":"Ecole Polytechnique Federale de Lausanne (EPFL)","correspondingAuthor":false,"prefix":"","firstName":"Shannon","middleName":"","lastName":"David","suffix":""},{"id":564796667,"identity":"0889bbd8-8cf5-4fd2-b071-c2cec47f4527","order_by":4,"name":"Paul Seguin","email":"","orcid":"","institution":"Swiss Federal Institute of Technology in Lausanne (EPFL)","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Seguin","suffix":""},{"id":564796668,"identity":"0908d5c5-069d-4819-a542-7224a39151bb","order_by":5,"name":"Olivier Schumpp","email":"","orcid":"","institution":"Agroscope","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Schumpp","suffix":""}],"badges":[],"createdAt":"2025-12-15 15:04:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8367630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8367630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99319433,"identity":"e3710f9a-ade5-412d-97c3-f1e333ae036f","added_by":"auto","created_at":"2025-12-31 16:37:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2113260,"visible":true,"origin":"","legend":"","description":"","filename":"DM2025AntiviralsManuscriptNatCom271125updated.docx","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/3d0f0a829642d60d5276113c.docx"},{"id":99280833,"identity":"9122e49f-0d66-4b22-8bc8-8c6ce22747fc","added_by":"auto","created_at":"2025-12-31 08:25:38","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7425,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25101689.json","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/049bced1e3ce07583adc59ab.json"},{"id":99318694,"identity":"f18d9d82-f43c-44c0-957f-22506572c54a","added_by":"auto","created_at":"2025-12-31 16:33:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":290000,"visible":true,"origin":"","legend":"","description":"","filename":"DM2025AntiviralsSupplem151225.docx","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/89126b7bbf1adb2d1f5b4292.docx"},{"id":99280839,"identity":"36af269a-6e07-46bc-960f-ac1d58cf8680","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126315,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS251016890enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/7cb14ba045ade5ecdf0443ef.xml"},{"id":99319734,"identity":"762822a6-d558-441b-99c4-2915d197a203","added_by":"auto","created_at":"2025-12-31 16:37:44","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1038953,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/9f0a6348599036c548947c78.png"},{"id":99280849,"identity":"03c05840-c570-4814-9908-d55467f9a2a6","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127816,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/f55c70c6e4bce5b03c3dbb21.png"},{"id":99280843,"identity":"b033a682-9668-41c3-afcb-c455ffd6211b","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72919,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/1b4e955bd725cffbdb6559df.png"},{"id":99280852,"identity":"3cb4b972-c25a-4172-b8fd-4f1fd1e5aaaa","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":327796,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/59602c730f6d5abeb795cb00.png"},{"id":99280848,"identity":"01ddce6e-136a-4fd7-904a-4a44005187da","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96286,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/ab3b5facd1466c8ec6fe9c47.png"},{"id":99321298,"identity":"a829aba7-2ad3-4277-bff2-e4208c76212c","added_by":"auto","created_at":"2025-12-31 16:39:19","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":327453,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/a7d96cea6efad982803d6fa1.png"},{"id":99318799,"identity":"22be5554-f53c-4757-87d5-eb6550681022","added_by":"auto","created_at":"2025-12-31 16:34:44","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":175532,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/a036521bbbe82af222efa446.png"},{"id":99280853,"identity":"75b85774-682e-48f5-b763-1452e44c12cb","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41959,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/230d9eef786e4136b3a0e90f.png"},{"id":99280840,"identity":"742c0ed2-a4b9-41c2-88d1-6b0c00a230db","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17816,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/c732045af53e278eab1d2ad0.png"},{"id":99280844,"identity":"0678d01f-44dd-405e-9303-fe60c9c296e3","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69838,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/da30a0d844f58c92030fb29d.png"},{"id":99319320,"identity":"9d85acd6-25bd-4b01-b982-56a9da4ccec5","added_by":"auto","created_at":"2025-12-31 16:36:56","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27766,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/a4f35199d30c85496eaf214f.png"},{"id":99280854,"identity":"9e1a818d-4317-4205-9c96-cb7690d4a961","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80151,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/0b3c096bd226501be35897c3.png"},{"id":99280857,"identity":"05f00411-5782-404b-8a83-f6f72dd4db09","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124138,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS251016890structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/110d34664fa3f833c8db2396.xml"},{"id":99280855,"identity":"42dfb8c1-d14a-4069-9544-061edb008433","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138903,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/554b511164b0112808206297.html"},{"id":99318929,"identity":"43c9b0f9-26cf-4f82-935b-eda14be81ed2","added_by":"auto","created_at":"2025-12-31 16:35:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1038953,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/5ba7471926e8f29307b53eb0.png"},{"id":99280836,"identity":"e6b11e36-8a92-4ed3-a156-866a29190cea","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic profiling of microbial communities in Swiss Alpine Lakes. Water\u003c/strong\u003e from each lake was used to assess the capacity of each lake’s microbial community to degrade the 31 different substrates present in Ecolog plates. Each lakewater was inoculated into an Ecolog plate and incubated for five days at room temperature. Optical density was measured at 590 nm and averaged for each substrate from three replicates (n=3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/f1734ec3a377f824d131c8d1.png"},{"id":99280832,"identity":"c6f73015-f6d1-4fa3-a002-94c1b8d104e7","added_by":"auto","created_at":"2025-12-31 08:25:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInactivation of Human viruses in selected Alpine Lakes. (A) \u003c/strong\u003eEchovirus 11 (E11) or \u003cstrong\u003e(B)\u003c/strong\u003e Influenza A virus (IAV) was inoculated into 0.8 mm filtered water from eight Alpine lakes and incubated for seven days at room temperature. Viral inactivation was determined by comparing the viral genome copies per ml (gc/ml) on day seven to day zero (Log C/C0) using quantitative PCR (qPCR). Each viral inactivation assay was performed in five replicates (n = 5). A one-way ANOVA with Tukey honest significant difference in R Studio was used for statistical analysis between viral decay values. Significant differences are indicated as follows: ***P \u0026lt; 0.001, **P \u0026lt; 0.01, and *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/bb239340599271c6bd47c594.png"},{"id":99319515,"identity":"772062b9-71f7-4790-bafb-4247a030f980","added_by":"auto","created_at":"2025-12-31 16:37:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":327796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhage susceptibility of Alpine bacterial isolates.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e An “Alpine Phage mix” was generated by mixing lakewater samples from each lake. In parallel, to isolate bacteria from each lake, one litre of lakewater was 0.45 mmfiltered. Filters were transferred to LB agar plates and incubated at room temperature for 72 hours. \u003cstrong\u003e(B)\u003c/strong\u003e A total of 223 bacterial isolates were recovered and identified using 16S gene sequencing. The number of bacterial isolates obtained varied per lake. \u003cstrong\u003e(C) \u003c/strong\u003eTo assess bacterial susceptibility to the Alpine Phage mix, each bacterial isolate was grown in LB and spiked with one ml of the mix. After five hours, bacterial cultures were recovered, spun down and filtered. DNA was extracted at 0 and 5 hours and qPCR against the g23 gene was used to compare phage titers between these two time points (Log C/C0).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/9f69d7b1e947884c942ff6e5.png"},{"id":99320432,"identity":"84dc33bd-2a38-406b-bbc0-3075fde244f9","added_by":"auto","created_at":"2025-12-31 16:38:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlpine synthetic communities inactivate human virus. \u003c/strong\u003eBacteria isolates were mixed to generate synthetic communities of 25 members. Communities 1 and 3 were created by mixing lakewater bacteria with less susceptibility to the “Alpine Phage Mix”. A control community was generated by mixing bacteria with the least susceptibility out of the 223 isolates. \u003cstrong\u003e(A)\u003c/strong\u003e To assess the inactivation capacity of each bacterial community, Echovirus 11 (E11) and Influenza A virus (IAV) were spiked into each community. Viral inactivation was determined (Log C/C0) using quantitative PCR (qPCR) as previously described after 4 days. Each viral inactivation assay was performed in five replicates (n = 5). \u003cstrong\u003e(B)\u003c/strong\u003eCommunity composition at the end of the experiment was determined by extracting DNA from each community and sequencing the 16S gene using Oxford Nanopore Technologies (ONT). A one-way ANOVA with Tukey honest significant difference in R Studio was used for statistical analysis between viral decay values. Significant differences are indicated as follows: ***P \u0026lt; 0.001, **P \u0026lt; 0.01, and *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/88d9157b19c7b3bd4de0bae7.png"},{"id":99320344,"identity":"05addf0e-7103-4491-9729-cb77bc468645","added_by":"auto","created_at":"2025-12-31 16:38:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":363021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial Pool 3 shows antiviral activity against Potato Virus X (PVX).\u003c/strong\u003e The antiviral activity of “Community 3” was assessed against Potato Virus X (PVX) and Pepino Mosaic Virus (PepMV). Both viruses were purified from tobacco plants and diluted into a PBS solution (A and D) or the “Community 3” (B and E). Virus only and Virus + Community 3 were incubated at room temperature for 4 days and used to infect tobacco plants. After 15 days, leaves were taken from each plant to perform an ELISA assay against both viruses (in duplicate). Plant controls were incubated and processed as treated plants (C and D).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/0d0355c264e2c1ed9f4a0a6b.png"},{"id":102962249,"identity":"186984ef-c32a-48fd-8e0d-4539529b61f9","added_by":"auto","created_at":"2026-02-19 04:06:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2855511,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/4c8c48a7-54c6-4234-8c38-7885ffedb100.pdf"},{"id":99280835,"identity":"4241d1d5-093d-4874-946f-ffc011f8c5e9","added_by":"auto","created_at":"2025-12-31 08:25:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":290000,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"DM2025AntiviralsSupplem151225.docx","url":"https://assets-eu.researchsquare.com/files/rs-8367630/v1/6bd0a0f0f892d95f21fbbcbe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Harnessing Alpine Lake Bacteria for the Development of Synthetic Communities with Broad-Spectrum Antiviral Activity","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAlpine lakes are unique ecosystems with extreme environmental conditions: low nutrient concentrations, low temperatures, ice cover during the wintertime, high UV radiation in summertime, short summertime and drastic light changes between the ice-covered and ice-free season [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite these harsh conditions, alpine lakes host diverse bacterial communities that play central roles in ecosystem functioning [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In aquatic ecosystems, phagotrophic protists and bacteriophages (also known as phages) are major biological factors shaping bacterial populations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, in alpine lakes bacterial communities are less influenced by protist grazing compared to low-land lakes due to the limited richness and diversity of the latest compared to non-alpine regions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Since bacteriophages are generally highly resistant to extreme environmental conditions, they likely represent the main biological factor shaping bacterial communities in alpine aquatic ecosystems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacteriophages are the most abundant microorganisms in aquatic systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. They lead to 20\u0026ndash;40% of bacteria mortality and exert a huge selective pressure on their bacteria hosts. Bacteria-viral interactions in this context, are a constant and dynamic back and forth battle [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Bacteria adapt their immune systems and develop mechanisms to resist bacteriophage infections, and bacteriophages frequently mutate to counteract bacteria adaptations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Bacteria have developed numerous mechanisms against bacteriophage infections and have clustered them in so-called genomic \u0026ldquo;defense islands\u0026rdquo; across their genomes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These defense mechanisms range from the well characterized bacterial CRISPR/Cas system shared by multiple bacterial species to the most recently discovered Bacteriophage Exclusion (BREX) system in \u003cem\u003eBacillus subtilis\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. They involve phage DNA silencing through small RNA molecules, DNA degradation by restriction enzymes, bacterial programmed cell dead to avoid viral proliferation, and the production of chemical antiviral molecules like viperins [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Noteworthy, bacterial and human viperins have structural and functional similarities. While bacteria use these molecules to fight bacteriophage infections, humans seem to also use them to fight virus infections [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacterial antiviral molecules and their mechanisms can be potentially exploited to our advantage against viral pathogens of multiple hosts. Climate change, urbanization and mobility increase the risk of infectious disease emergence [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Viral pathogens, in particular, have high mutation rates, large population numbers, short generation times and rapidly become resistant to antiviral drugs and disinfectants [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. There is a permanent lack of antiviral molecules effective against rapidly evolving and emerging human, animal and plant pathogens [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Aquatic ecosystems and, in general, extreme environments are potentially a rich source of microbial secondary metabolites with antimicrobial and antiviral activity. In the early 70s and 80s, Ward \u003cem\u003eet al.\u003c/em\u003e and Cliver \u003cem\u003eet al\u003c/em\u003e. described the antiviral activity of fresh water microorganisms and their proteases against Poliovirus, Echovirus-12, Rotavirus SA-11 and Coxsackievirus B5 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. More recently, our lab further characterized lakewater bacterial isolates and their proteolytic activity. We found a correlation between viral inactivation and extracellular metalloprotease production [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. As well, bacterial intra- and extra- cellular polysaccharides have shown antiviral activity against diverse viruses [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the potential of high-altitude aquatic environments as hotspots for the discovery of new antiviral compounds of bacterial origin remains largely unexplored.\u003c/p\u003e \u003cp\u003eDue to the extreme environmental conditions and the high abundance of bacteriophages in alpine lakes, we hypothesized that bacteria in these environments have developed complex and diverse antiviral mechanisms against bacteriophages that likely contribute to the inactivation of multiple viruses. Thus, we aimed to explore broad-spectrum viral inactivation by bacteria communities and isolates from Swiss Alpine lakes. In Switzerland, 60% of the land is covered by the Alps with over 1,500 lakes present in these high-altitude regions. We conducted sampling campaigns in 24 alpine lakes located above the tree line in the Swiss Alps and investigated the inactivation of two human viruses (Influenza A virus and Echovirus 11) in water from selected lakes. In addition, we identified and selected bacteriophage resistant isolates to build two synthetic communities with increased broad-spectrum antiviral activity against the two human viruses and two plant viruses (Potato Virus X and Pepino Mosaic Virus). Our findings highlight high-altitude lakes as unexplored reservoirs of microbial and metabolic diversity. They offer promising opportunities for the discovery of novel broad-spectrum antiviral mechanisms and bioactive compounds, and for the design of synthetic microbial consortia with potential applications in the biocontrol of viral pathogens.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eMicrobial abundances and metabolic diversity in Swiss alpine lakes.\u003c/b\u003e A total of 24 alpine lakes were sampled in the Swiss Alps from eight distinct regions in the canton of Valais and one region in the Bernese Oberland (Fig.\u0026nbsp;1A). The abundance of bacteria, eukaryotes and tailed bacteriophages was determined in each lake to identify those showing a higher ratio of bacteriophages to bacteria and eukaryotes. We found great variability of microbial abundances between samples (Fig.\u0026nbsp;1B). Bacteria concentrations (16S) ranged from 300 genome copies/liter (gc/l) in Lac Bleu (Arolla) to more than 10\u003csup\u003e8\u003c/sup\u003e gc/l in Spilsee (Bellwald). Eukaryotic DNA (18S) ranged from 100 gc/l in Lac Bleu to 10\u003csup\u003e9\u003c/sup\u003e gc/l in Stellisee and Leisee (Zermatt). The concentration of tailed bacteriophages (g23) varied between \u0026lt;\u0026thinsp;10 gc/l in Small Lac de Vaux (Verbier) to 10\u003csup\u003e7\u003c/sup\u003e gc/l in Lengsee (Bellwald). We calculated the ratio of tailed bacteriophages over the sum of bacteria and eukaryotes concentration (measured in gc/l) in each lake, shown in Fig.\u0026nbsp;1C. At the time of sampling, few of the lakes had particularly high ratios of tailed bacteriophages per bacteria and protists. Lakes Mossjesee, Guggisee, Marjelensee and Salanfe had ratios of 0.12, 0.15, 0.10 and 0.69, respectively, compared to an average ratio of 0.01 in the other 20 lakes. The ratio of tailed bacteriophages to bacteria and eukaryotes was 60-times higher in Lake Salanfe compared to the average ratio of the rest of the lakes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo characterize functional traits of the microbial communities in the sampled lakes that could provide insights into their antiviral activity, we assessed carbon metabolism using Biolog EcoPlates. These plates contain 31 compounds classified as carbohydrates, amino acids, carboxylic acids, amines or alcohols (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the number of metabolized carbon sources, we calculated Shannon diversity indexes to determine the metabolic diversities per each lake (Table S3). Lakes Bleu, Petit Aletsch 1 and Schwarzsee had the highest metabolic diversity (Shannon indexes\u0026thinsp;\u0026gt;\u0026thinsp;3). Microbial communities present in these three lakes metabolized substrates in all six categories (carbohydrates, amino acids, carboxylic acids, amines and alcohols). In contrast, lakes Stellisee and Leisee were the least metabolically diverse (Shannon indexes\u0026thinsp;\u0026lt;\u0026thinsp;1), as microbial communities from these lakes were unable to metabolize most of the substrates present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMicrobial communities in alpine lakes show antiviral activity against human viruses.\u003c/b\u003e We have previously reported that bacterial communities from Lake Geneva inactivate human enteric viruses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To confirm whether bacteria in Swiss alpine lakes also show antiviral activity against human viruses, we conducted inactivation experiments using two structurally different viruses: echovirus 11 (E11) and influenza A virus (IAV). For these assays, we selected four lakes with high proportion of tailed bacteriophages per bacteria and eukaryotes (Guggisee, Mossjesee, Marjelensee and Salanfe), and four lakes with low proportion (Lac Bleu, Schwartzsee, Small Lac de Vaux and Mittelsee) to evaluate whether elevated phage-to-host ratios are indicative of increased antiviral activity. We focused on the antiviral activity of bacteria present in the water. Therefore, the water used for these assays was filtered through a 0.8 \u0026micro;m membrane to remove protists, also known to inactivate human viruses [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed higher E11 inactivation compared to IAV in all the tested lakes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). E11 was inactivated\u0026thinsp;\u0026gt;\u0026thinsp;1 log10 in five out of eight of the lakes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) while IAV was inactivated around 1 log10 in all lakes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Out of the eight lakes, lake Mossjesee, an artificial reservoir resulting from glacial meltwater, had the greatest inactivation against both viruses (2.17 average log10 decay for E11; 1.03 log10 decay for IAV) over 7 days of incubation. E11 inactivation in water from this lake was significantly higher compared to lakes Salanfe and Mittelsee (P\u0026thinsp;=\u0026thinsp;0.0387 compared to Salanfe and P\u0026thinsp;=\u0026thinsp;0.0199 compared to Mittelsee). We did not observe a correlation between viral inactivation and the proportions of bacteriophages per bacteria and eukaryotes. However, two out of four of the chosen lakes with increased ratios of tailed bacteriophages (Mossjeesee and Marjelensee) showed the greatest inactivation against E11. Similarly, we did not identify any correlation between antiviral activity and lake metabolic diversities (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlpine lakes host bacteria with low susceptibility to bacteriophage infections.\u003c/b\u003e To identify and characterize specific bacteria with potential antiviral activity, we isolated single bacteria species from each lake regardless of the phage-to-host ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In total, we obtained 223 different bacteria isolates across the 24 alpine lakes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The number of bacterial isolates obtained varied per lake, ranging from 4 to 19. Lakes Guggisee and Oeschinensee yielded the highest number of isolates (19 and 16 cultures respectively), together accounting for approximately 15% of the total isolates. Lakes Stellisee and Wirbulsee yielded the lowest numbers of bacterial isolates (only 4 isolates from each of both lakes). Interestingly, Lakes Guggisee and Oeschinensee showed the lowest bacterial abundance (Fig.\u0026nbsp;1B), while lakes Stellisee and Wirbulsee were within the lakes with the greatest bacterial abundance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe hypothesized that bacterial isolates with lower susceptibility to bacteriophage infections would show increased antiviral activity against human and plant viruses. To identify bacteria with decreased susceptibility to bacteriophage infections and test our hypothesis, we created a \u0026ldquo;Phage Mix\u0026rdquo; by pooling virus fractions (0.22 \u0026micro;m filtered lakewater) from the surface water samples collected in the 24 lakes. This generated a phage cocktail that included a large abundance of diverse bacteriophages potentially capable of infecting most of the bacteria isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). All 223 isolates were grown independently and spiked with the alpine \u0026ldquo;Phage Mix\u0026rdquo;. After co-incubating bacteria isolates and phage mix, we compared the final concentration of bacteriophages to the initial concentration in the inoculum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Most of the isolates (148 specifically) showed susceptibility to the Phage Mix as indicated by an increase in the bacteriophages\u0026rsquo; titer after the incubation period. Conversely, we identified 75 isolates with decreased susceptibility indicated by a decrease in the concentration of bacteriophages after co-incubations. Lake Oeschinsee (9 isolates), Lac de Vaux (8 isolates), Leisee (8 isolates) and Lac de Salanfe (8 isolates) had the greatest number of phage resistant isolates. Interestingly, these lakes had the lowest metabolic diversity (Shannon 0.98 to 1.69) compared to other lakes. In terms of bacteriophage to bacteria and eukaryotes ratios, we did not observe any relation between bacteriophage resistant isolates and the calculated ratios. However, we noticed that Lac de Salanfe had the highest calculated ratios amongst all sampled lakes (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhage-resistant synthetic communities inactivate human and plant viruses.\u003c/b\u003e Previous studies performed in our laboratory showed greater viral inactivation in lakewater samples compared to single bacteria cultures, and a correlation between increased microbial diversity and enhanced inactivation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, we aimed to design microbial synthetic communities using isolates classified as \u0026ldquo;bacteriophage-resistant\u0026rdquo; and assess their antiviral activity against viral pathogens with different structures and hosts. We created two synthetic communities of 25 members each (communities 1 and 3 \u0026ndash; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In parallel, we created a control community using 25 bacteriophage-susceptible bacteria (Control community). The three communities were spiked with either E11 or IAV and incubated for 4 days. After co-incubation, the synthetic communities of bacteriophage-resistant bacteria (communities 1 and 3) had enhanced antiviral activity compared to the bacteriophage-susceptible (control) community (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In both cases, we observed significantly (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) more inactivation between the communities (1 and 3) and the control. In addition, we observed that E11 had greater inactivation compared to IAV (E11\u0026thinsp;~\u0026thinsp;3 log10; IAV\u0026thinsp;~\u0026thinsp;1 log10, in four days). Noteworthy, the synthetic communities showed greater inactivation against E11 as compared to the inactivation we previously observed in lakewater (~\u0026thinsp;2 log10 decay in seven days).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe determined the bacteria composition of the three communities (1, 3 and control) before viral inoculation using 16S amplicon sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We also confirmed the identity of individual bacteria using Sanger sequencing. We found high relative abundance of genera \u003cem\u003eBrevundimonas\u003c/em\u003e, \u003cem\u003eSerratia, Pseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e species across the three communities (control and communities 1 and 3). On the other hand, genera \u003cem\u003eAlcaligenes\u003c/em\u003e and \u003cem\u003eMicrobacterium\u003c/em\u003e species were shared between communities 1 and 3 but were not present in the control community. We also identified bacteria uniquely found in each community. \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eStaphylococcus\u003c/em\u003e sp. were uniquely present in community 1, and \u003cem\u003eAllorrhizobium\u003c/em\u003e species were uniquely found and abundant in community 3. Lastly, to identify some mechanisms behind the observed antiviral activity, we assessed protease secretion by individual bacteria from each community. Bacteria isolates were individually grown in skim milk agar (Figure S2A) and proteolytic activity was determined by measuring degradation halos around individual colonies. We did not observe a difference between the proteolytic activity of the community members present on each of the phage resistant and susceptible communities (Figure S2B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince our previous results indicated that the developed synthetic communities exhibit antiviral activity against bacteriophages and human viruses, we hypothesized that they may possess broad-spectrum antiviral properties. To test this hypothesis, we performed viral inactivation assays against Potato Virus X (PVX) and Pepino Mosaic Virus (PepMV) using synthetic community 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For these assays, this synthetic community was spiked with either plant virus, and then incubated at room temperature for four days. In parallel, both viruses were also diluted in PBS and incubated under the same conditions. These suspensions were used to mechanically inoculate tobacco plants and the infectivity of both viruses was assessed 19 days post-inoculation (dpi). We observed a decrease of PVX infectivity from 79% of plants infected with PVX alone to just 1% following the incubation with community 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). In contrast, PepMV showed no infectivity reduction in the presence of community 3. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and E). These results suggest that community 3 exhibits broad-spectrum antiviral activity but also displays some specificity in the extent of viral inactivation across different viruses. As controls, we included 10 untreated plants (five plants per virus) to evaluate crossed contamination in the greenhouse (Plant controls - Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and F) and observed one plant control positive for PVX. We also inoculated plants with community 3 to assess bacteria pathogenesis against plants and observed no pathogenicity. These results confirmed our working hypothesis and provide for the first time a link between resistance against phage infections and viral inactivation.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study we characterized broad-spectrum viral inactivation by bacteria communities and isolates from 24 Swiss Alpine Lakes. Across these lakes, we found heterogenous microbial abundances, diversity of carbon utilization and antiviral activity against two human viruses (E11 and IAV). Given the increased abundance of bacteriophages in alpine aquatic ecosystems, we hypothesized that bacteria populations in alpine lakes may have evolved antiviral mechanisms against bacteriophages that may be potentially harmful to other viral pathogens. To test our hypothesis, we generated a collection of alpine lake bacteria and used those classified as \u0026ldquo;bacteriophage-resistant\u0026rdquo; to assemble two synthetic communities that significantly inactivated E11, IAV and one plant virus (PVX) compared to a community built from bacteriophage-susceptible isolates. Although our research drew a relation between bacteriophage resistance and broad-spectrum antiviral activity, further research is necessary to identify and characterize shared molecular mechanisms.\u003c/p\u003e \u003cp\u003eOur results show that microbial abundances differed from one lake to another suggesting that abundances are influenced by environmental and/or biological factors acting at very local scale. Previous research showed that high altitude lakes have bacteria abundances and diversities greatly dependent on local characteristics of each lake (e.g. ionic concentrations, nutrients, temperature and pH) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although, identifying and characterizing the influence of these specific factors on microbial abundances was beyond the scope of this work, we found enhanced antiviral activity in bacterial fractions of waters collected from some lakes with high proportion of phages compared to other microorganisms. These findings are consistent with previous observations on the inactivation of human pathogens in freshwater samples were protists were also removed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the relationship between bacteriophage resistance and antiviral activity, we created a collection of 223 bacteria isolates from the 24 high-altitude Swiss lakes and assessed their susceptibility to a bacteriophage cocktail. We identified potential bacteriophage-resistance in 75 out of 223 isolates. The highest percentage of phage resistant bacteria were identified Lake Oeschinsee, Lac de Vaux, Leisee and Lac de Salanfe. Interestingly, Oeschinensee is the lake at the lowest altitude (1580 m), potentially experiencing the least extreme environmental conditions, and Lac Salanfe had the greatest phage to bacteria and eukaryotes ratio. Beyond these observations, we did not find any correlation between the microbial abundances or metabolic profile of these lakes and bacteriophage resistance. Our findings suggest that limited nutrients and high bacteriophage concentrations are not the only characteristics associated to bacteriophage resistance in bacteria populations. It is important to acknowledge that some methodological choices \u0026ndash;including media selection for bacterial isolation, possible absence of phages capable of infecting certain isolates in the Phage Mix [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and reliance on g23 qPCR as an indicator of total phage concentration \u0026ndash;may have influenced our results. Nevertheless, the identified bacterial subset represented promising candidates for further characterization and was used to develop synthetic communities with enhanced viral inactivation activity.\u003c/p\u003e \u003cp\u003eNoteworthy, using these candidates we built two synthetic bacterial communities with enhanced viral inactivation. We found that communities composed of bacteriophage-resistant isolates significantly inactivated E11 and IAV compared to a composed of bacteriophage-susceptible bacteria. This confirmed our working hypothesis and demonstrated for the first time a potential link between phage resistance and viral inactivation is drawn. We tested one of these communities against two plant viruses and found antiviral activity against only one virus (PVX), suggesting that bacterial viral inactivation is partially selective. Identification of taxa present in the synthetic communities revealed bacteria genera previously reported to possess antiviral activity. For example, a protease produced by \u003cem\u003eSerratia marcescens\u003c/em\u003e protected tobacco plants against Tobacco Mosaic Virus (TMV) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and secondary metabolites from \u003cem\u003eBacillus subtilis\u003c/em\u003e exhibit antiviral activity against RNA viruses and a pseudorabies virus (PVR) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While these antiviral compounds have been described in single isolates, their production and specific contribution to virus inactivation in mixed microbial communities remain unknown.\u003c/p\u003e \u003cp\u003eViral inactivation in freshwater was first described by Cliver et al. (1972) and Ward et al. (1986), who showed bacterial antiviral activity against enteroviruses and concluded that viral capsid proteins can be cleaved by proteolytic bacterial enzymes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Other studies on the fish pathogen Koi-Herpesvirus have shown significantly faster inactivation with bacteria present compared to bacteria free samples [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. They also suggest that proteases were likely responsible for the observed inactivation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Here, we were unable to find a correlation between viral inactivation and proteolytic activity of the community members. This suggests that either our assays did not capture the relevant proteases associated to viral inactivation or that additional antibacterial mechanisms\u0026mdash;chemical, enzymatic, or structural\u0026mdash;contribute to viral decay. Further work is needed to elucidate the specific mechanisms underlying viral inactivation by the enhanced synthetic communities described here and to characterize the contribution of each individual species in the community.\u003c/p\u003e \u003cp\u003eIn summary, we developed and applied an innovative strategy to construct two synthetic bacterial communities with enhanced broad-spectrum antiviral activity from environmental samples collected in 24 Swiss alpine lakes. We further demonstrated that alpine lakes exposed to extreme environmental conditions harbor bacterial populations with antiviral properties. This was consistent with our previous observations in lowland lakes such as Lake Geneva. Notably, our results provide the first evidence linking resistance to indigenous environmental bacteriophages with inactivation of viral pathogens infecting diverse hosts, highlighting potential avenues for future biotechnological applications. Future research will aim to elucidate the molecular mechanisms underlying the broad-spectrum viral inactivation reported here.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eAlpine Lake sampling and sample processing.\u003c/b\u003e The lakes chosen for this study were high altitude lakes located in the Swiss Alps above 2000 meters of altitude. All samples were collected between September and November 2021. The list of lakes sampled in this project as well as their sampling date and altitude are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. During each sampling campaign, two liters of surface water were collected using plastic containers made of polycarbonate and a telescopic sampling pole. Samples were kept at 4\u0026deg;C until further processing. Once in the laboratory, one liter of lakewater was filtered through 0.45 \u0026micro;m filters (Millipore\u0026trade;) using a conventional vacuum pump to retain protists, bacteria and bacteriophages (associated with the bacteria and organic matter). Water samples with higher turbidity and rich in suspended particles were first pre-filtered through 8.0 \u0026micro;m filters. The 0.45 \u0026micro;m filters were transferred to Bead Tubes of the DNeasy PowerWater kit (QIAgen, Germany). These tubes were frozen at -20\u0026deg;C until nucleic acid extractions were performed using the DNeasy PowerWater kit (QIAgen, Germany) following the manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMicrobial abundance quantification in alpine lakes.\u003c/b\u003e Quantitative polymerase chain reaction (qPCR) assays were used to determine the abundance of picoeukaryotes, bacteria and bacteriophages targeting 18S, 16S and g23 genes, respectively [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The 16S gene was targeted to quantify the abundance of bacteria using a Femto Bacterial DNA quantification kit (Zymo Research Corporation) following the manufacturer\u0026rsquo;s recommendations. The g23 gene encodes for a capsid protein of tailed bacteriophages and was used as a proxy of bacteriophage abundance given that no universal gene is available for viruses[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Table S2, shows the sequences of all the primers used in this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolic diversity of alpine lake communities.\u003c/b\u003e Biolog EcoPlates\u0026trade; were used to characterize the metabolic profile of bacteria communities found in the studied lakes. Briefly, 1x of M9 minimal media (Sigma-Aldrich) was supplemented with 2mM of MgSO4 (PanReac AppliChem), 0.1 mM of CaCl2 (Thermo Scientific) and 1X of trace metals (Teknova). The content of each well of the EcoPlate\u0026trade; was resuspended in 100 ul of supplemented M9 minimal. Then, 40 ul of lakewater were added on top of each well. Plates were incubated for 5 days at room temperature in the dark. Optical density of each well was measured at a wavelength of 590 nm using a microplate reader (BioTek \u0026ndash; Synergy Mx). Each carbon source is present in triplicate in the EcoPlate\u0026trade;. Average optical density was reported per carbon source for each lake. To calculate metabolic diversity, compounds that yielded an optical density\u0026thinsp;\u0026gt;\u0026thinsp;0.2 at 590 nm were used to calculate a Shannon index per lake using the following formula:\u003c/p\u003e \u003cp\u003eH = \u0026minus; Σ (p\u003csub\u003ei\u003c/sub\u003e * ln(p\u003csub\u003ei\u003c/sub\u003e))\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003eH\u003c/em\u003e is the Shannon diversity index, \u003cem\u003eΣ\u003c/em\u003e is the sum, \u003cem\u003ep\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the proportion of compounds with signal\u0026thinsp;\u0026gt;\u0026thinsp;0.2 nm and ln is the natural logarithm.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePropagation of human and plant viruses.\u003c/b\u003e Echovirus 11 (E11, Gregory strain, ATCC VR737) and Influenza A (IAV) virus were cultured in the laboratory using Buffalo Green Monkey Kidney (BGMK) and Madin-Darby Canine Kidney (MDCK) cells, respectively. Stocks were generated by infecting sub-confluent monolayers of cells, following previously established protocols [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For E11 purification, viral particles were harvested from the infected cells through a series of freeze-thaw cycles. Subsequently, to remove cellular debris, the suspensions underwent centrifugation at 3000 \u0026times; g for 5 minutes. Buffer exchange was performed into PBS using Amicon\u0026reg; Ultra filters in cycles of 4000 \u0026times; g for 10 minutes until viral suspension was clear. For IAV purification, culture supernatant from the infected cells was cleared by centrifugation (2,500 \u0026times; g, 10 min). Virus was concentrated and purified by pelleting through a 30% sucrose cushion at 112\u0026rsquo;400 \u0026times; g in a SW31Ti rotor (Beckman) for 90 min at 4◦C. The resulting pellets were soaked overnight at 4\u0026deg;C in PBS (ThermoFisher, 18912014) and then fully resuspended by pipetting up and down. Pepino Mosaic Virus (PepMV) and Potato Virus X (PVX) were obtained from tobacco plants (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e and \u003cem\u003eNicotiana tabacum Xanthi, respectively\u003c/em\u003e) mechanically inoculated with virus isolates from Agroscope collection in Changins (Nyon, Switzerland). Virus particles were semi-purified as previously described from symptomatic leaves stored at -20\u0026deg;C [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The abundance of particles was evaluated using a Tecnai G2 transmission electron microscope. The particles were diluted in 0.018 M phosphate-citrate buffer at pH 7 when required. All virus suspensions were aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnumeration and quantification of human viruses.\u003c/b\u003e The concentration of infectious viruses was determined using a most probable number (MPN) infectivity assay for E11 and plaque assays for IAV. For MPN, 5 replicates per sample were used as described previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Plaque assays were performed using MDCK cells as described previously [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor quantification of human viruses, qPCR targeting Influenza A Matrix or Echovirus 11 viral protein 1 were performed (Table S2). For both viruses, RNA extractions from samples were performed, using the QIAamp Viral RNA Mini extraction kit (52906, QIAGEN GmbH, Hilden, NW, Germany) according to manufacturer\u0026rsquo;s instructions. For E11 quantification, RT-qPCR was performed on a Mic qPCR Cycler (Bio Molecular Systems) using the One Step SYBR\u0026reg; PrimeScript\u0026trade; RT-PCR Kit (Takara). Each reaction (20 \u0026micro;L) contained 3 \u0026micro;L of template, 10 \u0026micro;L of 2\u0026times; One Step SYBR RT-PCR Buffer III, 0.4 \u0026micro;L of TaKaRa Ex Taq HS (5 U \u0026micro;L\u0026thinsp;\u0026minus;\u0026thinsp;1), 0.4 \u0026micro;L of PrimeScript RT enzyme mix II, 250 nM of each primer and 5.2 \u0026micro;L of water. The thermocycling conditions were as follows: RT at 42\u0026deg;C for 5 min, 10 s at 95\u0026deg;C, followed by 40 cycles of 95\u0026deg;C for 5 s, 52\u0026deg;C for 20 s and 60\u0026deg;C for 30 s. Each sample was run once, and the Cq values were determined using the micPCR software (v2.10.0; Bio Molecular Systems)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For IAV quantification, extracted nucleic acids were eluted in elution buffer, and stored at \u0026minus;\u0026thinsp;20◦C until analysis. Reverse-transcription and amplification were performed using the One Step PrimeScript\u003csup\u003e\u0026trade;\u003c/sup\u003e RT-PCR Kit (RR064A, Takara Bio Inc., Kusatsu, JP-25, Japan) using primers targeting the Influenza A Matrix gene (Table S2). The RT-qPCR mix was composed of 7.5 \u0026micro;L of 2x One-Step SYBR RT-PCR Buffer, 0.3 \u0026micro;L of Takara Ex Taq HS (5 U/\u0026micro;L stock), 0.3 \u0026micro;L PrimeScript RT enzyme mix, 0.3 \u0026micro;L forward and reverse primers (10 \u0026micro;M stocks), and 3.3 \u0026micro;L RNase-free water, to which 3 \u0026micro;L of extracted RNA sample was added. The RT-qPCR was performed in a Mic Real-Time PCR System (Bio MolecularSystems, Upper Coomera, QLD, Australia) with the following parameters: 2 min at 50 \u003csup\u003e◦\u003c/sup\u003eC, 10 min at 95\u0026deg;C, 15 seconds at 95\u0026deg;C followed by 60 sec at 60\u0026deg;C for 40 cycles. A final step from 55\u0026deg;C to 95\u0026deg;C at 0.3\u0026deg;C/s was performed for the melting curve analysis. A Gblock gene fragment (Gblock SA, Ath, WHT, Belgium), as described in Olive et al. (2022), was used to create a standard curve for quantification over a range of 10 to 10\u003csup\u003e7\u003c/sup\u003e genomic copies per microliter (GC/\u0026micro;l)[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eInactivation of human viruses in lakewater.\u003c/b\u003e Inactivation experiments were conducted in lakewater filtered through 0.8 \u0026micro;m (Millipore\u0026trade;) (to remove heterotrophic protists) as described above. Specifically, 50 ml of filtered water were spiked with 100 \u0026micro;l of IAV and E11 to the same initial concentration (10\u003csup\u003e5\u003c/sup\u003e genome copies per ml). Then, 5 ml of spiked water was placed in 6-well plates in three replicates. The plates were incubated for 7 days at room temperature and 500 \u0026micro;l aliquots were taken on days 0, 3 and 7. These aliquots were frozen at -20\u0026deg;C until analyzed. Viral nucleic acids were extracted using the QIAamp Viral RNA Mini extraction kit (52906, QIAGEN GmbH, Hilden, NW, Germany) and virus concentrations were determined by qPCR throughout the experiment or by cell culturing as described above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDevelopment of synthetic bacteria communities with antiviral activity.\u003c/b\u003e To develop synthetic bacteria communities with enhanced antiviral activity, we first aimed to identify specific bacteria species with antiviral properties in the lake water samples. To this end, we built a culture collection of isolates from all the sampled lakes. One liter of water from each lake was 0.45 \u0026#120583;m filtered. Filters were transferred to LB agar plates and incubated at room temperature for 72 hours. Then, single colonies were transferred to new LB plates. Serial transfers of isolated colonies into sterile LB plates were conducted until pure cultures were obtained. A total number of 223 pure bacterial isolates were obtained using this methodology. LB media was purchased from Invitrogen. Bacteria isolates were identified performing 16S rRNA gene sequencing using the primers 27 F and 786 R (Table S2). Samples were sent for conventional Sanger sequencing to Fasteris (Genesupport SA, Geneva, Switzerland).\u003c/p\u003e \u003cp\u003eTo identify which of the obtained isolates showed antiviral activity, we conducted phage susceptibility tests using the bacteriophage fraction obtained from each studied lake. Specifically, phage enrichments were conducted for each bacteria isolate obtained in this study (n\u0026thinsp;=\u0026thinsp;223) following state-of-the-art protocols [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The phage cocktail used to infect each bacteria isolate was a 1:1 mixture of all the control waters obtained from each lake which is called \u0026ldquo;Phage Mix\u0026rdquo; hereafter. This process was done to increase the chances for at least one phage type in the mix to be capable of infecting each bacteria isolate. To assess bacterial susceptibility to the \u0026ldquo;Phage Mix\u0026rdquo;, fresh cultures of each bacteria isolate were grown overnight in 5 ml of LB. Once bacteria cultures had an optical density at 600 nm around 0.6\u0026ndash;0.8 (exponential phase), they were spiked with 1 ml of the phage mix. The tubes were then incubated at room temperature for 15 min and then gently agitated (50 rpm) for 2 more hours. Then, chloroform was added to the bacteria-phage co-cultures (hereafter named \u0026ldquo;Phage enrichments\u0026rdquo;) which were centrifuged for 10 minutes at 5000 rpm and filtered at 0.2 \u0026micro;m to remove all bacteria. This process was done for each bacteria isolate.\u003c/p\u003e \u003cp\u003eTo compare phage susceptibility of bacteria isolates, the phage titer yield in the \u0026ldquo;Phage Mix\u0026rdquo; and in each enrichment was determined by qPCR targeting the g23 gene [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Bacteria cultures with phage concentrations equal or higher compared to the phage mix were considered \u0026ldquo;susceptible\u0026rdquo; and retained to build the control communities, while those yielding inferior concentrations were considered \u0026ldquo;resistant\u0026rdquo; and retained for the design of two different synthetic communities with enhanced antiviral properties. This approach assumes that resistant bacteria limit phage replication, so lower phage titers in the enrichment experiments reflect the bacteria\u0026rsquo;s ability to inhibit phage proliferation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlpine synthetic communities against human and plant viruses.\u003c/b\u003e To confirm the enhanced antiviral properties of the obtained synthetic communities, three groups of 25 bacterial isolates (2 synthetic communities with enhanced activity and one control group) were mixed and used to assess their inactivation activity against E11 and IAV. Before the inactivation assays, isolates were independently grown in LB overnight at room temperature to avoid competition and were mixed in equal parts in a final volume of 50 ml. Once the bacteria isolates were mixed together, inactivation experiments against E11 and IAV were conducted as previously described. Two bacterial pools were composed of 25 unique bacterial isolates with decreased susceptibility to the \u0026ldquo;Phage Mix\u0026rdquo; and one control was composed of 25 bacteria isolates with increased phage susceptibility. Specifically, 50 ml of mixed bacteria isolates were diluted 100x using sterile lakewater and spiked with 100 \u0026micro;l of IAV and E11 to the same initial concentration (10\u003csup\u003e5\u003c/sup\u003e genome copies per ml). Then, 5 ml of spiked media with the isolates was placed in 6-well plates in five replicates. The plates were incubated for 4 days at room temperature. For inactivation assays against plant viruses, the most inactivating community against human viruses was selected. All the community members were grown as previously described and mixed the day of the assay. Previously purified PepMV and PVX viruses were independently spiked into the bacteria solution and incubated at room temperature for four days. After incubation, the viral-bacterial solutions were filtered sterilized to remove bacteria and used to infect 75 tobacco plants as described [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A control group was simultaneously inoculated with a viral solution incubated for 4 days at room temperature in the absence of bacteria. Inoculated plants were maintained under controlled greenhouse conditions (approx. 20 days). A group of uninoculated plants were placed in the same green house as plant controls. In addition, a group of 16 plants were inoculated only with the bacterial pool as a control of bacteria pathogenesis. Virus detection was performed an ELISA test with antibodies from Bioreba AG (Reinach, Switzerland), with two leaves taken from each plant [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The infection threshold was determined as the mean value of the non-inoculated controls plus three standard deviations, following established methodology. For PVX and PepMV, this corresponds to 0.16 and 0.28 absorbance units, respectively, after 18 hours of incubation. The infected plants were further divided into two groups: one consisting of highly infected plants, whose absorbance signal exceeded the upper detection limit of the ELISA plate reader (4 units), and another consisting of moderately infected plants, whose absorbance signal fell between the infection threshold and the upper detection limit.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S amplicon sequencing of alpine synthetic communities.\u003c/b\u003e Once bacterial isolates were mixed, a sample from each community were taken for DNA extraction and 16S amplicon sequencing. Sample was 0.22 \u0026micro;m filtered and DNA from filters was extracted using DNeasy PowerWater Kit (Qiagen, Hilden, Germany) as previously described. DNA extracts and blanks were then used for 16S rRNA gene amplification using the 27F and 1492R (Table S2). The PCR reaction was carried out in a 50-\u0026micro;L volume containing 20 ng of input DNA and 25 \u0026micro;L of LongAmp Taq 2\u0026times; master mix (New England Biolabs, Ipswich, MS, USA). Each PCR product was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using Qubit\u0026trade; (dsDNA quantification, Broad Range). The sequencing library was prepared using the Native Barcoding Kit SQK-NBD114.24 (Oxford Nanopore Technologies, Oxford, UK) following the manufacturers\u0026rsquo; instructions. The library was loaded into a FLO-MIN106 flow cell (Oxford Nanopore Technologies) and sequenced using a MinION Mk1C sequencing device for 72 hours. Sequencing files were basecalled using Dorado (v0.9.1) basecaller. FASTQ files were input into the MetONTIIME workflow [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] to estimate bacterial relative abundance and assign taxonomy. Feature tables and count matrix were imported into R and analysed using the \u003cem\u003emicroeco\u003c/em\u003e package [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analysis and visualization.\u003c/b\u003e Analysis of all the data obtained during the project was done with the \u003cem\u003edyplr\u003c/em\u003e and \u003cem\u003etidyverse\u003c/em\u003e packages RStudio [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. All the plots were obtained in Rstudio using the \u003cem\u003eggplot\u003c/em\u003e package [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Correlation matrices and statistical analysis were performed using the Pearson correlation coefficient function in RStudio.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING AND ACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the project ANTIVIRALPS (\u0026ldquo;Screening for novel antiviral compounds in alpine and polar lakes\u0026rdquo;) funded by the EPFL Science Seed Fund 2021, and the project HYDROVIR (\u0026ldquo;Optimizing the use of alpine lake water bacteria with antiviral properties to combat viral diseases in hydroponic cultures\u0026rdquo;) funded by the EPFL ENABLE Grant, 2022. The authors gratefully acknowledge Nathalie Dubuis for her assistance with plant virus preparation and sample analysis in Agroscope.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eN. P. Adhikari, S. Adhikari, X. Liu, L. Shen, and Z. Gu, \u0026ldquo;Bacterial Diversity in Alpine Lakes: A Review from the Third Pole Region,\u0026rdquo; \u003cem\u003eJ. Earth Sci.\u003c/em\u003e, vol. 30, no. 2, pp. 387\u0026ndash;396, Apr. 2019, doi: 10.1007/s12583-018-1206-5.\u003c/li\u003e\n\u003cli\u003eH. Peter and R. Sommaruga, \u0026ldquo;Shifts in diversity and function of lake bacterial communities upon glacier retreat,\u0026rdquo; \u003cem\u003eISME J.\u003c/em\u003e, vol. 10, no. 7, pp. 1545\u0026ndash;1554, July 2016, doi: 10.1038/ismej.2015.245.\u003c/li\u003e\n\u003cli\u003eH. Br\u0026uuml;ssow, \u0026ldquo;Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity,\u0026rdquo; \u003cem\u003eMol. Microbiol.\u003c/em\u003e, vol. 65, no. 3, pp. 583\u0026ndash;589, Aug. 2007, doi: 10.1111/j.1365-2958.2007.05826.x.\u003c/li\u003e\n\u003cli\u003eJ. C. Vogt, J. L. Olefeld, C. Bock, J. Boenigk, and D. C. Albach, \u0026ldquo;Patterns of protist distribution and diversification in alpine lakes across Europe,\u0026rdquo; \u003cem\u003eMicrobiologyOpen\u003c/em\u003e, vol. 10, no. 4, p. e1216, Aug. 2021, doi: 10.1002/mbo3.1216.\u003c/li\u003e\n\u003cli\u003eE. Jończyk, M. Kłak, R. Międzybrodzki, and A. G\u0026oacute;rski, \u0026ldquo;The influence of external factors on bacteriophages\u0026mdash;review,\u0026rdquo; \u003cem\u003eFolia Microbiol. (Praha)\u003c/em\u003e, vol. 56, no. 3, pp. 191\u0026ndash;200, May 2011, doi: 10.1007/s12223-011-0039-8.\u003c/li\u003e\n\u003cli\u003eT. Sime-Ngando, \u0026ldquo;Environmental bacteriophages: viruses of microbes in aquatic ecosystems,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 5, July 2014, doi: 10.3389/fmicb.2014.00355.\u003c/li\u003e\n\u003cli\u003eK. E. Wommack and R. R. Colwell, \u0026ldquo;Virioplankton: Viruses in Aquatic Ecosystems,\u0026rdquo; \u003cem\u003eMicrobiol. Mol. Biol. Rev.\u003c/em\u003e, vol. 64, no. 1, pp. 69\u0026ndash;114, Mar. 2000, doi: 10.1128/MMBR.64.1.69-114.2000.\u003c/li\u003e\n\u003cli\u003eC. A. Suttle, \u0026ldquo;Marine viruses \u0026mdash; major players in the global ecosystem,\u0026rdquo; \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e, vol. 5, no. 10, pp. 801\u0026ndash;812, Oct. 2007, doi: 10.1038/nrmicro1750.\u003c/li\u003e\n\u003cli\u003eJ. E. Samson, A. H. Magad\u0026aacute;n, M. Sabri, and S. Moineau, \u0026ldquo;Revenge of the phages: defeating bacterial defences,\u0026rdquo; \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e, vol. 11, no. 10, pp. 675\u0026ndash;687, Oct. 2013, doi: 10.1038/nrmicro3096.\u003c/li\u003e\n\u003cli\u003eA. Stern and R. Sorek, \u0026ldquo;The phage‐host arms race: Shaping the evolution of microbes,\u0026rdquo; \u003cem\u003eBioEssays\u003c/em\u003e, vol. 33, no. 1, pp. 43\u0026ndash;51, Jan. 2011, doi: 10.1002/bies.201000071.\u003c/li\u003e\n\u003cli\u003eF. Tesson \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Systematic and quantitative view of the antiviral arsenal of prokaryotes,\u0026rdquo; \u003cem\u003eNat. Commun.\u003c/em\u003e, vol. 13, no. 1, p. 2561, May 2022, doi: 10.1038/s41467-022-30269-9.\u003c/li\u003e\n\u003cli\u003eS. Doron \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Systematic discovery of antiphage defense systems in the microbial pangenome,\u0026rdquo; \u003cem\u003eScience\u003c/em\u003e, vol. 359, no. 6379, p. eaar4120, Mar. 2018, doi: 10.1126/science.aar4120.\u003c/li\u003e\n\u003cli\u003eT. Goldfarb \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;BREX is a novel phage resistance system widespread in microbial genomes,\u0026rdquo; \u003cem\u003eEMBO J.\u003c/em\u003e, vol. 34, no. 2, pp. 169\u0026ndash;183, Jan. 2015, doi: 10.15252/embj.201489455.\u003c/li\u003e\n\u003cli\u003eA. Bernheim \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Prokaryotic viperins produce diverse antiviral molecules,\u0026rdquo; \u003cem\u003eNature\u003c/em\u003e, vol. 589, no. 7840, pp. 120\u0026ndash;124, Jan. 2021, doi: 10.1038/s41586-020-2762-2.\u003c/li\u003e\n\u003cli\u003eS. Kronheim \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A chemical defence against phage infection,\u0026rdquo; \u003cem\u003eNature\u003c/em\u003e, vol. 564, no. 7735, pp. 283\u0026ndash;286, Dec. 2018, doi: 10.1038/s41586-018-0767-x.\u003c/li\u003e\n\u003cli\u003eB. J. O\u0026rsquo;Hara, Z. K. Barth, A. C. McKitterick, and K. D. Seed, \u0026ldquo;A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome,\u0026rdquo; \u003cem\u003ePLOS Genet.\u003c/em\u003e, vol. 13, no. 6, p. e1006838, June 2017, doi: 10.1371/journal.pgen.1006838.\u003c/li\u003e\n\u003cli\u003eK. J. Helbig and M. R. Beard, \u0026ldquo;The Role of Viperin in the Innate Antiviral Response,\u0026rdquo; \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e, vol. 426, no. 6, pp. 1210\u0026ndash;1219, Mar. 2014, doi: 10.1016/j.jmb.2013.10.019.\u003c/li\u003e\n\u003cli\u003eR. E. Baker \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Infectious disease in an era of global change,\u0026rdquo; \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e, vol. 20, no. 4, pp. 193\u0026ndash;205, Apr. 2022, doi: 10.1038/s41579-021-00639-z.\u003c/li\u003e\n\u003cli\u003eT. H. Bacon, M. J. Levin, J. J. Leary, R. T. Sarisky, and D. Sutton, \u0026ldquo;Herpes Simplex Virus Resistance to Acyclovir and Penciclovir after Two Decades of Antiviral Therapy,\u0026rdquo; \u003cem\u003eClin. Microbiol. Rev.\u003c/em\u003e, vol. 16, no. 1, pp. 114\u0026ndash;128, Jan. 2003, doi: 10.1128/CMR.16.1.114-128.2003.\u003c/li\u003e\n\u003cli\u003eA. Carratal\u0026agrave;, H. Shim, Q. Zhong, V. Bachmann, J. D. Jensen, and T. Kohn, \u0026ldquo;Experimental adaptation of human echovirus 11 to ultraviolet radiation leads to resistance to disinfection and ribavirin,\u0026rdquo; \u003cem\u003eVirus Evol.\u003c/em\u003e, vol. 3, no. 2, July 2017, doi: 10.1093/ve/vex035.\u003c/li\u003e\n\u003cli\u003eQ. Zhong, A. Carratal\u0026agrave;, H. Shim, V. Bachmann, J. D. Jensen, and T. Kohn, \u0026ldquo;Resistance of Echovirus 11 to ClO\u003csub\u003e2\u003c/sub\u003e Is Associated with Enhanced Host Receptor Use, Altered Entry Routes, and High Fitness,\u0026rdquo; \u003cem\u003eEnviron. \u003c/em\u003e\u003cem\u003eSci. Technol.\u003c/em\u003e, vol. 51, no. 18, pp. 10746\u0026ndash;10755, Sept. 2017, doi: 10.1021/acs.est.7b03288.\u003c/li\u003e\n\u003cli\u003eC. S. Adamson, K. Chibale, R. J. M. Goss, M. Jaspars, D. J. Newman, and R. A. Dorrington, \u0026ldquo;Antiviral drug discovery: preparing for the next pandemic,\u0026rdquo; \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e, vol. 50, no. 6, pp. 3647\u0026ndash;3655, 2021, doi: 10.1039/D0CS01118E.\u003c/li\u003e\n\u003cli\u003eD. Cliver and J. Herrmann, \u0026ldquo;Proteolytic and microbial inactivation of enteroviruses,\u0026rdquo; \u003cem\u003eWater Res.\u003c/em\u003e, vol. 6, no. 7, pp. 797\u0026ndash;805, July 1972, doi: 10.1016/0043-1354(72)90032-2.\u003c/li\u003e\n\u003cli\u003eR. L. Ward, D. R. Knowlton, and P. E. Winston, \u0026ldquo;Mechanism of inactivation of enteric viruses in fresh water,\u0026rdquo; \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e, vol. 52, no. 3, pp. 450\u0026ndash;459, Sept. 1986, doi: 10.1128/aem.52.3.450-459.1986.\u003c/li\u003e\n\u003cli\u003eM.-H. Corre, V. Bachmann, and T. Kohn, \u0026ldquo;Bacterial matrix metalloproteases and serine proteases contribute to the extra-host inactivation of enteroviruses in lake water,\u0026rdquo; \u003cem\u003eISME J\u003c/em\u003e, vol. 16, no. 8, pp. 1970\u0026ndash;1979, Aug. 2022, doi: 10.1038/s41396-022-01246-3.\u003c/li\u003e\n\u003cli\u003eW. Chaisuwan \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;The Antiviral Activity of Bacterial, Fungal, and Algal Polysaccharides as Bioactive Ingredients: Potential Uses for Enhancing Immune Systems and Preventing Viruses,\u0026rdquo; \u003cem\u003eFront. Nutr.\u003c/em\u003e, vol. 8, p. 772033, Nov. 2021, doi: 10.3389/fnut.2021.772033.\u003c/li\u003e\n\u003cli\u003eM. Olive, C. Gan, A. Carratal\u0026agrave;, and T. Kohn, \u0026ldquo;Control of Waterborne Human Viruses by Indigenous Bacteria and Protists Is Influenced by Temperature, Virus Type, and Microbial Species,\u0026rdquo; \u003cem\u003eAppl Env. Microbiol\u003c/em\u003e, vol. 86, no. 3, pp. e01992-19, Jan. 2020, doi: 10.1128/AEM.01992-19.\u003c/li\u003e\n\u003cli\u003eA. Romanenko, H. Peter, J. Meibom, M. A. Borchardt, and T. Kohn, \u0026ldquo;Diversity of lake bacteria promotes human echovirus inactivation,\u0026rdquo; \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e, vol. 91, no. 2, pp. e02366-24, Feb. 2025, doi: 10.1128/aem.02366-24.\u003c/li\u003e\n\u003cli\u003eR. Wang \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Bacterial and Archaeal Communities within an Ultraoligotrophic, High-altitude Lake in the Pre-Himalayas of the Qinghai-Tibet Plateau,\u0026rdquo; \u003cem\u003eIndian J. Microbiol.\u003c/em\u003e, vol. 60, no. 3, pp. 363\u0026ndash;373, Sept. 2020, doi: 10.1007/s12088-020-00881-8.\u003c/li\u003e\n\u003cli\u003eR. S. Fujioka, P. C. Loh, and L. S. Lau, \u0026ldquo;Survival of Human Enteroviruses in the Hawaiian Ocean Environment: Evidence for Virus-Inactivating Microorganismst,\u0026rdquo; \u003cem\u003eAPPL Env. MICROBIOL\u003c/em\u003e, vol. 39, 1980.\u003c/li\u003e\n\u003cli\u003eS. A. Gatea Kaabi and H. K. Musafer, \u0026ldquo;New Phage cocktail against infantile Sepsis bacteria,\u0026rdquo; \u003cem\u003eMicrob. Pathog.\u003c/em\u003e, vol. 148, p. 104447, Nov. 2020, doi: 10.1016/j.micpath.2020.104447.\u003c/li\u003e\n\u003cli\u003eY. Qin \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Purification and Characterization of a Secretory Alkaline Metalloprotease with Highly Potent Antiviral Activity from Serratia marcescens Strain S3,\u0026rdquo; \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e, vol. 67, no. 11, pp. 3168\u0026ndash;3178, Mar. 2019, doi: 10.1021/acs.jafc.8b06909.\u003c/li\u003e\n\u003cli\u003eX. Wang, G. Hao, M. Zhou, M. Chen, H. Ling, and Y. Shang, \u0026ldquo;Secondary metabolites of Bacillus subtilis L2 show antiviral activity against pseudorabies virus,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 14, p. 1277782, Oct. 2023, doi: 10.3389/fmicb.2023.1277782.\u003c/li\u003e\n\u003cli\u003eL. Yuan, S. Zhang, J. Peng, Y. Li, and Q. Yang, \u0026ldquo;Synthetic surfactin analogues have improved anti-PEDV properties,\u0026rdquo; \u003cem\u003ePLOS ONE\u003c/em\u003e, vol. 14, no. 4, p. e0215227, Apr. 2019, doi: 10.1371/journal.pone.0215227.\u003c/li\u003e\n\u003cli\u003eD. O. Cliver, \u0026ldquo;Experimental Infection by Waterborne Enteroviruses,\u0026rdquo; \u003cem\u003eJ. Food Prot.\u003c/em\u003e, vol. 44, no. 11, pp. 861\u0026ndash;865, Nov. 1981, doi: 10.4315/0362-028X-44.11.861.\u003c/li\u003e\n\u003cli\u003eA. Amtmann \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Virucidal effects of various agents\u0026mdash;including protease\u0026mdash;against koi herpesvirus and viral haemorrhagic septicaemia virus,\u0026rdquo; \u003cem\u003eJ. Fish Dis.\u003c/em\u003e, vol. 43, no. 2, pp. 185\u0026ndash;195, Feb. 2020, doi: 10.1111/jfd.13106.\u003c/li\u003e\n\u003cli\u003eT. Shimizu, N. Yoshida, H. Kasai, and M. Yoshimizu, \u0026ldquo;Survival of Koi Herpesvirus (KHV) in Environmental Water,\u0026rdquo; \u003cem\u003eFish Pathol.\u003c/em\u003e, vol. 41, no. 4, pp. 153\u0026ndash;157, 2006, doi: 10.3147/jsfp.41.153.\u003c/li\u003e\n\u003cli\u003eL. A. Amaral-Zettler, E. A. McCliment, H. W. Ducklow, and S. M. Huse, \u0026ldquo;A Method for Studying Protistan Diversity Using Massively Parallel Sequencing of V9 Hypervariable Regions of Small-Subunit Ribosomal RNA Genes,\u0026rdquo; \u003cem\u003ePLoS ONE\u003c/em\u003e, vol. 4, no. 7, p. e6372, July 2009, doi: 10.1371/journal.pone.0006372.\u003c/li\u003e\n\u003cli\u003eT. Stoeck \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water,\u0026rdquo; \u003cem\u003eMol. \u003c/em\u003e\u003cem\u003eEcol.\u003c/em\u003e, vol. 19, no. s1, pp. 21\u0026ndash;31, Mar. 2010, doi: 10.1111/j.1365-294X.2009.04480.x.\u003c/li\u003e\n\u003cli\u003eF. Zhu, R. Massana, F. Not, D. Marie, and D. Vaulot, \u0026ldquo;Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene,\u0026rdquo; \u003cem\u003eFEMS Microbiol. Ecol.\u003c/em\u003e, vol. 52, no. 1, pp. 79\u0026ndash;92, Mar. 2005, doi: 10.1016/j.femsec.2004.10.006.\u003c/li\u003e\n\u003cli\u003eM. Mahillon, J. Brodard, I. Kellenberger, A. G. Blouin, and O. Schumpp, \u0026ldquo;A novel weevil-transmitted tymovirus found in mixed infection on hollyhock,\u0026rdquo; \u003cem\u003eVirol. J.\u003c/em\u003e, vol. 20, no. 1, p. 17, Jan. 2023, doi: 10.1186/s12985-023-01976-6.\u003c/li\u003e\n\u003cli\u003eS. C. David \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Inactivation mechanisms of influenza A virus under pH conditions encountered in aerosol particles as revealed by whole-virus HDX-MS,\u0026rdquo; \u003cem\u003emSphere\u003c/em\u003e, vol. 8, no. 5, pp. e00226-23, Oct. 2023, doi: 10.1128/msphere.00226-23.\u003c/li\u003e\n\u003cli\u003eO. Lariv\u0026eacute;, J. Brandani, M. Dubey, and T. Kohn, \u0026ldquo;An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes,\u0026rdquo; \u003cem\u003eJ. Virol. Methods\u003c/em\u003e, vol. 296, p. 114225, Oct. 2021, doi: 10.1016/j.jviromet.2021.114225.\u003c/li\u003e\n\u003cli\u003eG. Motos \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Dependence of aerosol-borne influenza A virus infectivity on relative humidity and aerosol composition,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 15, p. 1484992, Oct. 2024, doi: 10.3389/fmicb.2024.1484992.\u003c/li\u003e\n\u003cli\u003eN. \u0026Aacute;cs, M. Gambino, and L. Br\u0026oslash;ndsted, \u0026ldquo;Bacteriophage Enumeration and Detection Methods,\u0026rdquo; \u003cem\u003eFront. Microbiol.\u003c/em\u003e, vol. 11, p. 594868, Oct. 2020, doi: 10.3389/fmicb.2020.594868.\u003c/li\u003e\n\u003cli\u003eM. R. J. Clokie and A. M. Kropinski, Eds., \u003cem\u003eBacteriophages\u003c/em\u003e, vol. 501. in Methods in Molecular Biology, vol. 501. Totowa, NJ: Humana Press, 2009. doi: 10.1007/978-1-60327-164-6.\u003c/li\u003e\n\u003cli\u003eA. N. Adams and M. F. Clark, \u0026ldquo;Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses,\u0026rdquo; \u003cem\u003eJ. Gen. Virol.\u003c/em\u003e, vol. 34, no. 3, pp. 475\u0026ndash;483, Mar. 1977, doi: 10.1099/0022-1317-34-3-475.\u003c/li\u003e\n\u003cli\u003eA. Matoute \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Meat-Borne-Parasite: A Nanopore-Based Meta-Barcoding Work-Flow for Parasitic Microbiodiversity Assessment in the Wild Fauna of French Guiana,\u0026rdquo; \u003cem\u003eCurr. Issues Mol. Biol.\u003c/em\u003e, vol. 46, no. 5, pp. 3810\u0026ndash;3821, Apr. 2024, doi: 10.3390/cimb46050237.\u003c/li\u003e\n\u003cli\u003eC. Liu, Y. Cui, X. Li, and M. Yao, \u0026ldquo;\u003cem\u003emicroeco\u003c/em\u003e : an R package for data mining in microbial community ecology,\u0026rdquo; \u003cem\u003eFEMS Microbiol. Ecol.\u003c/em\u003e, vol. 97, no. 2, p. fiaa255, Jan. 2021, doi: 10.1093/femsec/fiaa255.\u003c/li\u003e\n\u003cli\u003eH. Wickham \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Welcome to the Tidyverse,\u0026rdquo; \u003cem\u003eJ. Open Source Softw.\u003c/em\u003e, vol. 4, no. 43, p. 1686, Nov. 2019, doi: 10.21105/joss.01686.\u003c/li\u003e\n\u003cli\u003eHadley Wickham, \u003cem\u003eggplot2: Elegant Graphics for Data Analysis\u003c/em\u003e. Springer-Verlag New York, 2016.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alpine lakes, microbial communities, viral inactivation, bacteriophage resistance, synthetic communities","lastPublishedDoi":"10.21203/rs.3.rs-8367630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8367630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn aquatic ecosystems, phagotrophic protists and bacteriophages are major biological factors shaping bacterial populations. However, the extreme environmental conditions and low nutrient concentrations in alpine lakes limit the abundance of protists. Thus, bacteriophages may represent the main biological factor shaping bacterial communities in these ecosystems. We hypothesized that alpine lake bacteria populations harbor diverse antiviral strategies against bacteriophages that could be used to fight against other viral pathogens. To evaluate the presence of bacteria with broad-spectrum antiviral activity in high altitude Swiss lakes, we developed a sampling campaign in 24 alpine lakes in Switzerland. We assessed the decay of two human viruses (echovirus 11 and influenza A virus) in lakewater and characterized the microbial abundance and metabolic diversity in the collected samples. In addition, we obtained a collection of 223 bacterial isolates and selected bacteriophage resistant bacteria to build synthetic communities with enhanced antiviral activity against the two tested human viruses, and one plant virus of commercial significance (Potato Virus X). This confirmed our hypothesis and drew for the first time a relation between bacteriophage resistance and viral inactivation. Our findings emphasize the importance of alpine lakes as hotspots of microbial diversity with unexplored biotechnological applications.\u003c/p\u003e","manuscriptTitle":"Harnessing Alpine Lake Bacteria for the Development of Synthetic Communities with Broad-Spectrum Antiviral Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 08:25:34","doi":"10.21203/rs.3.rs-8367630/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43b72700-2c64-4d44-a824-03a3521c26c4","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60135359,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":60135360,"name":"Biological sciences/Microbiology/Antimicrobials/Antiviral agents"}],"tags":[],"updatedAt":"2026-02-16T15:01:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 08:25:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8367630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8367630","identity":"rs-8367630","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}