Elvenn A Loret

Monica Lluesma

Aurianne Cassien

David Brinvillier

Pierre Henri Villard

Erwann P Loret
email: erwann.loret@univ-amu.fr

Erwann Loret

A marine virus controls the green algae Ulva lactuca proliferation in the bay of Marseille

Keywords: Ulva lactuca, green tides, nitrate, sea water, virus

Background. Ulva lactuca, a green macro algae, is a worldwide problem due to green tides observed in Western Europe (particularly in Brittany, west of France) and in Eastern China. It is a nitrophilic algae and surprisingly Ulva proliferation is not observed in the bay of Marseille (Provence, south of France) despite abundant sources of nitrate due to the Rhône River.

Methods and Results

. In this study we analyzed possible reasons why Ulva proliferation stops rapidly at the end of winter in the bay of Marseille. We show that nitrate concentration at Marseille is an average of 6 + 2 µM and this concentration is similar to what is observed in Brittany. Ulva collected in Brittany can grow and change its phenotype with sea water samples collected in Provence in winter but Breton Ulva become white (bleaching) with sea water samples collected in Provence in June 2018, 2019 and 2020. Breton Ulva bleaching is still observed after 0.2 µm sea water filtration and DAD HPLC shows a peak with a viral particle UV spectrum increasing with Ulva bleaching. Furthermore, epifluorescence microscopy analysis of SYBR-Gold pigment stain with 0.2 µm filtered sea water shows that virus-like particles increase to 6.5 million per ml with Ulva bleaching.

Conclusion.

This study suggests that a virus in bay of Marseille sea water induces Ulva bleaching and controls its proliferation. Sea water containing this virus, which is not harmful for mammals or other species including algae that are not Ulva, may be used to control Ulva proliferation.

Introduction

Ulva lactuca is a green macro alga that belongs to the phylum Chlorophyta [START_REF] Wichard | The green seaweed Ulva: a model system to study morphogenesis[END_REF]. U. lactuca called "sea lettuce" was first described by Linnaeus in the Baltic Sea in the eighteen century and has a belayed cell structure with a talus having a flat bladelike appearance [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. This alga is able to grow both with a holdfast on rocks or free floating. U. lactuca has also the capacity to reproduce with two methods, one being sexual and the other being from fragmentation of the talus [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. These two methods provide a capacity to rapidly proliferate and to cover the water surface decreasing the biodiversity even for other algae species [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. U. lactuca is a polymorphic alga regarding the degree of water salinity [START_REF] Hayden | Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera[END_REF] or symbiosis with bacteria [START_REF] Wichard | The green seaweed Ulva: a model system to study morphogenesis[END_REF]. This polymorphism led some authors to propose that different species may exist such as U. armoricana, U. fasciata, U. pertusa, U. prolifera, U. rigida, U. rotundata, etc… but a genetic analysis revealed that these different phenotypes were not due to genetic variations that would justify that different species other than U. lactuca may exist [START_REF] Rybak | Species of Ulva (Ulvophyceae, Chlorophyta) as indicators of salinity[END_REF]. These different phenotypes should be described as variants or clades of U. lactuca [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. We made the choice to use Ulva for the different U. lactuca clades used in this study. Ulva blooms are a great source of trouble in Brittany (west of France) because they damage marine ecosystems, impair local tourism and Ulva are difficult to valorize when they are collected on beaches [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. Ulva accumulate on beaches and its biodegradation produce acidic vapors (mainly H2S) that are deadly for animals and potentially for human beings [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. Another human health problem is due to amoeba proliferation on beaches. Amoeba is the natural scavenger for Ulva biodegradation and Amoebiasis is the second leading cause of death from parasitic disease worldwide [5]. Ulva is a nitrophilic algae and the reason of its proliferation was proposed to be due to intensive agriculture and fertilizers as a source of nitrate. However, the first Ulva bloom to be described was at Belfast (North of Ireland) at the end of the nineteen century and was due essentially to an increase of human wastes that are also rich in nitrates [START_REF] Letts | Report on green seaweeds and especially Ulva latissima in relation to the pollution of the waters in which they occur[END_REF]. Ulva blooms were studied in the Laguna of Venice from 1930 and a correlation was established with the increase of tourism and human wastes [START_REF] Schiffner | The algaee of the lagoon: Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae[END_REF][START_REF] Curiel | Macroalgael biomass and species variations in the lagoon of Venice (Northern Adriatic Sea, Italy): 1981-1998[END_REF]. An isotope study following a cyanobacteria proliferation in Florida revealed that the main source of nitrates was from human wastes instead of fertilizers [START_REF] Lapointe | Septic systems contribute to nutrient pollution and harmful algael blooms in the St. Lucie Estuary, Southeast Florida[END_REF]. Although green tides due to Ulva proliferation are observed worldwide, the Mediterranean seas appear to be protected from green tides due to Ulva proliferation or blooms. Even in the Laguna of Venice, Ulva blooms occur only during short periods for springtime since 1990 despite tourism and fertilizers carried through the Pô River [START_REF] Curiel | Macroalgael biomass and species variations in the lagoon of Venice (Northern Adriatic Sea, Italy): 1981-1998[END_REF]. Ulva blooms were never observed in the bay of Marseille. Marseille is located nearby the mouth of the Rhône River, which is a big river carrying large amounts of nitrate with a concentration up to 100 µM and an average flow rate of 9m 3 /sec [START_REF] Moriarty | The roles of resuspension, diffusion and biogeochemical processes on oxygen dynamics offshore of the Rhône River, France: a numerical modeling study[END_REF]. This study selected three main hypotheses to explain the control of Ulva proliferation. The first one is that the nitrate concentration in the bay of Marseille is much lower compare to Brittany coastal water. The second one is that Ulva in Brittany could have specificities to proliferate in Brittany and could not survive in Mediterranean seas. The third one is that a pollutant or a living organism could limit Ulva proliferation in Provence.

Materials & Methods

Sea water analyses and Ulva bleaching: Sea water samples were collected in surface in June 15 th and June 18 th 2018, April 15 th , June 4 th and June 10 th 2019 and June 8 th and June 12 th 2020 (n=7) in eight different spots in Brittany and Provence (Table 1). In Brittany (BR and PO) and in Provence (RN, WF, RS) were open coastal sea water but TR in Brittany is a fjord, PR in Provence is a marina and MU in Provence is a fjord (Calanque). Conductimetry was carried out with the INOLAB device (Weither, Germany). Acidity was tested with a CRISON pH Meter (Barcelona, Catalonia, Spain). The pH meter was calibrated before any measurement. Nitrate were measured with a METRHOM chromato ionic device (Berne, Switzerland) with a Metrosep column A supp 5 150/4 mm with 3.2 mM Na2CO3/1 mM NaHCO3 as solvent. Sea Water was diluted 1/8 and a standard was used to calibrate nitrates. Ulva were collected mainly for springtime in the Trieux fjord (TR) in the north coast of Brittany (48°46' N, 3°6'O). The green tubular algae (figure 1) formerly called Enteromorpha was collected in the same TR spot on November 2018. The incubation was carried out for one month at 20°C and day light exposure with sea water collected in June 2018 in the north bay (RN) of Marseille (43°18' N 5°16'E). For Ulva bleaching experiment, Ulva were collected at TR in June 15 th 2018, June 4 th 2019 and June 8 th 2020, then immediately sent in Provence. When Ulva were shipped at Marseille, they were green and healthy. They were cut in pieces of 1 cm 2 and put in sea water (40 ml) in tubes (n=25 for each spot) closed with a tape and let at day light (12 hours) with an average temperature of 25°C for one week. Controls were made with seawater collected in Brittany the very same day when Ulva were collected. Other experiments were carried out with Ulva collected in Provence at MU on April 15 th 2019 and Ulva collected in South Brittany (PO) on June 8 th 2020 (data not shown) Optical microscopy: Optical microscopy (10X) was performed on healthy Ulva lactuca before confluence, after acidic biodegradation and on white Ulva after five days with water sample collected in the PR spot in the bay of Marseille (see Table 1). Pictures were carried out with a camera Nikon D3100 coupled to a Nikon Eclipse Ti L100 microscope (Nikon, Tokyo, Japan). Diode Array Detection High Performance Liquid Chromatography (DAD HPLC): Sea water samples were filtered at 0.2 µm and were analyzed on a Beckman HPLC system gold device with a reverse phase (C8) column using H2O 0.1 % TFA (A) and CH3CN 0.1%TFA (B). The gradient was from 10 to 50 % B in 40 min, and then 10 min at 90 % B and 10 min at 10 % B. Diode Array Detector Beckman device was coupled after the injector. Flow rate was 0.8 ml/min. Fluorescence microscopy after SYBR staining. Mediterranean seas water without and with Ulva sample was filtrated through 0.22 µm membrane filter (Millex cat nº SLGP033RS) to remove cells and then through 0.02 µm Anodisc filters (Whitman cat nº WHA68096002) using a vacuum filtration system to collect viral particles. Then filters were stained with SYBR Gold dye (N',N'- 3-diamine) that binds reverently to DNA (Invitrogen cat nº S11494) at room temperature for 15 min in the dark, and washed three times with 500 µL of sterile 0.02 µmfiltered mQ water. Stained virus-like particles were observed with an epifluorescence Microscope Leica SP2.

dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]- N-propylpropane-1,

Results

Nitrate concentration is similar in Brittany and in Provence coastal water nearby the Rhône River: Ulva is naturally present in the bay of Marseille and appears each year in winter. Ulva grows rapidly from February to March and disappears for springtime. Ulva proliferation was never reported in the bay of Marseille and a first hypothesis could be that the nitrate concentration is lower compared to sea water in Brittany. Five spots called RN, WF, RS, PR and Mu nearby Marseille were selected and sea water samples were collected in June 2018, 2019 and 2020 and compared to three spots in Brittany (Table 1). Longitude and Latitude for the 8 spots are indicated in Table 1. TR and BR are in Northern Brittany coast near Paimpol while PO is in Southern Brittany coast near Nantes. From 2018, 2019 to 2020, Ulva collected in North Brittany (TR) was incubated with sea water from the five spots in Provence collected during springtime. The bay of Marseille is from 20 km of the mouth of Rhône River and North West winds (Mistral and Tramontane) blow regularly from Rhône River to Marseille. Table 1 shows that pH and conductimetry (related mainly to salinity) are lower in RN and WF regarding RS due probably to the influence of Rhône river. Table 1 shows that the concentration in nitrates in open coastal sea water is equivalent in Brittany (BR and PO) and in Provence (RN, WF, RS). However, nitrate concentration can be much higher in Brittany fjord (TR) or in marina (PR) in Provence. . This experiment illustrates the Ulva polymorphism that might be due either to a difference in salinity as previously described [START_REF] Hayden | Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera[END_REF] or other parameters such as day light.

Figure 1: Breton Ulva can grow in Mediterranean seas A, Ulva with a green tubular phenotype was collected in November 2018 in the Trieux fjord in the north coast of Brittany (TR see Table 1). B, the tubular morphology began to change after incubation at 20 +10°C and day light exposure with sea water collected in June 2018 in the RN spot in bay of Marseille (see Table 1). C, after one month the transformation was completed. This experiment shows the polymorphism of Ulva and the possibility for Breton Ulva to grow in Mediterranean seas water.

Breton Ulva bleaching in sea water samples collected in Provence. Natural biodegradation on beaches occurs when Ulva reach confluence inducing anoxia characterized by production of H 2 S [START_REF] Dominguez | Ulva lactuca, A Source of Troubles and Potential Riches[END_REF]. For this biodegradation, Ulva can become white due to dehydration. Ulva are intolerant to severe desiccation and the induced sub lethal stress due to reduced photosynthetic performance involves a loss of pigmentation [START_REF] Beer | Photosynthesis of Ulva sp. I. Effects of desiccation when exposed to air[END_REF]. However, this is a different phenomenon that we observed with Breton Ulva in sea water collected in the PR spot in bay of Marseille. Breton Ulva were turning white rapidly (bleaching) sometime in one day without dehydration. These experiments were carried out in 2018, 2019 and 2020 with sea water samples from the five spots in Provence and was reproducible each year (Table 1). No bleaching was observed with sea water collected in Brittany for springtime when Ulva proliferation occurs at its highest level. Regarding the five different spots in Provence, the number of tubes where bleaching was observed was not the same. Bleaching was observed mainly at PR and MU spots (Table 1). At PR, Ulva proliferation was observed only in winter while at MU Ulva was still observed at the beginning of springtime.

In tubes where Ulva could proliferate, confluence could be reached from one week to three weeks and acidity was detected. As observed in natural conditions Ulva remained green under biodegradation. No acidity was observed with Ulva bleaching and Ulva became white generally after 5 days. In tubes where Ulva could not grow, Ulva were becoming white under day light at 20°C in five days with no acidity detected, as shown in Figure 2A. This bleaching was not similar to white dehydrated Ulva observed in Brittany when Ulva was drying on the shore. Sea water from PR collected in June 2018 was kept from one month and six months before to be incubated again with Breton Ulva and bleaching was no longer observable after 6 months (data not shown). Chemical pollutants would have induced Ulva bleaching from D1 to D180. Other Breton algae (mainly brown such as Wrack) were tested and no bleaching was observed with sea waters from PR (data not shown). A pollutant would induce changes to these other algae and bleaching appears to be specific to Ulva. 0ptical microscopy showed that bleaching was not due to Ulva sporulation. Ulva bleaching was characterized with Ulva tissue becoming white in solution (Figure 2A). Optical microscopy showed that the tissue remained unaffected upon bleaching (Figure 2B) with a regular cell appearance similar to healthy Ulva that have a tall made of tight cells with chloroplasts in cytoplasm giving a green color to cells (Figure 2C). The white color in Figure 2B showed that cells were dead but this death was not due to a macro predator or stress due to environmental conditions as in dehydration that disrupted the tissue organization with liberation of chloroplasts in the interstitial middle as shown in Figure 2D. This was not a sporulation that could give a white color to cells. In that case, cells should become spherical with a large interstitial middle. Finally, when sea water was filtrated at 0.2 µm, Ulva bleaching could still occur showing that bleaching was not due to planktons, amoeba or bacteria having a size > 0.2 µm. Only a virus having a size < 0.2 µm could explain this rapid death of Ulva cells without tissue damage. Viral concentration increases in sea water with bleached Ulva Mediterranean seas water inducing bleaching was filtrated at 0.2 µm and then analyzed with Diode Array Detection coupled to High Performance Liquid Chromatography (DAD HPLC) providing a UV spectrum of each peak eluting at different times (Figure 3). A majority of peaks eluting between 5 to 45 min were characterized by UV spectra having a maximum absorption at 243 nm (Figure 3A) and were due to organic macromolecules called colloids [START_REF] Stemmann | Plankton and particle size and packaging: from determining optical properties to driving the biological pump[END_REF]. A 3D view of the DAD HPLC run showed that colloids were the major components of sea water filtrated at 0.2 µm (Figure 3B). Three peaks had different UV spectra. The peak indicated with a red arrow at 3.5 min (Figure 3C) was due to viral particles and was characterized by a first max. abs. at 266 nm (Figure 3D) due to nucleic acids and aromatic amino acids [START_REF] Beck | Comparison of UV-Induced Inactivation and RNA Damage in MS2 Phage across the Germicidal UV Spectrum[END_REF][START_REF] Porterfield | Simple and General Method for Determining the Protein and Nucleic Acid Content of Viruses by UV Absorbance[END_REF]. The two other peaks were free nucleic acids at 6 min (red arrow in Figure 3A) and free proteins at 45 min characterized respectively by a max. abs. at 260 and 280 nm (Fig 3A&B). When Breton Ulva were added to Mediterranean seas water for five days and after bleaching, the peak corresponding to virus increased significantly (Figure 3E). UV spectra (Figure 3D and 3F) showed that the max. abs. at 266 nm increased from 7 to 32 mAU. Interestingly, this peak increased while the colloid peaks decreased (due probably because Ulva was using colloids as nutriments). We observed a natural decay of 30% in one month of the peak corresponding to virus with DAD HPLC (data not shown). We observed also a good correlation with an increase of this virus peak in DAD HPLC and apparition of viral lyses in September 2019 both at RN and WF (data not shown). D Bleached Ulva release Virus-like particle: Provence water (MU) without and with Ulva was filtrated at 0.2 µm and then stained with aromatic compounds called SYBR Gold pigment binding on DNA and RNA. This pigment was currently used in virology to stain and visualize virus like particles (VLPs) observed in seawater and other aquatic samples [START_REF] Shibata | Comparison of SYBR Green I and SYBR Gold stains for enumerating bacteria and viruses by epifluorescence microscopy[END_REF]. Fluorescence microscopy after SYBR staining revealed a high viral production when Ulva was added to sea water (Figure 4). This high viral production was already significant when Ulva was still green. However, when Ulva became white the viral abundance reaches 6.5 x 10 8 viruses/ml, which was an atypical viral high concentration (Figure 4D). This experiment showed that viruses were actively produced and released with higher rates with Ulva bleaching.

Discussion

The average nitrate concentration in sea water worldwide and in Mediterranean seas is 1 µM. If nitrate concentration was the reason for the absence of Ulva proliferation at Marseille, we should have nitrate concentration up to 100 µM on Breton north coast where green tides are the most important in Western Europe particularly for springtime but such is not the case excepted in river or fjord (Table 1). Nitrate concentrations are variable regarding seasons. In Breton north coast there is an average of 5 µM at the marine station of Roskoff that went to almost 10 µM in winter and to 1 µM in summer in 2018 and 2019 [START_REF]Data from ''Service d'Observation en Milieu Littoral (SOMLIT)[END_REF]. Other parameters such as pH and conductimetry are also variable regarding season at Rockoff [START_REF]Data from ''Service d'Observation en Milieu Littoral (SOMLIT)[END_REF].

Our data at BR in Brittany North coast (Table 1) are in the range of the nitrate concentration observed at Roscoff and a same variability regarding season is observed at Marseille [START_REF]Data from ''Service d'Observation en Milieu Littoral (SOMLIT)[END_REF]. This variability regarding seasons was also observed in Galicia at the west of Spain [START_REF] Villares | Nitrogen and phosphorus in Ulva sp. in the Galician Rias Bajas (northwest Spain): Seasonal fluctuations and influence on growth[END_REF]. It is also important to point out that the origin of Ulva green tides does not necessarily come from Brittany coasts. Ulva proliferations are observed in the middle of North Atlantic and Ulva drift to Brittany due to dominant western winds in the North Atlantic Ocean [START_REF] Pinazo | MASSILIA: Modélisation de la baie de Marseille : Influence des apports Anthropiques de la métropole sur l'écosystème marin[END_REF]. Chlorophyll anomalies appear to be more and more frequent and the main cause of green tides could be due mainly to the global warming [START_REF] Pinazo | MASSILIA: Modélisation de la baie de Marseille : Influence des apports Anthropiques de la métropole sur l'écosystème marin[END_REF]. A very interesting survey carried out in bay of Marseille in 2007 and 2008 by IFREMER confirms our results showing that nitrate concentration could be similar in coastal seawater nearby Marseille and in North Brittany coast with nitrate concentration > 8 µM in June 2008 at Marseille [START_REF] Young | Ocean Acidification Accelerates the Growth of Two Bloom-Forming Macroalgaee[END_REF]. Although the nitrate concentration can be much higher in little rivers in the Brittany, our study shows that the huge flow rate of the Rhône River induces also high amounts of nitrates in the bay of Marseille.

Viruses are the most abundant biological entities in sea waters and can be found even in the bathypelagic (1000 to 2000 m) zone. The Mediterranean seas appear to have the highest concentration mainly in the epipelagic (5m) zone [START_REF] Roux | Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses[END_REF]. If prokaryotes and unicellular algae are the main viral hosts, only 9% of these viruses are sequenced [START_REF] Beck | Comparison of UV-Induced Inactivation and RNA Damage in MS2 Phage across the Germicidal UV Spectrum[END_REF]. Viruses are well known to control micro algae proliferation and begin to be discovered to control also macro algae proliferation such as Kelp [START_REF] Derelle | Life-cycle and genome of OtV5, a largeDNAvirus of the pelagic marine unicellular green algae Ostreococcus tauri[END_REF][START_REF] Derelle | Diversity of viruses infecting the green microalgae Ostreococcus lucimarinus[END_REF]. In Mediterranean seas, the proliferation of the green micro algae Ostreococcus tauri is controlled by a virus called OtV5 belonging to the Prasinovirus family [START_REF] Middelboe | Marine Viruses: Key Players in Marine Ecosystems Viruses[END_REF]. tauri was observed only with seawater close to the area where the host strain was isolated [START_REF] Mckeown | Phaeoviruses discovered in kelp (Laminariales)[END_REF]. It is assumed that most viruses decay, or are destroyed rapidly in the environment. Algae viruses may persist in the water through the establishment of latent infections where virus genomes remain dormant and archived within host cells. From December no viral lyses can be detected in the five spots that we selected in Provence (Table1). The decay of residual Ulva colonies both on the Provencal coasts and in the Rhône River due to lower day lights is probably the reason of the viral lyses decay. When sea waters reach 15°C at the end of winter at Marseille, Ulva is reappearing in PR due to its high nitrate concentration (Table 1) and therefore this Ulva specific virus. Winter remains both for Breton north coast and for Marseille the season where nitrate concentrations are the highest [START_REF]Data from ''Service d'Observation en Milieu Littoral (SOMLIT)[END_REF][START_REF] Young | Ocean Acidification Accelerates the Growth of Two Bloom-Forming Macroalgaee[END_REF]. However, middle temperatures and sunny winter days at Marseille makes possible for Ulva to shortly proliferate before apparition of viral lyses.

Conclusions

This study shows for the first time that a virus might control Ulva proliferation in the bay of Marseille and describes the experiments that made possible to reveal the origin of the Ulva bleaching with sea waters collected in Provence. The sequence and the characterization of this new virus will be soon published (Lluesma et al, manuscript in preparation). Metagenomic approaches that are independent of culturing and maintaining Ulva in the laboratory was the methodology that helped to identify and recover the genetic information of this new virus [START_REF] Mckeown | Phaeoviruses discovered in kelp (Laminariales)[END_REF]. The virus sequence will make possible to develop new tools such as droplet digital PCR to estimating the viral absolute abundance [START_REF] Martinez-Hernandez | Single-virus genomics reveals hidden cosmopolitan and abundant viruses[END_REF]. This virus could be used for an innovative ecological approach that might help to control Ulva proliferation and restore ecosystems in Western Europe and Eastern China.

Breton

  Ulva can grow in Mediterranean seas: Breton Ulva can grow rapidly with sea waters from Marseille (Fig 1). Ulva was collected in the Trieux fjord (North Brittany) with a green tubular phenotype (Fig 1A) and changed its morphology after one month at 20 + 10°C and day light exposure (Fig 1C)

Figure 2 :

 2 Figure 2: Comparison of bleached Ulva, regular Ulva and anoxia Ulva with optical microscopy. A Ulva became white in five days following incubation at 20°C and day light exposure with sea water from PR spot (Table 1). B. Optical microscopy (10X) of white Ulva. The white color is due to cellular death but Ulva tissue remains unaffected with a regular cell wall organization (fibers). C. Optical microscopy (10X) of healthy Ulva. D. Optical microscopy (10X) of Ulva after anoxia and acidic biodegradation. Ulva tissue is disrupted with release of chloroplasts in the interstitial middle that remain green in spite of anoxia.

Figure 3 :

 3 Figure 3: Virus particles increase with bleached Ulva. Mediterranean seas water (500 µl) filtrated at 0.2 µm and analyzed with DAD HPLC. Panel A shows peak elution times regarding wavelengths from 220 to 360 nm, the vertical red arrow shows the peak at 6 min that correspond to free nucleic acids while the horizontal green arrows outline the 260 nm wavelength. Panel B shows a 3D view of peak elution times regarding both wavelengths and milli Absorption Units (mAU). Panel C shows peak elution times at 260 nm without Ulva and the vertical red arrow indicates the peak at 3.5 min due to virus. Panel D shows the UV absorption spectrum from 220 to 360 nm of the peak at 3.5 min due to virus without Ulva with a max. abs of 3.5 mAU at 366 nm. Panel E shows peak elution times at 260 nm with Breton Ulva for five days and the vertical red arrow indicates the peak at 3.5 min that might be due to viral-particles. Panel F shows the UV absorption spectrum from 220 to 360 nm of the peak at 3.5 min that could be due to viral particles with Breton Ulva for five days with a max. abs of 32 mAU at 366 nm. The blue line indicates the increase of background absorptions.

Figure 4 :

 4 Figure 4: Fluorescence microscopy after SYBR staining. Sea water from Mediterranean seas inducing bleaching was incubated without Ulva (panel A), with white Ulva (panel B) and green Ulva (panel C). Panel D shows the Virus like particles amount. Sea water was filtrated at 0.2 µm.

Table 1 Springtime Sea Waters from Brittany (TR, BR & PO) and Provence (RN, WF, RS, PR & MU).

 1 In Brittany BR and PO and in Provence RN, WF and RS were open coastal sea water but TR in Brittany is a fjord, PR in Provence is a marina and MU in Provence is a calanque)

	(Location	TR	BR	PO	RN	WF	RS	PR	MU
	Latitude (N)	48°46' 48°43' 47°06'	43°18' 43°14' 43°15' 43°14' 43°09'
	Longitude	3°06'W 2°56'W 2°07'W	5°17'E 5°17'E 5°19'E 5°21'E 5°36'E
	Nitrate (µM)	24±583 7±3	11±2	5±2	6±1	5±2	24±5	9±3
	pH	7.9	8.0	8.1	7.9	8.0	8.0	8.0	8.0
	Salinity (mS)	30.9	45.6	48.1	57.4	56.1	57.9	57.8	58.1
	Bleaching* (%) 0	0	0	0	0	4±4	96±4	88±8

*Percentage of in vitro Breton Ulva bleaching with sea water samples collected for springtime in 2018, 2019 and 2020 (n=7). All experiments were carried out with Breton Ulva collected in Trieuc fjord (TR) in northern Brittany in 2018, 2019 and 2020 and 25 tubes for each sea water sample.

  The sequence of our new virus does not fit with the Prasinovirus family (Lluesma et al., manuscript in preparation). Bleaching experiments in Table 1 were carried out only with Ulva collected in north Brittany (TR). However, bleaching was also observed with Ulva collected in south Brittany (PO) and Ulva collected at MU in Provence for springtime 2020 (data not shown). Since 2019 we are doing seawater sampling in Provence every month and Ulva viral lyses can be different regarding season in the bay of Marseille. Viral lyses that are very important in PR for springtime become less important from September while viral lyses begin to be observed with water sample from RN, WF and RS (data not shown). It might be due to the absence of Ulva in PR in summer while Ulva proliferate in Rhône River and reach bay of Marseille. Detection of viral lyses activities against O.

Acknowledgements

EPL is grateful to VIRALGA to have made available data for this publication. EPL thanks Ian McBitter, Fabien Gerard and Claude Sarbac for reviewing the manuscript. EPL thanks Virgile Calvert and Patrick Rimbauld for fruitful discussions.


Carole Farre

Sara Viezzi

Alice Wright

Perrine Robin

Nathalie Lejal

Marisa Manzano

Jasmina Vidic

Carole Chaix
email: carole.chaix-bauvais@univ-lyon1.fr

Specific and sensitive detection of Influenza A virus using a biotin-coated nanoparticle enhanced immunomagnetic assay

Keywords: Immunoassay, Influenza A virus, Magnetic bead, Biotin-nanoparticle, Nucleoprotein, Diagnostics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Influenza viruses cause worldwide outbreaks and pandemics in humans and animals with high morbidity and mortality [START_REF] Miller | The Signature Features of Influenza Pandemics -Implications for Policy[END_REF]. Of the Influenza A, B, C and D viruses, Influenza A viruses mutate the most rapidly leading to the continuous apparition of new strains. The high antigenic flexibility and hence greater virulence increase the severity of illnesses and death rates in the event of a pandemic [2]. The seasonal subtypes of circulating Influenza A viruses (H1N1 and H3N2) cause 3-5 million human severe infections and between 290,000 and 650,000 fatal cases annually, mostly in elderly and immunocompromised individuals and in the young children [3,[START_REF] Khanna | Emerging influenza virus: A global threat[END_REF]. There is an urgent need to improve diagnostic of H1N1 and H3N2 viruses to better control future outbreaks.

Rapid diagnostic tests for influenza, mostly based on antigen detection, facilitate early detection and then improve clinical decision-making. Furthermore, such tests can serve as tools for disease monitoring. Most of the assays dedicated to rapid detection of the currently circulating subtypes of Influenza A viruses use monoclonal antibodies (mAbs) against the nucleoprotein (NuP) encoded by Influenza A viruses. The NuP protein is highly abundant, notably during Influenza A virus infections [START_REF] Moeller | Organization of the Influenza Virus Replication Machinery[END_REF][START_REF] Albo | Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein[END_REF]. It is also a highly conserved protein among the different subtypes of Influenza A viruses with a > 90% amino acid sequence identity [START_REF] Shu | Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990[END_REF]. Thus, the approach targeting the nucleoprotein A is generic for a large panel of Influenza A strains. For instance, Yang et al. produced two monoclonal antibodies against the avian Influenza A nucleoprotein [START_REF] Yang | Development and application of monoclonal antibodies against avian influenza virus nucleoprotein[END_REF]. The immunoassays targeting NuP proved to be robust and specific for 15 subtypes of avian Influenza A viruses. In 2020, an antibody microarray was developed from a preselection of 50 mAbs [START_REF] Plotnikova | Antibody microarray immunoassay for screening and differential diagnosis of upper respiratory tract viral pathogens[END_REF]. The diagnostic system detected and differentiated six clinically important upper respiratory tract pathogen groups, including Influenza A and B viruses by testing 25 mAbs against the nucleoprotein.

The first ELISA test based on immunocapture of the nucleoprotein was described by Chomel et al. [START_REF] Chomel | Rapid diagnosis of influenza infection of NP antigen using an immunocapture ELISA test[END_REF]. This immunocapture sandwich assay was found to be specific for a large panel of human and animal influenza viruses and no cross-reaction with other respiratory viruses was observed. A minimal amount of 5 ng mL -1 of purified viral NuP was detected using highly sensitive radioactive detection [START_REF] Coonrod | Etiological diagnosis of influenza A virus by enzymatic radioimmunoassay[END_REF]. In 2010, Chen et al. developed the Flu A DOT ELISA [START_REF] Chen | A rapid test for the detection of influenza A virus including pandemic influenza A/H1N1 2009[END_REF]. This test kit, which targets NuP in real samples of viruses, showed high sensitivity compared with the commercial kit, QuickVue Flu A from Quidel Corp., and sufficient specificity for all the variants of the Influenza A virus considered (LoD: 1.6x10 3 TCID50 mL -1 for the H5N1 strain). A one-step immunoassay with interferometric measurement was developed for the realtime detection of Influenza A nucleoprotein in samples [START_REF] Farris | Immuno-interferometric sensor for the detection of influenza A nucleoprotein[END_REF]. The technique enabled a level of detection of 1µg mL -1 NuP. This work aimed at developing a rapid and portable sensor for in situ detection of Influenza A viruses. More recently, a highly sensitive fluorescent europium nanoparticle (NP)-based immunoassay, ENIA, was developed by Zhang et al. [START_REF] Zhang | A Highly Sensitive Europium Nanoparticle-Based Immunoassay for Detection of Influenza A/B Virus Antigen in Clinical Specimens[END_REF]. ENIA proved to be accurate in real samples infected with both Influenza A and B viruses. It demonstrated an analytical sensitivity of 2.5x10 2 EID50 mL -1 for the A/Brisbane/59/07 H1N1 strain. Leirs et al. screened commercial antibodies against the Influenza A nucleoprotein using a digital ELISA microscopic approach. This imaging technique on the microscale proved ultrasensitive for detecting the recombinant Influenza A nucleoprotein spiked in buffer, reaching femtomolar detection limits. On the other hand, digital ELISA showed lower efficacy for purified viruses directly in the lysis buffer [START_REF] Leirs | Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital[END_REF]. A horseradish peroxidase (HRP)-labeled lateral flow immunoassay (HRP-LFIA) was recently developed for simultaneous detection of Influenza A and B viruses [START_REF] Zhang | An HRP-labeled lateral flow immunoassay for rapid simultaneous detection and differentiation of influenza A and B viruses[END_REF]. Four monoclonal antibodies, recognizing specific epitopes in nucleoproteins A and B, were evaluated. Compared with the real-time RT-PCR kit for diagnosis, HRP-LFIA was found to be sensitive and specific for the detection of Influenza A and B viruses [START_REF] Zhang | An HRP-labeled lateral flow immunoassay for rapid simultaneous detection and differentiation of influenza A and B viruses[END_REF]. No cross-contamination was observed with other common viral pathogens.

In another study, several europium detection-based LFIAs were evaluated and showed good performance in comparison to the RT-PCR test [START_REF] Yoo | Evaluation of high-throughput digital lateral flow immunoassays for the detection of influenza A/B viruses from clinical swab samples[END_REF]. Although conventional LFIA is simple to perform and provide rapid tests at a low cost, most LFIAs produce only a qualitative result (positive or negative) and are not really sensitive. To improve the performance of the assay, a microfluidic-based LFIA was recently developed [START_REF] Kim | Microfluidic immunoassay for point-of-care testing using simple fluid vent control[END_REF]. The device enabled a LoD of 0.1 ng mL - 1 NuP but its use requires high technical skills.

In this paper, we present a sensitive and easy-to-use magnetic bead-based enzyme-linked immunoassay (MELISA) for detecting the Influenza A virus in a clinical setting (saliva). A magnetic bead-based immunoassay for detection of the Influenza A virus via the nucleoprotein A has already been described in the literature [START_REF] Hung | Magnetic nanoparticlebased immunoassay for rapid detection of influenza infections by using an integrated microfluidic system[END_REF][START_REF] Lien | Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system[END_REF][START_REF] Noh | Pipetting-based immunoassay for pointof-care testing: Application for detection of the influenza A virus[END_REF]. A fluorescence immunoassay (FIA) together with a magnetic concentration were integrated into a microfluidic module and proved highly sensitive and selective for detecting the Influenza A/H1 and Influenza A/H3 viruses [START_REF] Lien | Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system[END_REF].

The microfluidic system afforded a 1,000-fold increase in sensitivity compared with a standard immunomagnetic-based FIA carried out according to a manual protocol. The LoD was 5x10 -4 HAU in 45 µL, which corresponds approximately to 10 4 PFU mL -1 [START_REF] Killian | Hemagglutination assay for the avian influenza virus[END_REF]. Although the assay demonstrated rapid and sensitive detection of the Influenza A infection, it was based on the use of a microsystem whose complex geometry makes it complicated and challenging to manufacture. HRP-LFIA associated with a preconcentration step of the NuP protein by magnetic beads improved 100-fold the sensitivity [START_REF] Noh | Pipetting-based immunoassay for pointof-care testing: Application for detection of the influenza A virus[END_REF]. The LoD was 4.7 µg mL -1 . Recently, Wu et al. developed a wash-free assay based on magnetic particle spectroscopy (MPS) and the self-assembly of magnetic NP to quantitatively detect H1N1 NuP [START_REF] Wu | Magnetic Particle Spectroscopy for Detection of Influenza A Virus Subtype H1N1[END_REF][START_REF] Su | Detection of Influenza a Virus in Swine Nasal Swab Samples With a Wash-Free Magnetic Bioassay and a Handheld Giant Magnetoresistance Sensing System[END_REF]. This rapid protocol reached the LoD of 0.25 µg mL -1 for NuP and 250 TCID50 mL -1 for Influenza A virus.

The biosensor sensitivity is of great importance to detect pathogens in environmental, agrifood or biological samples because pathogens are typically present in very low concentrations.

Therefore, various diagnostic assays using functionalized nanoparticles to amplify the signal and boost the sensitivity have been reported in recent years [START_REF] Bonnet | Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates[END_REF][START_REF] Chowdhury | Femtomolar Detection of Dengue Virus DNA with Serotype Identification Ability[END_REF][START_REF] Afonso | Electrochemical detection of Salmonella using gold nanoparticles[END_REF][START_REF] Sena-Torralba | Lateral flow assay modified with time-delay wax barriers as a sensitivity and signal enhancement strategy[END_REF]. Herein, we propose to evaluate the sensitivity increase of MELISA for Influenza A detection via the nucleoprotein A, using immunomagnetic beads for the capture step and biotin-coated nanoparticles (biotin-NP) to enhance the signal at the detection step.

MELISA is based on a simple operating process that is easy to implement. It only requires magnetic beads and biologics to elaborate the different reagents. In addition, it achieves good sensitivities compared to other microsystems such as lateral flow assays, and the immunomagnetic extraction allows direct analysis of biological samples. We selected a couple of commercial anti-nucleoprotein A antibodies to construct the sandwich assay. The monoclonal antibody 9G8 is a mouse monoclonal antibody that recognizes the NuP of the Influenza A virus [START_REF] Zu | In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives[END_REF]. This antibody was used to functionalize magnetic beads. The monoclonal antibody 5D8 showed broad reactivity against Influenza A subtypes H1, H2, H3, H5, H7, H9, and no cross-reactivity towards Influenza B and other respiratory viruses [START_REF] Liu | Sensitive detection of influenza viruses with Europium nanoparticles on an epoxy silica sol-gel functionalized polycarbonate-polydimethylsiloxane hybrid microchip[END_REF]. The latter was biotinylated to complete the sandwich test before HRP colorimetric detection.

The MELISA assay was carried out with a Streptavidin-HRP conjugate to detect purified NuP in buffer. We then used biotin-NP to amplify the signal for virus detection in complex matrices.

Experiments were performed in the lysis buffer complemented with saliva to simulate clinical samples. 50 nm diameter silica NP were functionalized via phosphoramidite chemistry, using a strategy of supported synthesis, adapting a protocol previously described by our group [START_REF] De Crozals | Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass[END_REF]. This well-controlled and reproducible method proved effective for NP functionalization with methylene blue-modified oligonucleotides [START_REF] Bonnet | Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates[END_REF] and proteins [START_REF] De Crozals | Granulocyte Colony-Stimulating Factor Nanocarriers for Stimulation of the Immune System (Part I): Synthesis and Biodistribution Studies[END_REF]. We used commercial biotin phosphoramidite for this NP functionalization. Then, the MELISA protocol with biotin-NP was optimized for nucleoprotein detection in virus samples. The LoD of MELISA was determined in a clinical setting. were obtained from Invitrogen and Molecular Probes, respectively (Thermo Fisher Scientific, Waltham, MA, USA). Influenza A NuP (9G8) and (5D8) antibodies were from Santa Cruz Biotechnology (St. Cruz, CA, USA). Rhodamine-coated silica nanoparticles were provided by Nano-H (Saint Quentin Fallavier, France). Oligonucleotides were synthetized using an Applied Biosystems 394 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA).

Material and methods

Reagents

Phosphoramidite synthons: Biotin

-TEG-phosphoramidite (1-dimethoxytrityloxy-3-O-(N- biotinyl-3-aminopropyl)-triethyleneglycolyl-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)- phosphoramidite); Spacer phosphoramidite 9 (9-O-dimethoxytrityl-triethylene glycol,1-[(2- cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite); dT-CE phosphoramidite (5'- dimethoxytrityl-2'-deoxythymidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)
and all synthesis reagents were purchased from Glen Research (Sterling, Virginia).

Saliva collection

Saliva samples were collected from healthy volunteers after thorough rinsing of the mouth with water no less than 1 h after a meal. Collection of saliva was carried using a 1.5-mL tube and diluted with PBS buffer. Saliva samples were stored at -20° C before use.

Scanning Electron Microscopy

Scanning electron microscopy (SEM) was performed on magnetic beads at an acceleration voltage of 5kV using a VEGA TESCAN SEM. Before analysis, samples were metallized with 5 nm thickness of gold.

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was performed on a Philips CM120 instrument operating at an acceleration voltage of 120kV (Centre Technologique des Microstructures, Lyon). Silica NP were observed after deposition of 5µL of diluted solution on a formvar-carbon coated copper grid and evaporation to dryness.

Nucleoprotein expression and production

The full-length nucleoprotein (NuP) gene of the H1N1 (strain A/WSN/33) influenza virus with a 6-His-tag at its C-terminal was cloned in the Escherichia coli expression vector pET22

(Novagen) and expressed in the BL-21 Rosetta DE3 E. coli cells (Stratagene) [START_REF] Tarus | Molecular Dynamics Studies of the Nucleoprotein of Influenza A Virus: Role of the Protein Flexibility in RNA Binding[END_REF]. Transformed cells were grown at 37 °C in 100 ml of Luria Bertani medium containing ampicillin to midexponential phase and then induced for 4 h by adding 1 mM isopropyl-β-Dthiogalactopyranoside at 37°C under shaking. NuP purification was performed as previously described [START_REF] Tarus | Molecular Dynamics Studies of the Nucleoprotein of Influenza A Virus: Role of the Protein Flexibility in RNA Binding[END_REF]. Briefly, after centrifugation, the bacterial pellet was resuspended in a lysis buffer (20 mM Tris at pH 7.4 with NaCl (50 mM or 300 mM), 5 mM imidazole, 1% Triton and 1 mg mL -1 lysozyme), sonicated and treated with 0.15 mg mL -1 RNAse A at 35 °C for 20 min in the presence of 10 mM Mg 2+ ions. The protein was purified by affinity chromatography using Nimagnetic beads (PureProteome, Millipore), following the procedure recommended by the manufacturer. The NuP was then dialysed against Tris buffer (20 mM Tris, 300 mM NaCl, pH 7.5). After dialysis, the protein concentration was determined using a molar extinction coefficient ε of 56,200 M -1 cm -1 at 280 nm. Fanny Blanc (INRAE, France). All inactivated viruses were stored at -80°C. Before inactivation, virus concentrations were determined in terms of MDCK cell infection. The initial titres of the viruses were 10 8 PFU mL -1 for H1N1 sample 1, 1.5x10 8 PFU mL -1 for H1N1 sample 2, 10 8 PFU mL -1 for H2N3. The control virus PRRSV was titrated at 10 7 PFU mL -1 .

Viral strains

Magnetic bead functionalization

Tosyl activated magnetic beads of 2.8 µm diameter (Dynabeads™ M-280, Invitrogen, US) were coated with 10 µg of Influenza A NuP (9G8) antibody per mg of beads for 1 h under gentle shaking, according to the protocol recommended by the manufacturer. Then, after 15 min passivation in BSA buffer (0.1 M sodium phosphate, 150 mM sodium chloride, 0.5% (w/v) BSA, pH 7.6), the magnetic beads were washed three times in 0.01 M sodium phosphate, 150 mM sodium chloride, 0.1% (w/v) BSA, pH 7.4 and stored at 4°C until the assays.

Biotin coating on silica NP

Rhodamine-coated silica nanoparticles were functionalized by a supported synthesis strategy developed by our group [START_REF] De Crozals | Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass[END_REF]. The functionalization was achieved in a DNA/RNA synthesizer using phosphoramidite chemistry. First, dT10-PEG2-dT10 was synthesized via the 1 µM coupling program of the instrument, reaching an average phosphoramidite coupling yield per cycle of 98%, then the biotin group was incorporated with a 10% coupling yield. Biotin incorporation was monitored at 498 nm using dimethoxytrityl quantification by UV-visible spectrophotometer.

Nanoparticles were then released from the CPG by incubating the support in 1 mL of DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) 0.1% (w/v) in water-acetonitrile 1/1 (v/v). The DBU solution was stirred in a thermomixer at 22°C for 1 h before recovering. A fresh DBU solution was added to the CPG suspension every hour. The released nanoparticles were washed with water (1 x 4 mL then 3 x 2 mL) and concentrated by centrifugation on a 30K Amicon Ultra filter (5000 g, 10 min). After the release kinetics, the nanoparticle concentration in each solution was quantified by UV-visible measurement at 560 nm (Fig. S1A andB). Finally, the NP were characterized by DLS (Fig. S2).

Biotin functionalization of Influenza A NuP (5D8) antibodies

The DSB-XTM Biotin Protein Labelling Kit (D-20655) was used. 100µL of 0.1 mg mL -1 5D8 antibody solution and 10 µL of 1 M sodium bicarbonate solution were transferred to a reaction tube. 1 µL of DSB-X biotin succinimidyl ester in DMSO solution was added. The reaction mixture was stirred for 90 min at RT. The biotinylated antibody was then purified on a purification resin by centrifugation.

ELISA protocol for Influenza A nucleoprotein detection

50 µL of 4 µg mL -1 solution of Influenza A NuP (9G8) antibody in PBS buffer was added to each well of a microtitration plate and incubated overnight at 4°C. The wells were washed three times using Washing buffer 0.5 (PBS, 0.5% (v/v) Tween 20, pH 7.4). 300 µL of PBS buffer with 5% (w/w) milk was added to each well and incubated at 37°C for 1 h under gentle shaking.

Then, the wells were washed three times using Washing buffer 0.5. 50 µL of Influenza A nucleoprotein in PBS buffer at different concentrations was added to each well, and the solutions were incubated at room temperature for 2 h under gentle shaking. Then, the wells were washed three times using Washing buffer 0.1 (PBS, 0.1% (v/v) Tween 20, pH 7.4). 50 µL of 0.5 µg mL -1 biotinylated Influenza A NuP (5D8*) antibody in PBS with 0.1% Tween 20 and 0.1% BSA was added before incubation at RT for 2 h. Then, the wells were washed three times using Washing buffer 0.1. 100µL of 1 µg mL -1 Streptavidin-HRP revelation solution was added to the wells before incubating for 45 min at room temperature under gentle shaking. The wells were washed three times with Washing buffer 0.1 before adding 100 µL of TMB and incubating the plate in the dark for 30 min at RT. Then, 100 µL of 2 M sulfuric acid was added to the wells to stop the reaction. Absorbance was measured at 450 nm and 550 nm. The absorbance values retained were A450nm -A550nm to take into consideration the optical imperfections of the plate. MELISA protocol for deactivated virus detection 1x10 6 magnetic beads previously coated with the Influenza A NP (9G8) antibody were first washed with PBS buffer three times. Deactivated virus solutions (PBS, 10% saliva) were incubated with the beads under gentle shaking at room temperature for 1 h. The beads were concentrated by means of a magnet and washed three times with Washing buffer 0.1.

MELISA protocol for Influenza A nucleoprotein detection

Conjugated biotin antibody 5D8* (0.5 µg mL -1 ) was added to the suspension in a Capture buffer (PBS, 0.1% Tween 20, 0.1% BSA) before shaking gently for 1 h. After three washing steps, Streptavidin-HRP revelation solution (100 µL, 1 µg mL -1 ) was added and the mixture was shaken at RT for 30 min at 1000 rpm in a thermomixer. The beads were washed three times with Washing buffer 0.1 before adding 100 µL of TMB and incubating in the dark for 30 min at RT. Finally, absorbance was read at 450 nm as previously described.

For the MELISA protocol using biotin-NP amplification, the same protocol was used for the antigen capture. Then, the two following steps were added for detection: biotin-NP (1x10 11 NP mL -1 in PBS, 0.1% (v/v) Tween 20) was added to the magnetic bead suspension before incubation for 30 min at 1000 rpm in a thermomixer. After three washing steps, Streptavidin-HRP revelation solution (100 µL, 1 µg mL -1 ) was added before incubating the suspension at room temperature for 30 min at 1000 rpm in a thermomixer. The beads were washed three times with Washing buffer 0.1 before adding 100 µL of TMB and incubating in the dark for 5 min at room temperature. Finally, Absorbance was read at 450 nm as previously described.

Results and Discussion

We first compared the performance of a magnetic bead-based immunoassay called MELISA with that of a more conventional ELISA assay for specific detection of the Influenza A nucleoprotein. Figure 1 illustrates the two strategies.

ELISA detection of NuP protein

A sandwich ELISA method for Influenza A nucleoprotein detection was performed using Streptavidin-HRP colorimetric detection. The assay was carried out with 1) the anti-Influenza A NuP antibody (9G8) for microwell coating and 2) the anti-Influenza A NuP biotinylated antibody (5D8*) as second antibody for detection. The experiments were carried out within a range of NuP concentrations from 0 µg mL -1 to 8 µg mL -1 . 9G8 and 5D8* antibodies were kept constant at 4 µg mL -1 and 0.5 µg mL -1 respectively, concentrations which have been determined

as suitable to obtain a measurable signal.

A positive control was performed in a well directly coated with 0.5 µg mL -1 of biotinylated antibody (5D8*). A strong colorimetric response was observed. A negative control with no 9G8 antibody coating on the bottom of the well and a blank corresponding to the full assay without the protein in the binding solution were also carried out.

The results are reported in Figure 2. A detection threshold of 0.6 OD was determined, corresponding to the average of three blank values plus three times the standard deviation. A LoD of 0.25 µg mL -1 for NuP was obtained with this ELISA format.

MELISA detection of NuP protein

The first step in the MELISA sandwich assay consisted of grafting the 9G8 antibody onto magnetic beads. Then, passivation of the bead surface was performed with BSA to reduce the signal-to-noise ratio. After grafting, the number of beads used for the assay was calculated considering the concentration of 9G8 antibody grafted to the beads, so a volume of beads containing the same amount of the 9G8 antibody as that used to coat the wells of the ELISA plate was used for each assay. 1x10 6 magnetic beads were therefore used at the capture step for each test. BSA was added to PBS to improve the washing steps and obtain the best signal-tonoise ratio.

The MELISA assays were carried out by varying the Influenza A NuP concentration from 0 to 4 µg mL -1 and the obtained data were compiled and averaged (Fig. 3). The capture step was performed with magnetic beads previously functionalized with the monoclonal antibody (9G8).

After NuP capture, the detection step consisted of a second binding operation with biotinylated 5D8* mAb and Streptavidin-HRP.

MELISA shows a strong response correlated with NuP detection. In the protocol, the beads were incubated with NuP protein solutions for 60 min. A plateau was reached at 0.5 µg mL -1 of protein. A detection threshold of 1.4 OD was determined, corresponding to the average of the three blank values plus three times the standard deviation. The results with MELISA show a NuP detection limit of 0.063 µg mL -1 , which is 4 times better than with ELISA. A range of linearity was observed between 0.063 and 1 µg mL -1 of NuP. The detection limit of MELISA is also better than that obtained by Wu et al using magnetic nanoparticles and direct measurement by spectroscopy (LoD of 0.25 µg mL -1 ) [START_REF] Wu | Magnetic Particle Spectroscopy for Detection of Influenza A Virus Subtype H1N1[END_REF] .

To optimize the time required to carry out the complete MELISA assay, we also investigated MELISA with different NuP and antibody incubation times. Incubation times of 30 min and 60 min were investigated to bind the capture antibody (9G8) with the NuP and the NuP with the second antibody (5D8*). The results are shown in Figure S3 (see supporting information). With a detection threshold calculated at 1.4 OD, the LoD reached by the faster test (incubation time of 30 min) was the same as that previously obtained (0.063 µg mL -1 ). However, the saturation plateau was reached more rapidly. The test response after 30 minutes incubation was more like a yes/no answer than a NuP concentration-dependent response. No progressive increase of the signal as a function of protein concentration was observed. This can be explained by a steric hindrance effect that impacted the kinetics. The binding reaction that occurs on the magnetic bead surface takes longer than in solution. Considering these results, we preferred to maintain an incubation time of 60 min for the following experiments with detection of whole Influenza viruses.

MELISA detection of H1N1 and H2N3 viruses

To determine MELISA performance for Influenza A virus detection, two different strains, H1N1 and H3N2, were studied, together with PRRSV, a porcine virus, whose nucleoprotein is not recognised by the antibodies.

ELISA was first performed with the H1N1 sample 1, following the protocol used for Influenza A NuP detection. Different dilutions of the mother solution of the virus were tested to estimate the lowest virus concentration that could be detected. The blank run with PBS instead of the virus gave a signal threshold (average of three blanks plus three times the standard deviation) of 0.5 OD. As shown on Figure 4, ELISA obtained a LoD of 1.2x10 7 PFU mL -1 for H1N1 sample 1.

The same sample was then analysed by MELISA (green curve, Fig. 4). The protocol was the same as for Influenza NuP detection. The detection threshold of 0.5 OD was determined as the average value of three blanks plus three times the standard deviation. MELISA shows a LoD of 2x10 5 PFU mL -1 for H1N1, which is approximatively 60 times more sensitive than with ELISA. As for the assays with purified NuP, MELISA increases the sensitivity of the assays run with whole viruses. MELISA was also carried out with different concentrations of H3N2 (blue curve, Fig. 4). As expected, a positive response was observed above a detection limit of 3x10 6 PFU mL -1 . MELISA shows good sensitivity for H1N1 and H3N2 detection but lacks sensitivity compared with the other immunomagnetic bead-based assays described in the literature [START_REF] Lien | Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system[END_REF][START_REF] Sayhi | Electrochemical detection of influenza virus H9N2 based on both immunomagnetic extraction and gold catalysis using an immobilization-free screen printed carbon microelectrode[END_REF][START_REF] Luo | Functional silica nanospheres for sensitive detection of H9N2 avian influenza virus based on immunomagnetic separation[END_REF]. The negative responses obtained for non-related PRRSV at different concentrations indicates the selectivity of MELISA for Influenza A viruses (Fig. 4).

Biotin-NP MELISA for Influenza A virus sensitive detection

To enhance MELISA's performance and push detection limits even further, we developed biotin-NP to amplify the positive signal at the detection step, as illustrated on Figure 5.

Biotin-NP development

Silica nanoparticles of 50 nm diameter were functionalized on surface with an oligonucleotidic strand bearing a biotin group at its extremity. The method used for NP functionalization was based on a solid-phase synthesis technology previously reported by our group [START_REF] De Crozals | Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass[END_REF].

Nanoparticles (NP) were first immobilized on a control pore glass (CPG) support. The resulting assembled nano-on-micro (NOM) support was characterized by Scanning Electron Microscopy (SEM) as shown on Fig. S4. The images show that the NP are grafted densely and homogeneously on the surface of CPG. Then, the NOM material was used in a DNA/RNA synthesizer to perform NP functionalization via phosphoramidite chemistry. This strategy provided highly functionalized nanoparticles. An oligonucleotidic spacer (ODN sequence: dT10-PEG2-dT10) was synthesized on the NP in order to distance the biotins from the NP surface.

Around 5000 ODNs per NP were estimated by measuring UV absorbance at 260 nm and 560 nm and quantifying the ODN per NP ratio in the suspension, a protocol described by Bonnet et al. [START_REF] Bonnet | Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates[END_REF] Then, the biotin group was incorporated. We decided to control the synthesis program on the instrument to obtain a 10% coupling yield for biotin incorporation. Using this strategy, we incorporated around 50 biotins per NP. This amount was optimized to achieve efficient binding of the biotin group during the MELISA assay. After NP release from the CPG support by DBU treatment and washing in water, the number of NP in the resulting suspension was estimated from the fluorescent NP quantification curve (Fig. S1C,D). The NP structure after release was controlled by TEM (Fig. 6A). This observation showed that NP morphology remained stable during synthesis.

We also confirmed the accessibility and reactivity of the biotins grafted onto the NP surface by SPR, via the biotin/avidin interaction. After immobilization of avidin on a SPR chip coated with a polyelectrolyte multilayer, we managed to observe the specific capture of the biotinylated nanoparticles (see supporting information, Fig. S5A). The NP specific anchoring on the avidin-functionalized SPR chip was also confirmed by fluorescence optical microscopy (surface in red on the picture, see supporting information, Fig. S5B).

Biotin-NP display by Electronic Microscopy

The magnetic beads used during the biotin-nanoparticle and immunomagnetic bead-based assay (Biot-NP MELISA) were analyzed by SEM.

For this experiment, the complete assay was carried out in the presence of nucleoprotein (0.25 µg mL -1 ) at the capture step (Fig. 6B). For comparison, a control was performed with PBS at the capture step (Fig. 6C). The images clearly show nanoparticles of the expected size of 50 nm decorating the magnetic bead surface in the positive assay carried out with the target protein.

This confirms the selectivity of the assay, with efficient binding of the biotin-NP on the magnetic beads through the NuP present in the sample.

Influenza A virus detection in a clinical setting

The biotin-nanoparticle and immunomagnetic bead-based assay (Biot-NP MELISA) was investigated for detection of H1N1 (samples 1 and 2), H3N2 and PRRSV viruses (Fig. 7).

To mimic clinical samples, the viral solutions were diluted in lysis buffer supplemented with 10% saliva. After the sandwich part of the assay with viruses, the detection step was carried out using highly biotinylated nanoparticles to increase the anchoring efficiency of the Streptavidin-HRP. This strategy provided a signal increase in the case of a positive response (Fig. 7). This improved sensitivity demonstrated the efficiency of the amplification strategy. Detection limits of 3x10 3 PFU mL -1 , 6x10 4 PFU mL -1 and 4x10 4 PFU mL -1 were calculated for H1N1 sample 1, sample 2, and H3N2, respectively. Use of biotin-NP at the detection step improved MELISA's sensitivity by a factor of 65 to 75. Detection limits below 10 4 PFU mL -1 and 10 5 PFU mL -1 were achieved for the H1N1 and H3N2 strains respectively, in the presence of 10% saliva to simulate a clinical setting. Good selectivity of the strains was also confirmed by the negative responses observed at different concentrations of PRRSV (Fig. 7).

The biotin-functionalized silica nanoparticles used in this work allowed a strong detection signal enhancement in MELISA immunological tests, which could fulfill the detection requirements for influenza A virus in saliva. The excellent reproducibility, accuracy and practicability of the proposed assay obtained in samples comparable to clinical, indicates the considerable potential of the present strategy. Biotin highly functionalized silica nanoparticles could be easily adapted to be a component of a variety of kits for detection of different analytes (DNA, enzyme, IgG, biomarkers). In addition, biotin-NP can be additionally functionalized to enable single step detection. For instance, this can be obtained with attaching organic dyedecorated antibodies or streptavidin-functionalized quantum dots conjugated to biotinylated antibodies. Overall, this technology is not limited to streptavidin directly labeled with HRP but could also work with labeled fluorophores via the streptavidin-biotin recognition.

Conclusions

In this study, a sensitive immunomagnetic bead-based assay (MELISA) was developed for Influenza A virus pan-reactive detection. The test was first compared with the standard ELISA assay and showed a significant improvement in sensitivity thanks to the magnetic capture and concentration of the nucleoprotein. Then, we developed a new MELISA assay (Biot-NP MELISA) based on the use of biotin-nanoparticles for signal amplification at the detection step and succeeded in detecting Influenza A/H1 and A/H3 viruses in a clinical setting. We demonstrated that biotinylated nanoparticles further increased sensitivity by a factor of 65-75.

Detection limits of 3x10 3 PFU mL -1 and 4x10 4 PFU mL -1 were achieved for the H1N1 and H3N2 strains respectively. Biot-NP MELISA sensitivity appears competitive compared with other immuno-assays developed with magnetic concentration at the capture step [START_REF] Lien | Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system[END_REF]. Our strategy allows capture of the viral nucleoprotein directly in solutions mimicking clinical samples, which is a huge advantage when developing assays with a fast time-to-result.

  Bovine serum albumin (BSA), Bradford reagent, ELISA Colorimetric TMB (3,3',5,5'tertamethylbenzidine) reagent, Tween 20, Streptavidin-HRP, 1,8-Diazabicyclo[5.4.0]undec-7ene (DBU), ELISA 5X Assay/Sample Diluent buffer B and high quality grade chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Phosphate-buffered saline (PBS) 10x was provided by Dominique Dutscher (Brumath, France). Sodium chloride was purchased from Laurylab (Brindas, France). Buffers were made with deionized water and sterilized by 0.22 µm filtration. Dynabeads® M-280 Tosylactivated and DSB-XTM Biotin Protein Labelling Kit (D-20655)

  Influenza A/WSN/33 (H1N1), influenza A/UDORN/72 (H3N2) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) Flanders13 (13V09) viruses inactivated by binary ethylenimine (BEI) were kindly provided by Dr. Nicolas Bertho (INRAE, France) and Dr.

1x10 6

 6 magnetic beads previously coated with the Influenza A NP (9G8) antibody were first washed with PBS buffer three times. Nucleoprotein solutions were incubated with the beads under gentle shaking at room temperature for 1 h. The beads were then washed three times with Washing buffer 0.1 (PBS, 0.1% (v/v) Tween 20, pH 7.4). Conjugated biotin antibody 5D8* (0.5 µg mL -1 ) was incubated in a Capture buffer (PBS, 0.1% (v/v) Tween 20, 0.1% BSA (w/w), pH 7.4) for 1 h under gentle shaking. After three washing steps, Streptavidin-HRP revelation solution was added before incubation at room temperature for 30 min at 1000 rpm in a thermomixer. The beads were washed three times with Washing buffer 0.1 before adding 100 µL of TMB and incubating in the dark for 30 min at RT. Finally, the supernatant was diluted with 100 µL of 2 M sulfuric acid and absorbance was read at 450 nm in a microplate reader (Perkin Elmer). A450nm -A550nm gave the correct value after suppression of the background signal.

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :

 1234 Fig. 1: Presentation of the ELISA and MELISA assays for NuP detection

Fig. 5 :Fig. 6 :Fig. 7 :

 567 Fig. 5: Presentation of MELISA (A) and Biot-NP MELISA (B) assays for Influenza A virus detection

Figure 1 Figure 4 23 Figure 5 Figure 6 25 Figure 7 Carole

 142356257 Figure 1

Acknowledgements

This work was supported by the ERASMUS program in the form of internships for Sara Viezzi and Alice Wright. The authors would like to express their gratitude to Sylvain Minot and François Bessueille for electronic microscopy (SEM) imaging. Thanks are also extended to Dr.

Conflict of interest

The authors declare they have no conflict of interest.

Nicolas Bertho (INRA, France) and Dr. Fanny Blanc (INRA, France) for providing us with viral strains. We thank Dr. Anny Slama-Schwok for initiating the production of recombinant NuP. Finally, the authors thank the Centre Technologique des Microstructures of Lyon 1 University for providing access to its TEM facilities.

Compliance with ethical standards

The saliva samples used in this work were blind samples, with no identification or any information required or registered, The informed consent was obtained from all volunteer participants included in the study. There was no association with a clinical trial.