Nicolas Gantzer

Isabelle Boudaud

Bertrand

H Hydrophobic

Adrien Brié

Christophe Gantzer

Nicolas Boudaud

Isabelle Bertrand
email: isabelle.bertrand@univ-lorraine.fr

Permeability to RNases:  Availability of hydrophobic domains:  RNases Free chlorine

Keywords: free chlorine, heat, MS2 bacteriophage, surface properties, hydrophobicity, charge free chlorine, heat, MS2 bacteriophage, surface properties, hydrophobicity, charge 44 45

HAL is

infectious particles and genome have led to their use as models to assess pathogenic enteric virus 121 behavior in water (Boudaud et al. 2012), in food (Deboosere et al. 2012;Bozkurt et al. 2015), and 122 on surfaces (Morin et al. 2015;[START_REF] Pelleieux | The effect of chlorination and hydrodynamic shear stress on 650 the persistence of bacteriophages associated with drinking water biofilms[END_REF] during treatment. Due to their structural 123 similarity with the main waterborne pathogenic viruses they have also been proposed as relevant 124 indicators of viral contamination in water (Cole, Long and Sobsey 2003;[START_REF] Ogorzaly | Relationship between F-specific RNA phage genogroups, 641 faecal pollution indicators and human adenoviruses in river water[END_REF] 125 Vergara et al. 2015) and shellfish [START_REF] Hartard | Rapid and sensitive method to assess human viral pollution in 600 shellfish using infectious F-specific RNA bacteriophages: Application to marketed products[END_REF]) using molecular and/or cell culture methods.

126

MS2 phage belonging to Levivirus genogroup I is the most studied of these FRNAPH, particularly 127 its physicochemical characteristics (Valegård et al. 1990;Langlet et al. 2008a;Dika et al. 2013a) 128 and the mechanisms of its inactivation [START_REF] Pecson | Quantitative PCR for determining the infectivity of bacteriophage 646 MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations 647 of an enzymatic treatment to reduce false-positive results[END_REF]Wigginton et al. 2012; 129 Sigstam et al. 2013). Thus, MS2 seems to be a relevant model for pathogenic enteric viruses, 130 especially for studying the mechanisms of inactivation by different factors, and it can be produced 131 in high concentrations, an essential prerequisite for some experimental methods focusing on 132 physicochemical characteristics such as electron microscopy.

133 Thus, it appears that even treatments commonly used on enteric viruses such as free chlorine 134 and heat need to be improved. Molecular-based methods allowing a more specific detection of 135 infectious enteric viruses after such treatments are also needed. Such improvements require a better 136 knowledge of the damage that occurs in enteric viruses during loss of infectivity. In such a context, 137 the present work aimed to optimize the conditions of use of free chlorine and heat for the 138 inactivation of enteric viruses and to evaluate their impact on the surface properties of MS2. The 139 impact of free chlorine used alone was first evaluated on MS2 phages because of the poor 140 knowledge about its effects on the surface properties of enteric viruses. After verification of the 141 preservation of the capsid of inactivated phages, we examined in greater detail the thermal stability 142 and the surface properties of MS2 phages exposed to chlorine. The weakening of the capsid 143 observed in the present work when free chlorine was applied alone and the increase in 144 hydrophobicity previously described for MS2 phages exposed to heat (Brié et al. 2016) perform further experiments focusing on hydrophobicity, in which heat treatment was applied to 146 MS2 phages previously exposed to chlorine. Since both free chlorine and heat have been shown to 147 act on the capsid, it might therefore be expected that their combination would increase their impact. Free chlorine and heat treatments 171 Three independent suspensions of purified phages at a concentration of 10 14 PFU mL -1 (250 µL in 172 1 mM PBS solution) (PFU: plaque-forming units) were exposed either to free chlorine alone, to heat 173 alone or to free chlorine followed by heat. To prepare our free chlorine stock solution, concentrated 174 sodium hypochlorite solution (5%, Acros Organics, Thermo Fisher Scientific) was diluted using 175 ultrapure water (> 18 MΩ cm -1 ). Determination of free chlorine concentration was performed 176 according to the DPD (N,N-diethyl-p-phenylenediamine, Acros Organics) colorimetric method (ISO 177 7393-2 1985). For exposure to free chlorine alone, the working concentrations of free chlorine were 178 100 and 200 mg.L -1 with a contact time of 10 min at 4°C. Residual free chlorine was immediately 179 quenched by adding two moles of sodium thiosulfate equivalent to one mole of free chlorine. The 180 heat treatments were performed at 55°C or 60°C for 10 min in pre-warmed Protein LoBind tubes 181 (Eppendorf, Hamburg, Germany) in a water bath. Samples were immediately placed on ice after 182 heat treatment to avoid further inactivation. When free chlorine was followed by heat, the phages 183 were exposed to 100 mg.L -1 of free chlorine for 10 min at 4°C and then to heat at 55°C or 60°C for 184 10 min. For each replicate, a part of the MS2 phage suspension was not exposed to free chlorine, 185 heat or to free chlorine followed by heat and was named control phages. Thus, there were nine 186 (n=3x3) control populations in this study. An RNase assay was used to evaluate if the capsid had maintained its integrity and was still able to 220 protect the virus genome [START_REF] Nuanualsuwan | Capsid functions of inactivated human picornaviruses and feline 634 calicivirus[END_REF]. Suspensions of MS2 phage, with a final 221 concentration of 10 7 gc mL -1 , were treated with RNase A at a final concentration of 1 µg mL -1 222 (AppliChem GmbH, Darmstadt, Germany) at 37°C for 60 min as previously described (Brié et al. The impact of free chlorine on the binding capacity of MS2 phage to its host cell E. coli Hfr K12 227 was evaluated as previously described (Wigginton et al. 2012;Brié et al. 2016). The binding

254

The binding assays were carried out as previously described (Brié et al. 2016). The binding capacity Orange. The Tm was determined by plotting the first derivative of the previous curve; it appeared 274 then as a peak (Loison et al. 2016). This method is also known to allow the expression of MS2 phage by the oxidant. Although there was a decrease in the thermal stability of the capsid we 320 did not find any changes in shape, size, and binding capacity, nor did the permeability to RNase 321 increase thus suggesting that the capsid of MS2 phage maintained its integrity after exposure to free 322 chlorine alone.

323

The decrease in the thermal stability after exposure to chlorine led us to expose MS2 phage to 324 chlorine (100 mg.L -1 ) and then to heat (55°C or 60°C). The infectivity decreased by -1.9 ± 0.3 log 10 325 for chlorine followed by heat treatment at 55°C and by -3.2 ± 0.6 log 10 for chlorine followed by 326 heat treatment at 60°C. For each treatment applied separately, the loss in infectivity reached -327 1.2 ± 0.3 log 10 after exposure to free chlorine (Table 1), -0.7 ± 0.1 log 10 after heat treatment at 55°C 328 (data not shown) and -1.1 ± 0.1 log 10 after heat treatment at 60°C (Brié et al. 2016). The highest 329 loss in infectivity was obtained when free chlorine was followed by heat treatment at 60°C (p 330 <0.01, Kruskal-Wallis test). This loss in infectivity was also higher than the sum of the decrease 331 values obtained with free chlorine and heat treatment at 60°C applied separately (p <0.01, Kruskal-332 Wallis test). This suggested that free chlorine improved the inactivation of MS2 phage by heat 333 treatment at 60°C. The RT-qPCR signal decreased only by -0.1 ± 0.4 log 10 for chlorine followed by 334 heat treatment at 55°C and by -0.3 ± 0.4 log 10 for chlorine followed by heat treatment at 60°C. The 335 concentrations in genome observed after these treatments were similar to those observed for MS2 336 phage exposed to 100 mg.L -1 free chlorine alone and also for control MS2 phage (p >0.01, Kruskal-337 Wallis test). Thus, the detection of the MS2 genome was not affected when free chlorine was 338 followed by heat. As observed for free chlorine alone, no modification of the shape of MS2 phage 339 was noted by TEM after exposure to free chlorine followed by heat (Fig. 1). The RNase treatment 340 applied after exposure to free chlorine (100 mg.L -1 ) followed by heat decreased the RT-qPCR signal 341 by -1.1 ± 0.2 log 10 for 55°C and -1.7 ± 0.4 log 10 for 60°C. The log removal of genome copies by 342 RNase treatment reached -0.3 ± 0.1 log 10 (Table 1) for free chlorine alone, -0.1 ± 0.1 log 10 (data treatment alone. The reduction in genome after chlorine followed by heat (55°C and 60°C) was treatment was observed for MS2 phage exposed to free chlorine followed by heat treatment at 60°C 348 (p <0.01, Kruskal-Wallis test). Overall, the increased effect of free chlorine followed by heat 349 treatment at 60°C on the loss in infectivity and on the capsid permeability to RNase was in 350 agreement with the free chlorine-induced weakening of the capsid suggested by DSF experiments. The electrophoretic mobility (µ) was represented as a function of the logarithm of ionic strength

354

(1.95, 19.5 and 195 mM) at pH 7.0 before and after exposure to free chlorine (100 and 200 mg.L -1 ) 355 in order to estimate the impact of free chlorine on the charge density of MS2 phage (Fig. 3A). A 356 decrease in the electrophoretic mobility was observed after exposure to free chlorine at ionic 357 strengths of 1.95 and 19.5 mM (p <0.01, Kruskal-Wallis test). On the other hand, no modification 358 of the electrophoretic mobility was observed after exposure to chlorine at an ionic strength of 195 359 mM (p >0.01, Kruskal-Wallis test). Therefore, a lower slope value was observed for chlorinated 360 phages than for control phages suggesting a decrease in the negative charge density of MS2 phage 361 exposed to free chlorine. The negative surface charge of total particles (i.e. RT-qPCR results) was 362 then estimated by binding to positively charged beads at different pH (i.e. 7.0, 4.0, 3.0, and 2.5) for 363 control and chlorinated phages (Fig. 3B). The lowest adhesion capacity of both control and 364 chlorinated phages on positively charged beads was obtained at pH 2.5 and 3.0 which are below the 365 IEP of MS2 phage. Conversely, the highest adhesion capacity was obtained at pH above the IEP 366 (i.e. 4.0 and 7.0). Thus, the IEP of MS2 phage was shown to be between 3 and 4. Interestingly, an 367 increase in the adhesion capacity to positively charged beads was observed for MS2 phages exposed Whitney test). Thus, the increase in the negative charge density of MS2 phage after exposure to free 370 chlorine seemed to be mainly linked to an increase in the external negative charge. The effect of free chlorine on the hydrophobicity of MS2 phage was then evaluated by DSF and by 375 its adhesion capacity to hydrophobic beads. The DSF profile (Fig. 2) showed the same fluorescence 376 intensity of SYPRO Orange at the beginning of the experiment for control and chlorinated phages, 377 meaning that free chlorine did not modify the overall expression of hydrophobic domains of the 378 capsid proteins. The expression of hydrophobic domains on the surface of MS2 particles was 379 compared between control and chlorinated phages using the adhesion ability to hydrophobic beads.

380

The experiments were performed at pH 7.0 and high ionic strength (10 mM Na 2 HPO 4 and 150 mM 381 NaCl) in order to minimize the influence of the global charge. No adhesion to hydrophobic beads 382 was observed for control phages and those exposed to chlorine (Fig. 4). Thus, the results obtained 383 using these two methods suggested that free chlorine did not change the expression of hydrophobic 384 domains of the MS2 capsid proteins or the permeability of the capsid to the probe.

385

Since we have previously described an increased hydrophobicity of MS2 phage after heat 386 treatment (Brié et al. 2016), DSF experiments were carried out after exposure to free chlorine (100 387 mg.L -1 ) followed by heat (55°C or 60°C) and were compared to 55°C and 60°C heat treatments 388 applied alone (Fig. 5). Similar DSF profiles with low initial fluorescence were obtained for both 389 control MS2 phages and those exposed to the 55°C heat treatment alone. The initial fluorescence 390 increased for MS2 phages exposed to the 60°C heat treatment alone and for those exposed to free 391 chlorine followed by heat treatment at 55°C and 60°C. The highest initial fluorescence value was 392 observed using 100 mg.L -1 free chlorine followed by 60°C heat treatment (p <0.01, Kruskal-Wallis

395

The increase in the initial fluorescence showed that free chlorine followed by heat led to the 396 expression of hydrophobic domains and/or increased the permeability of the capsid allowing the 397 probe to reach inside hydrophobic regions. The adhesion level on hydrophobic beads increased after 398 exposure to free chlorine followed by heat treatment at 55°C or 60°C (Fig. 4); thus showing an 399 increase in hydrophobic domains on the surface of MS2 particles. The highest adhesion level on 400 hydrophobic beads was observed after free chlorine followed by heat treatment at 60°C (-2.4 ± 0.5, 401 Fig. 4) (p <0.01, Kruskal-Wallis test). Remarkably, this increase in the adhesion capacity to 402 hydrophobic beads appeared to be specific to viral particles remaining infectious. The purpose of this study was to explore the impact of free chlorine alone or followed by heat on 406 the loss of infectivity and the modification of the physicochemical properties of MS2 phage used as 407 model of the main pathogenic enteric viruses. The impact of free chlorine on capsid proteins has 408 been previously described in a few studies focusing on their altered functions. However, 409 modification of the overall structure of viral particles may lead to modification of their surface 410 properties which govern their behavior in the environment. Moreover, the study of surface 411 properties may contribute to the understanding of the mechanisms involved in the inactivation 412 process and may also help in the development of detection methods specific for infectious viruses.

413

MS2 phage was first exposed to free chlorine alone. The loss of infectivity of MS2 phage 414 observed in our experiments after exposure to 100 and 200 mg.L -1 of free chlorine was similar to 415 that reported by Loison et al. (2016) for a Qβ phage suspension. Indeed, by applying sodium 416 hypochlorite at 1 mM (i.e. 74.4 mg.L -1 ) for 10 min at room temperature on a Qβ phage suspension 417 at an initial concentration of ≈ 10 12 PFU mL -1 , these authors observed a -1.7 log 10 reduction in 418 infectivity. In the present work, we observed that the binding to host bacteria was altered after exposure to 200 mg.L -1 of free chlorine only. Thus, the loss of infectivity was not due to the 420 alteration of the binding capacity after exposure to 100 mg.L -1 of free chlorine and was slightly due 421 to the alteration of this function after exposure to 200 mg.L -1 of free chlorine. Our observation of a 422 high concentration of free chlorine needed to observe an impact on binding to host cell was in 423 agreement with a previous study published by Wigginton et al. (2012). Indeed, these authors 424 applied a concentration of 2-3 mg.L -1 of free chlorine to an MS2 phage suspension of 10 11 PFU L -1 425 and observed alteration of the genome injection and extensive degradation of the maturation protein 426 (MP). This protein, which is present as a single copy in the capsid of MS2 phage, is clearly 427 involved in the binding of MS2 phage to the F-pilus of host bacteria and probably in the injection of 428 the genome into host cells. Dent et al. (2013) have proposed that the natural retraction of the F-pilus 429 could pull the MP from its location in the attached capsid, leaving a pore through which the phage 430 genome could exit into the cell. Thus, alteration of the MP by free chlorine may impact these two 431 functions depending on its intensity. The sensitivity of the capsid to oxidants has also been observed 432 for Qβ phage with the loss of its infectivity due either to capsid destabilization or to the 433 modification of capsid proteins especially the MP and the readthrough protein (Loison et al. 2016).

434

Considering the capsid of FRNAPH, the MP appears as one of the targets of free chlorine. Thus, 435 free chlorine weakens the capsid of MS2 phage and may alter the role of the MP in the genome 436 injection into host cells even at low concentrations. High concentrations of free chlorine seem to 437 further alter the MP with a reduced ability to bind to host cells.

438

In our experiments, the genome of MS2 phage was quantified using an RT-qPCR assay which 439 targets a fragment of ≈ 150 nucleotides and the RT-qPCR signal was found to be affected only after 440 exposure to 200 mg.L -1 of free chlorine. A few studies have shown that some regions of the viral 441 genome could be more sensitive than others to damage caused by free chlorine with significant 442 differences depending on the virus type (Li et al. 2002;[START_REF] Xue | Effects of chlorine and chlorine dioxide on human rotavirus infectivity 678 asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for 581 genome release[END_REF]. Free chlorine was shown 443 to induce genome damage on MS2 phage at levels comparable to singlet oxygen and UV. However, (Wigginton et al. 2012). For these authors, this suggested that the virucidal 446 effect due to genome damage was linked to the type of modification rather than its location.

447

No modification of the shape of MS2 phage was observed by TEM after exposure to free 448 chlorine. The mean size of MS2 phage before and after chlorination was similar to that previously 449 described (Valegård et al. 1990;Dika et al. 2013a). Qβ phage particles exposed to chlorine under 450 similar conditions were also demonstrated by TEM observations to maintain their shape (Loison et 451 al. 2016). Disruption of MS2 phage occurring after exposure to 72°C for 10 min has previously 452 been shown to be related to a significant increase in RNase sensitivity (Brié et al. 2016). In our 453 case, the limited effect of RNase treatment on RT-qPCR signal confirmed that the integrity of the 454 capsid was preserved after exposure to free chlorine under our experimental conditions. DSF was 455 then used to explore conformational modifications of the capsid that could not be revealed by TEM.

456

We observed a similar DSF profile to that obtained in our previous study (Brié et al. 2016) with only one Tm peak (66°C) before exposure to chlorine. The decrease in the Tm value after exposure 458 to free chlorine corresponded to a reduction in the denaturation temperature and suggested a 459 weakening of the capsid of MS2 phage by the oxidant by comparison to a previous study performed 460 on poliovirus (Robinson, Jesudhasan and Pfeiffer 2014). The weakening of the capsid might be 461 linked to the site-specific cleavage showed on the capsid protein of MS2 phage following exposure 462 to chlorine (Wigginton et al. 2012).

463

The reactions of hypochlorous acid with amino acids result in a wide variety of products such (Langlet et al. 2008a). The binding to positively and negatively charged 471 beads at different pH showed that the IEP of MS2 phage was between 3 and 4, which was in full 472 agreement with literature data in the field (Langlet et al. 2008b;[START_REF] Dika | Impact of internal RNA on aggregation 583 and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like 584 particles[END_REF]. MS2 phage 473 exposure to chlorine led to an increase in its adhesion capacity to positively charged beads at pH 474 above the IEP. Thus, the increase in the negative charge density of MS2 phage observed after 475 exposure to free chlorine resulted mainly from an increase in the external negative charge.

476

Hydrophobicity was the other surface property monitored after exposure to free chlorine. The 477 lack of adhesion to hydrophobic beads before exposure to free chlorine was in agreement with 478 previous studies performed with MS2 phage (Dika et al. 2013b;Brié et al. 2016). No adhesion to 479 hydrophobic beads was also observed after exposure to free chlorine. The DSF profile showed the 480 same fluorescence intensity of SYPRO Orange at the beginning of the experiment for control and 481 chlorinated phages. Thus, the results obtained with these two methods suggest that few hydrophobic 482 domains are naturally expressed on the surface of MS2 particles and that free chlorine does not 483 change the expression of hydrophobic domains of the MS2 capsid proteins or the permeability of 484 the capsid to the probe. An increase in hydrophobicity by chlorine has previously been described for 485 Qβ phage (Loison et al. 2016) and for specific proteins such as α2 chaperone (Wyatt et al. 2014) 486 and Bovine Serum Albumin (Ashrafi Kooshk et al. 2012). This suggests that the expression of 487 hydrophobic domains induced by chlorine depends on the composition and environment of the 488 protein.

489

Overall, the most significant effect we observed on MS2 phages exposed to free chlorine 490 alone was the decrease in the Tm in the DSF profile suggesting the weakening of the capsid. This 491 led us to explore the impact of free chlorine followed by heat on MS2 phages. Indeed, we had

495

An increased effect on the inactivation of MS2 phage and on the permeability of its capsid to 496 RNase was observed when the 60°C heat treatment was applied after chlorination. To our 497 knowledge, the virucidal effect of the combination of free chlorine and heat on enteric viruses and exposed to the 60°C heat treatment alone, the initial fluorescence of the DSF profile increased after 509 exposure to free chlorine followed by heat. The highest initial fluorescence value was observed 510 using free chlorine followed by a 60°C heat treatment. The increase in the initial fluorescence 511 showed that chlorine followed by heat led to the expression of hydrophobic domains and/or 512 increased the permeability of the capsid allowing the probe to reach inside hydrophobic regions.

513

The adhesion test to hydrophobic beads showed an increase in the expression of hydrophobic 514 domains at the surface of MS2 phages exposed to free chlorine followed by 60°C heat treatment.

515

Remarkably, this increase in the adhesion capacity to hydrophobic beads appeared to be specific to 516 viral particles remaining infectious as previously observed for MS2 phage exposed to heat alone 517 (Brié et al. 2016). After exposure to heat and free chlorine, infectious MS2 phages accounted for a small part of the total MS2 phage particles. Thus, our results showed that the permeability of the 519 capsid to the probe increased for both infectious and inactivated phages, whereas the increase in the 520 expression of hydrophobic domains at the surface was specific to phages remaining infectious after 521 treatment.

522

Overall, our results showed that free chlorine weakens the capsid of MS2 phage and alters the 523 functions of the MP at least for the highest concentration tested. The combination of heat and 524 chlorine has not been evaluated before for enteric phages or pathogenic viruses, but it has been 525 previously reported to be interesting for bacteria in foodstuffs. We observed that free chlorine 526 followed by heat increased the inactivation of MS2 phage and the structural modifications of the 527 capsid by comparison with these treatments applied separately. Our results suggested that the 528 oxidant should be applied before heat to have an optimal effect. Concerning the specific detection 529 of infectious viruses, the increase in permeability to enzymes such as RNases for inactivated phages 530 could offer new opportunities for such methods. In our opinion, the most important result 531 concerning free chlorine followed by heat is the specific increase in the expression of hydrophobic 532 domains at the surface of infectious MS2 phages. Indeed, this result suggested that hydrophobic One-sentence summary: Exposure of MS2 phage to free chlorine followed by heat leads to higher 11 inactivation, permeability to RNases, and availability of hydrophobic domains within the capsid 12 than does chlorine applied alone. fluorimetry. This phenomenon was confirmed when chlorination was followed by a heat treatment.

34

Indeed, an increase in the inactivation of MS2 phages and the permeability of their capsids to 35 RNases was observed. More interestingly, an increase in the expression of hydrophobic domains at 36 the phage surface was observed, but only for phages remaining infectious. The chlorine-caused weakening of the capsid suggested that, for an optimal use, the oxidant should be followed by heat.

38

The increased permeability to RNases and the expression of hydrophobic domains may contribute 39 to the development or improvement of molecular methods specific for infectious enteric viruses. hypochlorite revealed some hydrophobic parts of the capsid or increased the permeability of the 96 capsid allowing the probe to reach inside hydrophobic regions (Loison et al. 2016).

97

The study of the impact of heat and free chlorine on enteric viruses requires methods suited for 98 the specific detection of infectious viral particles, which are the only ones that pose a risk to human on the specific detection of oxidative damage (Tojo et al. 2013;Sano et al. 2015). However, their 111 optimal conditions of use such as the type of treatment (i.e. physical and/or chemical) and the 112 matrices (i.e. food and/or environmental matrices) still need to be defined. (Valegård et al. 1990;King et al. 2012). These MS2 phage belonging to Levivirus genogroup I is the most studied of these FRNAPH, particularly 127 its physicochemical characteristics (Valegård et al. 1990;Langlet et al. 2008a;Dika et al. 2013a) 128 and the mechanisms of its inactivation [START_REF] Pecson | Quantitative PCR for determining the infectivity of bacteriophage 646 MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations 647 of an enzymatic treatment to reduce false-positive results[END_REF]Wigginton et al. 2012; 129 Sigstam et al. 2013). Thus, MS2 seems to be a relevant model for pathogenic enteric viruses, The phages were then concentrated and purified as previously described (Brié et al. 2016). Briefly, 155 the phages were centrifuged twice (8,000 g, 20 min, 4°C) and the supernatant (≈ 1 L) was filtered 156 using a 0.22 µm filter unit (Stericup-GP Millipore Express Plus, 0.22 µm; Merck Millipore, Merck 157 KGaA, Darmstadt, Germany). After an ultracentrifugation step (Thermo Scientific A-621 fixed 158 rotor, 42,500 g, 18 h, 4°C; Thermo Fisher Scientific, Waltham, MA, USA), the pellets containing 159 the concentrated phages were resuspended in iodixanol solution (20% final, Optiprep; Axis-Shield, 160 Dundee, Scotland) and laid on a 40% iodixanol layer in a tube (Cone-Top PA tube 14 × 47 mm, 161 Thermo Fisher Scientific). The density gradient was formed upon centrifugation at 160,000 g for 162 7 h at 15°C (Thermo Scientific TH-641 swing rotor). After centrifugation, the MS2 phages were 163 gently removed with a syringe and dialyzed twice (one overnight, the other for 7 h) against 10 L of 164 PBS solution with a concentration of 1 mM (1 mM Na 2 HPO 4 and 14 mM NaCl), pH 7.4, using a 165 Float-A-Lyzer G2 dialysis device (MW100 kD; Spectrum Laboratories, Inc., Rancho Dominguez, 166 CA, USA). These working suspensions were kept at 4°C before use. Three working suspensions 167 were produced for this study in order to perform the experiments in triplicate on independent Three independent suspensions of purified phages at a concentration of 10 14 PFU mL -1 (250 µL in 172 1 mM PBS solution) (PFU: plaque-forming units) were exposed either to free chlorine alone, to heat 173 alone or to free chlorine followed by heat. To prepare our free chlorine stock solution, concentrated 174 sodium hypochlorite solution (5%, Acros Organics, Thermo Fisher Scientific) was diluted using 175 ultrapure water (> 18 MΩ cm -1 ). Determination of free chlorine concentration was performed 176 according to the DPD (N,N-diethyl-p-phenylenediamine, Acros Organics) colorimetric method (ISO 177 7393-2 1985). For exposure to free chlorine alone, the working concentrations of free chlorine were 178 100 and 200 mg.L -1 with a contact time of 10 min at 4°C. Residual free chlorine was immediately 179 quenched by adding two moles of sodium thiosulfate equivalent to one mole of free chlorine. The 180 heat treatments were performed at 55°C or 60°C for 10 min in pre-warmed Protein LoBind tubes 181 (Eppendorf, Hamburg, Germany) in a water bath. Samples were immediately placed on ice after 182 heat treatment to avoid further inactivation. When free chlorine was followed by heat, the phages 183 were exposed to 100 mg.L -1 of free chlorine for 10 min at 4°C and then to heat at 55°C or 60°C for 184 10 min. For each replicate, a part of the MS2 phage suspension was not exposed to free chlorine, 185 heat or to free chlorine followed by heat and was named control phages. Thus, there were nine 186 (n=3x3) control populations in this study. An RNase assay was used to evaluate if the capsid had maintained its integrity and was still able to 220 protect the virus genome [START_REF] Nuanualsuwan | Capsid functions of inactivated human picornaviruses and feline 634 calicivirus[END_REF]. Suspensions of MS2 phage, with a final 221 concentration of 10 7 gc mL -1 , were treated with RNase A at a final concentration of 1 µg mL -1

222

(AppliChem GmbH, Darmstadt, Germany) at 37°C for 60 min as previously described (Brié et al. The impact of free chlorine on the binding capacity of MS2 phage to its host cell E. coli Hfr K12 227 was evaluated as previously described (Wigginton et al. 2012;Brié et al. 2016). The binding Orange. The Tm was determined by plotting the first derivative of the previous curve; it appeared 274 then as a peak (Loison et al. 2016). This method is also known to allow the expression of 275 hydrophobic parts of the capsid and/or the permeability of the capsid to be investigated by the The analysis was performed using the values obtained for the three independent suspensions of 281 phages. Since the experimental values were not normally distributed, non-parametric tests were 282 performed using StatEL 2.7 (Ad Science). The explanatory variables were the exposure to free 283 chlorine alone, heat alone, and free chlorine followed by heat. For free chlorine alone, Mann-284 Whitney tests were performed to evaluate its effect on the infectivity (i.e. loss of infectivity, binding 285 capacity to host bacteria), structure (i.e. genome degradation, capsid permeability to RNase, thermal 286 stability), and hydrophobicity (i.e. initial adhesion of hydrophobic probe SYPRO Orange, binding 287 to hydrophobic probes), and the adhesion to positively charged beads by comparison between 288 control and chlorinated MS2 phages. A Kruskal-Wallis test was performed to compare the 289 electrophoretic mobility between control and chlorinated MS2 phages. The effect of free chlorine 290 followed by heat on the loss of infectivity, structure (i.e. genome degradation, capsid permeability 291 to RNase) and hydrophobicity (i.e. initial adhesion of hydrophobic probe SYPRO Orange, binding 292 to hydrophobic beads) was evaluated by comparison with free chlorine alone and heat alone using 293 Kruskal-Wallis tests. For all these tests, a p value of <0.01 was considered as significant. The effects of free chlorine applied alone on MS2 phage were evaluated for 100 and 200 mg.L -1

299

(Table 1). Infectivity decreased by -1.2 ± 0.3 log 10 for 100 mg.L -1 (p <0.01, Mann-Whitney test) 300 and by -3.5 ± 0.9 log 10 for 200 mg.L -1 (p <0.01, Mann-Whitney test). The binding capacity of MS2 301 phage to E. coli K12 Hfr was not altered after exposure to 100 mg.L -1 (-0.1 ± 0.1 log 10 ) (p >0.01, 302 Mann-Whitney test), whereas it decreased by -0.8 ± 0.1 log 10 after exposure to 200 mg.L -1 (p <0.01, 303 Mann-Whitney test). The free chlorine treatment at 100 mg.L -1 did not affect the detection of the 304 genome fragment of ≈ 150 nucleotides targeted with our RT-qPCR assay since the RT-qPCR signal 305 was not modified (p >0.01, Mann-Whitney test). However, the genome detection decreased by -306 0.5 ± 0.2 log 10 for 200 mg.L -1 (p <0.01, Mann-Whitney test). No modification of the shape of MS2 307 phage was observed by TEM for these two concentrations of free chlorine (Fig. 1). The mean size 308 of chlorinated MS2 phage was 20 ± 2 nm for 100 mg.L -1 (n=3×3) and 21 ± 1 nm for 200 mg.L -1 309 (n=3×3) which was similar to control phages (p >0.01, Mann-Whitney test). Moreover, the limited 310 effect of RNase treatment on RT-qPCR signal which decreased by less than -0.3 ± 0.1 log 10 with 311 the two concentrations of free chlorine (p >0.01, Mann-Whitney test) confirmed that the integrity of 312 the capsid was preserved under our experimental conditions. DSF was then used to explore 313 conformational modifications of the capsid that could not be revealed by TEM and to compare the 314 Tm (i.e. denaturation temperature) value of MS2 phages before and after exposure to free chlorine.

315

We observed only one Tm peak of 66.0 ± 0.1°C (n=3) before exposure to chlorine (Fig. 2). After 316 exposure to free chlorine for 10 min, a decrease in the Tm to 62.8 ± 0.1°C (n=3) for 100 mg.L -1 (p 317 <0.01, Mann-Whitney test) and 60.9 ± 0.7°C (n=3) for 200 mg.L -1 (p <0.01, Mann-Whitney test) 318 was noticed. This reduction in the denaturation temperature suggested a weakening of the capsid of MS2 phage by the oxidant. Although there was a decrease in the thermal stability of the capsid we 320 did not find any changes in shape, size, and binding capacity, nor did the permeability to RNase 321 increase thus suggesting that the capsid of MS2 phage maintained its integrity after exposure to free 322 chlorine alone.

RNase treatment reached -0.3 ± 0.1 log 10 (Table 1) for free chlorine alone, -0.1 ± 0.1 log 10 (data treatment alone. The reduction in genome after chlorine followed by heat (55°C and 60°C) was treatment was observed for MS2 phage exposed to free chlorine followed by heat treatment at 60°C 348 (p <0.01, Kruskal-Wallis test). Overall, the increased effect of free chlorine followed by heat 349 treatment at 60°C on the loss in infectivity and on the capsid permeability to RNase was in 350 agreement with the free chlorine-induced weakening of the capsid suggested by DSF experiments. The electrophoretic mobility (µ) was represented as a function of the logarithm of ionic strength

354

(1.95, 19.5 and 195 mM) at pH 7.0 before and after exposure to free chlorine (100 and 200 mg.L -1 ) 355 in order to estimate the impact of free chlorine on the charge density of MS2 phage (Fig. 3A). A 356 decrease in the electrophoretic mobility was observed after exposure to free chlorine at ionic 357 strengths of 1.95 and 19.5 mM (p <0.01, Kruskal-Wallis test). On the other hand, no modification 358 of the electrophoretic mobility was observed after exposure to chlorine at an ionic strength of 195 359 mM (p >0.01, Kruskal-Wallis test). Therefore, a lower slope value was observed for chlorinated 360 phages than for control phages suggesting a decrease in the negative charge density of MS2 phage 361 exposed to free chlorine. The negative surface charge of total particles (i.e. RT-qPCR results) was 362 then estimated by binding to positively charged beads at different pH (i.e. 7.0, 4.0, 3.0, and 2.5) for 363 control and chlorinated phages (Fig. 3B). The lowest adhesion capacity of both control and 364 chlorinated phages on positively charged beads was obtained at pH 2.5 and 3.0 which are below the 365 IEP of MS2 phage. Conversely, the highest adhesion capacity was obtained at pH above the IEP 366 (i.e. 4.0 and 7.0). Thus, the IEP of MS2 phage was shown to be between 3 and 4. Interestingly, an 367 increase in the adhesion capacity to positively charged beads was observed for MS2 phages exposed Whitney test). Thus, the increase in the negative charge density of MS2 phage after exposure to free 370 chlorine seemed to be mainly linked to an increase in the external negative charge. The effect of free chlorine on the hydrophobicity of MS2 phage was then evaluated by DSF and by 375 its adhesion capacity to hydrophobic beads. The DSF profile (Fig. 2) showed the same fluorescence 376 intensity of SYPRO Orange at the beginning of the experiment for control and chlorinated phages, meaning that free chlorine did not modify the overall expression of hydrophobic domains of the 378 capsid proteins. The expression of hydrophobic domains on the surface of MS2 particles was 379 compared between control and chlorinated phages using the adhesion ability to hydrophobic beads.

380

The experiments were performed at pH 7.0 and high ionic strength (10 mM Na 2 HPO 4 and 150 mM 381 NaCl) in order to minimize the influence of the global charge. No adhesion to hydrophobic beads 382 was observed for control phages and those exposed to chlorine (Fig. 4). Thus, the results obtained 383 using these two methods suggested that free chlorine did not change the expression of hydrophobic 384 domains of the MS2 capsid proteins or the permeability of the capsid to the probe.

385

Since we have previously described an increased hydrophobicity of MS2 phage after heat 386 treatment (Brié et al. 2016), DSF experiments were carried out after exposure to free chlorine (100 387 mg.L -1 ) followed by heat (55°C or 60°C) and were compared to 55°C and 60°C heat treatments 388 applied alone (Fig. 5). Similar DSF profiles with low initial fluorescence were obtained for both 389 control MS2 phages and those exposed to the 55°C heat treatment alone. The initial fluorescence 390 increased for MS2 phages exposed to the 60°C heat treatment alone and for those exposed to free 391 chlorine followed by heat treatment at 55°C and 60°C. The highest initial fluorescence value was 392 observed using 100 mg.L -1 free chlorine followed by 60°C heat treatment (p <0.01, Kruskal-Wallis The purpose of this study was to explore the impact of free chlorine alone or followed by heat on 406 the loss of infectivity and the modification of the physicochemical properties of MS2 phage used as 407 model of the main pathogenic enteric viruses. The impact of free chlorine on capsid proteins has 408 been previously described in a few studies focusing on their altered functions. However, 409 modification of the overall structure of viral particles may lead to modification of their surface 410 properties which govern their behavior in the environment. Moreover, the study of surface 411 properties may contribute to the understanding of the mechanisms involved in the inactivation 412 process and may also help in the development of detection methods specific for infectious viruses.

413

MS2 phage was first exposed to free chlorine alone. The loss of infectivity of MS2 phage 414 observed in our experiments after exposure to 100 and 200 mg.L -1 of free chlorine was similar to 415 that reported by Loison et al. (2016) for a Qβ phage suspension. Indeed, by applying sodium 416 hypochlorite at 1 mM (i.e. 74.4 mg.L -1 ) for 10 min at room temperature on a Qβ phage suspension 417 at an initial concentration of ≈ 10 12 PFU mL -1 , these authors observed a -1.7 log 10 reduction in 418 infectivity. In the present work, we observed that the binding to host bacteria was altered after exposure to 200 mg.L -1 of free chlorine only. Thus, the loss of infectivity was not due to the exposure to 200 mg.L -1 of free chlorine. A few studies have shown that some regions of the viral 441 genome could be more sensitive than others to damage caused by free chlorine with significant 442 differences depending on the virus type (Li et al. 2002;[START_REF] Xue | Effects of chlorine and chlorine dioxide on human rotavirus infectivity 678 asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for 581 genome release[END_REF]. Free chlorine was shown 443 to induce genome damage on MS2 phage at levels comparable to singlet oxygen and UV. However, (Langlet et al. 2008a). The binding to positively and negatively charged 471 beads at different pH showed that the IEP of MS2 phage was between 3 and 4, which was in full 472 agreement with literature data in the field (Langlet et al. 2008b;[START_REF] Dika | Impact of internal RNA on aggregation 583 and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like 584 particles[END_REF]. MS2 phage 473 exposure to chlorine led to an increase in its adhesion capacity to positively charged beads at pH 474 above the IEP. Thus, the increase in the negative charge density of MS2 phage observed after 475 exposure to free chlorine resulted mainly from an increase in the external negative charge.

476

Hydrophobicity was the other surface property monitored after exposure to free chlorine. The 477 lack of adhesion to hydrophobic beads before exposure to free chlorine was in agreement with 478 previous studies performed with MS2 phage (Dika et al. 2013b;Brié et al. 2016). No adhesion to 479 hydrophobic beads was also observed after exposure to free chlorine. The DSF profile showed the 480 same fluorescence intensity of SYPRO Orange at the beginning of the experiment for control and 481 chlorinated phages. Thus, the results obtained with these two methods suggest that few hydrophobic 482 domains are naturally expressed on the surface of MS2 particles and that free chlorine does not 483 change the expression of hydrophobic domains of the MS2 capsid proteins or the permeability of 484 the capsid to the probe. An increase in hydrophobicity by chlorine has previously been described for 485 Qβ phage (Loison et al. 2016) and for specific proteins such as α2 chaperone (Wyatt et al. 2014) 486 and Bovine Serum Albumin (Ashrafi Kooshk et al. 2012). This suggests that the expression of 487 hydrophobic domains induced by chlorine depends on the composition and environment of the 488 protein.

489

Overall, the most significant effect we observed on MS2 phages exposed to free chlorine 490 alone was the decrease in the Tm in the DSF profile suggesting the weakening of the capsid. This 491 led us to explore the impact of free chlorine followed by heat on MS2 phages. Indeed, we had

495

An increased effect on the inactivation of MS2 phage and on the permeability of its capsid to 496 RNase was observed when the 60°C heat treatment was applied after chlorination. To our 497 knowledge, the virucidal effect of the combination of free chlorine and heat on enteric viruses and exposed to the 60°C heat treatment alone, the initial fluorescence of the DSF profile increased after 509 exposure to free chlorine followed by heat. The highest initial fluorescence value was observed 510 using free chlorine followed by a 60°C heat treatment. The increase in the initial fluorescence 511 showed that chlorine followed by heat led to the expression of hydrophobic domains and/or 512 increased the permeability of the capsid allowing the probe to reach inside hydrophobic regions.

513

The adhesion test to hydrophobic beads showed an increase in the expression of hydrophobic 514 domains at the surface of MS2 phages exposed to free chlorine followed by 60°C heat treatment.

515

Remarkably, this increase in the adhesion capacity to hydrophobic beads appeared to be specific to 516 viral particles remaining infectious as previously observed for MS2 phage exposed to heat alone 517 (Brié et al. 2016). After exposure to heat and free chlorine, infectious MS2 phages accounted for a small part of the total MS2 phage particles. Thus, our results showed that the permeability of the 519 capsid to the probe increased for both infectious and inactivated phages, whereas the increase in the 520 expression of hydrophobic domains at the surface was specific to phages remaining infectious after 521 treatment.

522

Overall, our results showed that free chlorine weakens the capsid of MS2 phage and alters the 523 functions of the MP at least for the highest concentration tested. The combination of heat and 524 chlorine has not been evaluated before for enteric phages or pathogenic viruses, but it has been 525 previously reported to be interesting for bacteria in foodstuffs. We observed that free chlorine 526 followed by heat increased the inactivation of MS2 phage and the structural modifications of the 527 capsid by comparison with these treatments applied separately. Our results suggested that the 528 oxidant should be applied before heat to have an optimal effect. Concerning the specific detection 529 of infectious viruses, the increase in permeability to enzymes such as RNases for inactivated phages 530 could offer new opportunities for such methods. In our opinion, the most important result 531 concerning free chlorine followed by heat is the specific increase in the expression of hydrophobic 532 domains at the surface of infectious MS2 phages. Indeed, this result suggested that hydrophobic 533 surfaces could be used to capture specifically infectious viruses before their detection using 534 molecular methods. Finally, the modifications observed on the capsid with our treatments underline 535 the need for further studies focusing on the structural modifications that occur during the viral life 536 cycle and also during inactivation treatments in order to better understand the mechanisms of virus 537 inactivation resulting from capsid damage. and 150 mM NaCl) for control MS2 phages (before treatment) and those exposed to free chlorine alone (100 and 200 mg.L -1 for 10 min) or to free chlorine (100 mg.L -1 for 10 min) followed by heat (55°C or 60°C for 10 min). Three independent experiments were performed.

Error bars indicate standard deviations. C X , concentration after binding assay; C 0 , initial concentration.

Figure 5. SYPRO Orange signature of control MS2 phages (before treatment) and those exposed to heat alone (55°C and 60°C for 10 min) or to free chlorine (100 mg.L -1 for 10 min) followed by heat (55°C or 60°C for 10 min). The curve represents one of three independent experiments. Fluorescence intensity is given in arbitrary units (10 6 × AU).

  phage (ATCC, 15597-B1) was done according to the standard procedure (ISO

  were counted after treatment (I) and without treatment (I 0 ) by the plaque 190 assay method using the double-agar-layer method (ISO 10705-1 1995) with E. coli K-12 Hfr as host 191 cells. Three to five decimal dilutions of the MS2 phage suspensions were dropped in duplicate onto 192 agar plates in order to have a dilution resulting in the enumeration of 10 to 100 plaques per Petri 193 dish. Infectious concentration was expressed in PFU mL -1 . The final concentration of purified MS2 194 phages was ≈ 10 14 PFU mL -1 . The loss in infectivity due to each treatment was estimated by ܗܔ . of potential degradation of the viral genome by free chlorine and/or heat. Thus, the 199 genome of MS2 phage was quantified after each treatment (C) and without treatment (C 0 ).The 200 genome degradation by the treatment was estimated by ܗܔ . Prior to the RT-qPCR assays, 201 extractions of viral RNA (50 µL) were performed using the QIAamp viral RNA kit (Qiagen, 202 Hilden, Germany) according to the manufacturer's recommendations. The extracts (100 µL) were

  228capacity of MS2 phage to E. coli was estimated by ܗܔ where Q is the viral RNA found on the 229 bacteria after free chlorine treatment and Q 0 without treatment. mobility (µ) measurements were performed in order to evaluate the impact 233 of free chlorine on the overall structure and charge density of MS2 phage, respectively. The 234 experiments were achieved in disposable capillary cells (Malvern Instruments, Worcestershire, UK) 235 at 23°C ± 0.1°C and pH 7.0 using a Zetasizer Nano ZS instrument (Malvern Instruments, He-Ne 236 red laser, wavelength 633 nm) driven by Dispersion Technology Software (Malvern Instruments) as 237 previously described (Langlet et al. 2008a). The slope of the electrophoretic mobility as a function 238 of the logarithm of ionic strength (1.95, 19.5 and 195 mM) reflects the ratio between the density of 239 electric charges located within the particle and that of charges distributed at the outer virus 240 periphery. A decrease in the slope value shows a decrease in the charge density of the virus (Langlet 241 et al. 2008a). The experimental conditions were identical to those previously described (Brié et al. 242 2016). For each condition, measurements were obtained by means of three independent experiments 243 with triplicate measures in each to ensure repeatability of the measurements. performed to study the variation in hydrophobicity or negative charge at the 247 surface of MS2 phages. Magnetic beads were used in these experiments: hydrophobic beads were 248 coated with polystyrene (PolySciences Europe GmbH, Hirschberg, Germany) and positively 249 charged beads were BcMag PSA (Bioclone, San Diego, CA, USA). As recommended by the 250 manufacturers, the beads were first washed twice in the buffer to be used in the assay which was 251 either 10 mM PBS (10 mM Na 2 HPO 4 and 150 mM NaCl) at pH 7 for hydrophobic beads or 50 mM 252 PBS (50 mM Na 2 HPO 4 and 20 mM NaCl) at different pH values (2.5, 3.0, 4.0, and 7.0) for ion 253 exchange beads. The positively charged beads exhibited a pKa of ≈ 10.

  255of MS2 phage to beads was estimated by ܗܔ where C X is the concentration of either infectious 256 phages or viral RNA after the binding assay in suspension and C 0 the initial concentration of either 257 infectious phages or viral RNA in suspension. treatment (5.10 14 -10 15 gc mL -1 ). The temperature was increased for all replicates from 270 20°C to 95°C on a 1% stepwise gradient corresponding to ≈ 1°C min -1 with fluorescence 271 measurements. The sum of the fluorescence intensities obtained with all the filters was plotted 272 against temperature (Excel software) to obtain the maximum fluorescence intensity of SYPRO 273

275

  hydrophobic parts of the capsid and/or the permeability of the capsid to be investigated by the 276 fluorescence intensity of SYPRO Orange at the beginning of the DSF experiment (Loison et al. 277 2016).

  performed using the values obtained for the three independent suspensions of 281 phages. Since the experimental values were not normally distributed, non-parametric tests were 282 performed using StatEL 2.7 (Ad Science). The explanatory variables were the exposure to free 283 chlorine alone, heat alone, and free chlorine followed by heat. For free chlorine alone, Mann-284 Whitney tests were performed to evaluate its effect on the infectivity (i.e. loss of infectivity, binding 285 capacity to host bacteria), structure (i.e. genome degradation, capsid permeability to RNase, thermal 286 stability), and hydrophobicity (i.e. initial adhesion of hydrophobic probe SYPRO Orange, binding 287 to hydrophobic probes), and the adhesion to positively charged beads by comparison between 288 control and chlorinated MS2 phages. A Kruskal-Wallis test was performed to compare the 289 electrophoretic mobility between control and chlorinated MS2 phages. The effect of free chlorine 290 followed by heat on the loss of infectivity, structure (i.e. genome degradation, capsid permeability 291 to RNase) and hydrophobicity (i.e. initial adhesion of hydrophobic probe SYPRO Orange, binding 292 to hydrophobic beads) was evaluated by comparison with free chlorine alone and heat alone using 293 Kruskal-Wallis tests. For all these tests, a p value of <0.01 was considered as significant.

  343not shown) for 55°C heat treatment alone and -0.8 ± 0.2 log 10 (Brié et al. 2016) for 60°C heat 344

  chlorine on the charge of MS2 phage particles 353

  368to the two concentrations of free chlorine in comparison with control phages (p <0.01, Mann-

  393test). The decrease in the fluorescence of the DSF profiles observed between 20°C and 60°C could 394 to probe quenching by water (Cimmperman and Matulis 2011;Loison et al. 2016).

  replication function played a lesser role in the overall inactivation than in the other two 445 inactivation treatments

464

  as sulfinic/sulfonic acids, methionine sulfone, and N-chlorinated species(Pattison, Davies and 465 Hawkins 2012) which may modify the electrostatic charge of viral particles. In our study, the 466 charge density was evaluated by measuring the electrophoretic mobility as a function of ionic 467 strength which suggested a decrease in the negative charge density of MS2 phage exposed to free 468 chlorine. On a qualitative level, it meant that free chlorine could change the negative charge density 469 particles by increasing their external negative charge and/or by decreasing their 470 internal negative charge

  492previously shown that heat significantly impacts the structure of the capsid with disruption of MS2 493 high temperatures (72°C) and transient expression of hydrophobic domains at 494 lower temperatures (60°C)(Brié et al. 2016).

  498phages has not been studied before. Indeed, chlorine-induced inactivation of enteric viruses and 499 phages in suspension in water has only been studied for temperatures below 30°C. The combined 500 effect of temperature and chlorination in foodstuffs has been more closely studied on bacteria, 501 showing an increase in inactivation compared to chlorine applied alone (Klaiber et al. 2005; Choi et 502 al. 2016). Moreover, these authors have shown that warm water (50°C) or dry heat (80°C) and 503 chlorine can be combined without altering the organoleptic quality or the viability of vegetables or 504 seeds. 505 Since hydrophobicity is known to be the main surface property affected by exposure of MS2 506 phages to heat (60°C) (Brié et al. 2016), a significant change in this parameter was expected when 507 free chlorine was followed by heat. As previously described by Brié et al. (2016) for MS2 phages 508

  518

  533surfaces could be used to capture specifically infectious viruses before their detection using 534 molecular methods. Finally, the modifications observed on the capsid with our treatments underline 535 the need for further studies focusing on the structural modifications that occur during the viral life 536 cycle and also during inactivation treatments in order to better understand the mechanisms of virus 537 inactivation resulting from capsid damage.

12

  processing technologies. Compr Rev Food Sci Food Saf 2010;9:3-20. 604 International Organization for Standardization. Water quality: Determination of free chlorine and 605 total chlorine. Part 2: Colorimetric method using N,N-diethyl-1,4-phenylenediamine, for routine treatment with intercalating dyes. Water Res 2016;91:68-76. Université de Lorraine, CNRS, LCPME, Nancy, F-54000, France 6 Food Safety Department, ACTALIA, Saint Lô, F-50000, France.

  Isabelle Bertrand, Laboratoire de Chimie Physique et Microbiologie pour 22 l'Environnement (LCPME), UMR 7564 CNRS Université de Lorraine, Faculté de Pharmacie, 5 rue 23 Albert Lebrun, BP 80403, 54001 Nancy, France.

24Page

  Tel:; E-mail: isabelle.bertrand@univ-lorraine.fr 25 and heat are the most commonly used sanitizer in water industry and 28 treatment in the food industry, respectively. Using MS2 phages as models for enteric viruses, we 29 investigated the impact of free chlorine and heat on their physicochemical properties. Free chlorine 30 was first evaluated alone. No increase in either capsid permeability or hydrophobicity was 31 observed. The negative surface charge slightly increased suggesting molecular changes in the 32 capsid. However, a weakening of the capsid by chlorine was suggested by differential scanning 33

  Heat-induced modification of the electrostatic charge is less obvious. Indeed, a decrease in 71 the isoelectric point (IEP) has been observed for poliovirus heated at 56°C and linked to the loss of 72 viral RNA genome and capsid protein VP4[START_REF] O'brien | Structural and compositional changes associated with chlorine inactivation 636 of polioviruses[END_REF] Breindl 1971). Only a 73 slight decrease in the global negative charge has been reported for MS2 phages after exposure to 7460°C (Brié et al. 2016). The structural changes in the capsid proteins caused by heat seem to alter 75 the structure specifically involved in host cell binding, and to be linked to modification of 76 hydrophobic domain expression.77Free chlorine has been shown to alter both genome-and protein-mediated functions of MS2 78 phage, more specifically genome injection and replication, but not its binding capacity to host cells 79(Wigginton et al. 2012). Free chlorine has also been shown to affect proteins of pathogenic enteric 80 viruses such as picornaviruses (Nuanualsuwan and Cliver 2003) and adenovirus (Page, Shisler and 81 Mariñas 2010). Moreover, the loss of genome replication has been shown to coincide with extensive 82 genome damage (Wigginton et al. 2012). The reaction of free chlorine with amino acids 83 constituting the capsid proteins leads to the formation of numerous products such as carbonyl 84 groups resulting from the oxidization of lysine, arginine, threonine, and proline. A relationship 85 between the number of carbonyl groups and the rate of free chlorine-induced inactivation has been 86reported for enteric viruses belonging to Astroviridae, Reoviridae, Caliciviridae, and 87 Picornaviridae(Sano et al. 2010; Tojo et al. 2013; Sano et al. 2015). The oxidization of amino 88 acids by chlorine could also result in modifications of the electrostatic charge, which, together with 89 hydrophobicity, is the major component of the surface properties of virus particles. To our 90 knowledge, the electrostatic charge has not previously been explored for enteric viruses exposed to 91 free chlorine. Concerning the hydrophobicity of chlorine-exposed viruses, a recent study has shown 92 that exposure to sodium hypochlorite modified the profile of the hydrophobic probe SYPRO 93 Orange in Qβ phage belonging to F-specific RNA phage genogroup III. Indeed, an increase in 94 hydrophobicity was observed at the beginning of the experiment; this meant that sodium

  99health. Indeed, studies performing a simultaneous and quantitative comparison between infectivity 100 loss and genome degradation have showed a higher persistence of viral genome after heat treatment 101(Bertrand et al. 2012) and chlorination[START_REF] Pelleieux | The effect of chlorination and hydrodynamic shear stress on 650 the persistence of bacteriophages associated with drinking water biofilms[END_REF]. Thus, the detection of viral genomes 102 by molecular methods used alone does not provide information about infectivity (Girones et al. 103 2010). Human noroviruses (NoV) and hepatitis A virus (HAV) are considered as the main 104 pathogenic enteric viruses. However, the detection of their infectious particles remains challenging 105 although cell culture methods have been very recently proposed for noroviruses (Jones et al. 2015; 106 Ettayebi et al. 2016). Various methods which could be combined with molecular approaches have 107 been evaluated in order to favor the detection of only infectious viruses. Most of them are based on 108 the increased permeability of the capsid to enzymes or intercalating dyes such as RNases, ethidium 109 monoazide (EMA), or propidium monoazide (PMA) (Prevost et al. 2016; Randazzo et al. 2016), or 110

113F

  -specific RNA phages (FRNAPH) belong to the family Leviviridae and are classified in two 114 genera, Levivirus and Allovivirus, each divided into two genogroups. These phages exhibit 115 numerous similarities with NoV and HAV in terms of size (20 -30 nm), genome (positive-sense 116 single-stranded RNA), and overall capsid structure

117

  phages infect bacteria with F pili, such as Escherichia coli, in the gut of mammals and are released 118 in environments exposed to fecal contamination similarly to enteric viruses. Their physicochemical 119 and biological characteristics and the easy-to-apply methods available for the quantification of their 120 genome have led to their use as models to assess pathogenic enteric virus 121 behavior in water (Boudaud et al. 2012), in food (Deboosere et al. 2012; Bozkurt et al. 2015), and 122 on surfaces (Morin et al. 2015; Pelleieux et al. 2016) during treatment. Due to their structural 123 similarity with the main waterborne pathogenic viruses they have also been proposed as relevant 124 indicators of viral contamination in water (Cole, Long and Sobsey 2003; Ogorzaly et al. 2009; 125 Vergara et al. 2015) and shellfish (Hartard et al. 2017) using molecular and/or cell culture methods.

  126

  130especially for studying the mechanisms of inactivation by different factors, and it can be produced 131 in high concentrations, an essential prerequisite for some experimental methods focusing on 132 physicochemical characteristics such as electron microscopy.133Thus, it appears that even treatments commonly used on enteric viruses such as free chlorine 134 and heat need to be improved. Molecular-based methods allowing a more specific detection of 135 infectious enteric viruses after such treatments are also needed. Such improvements require a better 136 knowledge of the damage that occurs in enteric viruses during loss of infectivity. In such a context, 137 the present work aimed to optimize the conditions of use of free chlorine and heat for the 138 inactivation of enteric viruses and to evaluate their impact on the surface properties of MS2. The 139 impact of free chlorine used alone was first evaluated on MS2 phages because of the poor 140 knowledge about its effects on the surface properties of enteric viruses. After verification of the 141 preservation of the capsid of inactivated phages, we examined in greater detail the thermal stability 142 and the surface properties of MS2 phages exposed to chlorine. The weakening of the capsid 143 observed in the present work when free chlorine was applied alone and the increase in 144 hydrophobicity previously described for MS2 phages exposed to heat (Brié et al. 2016) led us to focusing on hydrophobicity, in which heat treatment was applied to 146 MS2 phages previously exposed to chlorine. Since both free chlorine and heat have been shown to 147 act on the capsid, it might therefore be expected that their combination would increase their impact. phage (ATCC, 15597-B1) was done according to the standard procedure (ISO 152 10705-1 1995) (without the chloroform step) using Escherichia coli Hfr K12 (ATCC, 23631) as 153 host cells.

  154

  were counted after treatment (I) and without treatment (I 0 ) by the plaque 190 assay method using the double-agar-layer method (ISO 10705-1 1995) with E. coli K-12 Hfr as host 191 cells. Three to five decimal dilutions of the MS2 phage suspensions were dropped in duplicate onto 192 agar plates in order to have a dilution resulting in the enumeration of 10 to 100 plaques per Petri 193 dish. Infectious concentration was expressed in PFU mL -1 . The final concentration of purified MS2 194 phages was ≈ 10 14 PFU mL -1 . The loss in infectivity due to each treatment was estimated by ܗܔ . -quantitative Polymerase Chain Reaction (RT-qPCR) method was used 198 for the detection of potential degradation of the viral genome by free chlorine and/or heat. Thus, the 199 genome of MS2 phage was quantified after each treatment (C) and without treatment (C 0 ).The 200 genome degradation by the treatment was estimated by ܗܔ . Prior to the RT-qPCR assays, 201 extractions of viral RNA (50 µL) were performed using the QIAamp viral RNA kit (Qiagen, 202 Hilden, Germany) according to the manufacturer's recommendations. The extracts (100 µL) were 203 immediately stored at -80°C before quantification. Quantification of the phage genome was 204 performed by specific a RT-qPCR assay targeting a genome fragment of ≈ 150 nucleotides as 205previously described[START_REF] Ogorzaly | Development of real-time RT-PCR methods for specific detection of F-638 specific RNA bacteriophage genogroups: application to urban raw wastewater[END_REF]. Two negative controls and one positive control 206 (i.e. known quantity of MS2 genome) were added to each RT-qPCR analysis. RT-qPCR efficiency 207 ranged from 80% to 95%. Determination of MS2 genome copies per mL (gc mL -1 ) used for the 208 standard curve was performed as previously described(Brié et al. 2016). The final concentration of 209 purified MS2 phages was ≈ 10 15 gc mL -1 . microscopy (TEM) was used to visualize virus disruption or major structural 213 modifications. The samples were adsorbed onto grids and negatively stained for 1 min with 2% 214 phosphotungstic acid at pH 7.4 previously filtered with a 0.22 µm membrane filter. The 215 examination was carried out using a CM200 transmission electron microscope (Philips, Andover, 216 MA, USA) operating at 200 kV accelerating voltage and with a ×38,000 magnification.

  228capacity of MS2 phage to E. coli was estimated by ܗܔ where Q is the viral RNA found on the 229 bacteria after free chlorine treatment and Q 0 without treatment.

  mobility (µ) measurements were performed in order to evaluate the impact 233 of free chlorine on the overall structure and charge density of MS2 phage, respectively. The 234 experiments were achieved in disposable capillary cells (Malvern Instruments, Worcestershire, UK) 235 at 23°C ± 0.1°C and pH 7.0 using a Zetasizer Nano ZS instrument (Malvern Instruments, He-Ne 236 red laser, wavelength 633 nm) driven by Dispersion Technology Software (Malvern Instruments) as 237 previously described (Langlet et al. 2008a). The slope of the electrophoretic mobility as a function 238 of the logarithm of ionic strength (1.95, 19.5 and 195 mM) reflects the ratio between the density of 239 electric charges located within the particle and that of charges distributed at the outer virus 240 periphery. A decrease in the slope value shows a decrease in the charge density of the virus (Langlet 241 et al. 2008a). The experimental conditions were identical to those previously described (Brié et al. 242 2016). For each condition, measurements were obtained by means of three independent experiments 243 with triplicate measures in each to ensure repeatability of the measurements.

  244

  performed to study the variation in hydrophobicity or negative charge at the 247 surface of MS2 phages. Magnetic beads were used in these experiments: hydrophobic beads were 248 coated with polystyrene (PolySciences Europe GmbH, Hirschberg, Germany) and positively 249 charged beads were BcMag PSA (Bioclone, San Diego, CA, USA). As recommended by the 250 manufacturers, the beads were first washed twice in the buffer to be used in the assay which was 251 either 10 mM PBS (10 mM Na 2 HPO 4 and 150 mM NaCl) at pH 7 for hydrophobic beads or 50 mM 252 PBS (50 mM Na 2 HPO 4 and 20 mM NaCl) at different pH values (2.5, 3.0, 4.0, and 7.0) for ion 253 exchange beads. The positively charged beads exhibited a pKa of ≈ 10. 254 The binding assays were carried out as previously described (Brié et al. 2016). The binding capacity 255 of MS2 phage to beads was estimated by ܗܔ where C X is the concentration of either infectious 256 phages or viral RNA after the binding assay in suspension and C 0 the initial concentration of either 257 infectious phages or viral RNA in suspension.

  fluorimetry (DSF) was performed using the hydrophobic probe SYPRO 261 Orange. This probe has a high affinity for hydrophobic domains (Niesen, Berglund and Vedadi 262 2007), which are unmasked when proteins unfold. DSF was used to compare the thermal stability of 263 MS2 phage, as measured by the denaturation temperature (Tm). Thermal melting curves of MS2 264 phage were obtained in 96-well plates using StepOne Plus qPCR System (Life Technologies, 265 Thermo Fisher Scientific) and the fluorescent dye SYPRO Orange, 5,000 × concentrate, supplied in 266 DMSO (Invitrogen, Thermo Fisher Scientific). Reactions were performed in a 25 µL final volume 267 containing 5 µL of buffer (150 mM Tris-HCl, pH 8), 2 µL of SYPRO Orange solution 62.5 × (final 268 concentration = 5 ×), 16 µL of ultrapure water (> 18 MΩ cm -1 ), and 2 µL of MS2 suspension from 269 treatment (5.10 14 -10 15 gc mL -1 ). The temperature was increased for all replicates from 270 20°C to 95°C on a 1% stepwise gradient corresponding to ≈ 1°C min -1 with fluorescence 271 measurements. The sum of the fluorescence intensities obtained with all the filters was plotted 272 against temperature (Excel software) to obtain the maximum fluorescence intensity of SYPRO 273

276

  fluorescence intensity of SYPRO Orange at the beginning of the DSF experiment (Loison et al. 277 2016).

  294

  319

  343not shown) for 55°C heat treatment alone and -0.8 ± 0.2 log 10 (Brié et al. 2016) for 60°C heat

  chlorine on the charge of MS2 phage particles 353

  368to the two concentrations of free chlorine in comparison with control phages (p <0.01, Mann-

  Fig. 4) (p <0.01, Kruskal-Wallis test). Remarkably, this increase in the adhesion capacity to

  419

  replication function played a lesser role in the overall inactivation than in the other two particles by increasing their external negative charge and/or by decreasing their 470 internal negative charge

  492previously shown that heat significantly impacts the structure of the capsid with disruption of MS2 high temperatures (72°C) and transient expression of hydrophobic domains at 494 lower temperatures (60°C)(Brié et al. 2016).

498

  phages has not been studied before. Indeed, chlorine-induced inactivation of enteric viruses and 499 phages in suspension in water has only been studied for temperatures below 30°C. The combined 500 effect of temperature and chlorination in foodstuffs has been more closely studied on bacteria, 501 showing an increase in inactivation compared to chlorine applied alone (Klaiber et al. 2005; Choi et 502 al. 2016). Moreover, these authors have shown that warm water (50°C) or dry heat (80°C) and 503 chlorine can be combined without altering the organoleptic quality or the viability of vegetables or 504 seeds. 505 Since hydrophobicity is known to be the main surface property affected by exposure of MS2 506 phages to heat (60°C) (Brié et al. 2016), a significant change in this parameter was expected when 507 free chlorine was followed by heat. As previously described by Brié et al. (2016) for MS2 phages 508

  518

  supported by the "Conseil Régional de Basse Normandie" and coordinated by 548 ACTALIA within the scope of the project CapsiVir. This study, labeled by the competitiveness 549 cluster VALORIAL, was also supported by the Joint Technological Unit ACTIA VIROcontrol 550 (partnership between ACTALIA and LCPME) and the "Syndicat des Fabricants de Produits 551 Végétaux Frais Prêts à l'Emploi" (cluster of ready-to-eat food industries: Florette, Bonduelle, 552 Crudettes, and Rosée des Champs). inert and vegetal food surfaces. Food Microbiol 2012;32:48-56.

  processing technologies. Compr Rev Food Sci Food Saf 2010;9:3-20. 604 International Organization for Standardization. Water quality: Determination of free chlorine and 605 total chlorine. Part 2: Colorimetric method using N,N-diethyl-1,4-phenylenediamine, for routine 606 control purposes. International Organization for Standardization, Geneva, Switzerland. ISO Martin H, Soumet C et al. Comparison of the virucidal efficacy of peracetic acid, 629 potassium monopersulphate and sodium hypochlorite on bacteriophages P001 and MS2. treatment with intercalating dyes. Water Res 2016;91:68-76.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. SYPRO Orange signature of MS2 phages exposed to free chlorine. Effect of free

Figure 3 .

 3 Figure 3. Electrostatic charge of control MS2 phages (before treatment) and those exposed to free chlorine (100 and 200 mg.L -1 for 10 min). (a) Electrophoretic mobility (µ) as a function

Figure 4 .

 4 Figure 4. Binding to hydrophobic, magnetic beads at pH 7 in 10 mM PBS (10 mM Na 2 HPO 4

Table 1 :

 1 Figure 1

  Figure 2

  Figure 4

  led us to

	F o r
	P e
	e r
	R e
	v
	i e
	w
	145
	7

ScholarOne Support 1-434/964-4100

<0.01, Kruskal-Wallis test). Moreover, the highest log removal of genome copies by RNase

to the alteration of this function after exposure to 200 mg.L -1 of free chlorine. Our observation of a

effect due to genome damage was linked to the type of modification rather than its location.

Microbiol 2015;119:655-65.

ScholarOne Support 1-434/964-4100 FEMS Microbiology Ecology

ACKNOWLEDGMENTS ACKNOWLEDGMENTS

Enteric viruses and phages are exposed to various inactivating factors in water, in foodstuffs, 47 and on surfaces. Among them, heat and chlorine are one of the most cost-effective and easy-to-48 apply treatments and sanitizers in the food and water industries. The level of inactivation of enteric 49 viruses by heat has been widely monitored in food and environmental matrices in which it is 50 considered as one of the main inactivating factors (Hirneisen et al. 2010;Bertrand et al. 2012).

51

Chlorine is a major disinfection treatment of water and food for its broad-spectrum antimicrobial 52 activity (Hirneisen et al. 2010;Boudaud et al. 2012;[START_REF] Girard | Efficacy of oxidizing disinfectants at inactivating murine 596 norovirus on ready-to-eat foods[END_REF]Meireles et al. 2016).

53

Although virus inactivation by heat or chlorine has been evaluated in numerous studies, the 54 mechanisms leading to non-infectious virus particles still remain partially understood. Comparison 55 of the impact of different inactivating treatments on virus functions (i.e. host cell binding, genome 56 injection and replication), using MS2 phage as model of pathogenic enteric viruses, has revealed 57 differences in the mechanisms of inactivation between physical treatments such as heat and 58 chemical oxidants such as free chlorine, but also within treatment groups (Wigginton et al. 2012).

59

Heat induces structural and conformational changes of the capsid proteins leading to at least 60 inactivation through alteration of the binding capacity to host cells, modification of surface 61 properties, and increase in permeability to enzymes and intercalating dyes. These findings have 62 been reported for poliovirus, hepatitis A virus, feline calicivirus, and MS2 phage (Breindl 1971; 63 [START_REF] Nuanualsuwan | Capsid functions of inactivated human picornaviruses and feline 634 calicivirus[END_REF][START_REF] Pecson | Quantitative PCR for determining the infectivity of bacteriophage 646 MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations 647 of an enzymatic treatment to reduce false-positive results[END_REF]Wigginton et al. 2012;Brié et al. 64 2016). Heat has been shown to have, if any, only a very low impact on the virus genome 65 (Wigginton et al. 2012). Hydrophobicity seems to be the surface property the most affected by heat 66 treatment. Indeed, the expression of hydrophobic domains has been reported for MS2 phages 67 exposed to heat but for remaining infectious particles only (Brié et al. 2016). An increase in 68 hydrophobicity has also been observed for poliovirus exposed to 50°C, which has been linked to the 69 externalization of the N-terminal region of viral protein VP1 [START_REF] Fricks | Cell-induced conformational change in poliovirus: externalization of the 594 amino terminus of VP1 is responsible for liposome binding[END_REF]Curry et al. 70 The decrease in the thermal stability after exposure to chlorine led us to expose MS2 phage to 324 chlorine (100 mg.L -1 ) and then to heat (55°C or 60°C). The infectivity decreased by -1.9 ± 0.3 log 10 325 for chlorine followed by heat treatment at 55°C and by -3.2 ± 0.6 log 10 for chlorine followed by 326 heat treatment at 60°C. For each treatment applied separately, the loss in infectivity reached -327 1.2 ± 0.3 log 10 after exposure to free chlorine (Table 1), -0.7 ± 0.1 log 10 after heat treatment at 55°C 328 (data not shown) and -1.1 ± 0.1 log 10 after heat treatment at 60°C (Brié et al. 2016). The highest 329 loss in infectivity was obtained when free chlorine was followed by heat treatment at 60°C (p 330 <0.01, Kruskal-Wallis test). This loss in infectivity was also higher than the sum of the decrease 331 values obtained with free chlorine and heat treatment at 60°C applied separately (p <0.01, Kruskal-332 Wallis test). This suggested that free chlorine improved the inactivation of MS2 phage by heat 333 treatment at 60°C. The RT-qPCR signal decreased only by -0.1 ± 0.4 log 10 for chlorine followed by 334 heat treatment at 55°C and by -0.3 ± 0.4 log 10 for chlorine followed by heat treatment at 60°C. The 335 concentrations in genome observed after these treatments were similar to those observed for MS2 336 phage exposed to 100 mg.L -1 free chlorine alone and also for control MS2 phage (p >0.01, Kruskal-337 Wallis test). Thus, the detection of the MS2 genome was not affected when free chlorine was 338 followed by heat. As observed for free chlorine alone, no modification of the shape of MS2 phage 339 was noted by TEM after exposure to free chlorine followed by heat (Fig. 1). The RNase treatment 340 applied after exposure to free chlorine (100 mg.L -1 ) followed by heat decreased the RT-qPCR signal 341 by -1.1 ± 0.2 log 10 for 55°C and -1.7 ± 0.4 log 10 for 60°C. The log removal of genome copies by high concentration of free chlorine needed to observe an impact on binding to host cell was in 423 agreement with a previous study published by Wigginton et al. (2012). Indeed, these authors 424 applied a concentration of 2-3 mg.L -1 of free chlorine to an MS2 phage suspension of 10 11 PFU L -1 425 and observed alteration of the genome injection and extensive degradation of the maturation protein 426 (MP). This protein, which is present as a single copy in the capsid of MS2 phage, is clearly 427 involved in the binding of MS2 phage to the F-pilus of host bacteria and probably in the injection of 428 the genome into host cells. Dent et al. (2013) have proposed that the natural retraction of the F-pilus 429 could pull the MP from its location in the attached capsid, leaving a pore through which the phage 430 genome could exit into the cell. Thus, alteration of the MP by free chlorine may impact these two 431 functions depending on its intensity. The sensitivity of the capsid to oxidants has also been observed 432 for Qβ phage with the loss of its infectivity due either to capsid destabilization or to the 433 modification of capsid proteins especially the MP and the readthrough protein (Loison et al. 2016).

Considering the capsid of FRNAPH, the MP appears as one of the targets of free chlorine. Thus, 435 free chlorine weakens the capsid of MS2 phage and may alter the role of the MP in the genome 436 injection into host cells even at low concentrations. High concentrations of free chlorine seem to 437 further alter the MP with a reduced ability to bind to host cells.

438

In our experiments, the genome of MS2 phage was quantified using an RT-qPCR assay which 439 targets a fragment of ≈ 150 nucleotides and the RT-qPCR signal was found to be affected only after No modification of the shape of MS2 phage was observed by TEM after exposure to free 448 chlorine. The mean size of MS2 phage before and after chlorination was similar to that previously 449 described (Valegård et al. 1990;Dika et al. 2013a). Qβ phage particles exposed to chlorine under 450 similar conditions were also demonstrated by TEM observations to maintain their shape (Loison et 451 al. 2016). Disruption of MS2 phage occurring after exposure to 72°C for 10 min has previously 452 been shown to be related to a significant increase in RNase sensitivity (Brié et al. 2016). In our 453 case, the limited effect of RNase treatment on RT-qPCR signal confirmed that the integrity of the 454 capsid was preserved after exposure to free chlorine under our experimental conditions. DSF was 455 then used to explore conformational modifications of the capsid that could not be revealed by TEM.

456

We observed a similar DSF profile to that obtained in our previous study (Brié et al. 2016) with 457 only one Tm peak (66°C) before exposure to chlorine. The decrease in the Tm value after exposure 458 to free chlorine corresponded to a reduction in the denaturation temperature and suggested a 459 weakening of the capsid of MS2 phage by the oxidant by comparison to a previous study performed 460 on poliovirus (Robinson, Jesudhasan and Pfeiffer 2014). The weakening of the capsid might be 461 linked to the site-specific cleavage showed on the capsid protein of MS2 phage following exposure 462 to chlorine (Wigginton et al. 2012).

463

The reactions of hypochlorous acid with amino acids result in a wide variety of products such 464 as sulfinic/sulfonic acids, methionine sulfone, and N-chlorinated species (Pattison,Davies and 465 Hawkins 2012) which may modify the electrostatic charge of viral particles. In our study, the 466 charge density was evaluated by measuring the electrophoretic mobility as a function of ionic 467 strength which suggested a decrease in the negative charge density of MS2 phage exposed to free 468 chlorine. On a qualitative level, it meant that free chlorine could change the negative charge density 469


J Deng

J M Pearce

V Vlahos

M L Albert

R W Collins

C R Wronski

A NEW APPROACH TO THE ANALYSIS OF FORWARD BIAS DARK CURRENT-VOLTAGE CHARACTERISTICS OF A-SI:H SOLAR CELLS

Generally the dark forward bias current voltage (JD-V) characteristics of a-Si:H solar cells are analyzed without clearly separating their contributions due to carrier recombination in the bulk from that at the p/i interface regions nor those imposed by carrier injection from the p and n contacts. Furthermore their exponential regimes are interpreted and fitted with constant diode quality factor n with modeling which is based on many fitting parameters that have not been reliably established. A new approach has been developed for the analysis of JD-V characteristics for the entire voltage range relevant to the operation of solar cells, which has allowed the three contributions to be identified and characterized. It is based on the analysis of their bias dependent differential diode quality factors, n(V), from which important information on the energy distribution of the defect states in the i-layers has been obtained. Results are presented and discussed here for p-i-n a-Si:H solar cells having sufficiently low p/i interface recombination so that the limitations by the bulk recombination on open circuit voltage, Voc, can be identified. These results are then correlated with the defect state distributions previously obtained from the analysis on differential diode quality factor n(V) characteristics for hydrogen diluted and undiluted intrinsic layers both in the annealed state as well as after introducing light induced defects.

INTRODUCTION

A good understanding of the defect states in the intrinsic layers of a-Si:H solar cells and the limitations imposed by different device loss mechanisms on their performance and stability is critical in the optimization of constituent materials as well as cell structures for the further improvement of the already high performance of these solar cells. The conventional measurement techniques and analysis used in such investigations are primarily based on the analysis of light current-voltage (I-V) parameters such as fill factor ( FF), open circuit voltage (Voc), and short circuit current (Jsc), measured under 1 sun illumination. These parameters are inherently complicated, being affected by the contributions from different parts of the cells such as bulk i-layer, p/i interface as well as n and p contacts, which are difficult to separate and thus to quantify. In order to overcome these limitations new approaches are being developed. They include the systematic analysis on carefully designed cell structures of forward bias dark current (JD-V) characteristics which are extensively used in characterizing crystalline solar cells. In the past, little attention was given to JD-V characteristics of a-Si:H based solar cells because of reported lack of correlation between light and dark I-V's and the predictions of the defect pool model for the presence of very high densities near the n and p contacts and nonuniform distributions of defect states across the i-layer [START_REF] Crandall | Proc. 21 st IEEE PVSC[END_REF]. Recently however, equivalence between the carrier recombination in the JD-V and light I-V characteristics has been established from the superposition between Jsc-Voc and JD-V characteristics over a wide range of illumination intensities [START_REF] Deng | [END_REF]3]. It has also been shown that in solar cells with high quality materials and optimized cell structures the defect state densities are approximately uniform across the entire bulk i-layer [4]. Consequently, it has been possible through the new approaches to the analysis of JD-V characteristics to obtain valuable information about the contributions of p/i interface recombination and potential barriers at the p and n contacts, as well as bulk recombination. A key factor of the new approach is the analysis of differential diode quality factors, n(V), characteristics whose presence has not been previously recognized and allow direct information to be obtained about the nature and densities of the defect states in the ilayers [5]. The advantages of this new approach are illustrated here with successful application of the results on JD-V characteristics to the results on Voc's under various illumination intensities in the cells where the p/i interface recombination is minimized and the carrier transport is dominated by the recombination in the bulk ilayers. Excellent correlation is found between the JD-V characteristics and Voc's for cells with different bulk ilayers in their annealed states as well as after light induced degradation. In addition, the distinct differences observed in the degradation kinetics of 1 sun Voc between the cells having hydrogen diluted (protocrystalline) and undiluted i-layers are correlated with the differences in their defect state distributions between these two materials as indicated by the analysis carried out previously on the differential diode quality factors n(V) [5].

EXPERIMENTAL DETAILS

The p (a-SiC:H)/ i (a-Si:H)/ n (µc-Si) (superstrate) a-Si:H solar cells used in this study were fabricated by RF plasma enhanced chemical vapor deposition at a 1404 0-7803-8707-4/05/$20.00 ©2005 IEEE.

substrate temperature of 200°C under conditions described previously [6]. The bulk i-layers in the cell structures were deposited using SiH4 diluted with H2, with R = [H2]/ [SiH4] of either 10 (diluted) or 0 (undiluted). To minimize the contributions of shunts, cell str ucture areas of 0.02 cm 2 were used, which were defined by removing the uncovered top n µc-Si:H layers with reactive ion etching. The JD-V characteristics measurements were carried out at temperatures that were controlled to within ±0.1 o C and a three-probe technique was used to eliminate any effects of extraneous series resistance. Care was also taken to ensure that the steady-state currents were measured at low biases and that no defects were introduced by double injection in the high forward bias regions. The annealed states were achieved by heating the cells for 4 hours at 170°C and the light induced defects were introduced using illumination generated by ELH lamp which has light intensity equivalent to that of 1 sun illumination. The illumination intensities lower than that of 1 sun was obtained with various combinations of neutral density filters.

RESULTS

It has been shown that in the cells where bulk recombination dominates the current and the defect state distribution is essentially uniform across the bulk i-layer, there is a dependence of the JD-V characteristics on the bulk i-layer thickness as a result of the scaling of the electric fields [4]. One natural extension to this is that because of the higher recombination in the thicker cells there should also be a corresponding reduction in their 1 sun Voc's. This requires that the bulk recombination dominates the carrier transport not only at relatively low biases but also at high biases up to 1 sun Voc. That this is indeed the case is illustrated in Fig. 1 which shows the results on the thickness dependence of 1 sun Voc as well as JD-V characteristics for p-i-n cells having the same structure but different thickness of R=0 bulk i-layers. In these cells the recombination at the p/i interfaces is minimized by having a 200Å of R=40 protocrystalline ilayer in the p/i region. It can be seen in the insert of Fig. 1 that the 1 sun Voc decreases systematically with the increasing thickness of the bulk i-layer where a decrease of ~30mV (from about 0.89 to 0.86V) occurs in Voc for an increase of a factor of 2 in the dark currents at low biases. This 30mV decrease in Voc is consistent with the increase in the dark currents whose increase for this change in voltage corresponds to the "effective" diode quality factor of ~1.6 present in these cells [5]. It is important to note here that these Voc's were measured not with fixed illumination intensities but instead with a fixed Jsc of 10 mA/cm 2 (equivalent to 1 sun Jsc for the cells with 0.4µm thick i-layer) to eliminate the effect from the changes in light absorption due to the differences in the i-layer thickness. In thicker cells the increases in the dark current at voltages corresponding to 1 sun Voc cannot be directly observed because they become limited by carrier injection over the potential barriers in the i-layers adjacent to the p and n contacts [4]. The domination of these Voc's by bulk recombination currents at biases corresponding to 1 sun Voc in the cells studied could be further confirmed with the degradation kinetics of the 1 sun Voc. Shown in Fig. 2 is the degradation of the 1 sun Voc under 1 sun illumination at 25 o C for the cell in Fig. 1 having the 0.4µm thick i-layer. It can be seen that there is an immediate decrease in the 1 sun Voc which is a clear indication that there are no significant contributions from p/i interface recombination. After 100 hours of degradation there is a decrease in Voc of around 50 mV that is consistent with the changes in the JD-V characteristics of the same cell as shown in Fig. 3. In Fig. 3 there is an increase in the recombination current of a factor of ~3 at a bias that corresponds to the 1 sun Voc. Annealed state 1 sun 100 hours 1 sun V oc Fig. 3: JD-V characteristics of the cell in Fig. 2 in the annealed state and after 100 hours of 1 sun illumination.

Similar correlations between the degradation of Voc's and JD-V characteristics are also obtained for the cells with R=10 protocrystalline i-layers. This is illustrated in Figs. 4 and5 with results on a p-i-n cell with 0.4µm thick R=10 bulk i-layer and 200Å thick R=40 p/i interface. In Fig. 4 the decreases in Voc's measured under light intensities from 1 to 10 -4 sun, are shown as a function of the degradation time under 1 sun illumination. It can be seen that as the light intensity and Voc are lowered there are corresponding changes in both the rate and magnitude of the voltage decreases in reaching a degraded steady state after ~100 hours of illumination. For the illumination intensity of 10 -4 sun, corresponding to an initial Voc of 0.62V, there is a decrease of ~ 70 mV in Voc. For 1 sun illumination the degradation is less than 10 mV, a value that is also significantly sm aller than the 50 mV degradation of the 1sun Voc for the cell with R=0 i-layer after the same degradation. This small degradation at such a high illumination level cannot be due to the limitations imposed by p/i interface recombination since a similar dependence of 1 sun Voc on thickness was also found for the cells with R=10 i-layers. The degradation in Voc's in Fig. 4 can be directly correlated with the changes in JD-V characteristics shown in Fig. 5. In Fig. 5 there are systematic increases in the dark currents with illumination time just as in the corresponding Vos's. The changes in the currents which are larger at the lower biases correspond to the higher decreases in Voc's measured at the lower illumination intensities. Unlike the large change in the cell with the R=0 i-layer, there is hardly any discernable changes in the dark current at the levels equivalent to 1 sun Jsc. This cannot be completely due to the carrier injection limitations imposed by the potential barriers since these limitations occur at similar current levels for both types of the cells. The difference in the 1 sun Voc degradation between the cells with the R=10 and R=0 ilayer thus clearly points to a distinct difference in their gap state distributions upon light induced degradation. Annealed state 1 sun 30 mins 1 sun 10 hrs 1 sun 100 hrs R=10 Bulk with R=40 p/i 1 sun V oc Fig. 5: JD-V characteristics of the cell in Fig. 4 in the annealed state and after light induced degradation.

DISCUSSION

Several conclusions can be drawn from the above experimental results. First, in these cells the currents are dominated by bulk recombination up to bias levels equivalent to 1 sun Voc. This is significant since then the results obtained on the JD-V characteristics can be directly applied to the analysis of solar cell performance. Second, there are large light induced changes in the densities and distributions of the defect states in both the undiluted and diluted protocrystalline i-layers associated with Staebler-Wronski Effect (SWE). Thirdly, the distributions of gap states in the two i-layers are distinctly different, thus leading to the differences in the corresponding solar cell performance and stability.

In order to understand the relation between solar cell parameters such as Voc and the distribution of gap states in the i-layers it is necessary to identify the differences present in the two i-layer materials. The essential features of the distributions for both the undiluted and diluted i-layers have been obtained from the analysis on the n(V) characteristics [5]. In both cases they are consistent with a Gaussian-like distribution around midgap followed by an exponentially rising distribution away from the midgap which could also be regarded as the rising edge of a second Gaussian-like distribution. Similar Gaussian-like distributions have been proposed in several previous studies [7][START_REF] Jiao | Proc. 25 th IEEE PVSC[END_REF][START_REF] Günes | [END_REF][10]. There is however a major difference between the undiluted and diluted i-layer where in the former case the rising edge of the second Gaussian-like distributions are much less steep. Furthermore, after the introduction of light induced defects, the distributions in both i-layers broaden with a much smaller broadening occurring in the case of the protocrystalline i-layer. For both cases there is also a large increase in the density of states around midgap which causes the large increases in the dark currents under low forward biases and the large decreases in the Voc's under illumination intensities much smaller than 1 sun. On the other hand, in the case of the dark currents under high forward bias and Voc under sufficiently high illumination intensity, the splitting between the two quasi-Fermi levels are sufficiently large as to include as recombination centers not only the gap states around midgap but also those located further away from the midgap. For the cell with protocrystalline i-layer, due to the narrower Gaussian-like distributions in the annealed and degraded states, the positions of quasi-Fermi levels at 1 sun Voc are in a region where the native defects away from midgap play a dominant role in the recombination. This then explains the very small degradation of 1 sun Voc while there is significant degradation for the Voc's under the lower intensities of illuminations. On the other hand, for the cell with undiluted i-layer, due to the broad distribution away from midgap the contribution from the light induced defects increases the recombination over that of the native defect states alone and accounts for the significant degradation in 1 sun Voc. It is very important to note here that for cells having both undiluted and protocrystalline i-layers the degradation in the 1 sun Voc's are much smaller than that expected if these Voc's were limited purely by the recombination through the neutral dangling bonds, with states located around midgap and detected by electron-spin resonance. This is a further indication for the gap states other than the neutral dangling bonds determining cell performance [11,12] and the key role of the Gaussian-like distributions away from midgap identified from the analysis of the n(V) characteristics.

CONCLUSIONS

It has been shown from the JD-V characteristics it is possible to identify and quantify the limitat ion imposed on 1 sun Voc by bulk recombination. As a consequence, it has been possible to relate the light induced changes in JD-V characteristics to those in 1 sun Voc for cells with undiluted and protocrystalline i-layers. In addition, the new approach in the analysis of JD-V characteristics in terms of differential diode quality factor n(V) has allowed these differences to be directly related to those in the distributions of gap states in the corresponding i-layers. The results also demonstrate that JD-V characteristics offer a new and powerful probe for characterizing mechanisms limiting the performance and stability of a-Si:H solar cells as well obtaining direct information about the distribution of gap states in the intrinsic layers. They also point out the key reason why it is not possible to obtain correlation between solar cell characteristics and neutral dangling bond densities. It is therefore important to take advantage of this powerful probe in guiding systematic approach to further improving performance of a-Si:H based solar cells as well as obtaining new insights into SWE.

Fig. 1 :Fig. 2 :

 12 Fig.1: Thickness dependence of 1 sun Voc and JD-V characteristics for p-i-n cells having the same structure but different thickness of R=0 bulk i-layers.

Fig. 4 :

 4 Fig. 4: Degradation of Voc's for a p-i-n cell with 0.4µm thick R=10 bulk i-layer and 200Å thick R=40 p/i interface measured under light intensities from 10 -4 to 1 sun.

ACKNOWLEDGEMENTS

This research was supported by the National Renewable Energy Laboratory (NREL) under subcontract NDJ-2-30630-01.