assessing innovation, and disseminating knowledge, these activities being organised to take up the challenges of

This study shows that, from a purely metrological point of view, the ramp test and the psychophysical method give the highest correlation coefficients. The second advantage of the ramp test is its renown among trade professionals who have been using it for categorising floor coverings for several decades now, regardless of the types of flooring and of their fields of use. However, implementing it suffers from major drawbacks, such as the need to use test subjects and the impossibility of taking measurements on site.

The LabINRS method gives correlation coefficients with the psychophysical method that are very good and that are almost equivalent to those obtained with the ramp test. In addition, it is very simple to implement; however, it suffers from the drawback of not making it possible for measurements to be taken in situ.

Conversely, due to its very simple operating principle and to it being easy implement, the pendulum SRT is the best suited to taking measurements in situ. Its renown is high as it has been in use for many years for qualifying road surfaces and pedestrian surfaces. However, the correlation coefficients with the psychophysical method are low, raising questions as to its capacity to rank floor coverings as a function of slipperiness. In addition, it does not make it possible to qualify correctly all types of floor covering, e.g. coverings with macro-relief.

As for PFT, the correlation coefficients with the psychophysical method are very satisfactory. Even though it was originally designed to qualify road surfaces, INRS has considerable experience in using it for qualifying pedestrian surfaces, in particular such surfaces that are designed for use in the food industry. It makes it possible to take measurements in the laboratory and in situ, on any type of floor covering.

-OUTLINE OF REASONS AND OBJECTIVES

According to the statistics of France's National Health Insurance Fund for Salaried Employees (Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés), falls on the same level represent about one quarter of occupational accidents resulting in sick leave. Loss of balance due to slipping is one of the causes of such falls. For some twenty years now, INRS (France's National Research and Safety Institute for the Prevention of Occupational Accidents and Diseases) has been using two testing methods namely the LabINRS, that it developed, and the PFT (Portable Friction Tester), for evaluating the coefficients of friction of floor coverings. They make it possible, respectively, to conduct measurements in the laboratory and in situ [1,2,3].

These two testing methods are essentially known and used for characterising the performance of slip resistance of flooring in workplaces in the food industry.

A European standardisation group recently worked on preparing a Technical Specification [4] representing the current state of the art of the most commonly used test methods for determining slip resistance of pedestrian surfaces. That document has been incorporated into the collections of French experimental standards, with a view to supplementing and to ultimately replacing the experimental standard XP P 05-010 which addresses the same topic. The two most commonly used assessment methods selected by the European Committee for Standardization (CEN) for characterising new products in the laboratory are the Ramp Test [5] taken from German Standard DIN 51130 [START_REF]Testing of floor coverings -Determination of the anti-slip properties -Workrooms and fields of activities with slip danger; Walking method -Ramp test[END_REF], and whose principle has been taken up in Standard XP P 05-010 [START_REF]Floor coverings -Determination of slip resistance using the ramp test[END_REF], and the pendulum Skid Resistance Tester (SRT) developed in Great Britain and described in Standard EN 13036-4 [START_REF]Road and airfield surface characteristics -Test methods -Part 4 : method for measurement of slip/skid resistance of a surface : the pendulum test[END_REF].

Assessment by using the ramp test is performed with human testers. This method cannot be used in situ and is applicable only for laboratory characterisation of samples of floor coverings. And yet the slip resistance of such coverings need to be tested in situ, in particular coverings that are cast on site, in order to check that their coefficients of friction are similar to the coefficients of friction measured on the samples in the laboratory. The coefficients of friction depend on the surface characteristics (roughness, homogeneity, evenness) that in turn depend essentially on the quality of fitting or of casting of the coverings.

The pendulum SRT is transportable and makes it possible to take both laboratory measurements and also in situ measurements. However, its measurement distance is only 12 cm. It can be necessary to perform quite large numbers of measurement operations in order to characterise a floor covering in situ.

The Ramp Test and SRT methods are in common use in France for assessing the slip resistance of pedestrian surfaces respectively initially in the laboratory, and in-situ in a variety of premises and of sectors of activity.

The aims of this study were thus to compare the four methods, to assess the practical aspects of each of them, and to define the limits of them. To be relevant, the 4 measurement methods need to be sufficiently representative of the slip perceived during walking, and to be sufficiently discriminatory with respect to slips. INRS developed a psychophysical rating method, the aim of which was to make it possible to establish reference rankings based on "perceived slipperiness". Those rankings were to be compared with the rankings obtained using the metrological methods.

This study was conducted in partnership between INRS and CSTB (Centre Scientifique et Technique du Bâtiment). The aim of this collaboration was to define a common position and to enable that position to be defended more effectively in the working groups and on the standardisation committees. p. 5

-METHODOLOGY

Methods studied

In order to meet the objectives of the study, measurements were performed in the laboratory using the 4 methods on a panel of floor coverings so as to rank them from the least slippery to the most slippery.

The tests were distributed in the following manner:

 for the laboratory equipment: the ramp tests were conducted at CSTB, and the LabINRS tests were conducted at INRS;  for the equipment serving for taking the in situ and the laboratory measurements: the PFT tests were conducted at INRS, and the SRT tests were conducted at CSTB.

The principles and operating modes of the 4 methods are described in Appendix 1.

Floor coverings

Fifteen floor coverings were chosen. That quantity made it possible to obtain a sufficiently broad panel of coefficients of friction and of different materials representatives of the floor coverings used in premises in which there is a risk of slipping over. Each sample of floor covering was identified by a letter given in Appendix 2.

Materials rubbing on the flooring

For each test method, the types of material that came into contact with the floor coverings and therefore that were subjected to friction were the materials commonly used in the operating mode or in the corresponding test standard (cf. Appendix 1). In the particular case of testing in the barefoot configuration by using the Ramp Test method, the test persons' feet were directly in contact with each test covering.

Contaminants

Some floor coverings are slippery only if a contaminant or pollutant is present between the shod foot or the bare foot and the floor covering. Two contaminants were chosen that represented a majority of situations encountered at workplaces or other premises for pedestrian use:

mineral oil, of SAE viscosity grade 10W30. That contaminant is representative of the situations in which flooring is soiled with greasy substances; -water solution containing 1 g/l of neutral wetting agent (sodium dodecyl sulphateor lauryl sulphate 98%). That contaminant is representative of the situations in which flooring is polluted with dirty or soapy water.

Psychophysical rating method

INRS has developed a subjective rating method [START_REF] Marchal | Evaluations psychophysiques en laboratoire de la glissance de revêtements de sol (Laboratory psychophysical evaluations of slipperiness of floor coverings)[END_REF] for subjectively rating the slipperiness of floor coverings. It is derived from Thurstone's method of pairwise comparisons. A test installation (cf. Figure 1) was designed and manufactured by INRS. It enables a test person (wearing a harness and a lifeline for safety) to rate the slipperiness of floor coverings by following a defined protocol. All of the floor coverings are installed on the floor as shown in Figure 2. The principle is to move a sample e1 past the (n-1) other samples so as to compare it with them. Then, sample e1 is replaced with sample e2 that can be compared with the (n-1) samples, and so on. The floor coverings were rated in the presence of a contaminant successively in the two following configurations:

with mineral oil, of SAE viscosity grade 10W30, and with the test persons wearing specific safety footwear: New 1000 of the "Gaston Mille" brand. The coefficients of friction of all of the items of footwear was measured as soon as they were received using Standard EN 13287 [START_REF]Personal protective equipment -Footwear -Test method for slip resistance[END_REF] to check their grip characteristics; -with water solution containing 1 g/l of neutral wetting agent (lauryl sulphate 98%) in water coming from the water supply network, with the test persons being barefoot. This psychophysical rating method made it possible to obtain rankings for the 15 floor coverings based on "perceived slipperiness" as perceived by the test persons.

Comparison of the methods

Metrological aspects

The methods studied give rankings for the floor coverings on the basis of numerical values obtained using the assessment or rating method of each of them:

psychophysical rating: score; The rankings were analysed in order to check:

-
the Spearman's correlation of each method with the reference rankings; -the Spearman's correlation of the various methods with one another.

A descriptive analysis was conducted to explain the correlation coefficients, to highlight the specific differences in the rankings of the floor coverings depending on the methods, and to check their slippery/not slippery rankings on the basis of the specifications of each method.

Practical aspects

During the laboratory tests, the practical aspects of use of the 4 methods were assessed to estimate their advantages and their drawbacks. That assessment was supplemented with in situ tests using 2 "transportable" methods. The sites chosen, with the help of the French Occupational Health and Pension Insurance Fund (CARSAT) for the Burgundy and Franche-Comté Regions, were chosen to enable measurements to be taken under a variety of conditions and on a variety of different floor coverings. INRS conducted the PFT tests and CSTB performed the pendulum SRT tests, in the presence of one of the two contaminants (oil or water solution), chosen depending on the activity of the premises. All of these tests enabled an overall comparison of the 4 method to be made.

-MAIN RESULTS

Psychophysical method

Reference rankings

Reference [START_REF] Marchal | Evaluations psychophysiques en laboratoire de la glissance de revêtements de sol (Laboratory psychophysical evaluations of slipperiness of floor coverings)[END_REF] presents the rankings of the floor coverings, from the least slippery to the most slippery, obtained per test person and per contaminant. They made it possible to establish the reference rankings (cf. Table 1) by adding up, for each floor covering, the scores attributed by the 12 test persons depending on the contaminants.

Ranking with oil as the contaminant

Ranking with water solution as the contaminant

least slippery L X X L A N B A N B K D R C S K D S C H E R I E H I U U most slippery M M
Table 1. Rankings of the floor coverings with 12 test persons.

p. 9

Hierarchical rankings

The dendrograms (cf. Figures 3 and4) were plotted on the basis of the rankings of the 12 test persons, for each contaminant. They make it possible to show indistinctness, i.e. floor coverings that are substantially similar in terms of slipperiness according to the test persons. This information is useful for putting into perspective any inversions that might be observed when comparing the rankings obtained using the different measurement methods studied. Some indistinct pairings are highlighted by the dendrograms and these need to be taken into account when comparing the 4 methods studied.

Summary and conclusion of the psychophysical ratings

The consistency of the rankings between the test persons was checked and the number of test persons was validated [START_REF] Marchal | Evaluations psychophysiques en laboratoire de la glissance de revêtements de sol (Laboratory psychophysical evaluations of slipperiness of floor coverings)[END_REF]. The analyses show that the rankings of the 15 floor coverings, obtained from the psychophysical ratings conducted by the 12 test persons with oil and water solution as contaminants are sufficiently robust to be used as references and to be compared with the rankings obtained using the 4 methods studied.

Comparison of the studied methodsmeasurements with oil

Table 2 shows the summary of the results of the measurements of all of the floor coverings using the 4 metrological methods and the psychophysical method, in the presence of oil. In addition, for each method, it gives the ranks of the samples. On the basis of Table 2, Figure 5 gives a matrix of graphs comparing, in pairwise manner, the floor covering rankings obtained using the various methods. In each cell, the axis of abscissas gives the rank obtained by the method of the column in question, and the axis of the ordinates gives the rank obtained by the method of the row in question.

Sample

Ramp Oil p. 11 Comparison of the rankings of the 3 methods comprising the ramp test, the PFT method, and the LabINRS method gives linear links with the psychophysical ratings; the rankings are quite similar. Comparison between the ranks of the psychophysical ratings and the ranks obtained with the pendulum SRT gives a cluster of points in the top portion; the rankings are different.

Mutual comparison of the 3 methods comprising the ramp test, the PFT method and the LabINRS method gives linear links. These methods rank the floor coverings in quite similar manner. Regardless of the method with which it is compared, the pendulum SRT method does not have any very visible link. The graphs show 2 clusters of points: one for the slippery floor coverings and one for those considered as having grip. PFT, Oil p. 12 strength of the association between the methods. At the level of confidence 99%, all of the methods are correlated, except for the pendulum SRT with the psychophysical rating method. The numerical values confirm the graphical analyses.

Psy, Oil

Figure 6 shows a graphical comparison of the rankings and of the levels of slipperiness of the floor coverings that were obtained by the various methods and by the psychophysical ratings. The values used in the graph show the slip indices of 15 floor coverings with oil. The grey zone represents the limit or boundary between slippery and not slippery, namely:

-PFT  0.30  10% (INRS limit) -LabINRS  0.30  10% (INRS limit) -Ramp  20°  10% PC20 (XP P05-011 limit) -Pendulum SRT  35  10% (NF EN 1339 limit). On the basis of the dendrogram, it is possible to consider 5 clusters (L, X), (A, B, N), (K, R, S), (D, C), and (E, H, I, M & U). The cluster of floor coverings E, H, I, M and U, which was deemed to be the most slippery by the psychophysical rating method, remained regardless of the method. This graph confirms that the pendulum SRT, the PFT, and, to a lesser extent, the LabINRS could not distinguish between these 5 floor coverings; their levels of slipperiness are less than the sensitivity of these three methods. Conversely, the ramp test does discriminate between them. For the other samples, the ramp test, the PFT, and the LabINRS kept the clusters, except for the sample S. The pendulum SRT inverts the ranks of certain non-slippery coverings from one cluster to another.

Comparison of the studied methodsmeasurements with the water solution

Table 4 shows the summary of the results of the measurements of all of the floor coverings using the 4 metrological methods and the psychophysical method, in the presence of water solution. In addition, for each method, it gives the ranks of the samples. p. 14

Sample

On the basis of Table 4, Figure 7 gives a matrix of graphs comparing, in pairwise manner, the floor covering rankings obtained using the various methods.

Figure 7. Matrix of graphs of the comparisons of the methods, with water solution.

The graphs plotted on the basis of the values from the ramp test show alignments of points in the top portions corresponding to the maximum limit of that method. The samples assessed at 35° all have the same rank.

Figure 7 shows that the 4 metrological methods substantially give linear links with the psychophysical ratings: the rankings are quite similar. Only the pendulum SRT compared with the psychophysical ratings shows an elongate cluster of points in the portion of the floor coverings with grip; this highlights some rank inversions.

Mutual comparison of the 4 metrological methods substantially gives linear links: the rankings are quite similar. Only the pendulum SRT shows an elongate cluster of points in the portion of the floor coverings with grip compared to the other three metrological methods; this highlights some rank inversions. p. 15 strength of the association between the methods. At the level of confidence 99%, all of the methods are correlated. Figure 8 shows a graphical comparison of the rankings and of the levels of slipperiness that were obtained by the various methods relative to the reference rankings obtained on the basis of the psychophysical ratings. The graph is plotted in the same manner as the graph of Figure 6.

Psy

Figure 8. Comparison of the slipperiness measurements depending on the methods.

Figure 8 shows overall consistency for all of the methods for ranking the floorings in terms of slipperiness or non-slipperiness. Only sample R was rated as slippery by the test persons while being defined as having grip by the 4 studied methods. The explanation is to be found in the fact that its surface was not entirely even. A layer of contaminant laid stagnant on it and gave rise to a phenomenon of aquaplaning during the psychophysical rating movements. Apart from sample R, the order obtained with the psychophysical ratings is substantially the same with the 4 studied methods, and confirms the high correlation coefficients.

Assessment of the practical aspects

Apparatus for assessing the level of slipperiness of a floor covering needs to satisfy not only metrological requirements, but also practical requirements. The following rating system was used for assessing the practical capacity of the apparatus to measure coefficient of friction: Table 6 shows that none of the devices assessed satisfies all of the requirements. Conversely, it shows that the pendulum SRT and the PFT have indisputable advantages as regards practical aspects.

 ++ very good  + good  0 sufficient  -insufficient  --
p. 17

-DISCUSSION AND CONCLUSIONS

Test methods

To assess the metrological methods, INRS developed a method for psychophysically rating the "perceived" slipperiness of floor coverings. This method made it possible to establish reference rankings per contaminant for the 15 floor coverings, from the least slippery to the most slippery. Twelve test persons, spread over 4 campaigns of tests, took part in these ratings. Analysis showed that they were in close agreement as regards the comparison of the 15 floor coverings. Hierarchical rankings obtained from the dendrogram made it possible to establish clusters of floor coverings and to highlight a few indistinct pairings in terms of slipperiness that were useful for comparing the studied methods.

This study shows that, from a purely metrological point of view, the ramp test and the psychophysical method give the highest correlation coefficients. The second advantage of the ramp test is its renown among trade professionals who have been using it for categorising floor coverings for several decades now, regardless of the types of flooring and of their fields of use. However, implementing it suffers from major drawbacks, such as the need to use test subjects and the impossibility of taking measurements on site.

The LabINRS method gives correlation coefficients with the psychophysical method that are very good and that are almost equivalent to those obtained with the ramp test. In addition, it is very simple to implement; however, it suffers from the drawback of not making it possible for measurements to be taken in situ.

Conversely, due to its very simple operating principle and to it being easy implement, the pendulum SRT is the best suited to taking measurements in situ. Its renown is high as it has been in use for many years for qualifying road surfaces and pedestrian surfaces. However, the correlation coefficients with the psychophysical method are low, raising questions as to its capacity to rank floor coverings as a function of slipperiness. In addition, it does not make it possible to qualify correctly all types of floor covering, e.g. coverings with macro-relief.

As for PFT, the correlation coefficients with the psychophysical method are very satisfactory. Even though it was originally designed to qualify road surfaces, INRS has considerable experience in using it for qualifying pedestrian surfaces, in particular such surfaces that are designed for use in the food industry. It makes it possible to take measurements in the laboratory and in situ, on any type of floor covering.

Risk prevention / Standardisation

Currently, with the two methods proposed in French Standard XP P05-010 and in European Technical Specification XP CEN/TS 16165, it is impossible to define a model for predicting the values obtained using the pendulum SRT on the basis of the values measured using the ramp test or vice versa. With oil as the contaminant, it was observed that a floor covering from the panel that was assessed as having grip when the pendulum SRT was used could be considered to be slippery when the ramp test is used, and vice versa with some other floor covering. Such a situation is difficult to accept for standardisation.

Floor covering manufacturers and fitters, clients and operators, and occupational risk preventers wish to have the means to enable them to measure the coefficients of friction of floor surfaces in the laboratory and in situ with the possibility of comparing the values easily:

-It has been observed that, due to wear and/or to the cleaning and maintenance protocol used, the surfaces of certain floor coverings can change significantly. Since p. 18

wear is a function of use and of cleaning & maintenance, it therefore appears necessary to enable them to take measurements for monitoring the slip resistance performance periodically and on a case-by-case basis (and therefore in situ). -The grip qualities of floor coverings that are cast are directly related to the quality of the casting and can vary from one site to another and from one zone to another on the same site. This is therefore another reason for the need to take measurements in situ.

The PFT satisfies these various constraints. It would appear judicious to propose PFT for standardisation, as the means for measuring the dynamic coefficient of friction. It offers the advantage of being as metrologically satisfactory as the Ramp Test and of being as practical the pendulum SRT.

The pendulum SRT could also satisfy the constraints subject to properly targeting the field of use (type of flooring, type of surface, etc.) for which the link with the Ramp Test or with the LabINRS could be established satisfactorily.

Although the LabINRS and the Ramp Test are less practical to use, they remain metrologically satisfactory as laboratory reference methods with a view to determining the initial standard slip resistance performances of floor coverings.

Prospects

The experience acquired by INRS using the PFT and the LabINRS in the food industry sector could be extended to other sectors of activity such as the metallurgical industry, automobile garages, etc. In addition, this study has made it possible to show that it is possible to use the PFT with the two contaminants proposed by the experimental standard. It therefore appears apposite to further extend its scope of action to other sectors of activity for evaluation in situ.

Furthermore, CSTB has significant past experience with the Ramp Test. Advantageous use could be made of that experience so as ultimately to develop the initial slip resistance assessments in sectors other than industry or than the food industry, or for barefoot use.

APPENDIX 1: TEST EQUIPMENT

Measurements using the Ramp Test method

The inclined-plane (or inclinable-plane) or ramp test method makes it possible to assess the slip resistance of a floor covering. The test bench used is the inclined plane or ramp (Figure 13) initially designed and developed in Germany.

The principle of the ramp test method is based on having a human tester walk forwards and backwards over the sample of floor covering which is inclined at an increasing angle α relative to the horizontal, until a slip starts to occur. The test bench is equipped with a system making it possible to incline the sample of floor covering relative to the horizontal at a maximum speed of 1° per second. An angle sensor makes it possible to measure the angle of inclination with accuracy of 0.5°, but the angle it indicates is not visible to the test person.

The test person is equipped with a harness and with a fall arrester in order to be protected in the event of sudden slippage. With the flooring sample initially in the horizontal position, the test person walks forwards and backwards several times while taking half steps (each half step being equivalent to half the size of the test person's shoe), and while, as far as possible, walking at a gait of 2.4 half-steps per second, and then the test person increments the angle of inclination. This procedure is repeated until the instability limit angle is reached, or until a forward slip is obtained. The corresponding angle α is then noted to within 0.1°. The test result can also be expressed as a coefficient of friction µ by the relationship µ = tg α. p. 21

Measurements using the pendulum SRT

The pendulum SRT (Skid Resistance Tester) was developed by the "Transport and Road Research Laboratory" in Great Britain. The aim of the method is to measure the grip of a floor covering by means of a slider coated with an elastomer material. This instrument is portable.

The elastomer slider, which has a surface of area 76 mm x 25 mm is mounted at the end of a metal arm that can swing pendulum-like about a horizontal axis.

Released with the arm horizontal, the slider comes to rub against the floor covering over a distance of 127 mm (Figure 14). A spring system incorporated into the arm makes it possible, during the rubbing, to apply a constant force F N of 22 N, normal to the test surface.

The height to which the pendulum swings depends on the energy absorbed by the friction during the rubbing, and is indicated by a needled placed in front of a dial with graduations in the form of Pendulum Test Value (PTV) units.

The basic operating modes for taking the measurements, preparing the slider, and checking the pendulum SRT are described in the tests standard EN 13036-4.

The slider used for taking the measurements is made of elastomer of the four S (Standard, Simulated Shoe Sole) type, also known as Slider 96 rubber, having Shore A hardness of 96 ± 5.

For each sample of flooring and for each contaminant, at least 3 series of 5 measurements are taken at various different locations. The result taken into account for any flooring sample is the arithmetic mean computed on the basis of the measurements taken at the 3 locations.

For the tests performed in the context of this study, the samples were measured successively with 10W30 mineral oil as the contaminant and then with lauryl sulphate solution as the contaminant. The contaminant was placed in a sufficient quantity on the flooring sample to completely cover the friction zone over which the slider rubs. p. 22

Measurements using the "PFT"

The Portable Friction Tester (PFT) is a tribometer developed by VTI (the Swedish Road and Traffic Research Institute) for measuring the dynamic friction coefficient (µd) of road or other surfaces (Figure 11). It is transportable and enables measurements to be taken in situ and over long distances.

The measurement is based on the principle of a braked wheel. When the instrument is in motion, the two rear wheels of the PFT drive gearing via a chain. The gearing implies a smaller speed of rotation to the front test wheel, thereby generating a slip resistance force between the surface or the flooring and the test wheel. That force activates a sensor that generates an electrical signal that is proportional to the slip resistance. The test wheel is covered with a smooth elastomer strip of formulation identical to the formulation of a safety footwear sole, whose Shore hardness is 80 ± 5. A tank of contaminant (oil or lauryl sulphate solution) situated above the test wheel provides lubrication between that wheel and the surface or floor covering. The dynamic friction coefficient µd is measured at stabilised speed. It is equal to the ratio of the modulus of the horizontal slip resistance force Ft to the modulus of the flooring vertical reaction force Fn at the point of contact of the test wheel: µd = Ft/Fn For laboratory measurements, the tests are performed at a temperature of 23 ± 5°C. As for measurements in situ, they are taken at ambient temperature. The floor covering is cleaned before the testing. At least three passes are performed without measurements being taken, in order to lubricate the measurement zone uniformly.

During the measurement, the instrument is pushed at constant speed (about 0.4 metres per second, as checked on the speed indicator). Five measurements are taken on each sample. The result taken into account is the arithmetic mean computed over the 5 measurements.

For the tests performed in the context of this study, the samples were measured successively with 10W30 mineral oil as the contaminant and then with lauryl sulphate solution as the contaminant. p. 23

Measurements using the "LabINRS" bench

The LabINRS method draws its inspiration from measuring the dynamic coefficient of friction of safety footwear. It makes it possible to assess the slip resistance of a floor covering (Figure 16). The test consists in measuring the dynamic coefficient of friction between a model of sole and the sample of floor covering. This coefficient is the ratio of the modulus of the friction force Ft that opposes the movement to the modulus of the force Fn normal to the friction plane: µd = Ft/Fn The testing was performed in the presence of a contaminant. The model of sole, laid flat, is made up of two smooth elastomer sliders (the elastomer being polychloroprene of formulation identical to that of a safety footwear sole) whose Shore hardness is 80 ± 5. It bears a load of 600 N. The semi-cylindrical shape of the elastomer sliders facilitates penetration of the contaminant and prevents the elastomer from catching on and being braced against pieces in relief on the floor coverings.

The sample of flooring is moved in reciprocating rectilinear motion. The dynamic friction force is measured at the instant when the speed reaches its maximum, i.e. 0.2 metres per second. It suffices to divide this value by the vertical load of 600 N in order to obtain the dynamic coefficient of friction µd.

The tests are performed at a temperature of 23 ± 5°C. The sample is cleaned before the testing. The contaminant is placed in a sufficient quantity on the flooring sample to completely cover the friction zone over which the sliders rub. Ten cycles are performed before the recording sequence is triggered. The variation in the friction force is then recorded over 2 cycles.

At least five tests are conducted on each sample, with each contaminant, in order to assess the coefficient of friction of the floor covering. The result taken into account is the arithmetic mean computed over the 5 measurements.

For the tests performed in the context of this study, the samples were measured successively with 10W30 mineral oil as the contaminant and then with lauryl sulphate solution as the contaminant.

Figure 1 .

 1 Figure 1. Drawing of the setup for subjectively rating floor coverings.

Figure 2 .

 2 Figure 2. Layout of the floor coverings during the tests.

  ramp test: angle [°] as from which the test person slips; -pendulum SRT: Pendulum Test Value [PTV] corresponding to the height to which the pendulum swings after being released; -PFT: dynamic friction coefficient [µd]; -LabINRS: dynamic friction coefficient [µd].

Figure 3 .

 3 Figure 3. Dendrogram of the ranking with 12 test persons and oil as the contaminant.

Figure 4 .

 4 Figure 4. Dendrogram of the ranking with 12 test persons and water solution as the contaminant

Figure 5 .

 5 Figure 5. Matrix of graphs of the comparisons of the methods, with oil.

Figure 5

 5 Figure5shows that the graphs of the PFT and of the pendulum SRT present alignments of points in their bottom portions. The floor coverings in question have levels of slipperiness less than the sensitivity of the 2 methods and the ranks that are attributed to them are equal.

Figure 6 .

 6 Figure 6. Comparison of slipperiness measurements depending on the methods.

Figure 6

 6 Figure6shows overall consistency for all of the methods in ranking the floorings in terms of slipperiness or non-slipperiness, except for the samples S and N and for the pendulum SRT.

For 1 :Figure 9 .

 19 Figure 9. Diagram showing the principle of the inclinable plane (ramp).

Figure 10 .

 10 Figure 10. Diagram showing the principle of the pendulum SRT.

Figure 11 .

 11 Figure 11. Diagram showing the principle of the PFT (Portable Friction Tester).

Figure 12 .

 12 Figure 12. Diagram showing the principle of the LabINRS method.

  Test persons walk on a particular type of floor covering while generating accelerations, skids, and turns following an established protocol, and then they repeat those operations on another type of covering. When they have finished, each test person gives a comparative opinion on the perceived performance of the two types of floor covering: the test person designates the slipperier of the two or indicates they consider them to be equivalent. A comparisons record is published per test person and per contaminant. That record makes it possible to give scores to the floor coverings of the panel, and to rank them from the least slippery to the most slippery. The data processing is explained in Appendix 3. A mean ranking is established for each floor covering by adding up the scores given by the various test persons. Twelve test persons took part in the tests. They had to be aged from 18 years to 60 years, without any particular specification as to sex and body measurements.

	The consistency of the ratings was validated by establishing the Spearman's correlations of
	the test persons in pairs, and by establishing the Kendall's coefficient of concordance for all
	of the test persons. In addition, the number of test persons was validated by comparing the
	mean rankings obtained with 12 test persons and the mean rankings obtained with 9 test
	persons.

Rank [PTV] Rank [µd] Rank [µd]

		Ramp, Oil	SRT, Oil	PFT, Oil	LabINRS, Oil	Psy, Oil
	ref.	[°] Rank [score] Rank
	M	2	1	13	5	0	3	0	1	2	1
	U	3	2	11	1	0	3	0	1	20.5	2
	H	6	4	11	1	0	3	0.01	4	23.5	3
	I	5	3	13	5	0	3	0.03	5	37.5	4
	E	8	5	13	5	0	3	0.008	3	43	5
	C	15	8	13	5	0.1	7	0.19	7	69.5	6
	D	10	6	13	5	0	3	0.09	6	71.5	7
	S	10	6	44	13	0.24	9	0.31	9	93.5	8
	R	17	10	41	12	0.29	10	0.34	10	97	9
	K	17	10	36	10	0.2	8	0.27	8	98	10
	N	24	13	28	8	0.31	11	0.36	11	121	11
	B	20	12	45	15	0.45	12	0.38	12	128.5	12
	A	19	11	36	10	0.46	13	0.42	13	135.5	13
	X	26	14	45	15	0.5	15	0.49	14	155	14
	L	30	15	36	10	0.5	15	0.53	15	165	15

Table 2 .

 2 Results of the slipperiness measurements with the various studied methods in the presence of oil

Table 3 .

 3 Spearman's correlation between the methods.

		Ramp, Oil	SRT, Oil	PFT, Oil	LabINRS,
					Oil
	Psy, Oil	0.92	0.58	0.82	0.88
	Ramp, Oil		0.74	0.93	0.95
	SRT, Oil			0.83	0.82
	PFT, Oil				0.97
	LabINRS, Oil

Table 3

 3 gives the Spearman's rank correlation coefficients between each pair of methods. These correlation coefficients vary over the range 0.58 to 0.97 and they measure the

	Psy, Oil
	Ramp, oil
	SRT, Oil
	LabINRS, Oil

Table 4 .

 4 Results of the slipperiness measurements with the various studied methods in the presence of water solution.

								LabINRS,	Psy, Water
	ref.	Ramp, Water [°] Rank [PTV] Rank SRT, Water	PFT, Water [µd] Rank	Water [µd] Rank [Score] Rank
	M	14	2	10	1	0.11	1	0.11	1	0.5
	U	12	1	15	2	0.19	3	0.21	3	19.5
	I	16	3	19	3	0.35	5	0.32	4	20.5
	E	17	4	28	5	0.34	4	0.33	5	51.5
	R	27	10	61	10	0.59	11	0.56	9	53
	H	17	4	25	4	0.17	2	0.12	2	60
	S	23	8	62	11	0.42	6	0.52	8	73
	K	21	7	48	8	0.55	9	0.45	6	79
	C	19	6	42	7	0.56	10	0.59	10	88
	D	23	8	37	6	0.53	7	0.50	7	106	10
	B	31	11	68	15	0.54	8	0.67	11	122	11
	A	35	14	65	14	0.62	12	0.77	13	126.5	12
	N	35	14	54	9	0.69	13	0.73	12	143	13
	L	35	14	64	12	0.93	15	1.03	15	155.5	14
	X	35	14	65	14	0.83	14	0.97	14	162	15

Table 5 .

 5 Spearman's correlation between the methods.

		Ramp,	SRT,	PFT,	LabINRS,
	, Water	Water	Water	Water	Water
	Psy, Water	0.91	0.81	0.86	0.90
	Ramp, Water		0.90	0.90	0.93
	SRT, Water			0.78	0.88
	PFT, Water				0.95
	LabINRS, Water

Table 5

 5 gives the Spearman's rank correlation coefficients between each pair of methods. These correlation coefficients vary over the range 0.78 to 0.95 and they measure the

	Psy, Water
	Ramp, Water
	SRT, Water
	PFT, Water
	LabINRS, Water

Table 6 .

 6 Comparison of the practical aspects.

	very insufficient
	 / Not applicable
		Ramp	PFT LabINRS pendulum
		Test			SRT
	Size	--	++	--	++
	Weight	--	+	--	+
	Possibility of being transported (measurement	--	++	--	++
	in situ)
	Independence of the power supply	--	+	--	++
	User-friendliness / Intuitiveness	--	++	+	+
	Mode of use	--	++	++	0
	Ease of cleaning/maintenance	0	0	0	++
	Mechanical reliability	0	0	-	0
	Electronic reliability	0	-	-	++
	Measurement of floor coverings having macro	++	++	-	--
	relief
	Measurement area	-	++	-	--
	Ease of implementation	-	++	+	0
	Measuring time	--	++	0	++
	Influence of the test technician	++	++	++	0
	Influence of the human test person	-	/	/	/
	Ease of calibration	0	0	-	+
	Ease of adjustment	--	++	++	0
	Renown / Standardisation	++	-	--	++
	Purchase price	-	+	--	++
	Cost price of the measurement	-	+	+	+

A publicly funded institution working for innovation in the building trade, CSTB (Centre Scientifique et Technique du Bâtiment), France's Scientific and Technical Centre for the Building Trade has four key activities: conducting research, providing expertise, sustainable development in the building world. improve the quality and the safety of buildings.

INRS

 8shows an example of a matrix plotted on the basis of the psychophysical assessment results. This matrix makes it possible to obtain, for a test person and for a contaminant, the score of each covering and the rankings of the 15 test floor coverings. Each cell of the matrix gives the result of the comparison of the floor covering of row i with the floor covering of column j. A value is then assigned to the corresponding cell: 1 : if i is estimated to be more slippery than j 0.5 : if i is estimated to be equivalent to j 0 :

if i is estimated to be less slippery than j

The "total" column gives the sum of the values of any one row. It makes it possible to assign a score to the various floor coverings. These scores are used to define the rankings, from the highest to the lowest, i.e. from the most slippery floor covering to the least slippery floor covering.

Example of an assessment matrix for a test person and for a contaminant.


First International Summer School on Runtime Verification

First International Summer School on Runtime Verification as part of the ArVi COST Action 1402

Christian Colombo 1 and Yliès Falcone 2

1 University of Malta, Maltachristian.colombo@um.edu.mt 2 Univ. Grenoble-Alpes, Inria, LIG, F-38000 Franceylies.falcone@imag.fr Abstract. This paper briefly reports on the first international summer school on Runtime Verification: Branches of practical topics rooted in theory, co-organized and sponsored by COST Action IC1402 ArVi which was held September 23-25, Madrid, Spain as part of the 16th international conference on Runtime Verification (RV 2016).

Runtime Verification [1, 3-5, 2] is an umbrella term usually denoting the languages, techniques, and tools related to the verification of system executions against formally-specified behavioral properties. This field of research has been mainly represented by the Runtime Verification (RV) conference [START_REF] Havelund | Verify your runs[END_REF] which was held yearly for the last 16 years. As the field is growing and the techniques are becoming more and more mature, there is a pressing need in the community for documentation and lecture material to help students and practitioners entering the field, in spite of the existing (incomplete) tutorials and short overviews of the field. We foresee the organization of this summer school as one of the steps towards achieving this goal. By organizing the summer school, we wanted to build a short theoretical and practical program allowing to give in 3 days the necessary introductory knowledge to a practitioner or student entering the field.

Objectives. As the name of the summer school suggests, the summer school aimed to provide a balance of theory and practice: In the theoretical aspect, while all the core concepts were covered, participants were also exposed to cutting edge advances in the field. At the same time, the summer school was very hands-on and students followed up with practical work in the evenings so that by the end of the summer school, participants had created their own basic runtime verification tool.

For PhD students and researchers entering the field of RV, the school was a great opportunity to get to know other people working in the area, to meet distinguished scholars, and to establish contacts that may lead to research collaborations in the future. For people coming from industry, the school provided an exposition of the major challenges as well as possible solutions to the application of RV in industry, an exposition to the major tools, as well as the basics of RV tool-building. Program overview. The Summer School was organised over three days with a series of lectures from international experts during the day and a follow up practical session in the evening to enable the participants to incorporate the covered knowledge into their tool (see Tables 123). The first day covered the fundamentals of runtime verification: starting with the basic concept of what is runtime verification, moving on to instrumentation techniques, and property specification languages. The second day covered the major practical aspects of runtime verification: handling data through monitor parametrisation, monitoring concurrency errors, and performance issues of monitors. The second day ended with a session on RV tools, giving the participants time to try tools and interact with their creators. Finally, the third day covered advanced and cutting-edge research in the field with topics ranging from runtime enforcement to the combination of static and dynamic analysis, and from monitoring of distributed and transaction-based systems to low-level hardware monitoring.

Table 1 .

 1 Programme overview -Day 1 -23rd September

	Time	Topic

Table 2 .

 2 Programme overview -Day 2 -24th September

	Time	Topic	Lecturer
	09:00 09:45 Handling data 09:45 10:30 in user-provided specifications	K. Havelund
	11:00 11:45 Monitoring concurrency errors: 11:45 12:30 deadlocks, atomicity violations, and data races	J. Lourenco
	14:00 14:45 Performance issues and optimizations 14:45 15:30	G. Reger
	16:00 16:45 Hands on 16:45 17:30	C. Colombo
	18:00	Optional further assistance with hands on

Table 3 .

 3 Programme overview -Day 3 -25th September The following researchers lectured at the summer school: -Prof. Wolfgang Ahrendt, Chalmers University of Technology and University of Gothenburg (Sweden). -Prof. Ezio Bartocci, TU Wien (Austria). -Prof. Borzoo Bonakdarpour, University of MacMaster (Canada). -Dr. Marius Bozga, CNRS, Vérimag (France). -Dr. Christian Colombo, University of Malta (Malta). -Dr. Yliès Falcone, University of Grenoble (France). -Dr. Adrian Francalanza, University of Malta (Malta). -Dr. Klaus Havelund, NASA Jet Propulsion Laboratory (USA). -Prof. Martin Leucker, University of Lübeck (Germany). -Prof. Joao Loureno, Universidade Nova de Lisboa (Portugal). -Dr. Dejan Nickovic, Austrian Institute of Technology (Austria). -Prof. Gordon Pace, University of Malta (Malta). -Dr. Giles Reger, University of Manchester (UK).

	Time	Topic

Acknowledgment. We would like to warmly thank all the researchers for their lectures and all the participants to the summer school. We hope that the summer school will be continued in the future by becoming a regular event.

This summer school is based upon work from COST Action ARVI IC1402, supported by COST (European Cooperation in Science and Technology). The organizers are grateful to the COST association for sponsoring the summer school.


Aure Saulnier

Isabelle Pelletier

Karine Labadie

Florence Colbère-Garapin

Complete cure of persistent virus infections by antiviral siRNAs

Keywords: RNAi, interference, PTGS, silencing, siRNA, enterovirus, poliovirus, persistent infection, chronic infection, antiviral. Complete cure of persistent virus infections by antiviral siRNAs RNAi, interference, PTGS, silencing, siRNA, enterovirus, poliovirus, persistent infection, chronic infection, antiviral

Complete cure of persistent virus infections by antiviral

siRNAs.

INTRODUCTION

Post-transcriptional gene silencing (PTGS) is a natural, conserved mechanism, present in most eukaryotes and conferring antiviral immunity in plants [START_REF] Voinnet | RNA silencing as a plant immune system against viruses[END_REF]. T. Tuschl and coll. [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF] demonstrated that synthetic 21 nucleotide (nt)-long RNA duplexes transfected into cultured cells silenced genes in a sequence-specific manner. These synthetic siRNAs, with 2 nucleotide 3' overhangs, mimic small interfering RNAs (siRNAs) produced by the ribonuclease Dicer by digestion of large double-stranded (ds) RNAs. After incorporation into the RNA-induced silencing complex (RISC), one of the siRNA strands acts as guide and selects the homologous target in the mRNA, which is then cleaved and degraded by RNases. One characteristic of siRNAs is that for activity, the guide (or antisense) strand must be perfectly base-paired to its target in the corresponding RNA [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF]. However, G:U wobble base pairs do not necessarily alter target specificity, probably because they have the same thermodynamic stability as Watson-Crick base pairs, and C:A mismatches are in some cases tolerated [START_REF] Du | A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites[END_REF][START_REF] Jacque | Modulation of HIV-1 replication by RNA interference[END_REF][START_REF] Saxena | Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells[END_REF]. Other small RNAs, named micro RNAs (miRNAs), are also dependent on Dicer for processing, and target mRNA for cleavage or translational repression [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]; it has been shown that siRNAs can also function as miRNAs [START_REF] Doench | siRNAs can function as miRNAs[END_REF].

Guidelines for the choice of siRNA sequences have been published [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF][START_REF] Ui-Tei | Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference[END_REF]. The effect of siRNAs transfected into mammalian cells is transient: it generally does not last more than a few days [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF], and it has been suggested that the continued presence of mRNA may be needed for persistence of siRNAs [START_REF] Song | Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages[END_REF]. DNA vectors and repeated transfections have been used to sustain silencing activity [START_REF] Schubert | Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs[END_REF][START_REF] Xia | siRNA-mediated gene silencing in vitro and in vivo[END_REF]. We chose to use repeated transfections in this study because the transfer of siRNAs may be associated with fewer safety concerns than the use of DNA vectors in future therapeutic strategies.

Plus strand RNA viruses appear to be attractive targets for siRNAs (reviewed in [START_REF] Colbère-Garapin | Silencing viruses by RNA interference[END_REF]), as their genome functions as both mRNA and replication template. In particular, poliovirus (PV), an enterovirus, has a positive strand RNA genome enclosed in an icosahedral capsid composed of the four proteins VP1 to VP4 [START_REF] Stanway | Molecular and biological basis of picornavirus taxonomy[END_REF]. The large coding region of PV is flanked by 5' and 3' non-coding (NC) regions comprising signals involved in the initiation of viral replication. The PV genome encodes a polyprotein, precursor of capsid and nonstructural proteins, including the 3D RNA polymerase (3Dpol). Gitlin et al. [START_REF] Gitlin | Short interfering RNA confers intracellular antiviral immunity in human cells[END_REF] used RNA interference targeting the capsid protein region and the 3Dpol region of the PV genome in lytic infections; they showed that the viral yield of a one-step growth cycle was much lower in cells transfected by antiviral siRNAs before infection, than in cells transfected by negative control siRNAs. Viral escape as a result of exposure to single antiviral siRNA has been observed in several studies [START_REF] Schubert | Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs[END_REF][START_REF] Gitlin | Short interfering RNA confers intracellular antiviral immunity in human cells[END_REF][START_REF] Westerhout | HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome[END_REF]. The emergence of escape mutants is facilitated by the heterogeneity of viral RNA genomes (quasispecies). For PV and another picornavirus, human hepatitis A virus, HAV, combinations of siRNAs were more effective than single siRNAs [START_REF] Gitlin | Short interfering RNA confers intracellular antiviral immunity in human cells[END_REF][START_REF] Kanda | Interference of hepatitis A virus replication by small interfering RNAs[END_REF].

For most experiments investigating silencing viruses by siRNAs, a preventive approach was used: siRNAs were transfected before virus infection [START_REF] Gitlin | Short interfering RNA confers intracellular antiviral immunity in human cells[END_REF][START_REF] Bitko | Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses[END_REF]. Recently, however, successful therapies based on treatment with siRNA have been reported [START_REF] Uprichard | Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs[END_REF][START_REF] Yuan | Inhibition of Coxsackievirus B3 replication by small Interfering RNAs requires perfect sequence match in the central region of the viral positive strand[END_REF].

Nevertheless, to the best of our knowledge, complete cure of cells has never been obtained in therapeutic assays. Persistent virus infections are major medical and veterinary problems. Examples of agents causing persistent infections include plus-strand RNA viruses such as human hepatitis C virus (HCV) and foot-and-mouth disease virus (FMDV). In the absence of a suitable model for growing HCV, the best results of long-term experiments with cell lines transduced by HCV replicons demonstrated that expression of HCV RNA is substantially reduced by specific siRNAs [START_REF] Randall | Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs[END_REF]. Inhibition of FMDV by specific siRNAs has been successful in lytic infections in vitro and in acute infections in vivo [START_REF] Yan | RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice[END_REF][START_REF] Kahana | Inhibition of foot-and-mouth disease virus replication by small interfering RNA[END_REF], but the effect of these siRNAs during persistent infections has not been reported.

There are several difficulties associated with the development of therapies in in vitro models mimicking some aspects of in vivo persistent virus infections: the treatment is applied late after the initial infection, the ratio of infectious virus to cells may be high, the presence of virus mutants is a common feature of persistent infections [START_REF] Colbère-Garapin | Persistent infection by picornaviruses[END_REF], the ratio of plus to minus-strand RNA may be close to one, suggesting that the majority of viral RNA in cells may be in a double stranded form [START_REF] Girard | Restriction of poliovirus RNA replication in persistently infected nerve cells[END_REF], and finally the cure has to be complete to avoid reinfection of the entire culture by a few virus particles. Some picornaviruses establish persistent infections in their natural animal hosts, and others are suspected to be responsible for chronic human diseases [START_REF] Colbère-Garapin | Persistent infection by picornaviruses[END_REF]. Immunodeficient individuals can be chronically infected by PV Sabin strains [START_REF] Maclennan | Failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man[END_REF], and PV mutants isolated from the intestine of patients after several months of chronic infection can persistently infect human enterocyte-like cells in vitro [START_REF] Labadie | Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells[END_REF]. We have developed several models for studying the molecular mechanisms contributing to persistent enterovirus infections [START_REF] Colbère-Garapin | Persistent infection by picornaviruses[END_REF][START_REF] Pelletier | Molecular mechanisms of poliovirus persistence: key role of capsid determinants during the establishment phase[END_REF].

Here, we report an investigation of the possibility of curing persistent PV infections by treatment with specific antiviral siRNAs several months after infection. Despite the high titers of virus produced by these cells and the presence of PV mutants, repeated treatment with a mixture of two siRNAs induced complete cure of the majority of persistently PVinfected cultures, and our findings are consistent with a mechanism of post-transcriptional silencing.

RESULTS AND DISCUSSION

SiRNAs directed to the PV 5'NC region and 3Dpol gene reduced the yield of lytic and persistent PV strains.

The reference PV type 3 Sabin strain (Sabin 3) is fully lytic, whereas the L2-2 strain can establish persistent infections in HEp-2 cells at low multiplicity of infection (m.o.i.) [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF].

For effective virus silencing while limiting the risk of silencing undesired cellular genes, we used a combination of only two antiviral siRNAs, targeting sequences separated by about 6200 nt on the viral genome, that meet the criteria defined by Elbashir et al. [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF] for siRNA targets. One target is in the 5'NC region of the PV type 3 genome between nucleotide (nt) 687 and 709, outside of the highly structured internal ribosomal entry site (IRES) and upstream from the initiator AUG codon (Fig. 1). The other target is in the 3Dpol region, between nt 6908 and 6930, and is conserved among PV serotypes and some other enteroviruses. The corresponding amino acid sequence is part of interface I of 3Dpol oligomers. We assumed that virus displaying mutations in the 3Dpol target would not be viable or would have a reduced growth capacity, and therefore the frequency of emergence of escape mutants would be low. Sabin 3 and L2-2 have identical genomic sequences in both target regions [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF]. HEp-2 cells were transfected with siRNAs with Lipofectamine2000 and the growth of the Sabin 3 strain inoculated at an m.o.i. of 10 -2 ID 50 per cell was assessed, as described in Materials and Methods. Negative controls included an irrelevant siRNA, siRNA-IRR, and a transfection mixture free of siRNA (Fig. 1). Twentyfour hours post-infection (pi), siRNA-5'NC reduced the yield of progeny virus by more than 95%, whereas siRNA-IRR had no significant effect. This result is in agreement with those of Gitlin et al. [START_REF] Gitlin | Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches[END_REF], who inserted a let-7 microRNA sequence, acting as an siRNA, at position 703 of the PV type 1 genome. These results suggest that the 5'NC region downstream from the IRES, around nt position 700, can be effectively targeted by siRNAs without significant inhibition by ribosomal scanning during the initiation of translation. The silencing by siRNA-3Dpol was slightly less effective than that by siRNA-5'NC 24 h pi, possibly because of a secondary structure in the 3Dpol target region. It reduced the yield of progeny virus by more than 80%. However, at earlier times post-infection, siRNA-3Dpol was at least as efficient as siRNA-5'NC (Fig. 1). When used together, the mixture (Mix) of siRNA-5'NC and siRNA-3Dpol reduced the yield by 99.9%, 24 h pi. These results suggest that at least 99.9% of cells were effectively transfected with siRNAs in a single transfection. At a 100-fold higher m.o.i. (1 ID 50 /cell), the reduction of the yield of Sabin 3 by siRNA-5'NC and siRNA-3Dpol was 94 and 80%, respectively, but the effect of the Mix was similar to that of siRNA-5'NC alone (Fig. 2A). Similar results were obtained with the persistent PV strain, L2-2 (Fig. 2A).

To determine whether anti-PV siRNAs can have a therapeutic effect on a persistent PV infection, HEp-2 cells were infected persistently with L2-2 as described previously [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF].

A persistently L2-2-infected cell line, HEp/L2-2/6, was used for most of the subsequent experiments: four months pi, the virus titer in the culture medium of HEp/L2-2/6 remained high, between 10 6 and 10 7 ID 50 /ml. Experiments were performed 4 to 6 months pi, during which period the genome of the virus, L2-2m, produced by HEp/L2-2/6, had a mutation (C>U) at nt 698 with respect to the quasispecies consensus sequence. This mutation was in the middle of the target of siRNA-5'NC, whereas the target sequence of siRNA-3Dpol was not mutated. The mutation at position 698 in the genome of L2-2m may correspond to an adaptation of L2-2 to host HEp-2 cells; it created a wobble base pair between the viral genome and the guide strand of siRNA-5'NC, close to the predicted cleavage site of the RISC complex. L2-2m virus was used to infect HEp-2 cells pretreated with siRNA-5'NC, and the yield of progeny virus was 75% lower than that for negative controls; the yield from cells pretreated with the Mix was more than 85% lower (Fig. 2A). The silencing effects on L2-2m of the Mix and Mixm (a mixture of siRNA-5'NCm, strictly homologous to the 5'NC target of L2-2m, and siRNA-3Dpol) were not significantly different. Therefore, the multiplication of L2-2m was silenced by each of anti-PV siRNAs and by their mixtures.

Although it was once reported that some mutations in the target sequence resulting in G :U base pair are sufficient for the virus to escape siRNA inhibition [START_REF] Gitlin | Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches[END_REF], it seems probable that this wobble base pair between PV RNA and the guide strand of siRNA-5'NC, created by the mutation, was recognized as an authentic base pair by the RNA interference machinery, as in other instances [START_REF] Jacque | Modulation of HIV-1 replication by RNA interference[END_REF][START_REF] Saxena | Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells[END_REF]. Consistent with this possibility, three other siRNAs targeting the same part of the 5'NC region, but corresponding to mutations C>A and C>G at nt 698 of the target, and a 5 nt central mismatch, had little or no silencing activity on L2-2m (Fig. 3). This suggests that silencing of L2-2m RNA by siRNA-5'NC involved RNA degradation rather than inhibition of translation. SiRNA-5'NCm had a moderate silencing effect on Sabin 3 and L2-2 (Fig. 2A), presumably a consequence of the central C:A mismatch between viral RNA and the guide strand of siRNA-5'NCm. Consistent with this result, the Mixm was slightly less effective than the Mix against the multiplication of these viruses. This result confirms that A:C mismatches are in some cases tolerated [START_REF] Du | A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites[END_REF].

In all of these short-term infections, the negative control siRNA-IRR did not result in a significantly lower viral yield of Sabin 3, L2-2 or L2-2m, than when no siRNA was present (Fig. 123). This strongly suggests that the silencing induced by PV-specific siRNAs did not result from activation of Toll-like receptors.

To confirm that the antiviral effects of siRNAs targeting the 5'NC and 3Dpol regions were not the result of modulation of the interferon pathway, we used Vero cells that have a deletion in the type-1 interferon locus [START_REF] Mosca | Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis[END_REF]. Each of the antiviral siRNAs (siRNA-5'NC, siRNA-5'NCm and siRNA-3Dpol) inhibited PV strains Sabin-3 and L2-2m similarly in Vero cells and in HEp-2 cells (Fig. 2A andB). This strongly suggests that the antiviral effect did not result from a generalized interferon response.

Viral inhibition by specific siRNAs in infected cells correlates with a decrease in the amount of viral RNA.

Viral RNA in short-term, lytic, and long-term, persistent, infections of HEp-2 cells was analyzed by Northern blotting to investigate whether the antiviral effect of siRNA-5'NC, siRNA-5'NCm and siRNA-3Dpol correlated with a decrease in the amount of viral RNA.

For short-term infections, we used two viral strains: L2-2, the strain initially used to establish the persistent infection of HEp/L2-2/6; and L2-2m, the mutated virus produced by this cell line a few months post-infection. HEp-2 cells were pretreated with siRNAs and then infected with each virus at a m.o.i. of 10 ID 50 /cell, as described in Materials and Methods. Five hours after infection, as expected, viral RNA was clearly detected with the same electrophoretic mobility as genomic PV RNA in cells treated with the negative control siRNA-IRR (Fig. 4A). In contrast, there was very much less viral RNA in cells treated with siRNA-5'NC, siRNA-5'NCm or siRNA-3Dpol, the Mix or Mixm. Similar results were obtained with the Sabin 3 strain (not shown). In cells transfected with siRNA-5'NC and infected with L2-2m, the amount of viral RNA was one third of that in cells transfected with siRNA-IRR. This reduction was very similar to those obtained with siRNA-5'NCm and siRNA-3Dpol, which are strictly homologous to the target of the L2-2m genome (Fig. 4A andB). These results suggest that siRNA-5'NC, like siRNA-5'NCm, induced L2-2m RNA degradation despite the wobble base pair in the middle of the target. However, inhibition of translation cannot be excluded, because PV replication depends on early translation of the viral genome.

In persistently infected HEp/L2-2/6 cells, 6 months post-infection, PV RNA was detected in cells treated with the negative control siRNA-IRR, as in lytic infections.

Twenty-four hours after transfection of HEp/L2-2/6 cells with antiviral siRNA-5'NC, -5'NCm, -3Dpol, the Mix and Mixm, the amount of viral RNA fell by 53 to 77% (Fig. 4C andD). Thus, in both lytically and persistently PV-infected cells, antiviral siRNA treatment induced a decrease in the amount of viral RNA, and the results are consistent with a mechanism of RNA interference.

A mixture of both siRNAs directed to the PV 5'NC region and 3Dpol-encoding region has a therapeutic effect on persistent L2-2 infections.

The preexistence of viral sequence diversity may affect siRNA effectiveness in persistent infections, as there could have been hundreds or thousands of virus generations since the initial infection [START_REF] Leonard | Computational design of antiviral RNA interference strategies that resist human immunodeficiency virus escape[END_REF] and the emergence of viral mutants may contribute to the mechanism of persistence (reviewed in [START_REF] Colbère-Garapin | Persistent infection by picornaviruses[END_REF]). The therapeutic effect of antiviral siRNAs was assayed on HEp/L2-2/6 cells 4 to 6 months pi. We used the mixtures rather than single siRNAs to prevent the emergence of escape mutants. Negative controls included siRNA-IRR and no siRNA. Any strategy aimed at curing persistent infections must reach the maximum of cells in cultures: it was thus important to transfect all of the cells (5X10 5 ) in each culture. The relative yield of progeny virus produced by HEp/L2-2/6 cells, 7 and 14 days after a single transfection with either the Mix or Mixm, was not significantly reduced, whereas the mean values for virus production, 14 days after a DT with either the Mix or Mixm, were 5±3% and 8±2%, respectively, of that in cultures transfected with siRNA-IRR Although the effect of chemically synthesized siRNAs is usually transient and vanishes after a few days, virus production decreased for about 20 days in all three DT cultures treated with antiviral siRNAs (Fig. 5). This agrees with the hypothesis proposed by Song et al. [START_REF] Song | Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages[END_REF] that the continued presence of target mRNA may be needed for persistence of siRNA. Furthermore, the mechanism of viral persistence in HEp-2 cells relies on an equilibrium between a lytic and an abortive infection [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF]; and another, non-exclusive hypothesis is that the prolonged effect of antiviral siRNAs in persistently PV-infected cells may be a consequence of siRNAs modifying this equilibrium.

In two HEp/L2-2/6 cultures treated with the Mix, virus production first declined after transfection, but returned after three weeks to the initial level (Fig. 5, upper panel), suggesting either that some viral RNA and/or infected cells had escaped antiviral siRNAs, or that escape PV mutants had emerged. To prevent this increase in virus production, we performed a second round of DT with the Mix or control siRNA-IRR, 20 days after the first transfections. Cultures transfected four times were noted QT for quadruple transfection.

One culture, not cured by a single set of DT, stopped producing virus after the second set of DT with the Mix, 58 days after the first one (Fig. 5, lower panel, culture labeled Mix 2). This suggested that the percentage of cultures cured would be higher after four than after two transfections with the Mix.

Four successive transfections with either the Mix or Mixm were also performed: the three siRNA-IRR QT cultures remained productive, whereas for each mixture, all three QT cultures stopped producing virus (data not shown), confirming that the Mix and Mixm were similarly effective in HEp/L2-2/6 cultures. A total of 6 experiments similar to that presented in Fig. 5 were performed (Fig. 6): 72 % of HEp/L2-2/6 cultures stopped producing virus after four transfections with the Mix of antiviral siRNAs, whereas no cures were observed after mock-transfections without siRNAs. Most siRNA-IRR-treated cultures continued producing virus so the role of activation of some Toll-like receptors by siRNAs in the cure of persistently infected cells seems improbable, but cannot be excluded. The percentage of cures was significantly higher for QT cultures transfected by the Mix than for both cultures transfected by siRNA-IRR and mock-transfected cultures.

We tested for the presence of PV RNA in HEp/L2-2/6 cultures, which stopped producing virus after transfection with antiviral siRNAs. We used RT-nested PCR of the 5'NC region of the PV genome to determine whether these cultures were completely cured and free of viral genomes. The sensitivity of the method was 0.1 fg of PV RNA, corresponding to about 25 viral genomes or one viral genome per 6000 cells. We tested cultures that had stopped producing infectious virus for at least one month (4 DT and 13 QT treated with the Mix, 2 DT and 6 QT treated with the Mixm). No viral RNA was detected in these cultures and they were therefore considered to be completely cured (Fig. 7). Thus, after four transfections with the mixtures of antiviral siRNAs, the majority of HEp/L2-2/6 cultures were cleared of both infectious virus particles and viral genomes.

Three of the cured cultures (1 DT and 2 QT treated with the Mix) were tested for their permissivity to PV infection: the progeny yields of Sabin 3 and L2-2m were similar (4 10 8 to 7 10 8 ID 50 /ml) 24 h pi at an m.o.i. of 1 ID 50 /cell. When the cured cells were pretreated either with the Mix or Mixm 24 h before de novo infection, a 3 log-reduction in viral yield was observed (data not shown). These results indicated that the cured cells were fully permissive to PV infection, like HEp-2 cells; they were also highly susceptible to siRNA transfection, because antiviral siRNAs could efficiently prevent PV multiplication in these cells.

Persistently PV-infected cultures that escaped antiviral siRNAs did not produce PV escape mutants.

Even after four transfections with antiviral siRNAs, virus production sometimes rose abruptly about 50 days after the first DT (Fig. 5). To investigate whether PV escape mutants had emerged, the genome of viruses produced by 5 DT and 1 QT Mix-treated, but uncured HEp/L2-2/6 cultures were sequenced in the regions targeted by siRNAs. No mutation was detected, suggesting that no escape mutations had been selected in the viral genomes at any time during the experiments (75 days), at least in targeted regions. It was possible that mutations had appeared in other regions of the viral genome, and modified the secondary structure of targeted region, as described for HIV-1 [START_REF] Westerhout | HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome[END_REF]. To test for this, the susceptibility of one virus (L2-2m/DT) from a DT culture and one virus from a QT culture to silencing by siRNA-5'NC, siRNA-5'NCm and siRNA-3Dpol was studied in HEp-2 cells, 24 h pi at a m.o.i. of 1 ID 50 per cell (Fig. 2C). Both these viruses remained fully susceptible to silencing by each of the 3 antiviral siRNAs, confirming that they were not escape mutants. This strongly suggests that, even if mutations appeared in viral genomes outside of targeted regions during siRNA treatment, they did not confer resistance to siRNA. Therefore, the rare failures of antiviral siRNA treatment were probably due to a small number of cells escaping transfection by antiviral siRNAs and re-populating and/or re-infecting the entire culture after several weeks. This agrees with the higher therapeutic efficacy of QT than that of DT. It seems likely therefore that 100% of cultures could be cured by application of a larger number of repeat treatments.

In conclusion, these results indicate that the strategy of repeated transfections with different antiviral siRNAs is sufficient to prevent the emergence of virus escape mutants in persistently infected cultures. They strongly suggest that antiviral siRNAs inhibited viral multiplication in both lytically and persistently infected cells by a mechanism of RNA interference. This is, to our knowledge, the first demonstration of the complete cure by specific antiviral siRNAs of entire cultures of cells productively infected by a human virus.

We also show that antiviral siRNAs can completely cure cells persistently infected by virus for several months.

MATERIALS AND METHODS

Cells and viruses

The human epithelial tumor cell line HEp-2 and the monkey kidney cell line Vero (clone VC10, [START_REF] Colbère-Garapin | Late transient expression of human hepatitis B virus genes in monkey cells[END_REF]) were propagated at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf serum (NCS). The vaccine Sabin 3 strain (P3/Leon12a 1 b), lytic in these cell lines, was used as a reference PV strain. The L2-2 PV mutant was isolated and described previously [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF]. Virus stocks were prepared in HEp-2 cells, at 34°C and 37°C for the Sabin 3 and L2-2 PV strains, respectively. Titers were determined in 96-well plates, using an endpoint micromethod [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF]. All experiments in cells were carried out at 37°C. To establish persistent infections, HEp-2 cells were infected with L2-2 at a m.o.i. of 10 -4 infectious doses 50 (ID 50 ) per cell and grown at 37°C, in DMEM-2% fetal calf serum (FCS) for the first week, and thereafter in DMEM-10% NCS as described [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF]. Persistently L2-2-infected cultures were named HEp/L2-2 followed by the number of the culture. Most experiments were performed with the HEp/L2-2/6 culture, in which no spontaneous cure was ever observed. After four months of culture, HEp/L2-2/6 was found to produce a mutant, L2-2m, with a point mutation at nt position 698 (C>U).

siRNAs.

The choice of the 21 nt-long sequences was made with the help of the Fuzznuc program (http://bioweb.pasteur.fr/seqanal/interfaces/fuzznuc.html) according to the recommendations of T. Elbashir and coll. [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF]. Oligoribonucleotides were synthesized either by Genset-oligos, or by C. Gouyette (PT 7, Institut Pasteur). SiRNA-5'NC (nt 687-709) and siRNA-3Dpol (nt 6908-6930) are strictly homologous to the corresponding segments of the genome of PV strains Sabin 3 and L2-2. An equimolecular mixture of siRNA-5'NC and siRNA-3Dpol was made and named Mix. SiRNA-5'NCm differs from siRNA-5'NC by a (C>U) nt change corresponding to the target sequence of L2-2m. Two other siRNAs with either an A or a G at the same central position on the sense strand, that is a U or a C on the guide strand, were also produced and named siRNA-5'NCm U and siRNA-5'NCm C , respectively (Fig. 3). An additional siRNA, siRNA-5'NCm 5 , with a 5 nt central mismatch with the target was used. A negative irrelevant control, siRNA-IRR, (5'CACCGACGUCGACUGUUGG d T d T3') with the inverted sequence of exon 8 of CD155 (nt 2402-24) was included in all experiments. To obtain duplex siRNAs, sense and antisense strands at a concentration of 20 mM were annealed, as described [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF]. Annealing of oligoribonucleotides was verified by electrophoresis in a 4% NuSieve gel, and the amount of siRNAs was verified with a Fluor-S Multi-Imager (Biorad).

Transfection and infection of cells.

SiRNA transfections by Lipofectamine 2000 (Invitrogen) and other transfection agents were performed and their efficiencies were compared (see Supplementary Material).

Cells were grown in OptiMEM I Glutamax (abbreviated here to OptiMEM) (Gibco BRL) containing 10% NCS, without antibiotics, for 24 h in 24-well plates, until they reached 90-95% confluence. Cells were then washed and transfected with 15 pmoles siRNAs (final concentration of each siRNA: 25 nM), mixed with 3 µl of Lipofectamine 2000 in 100 µl of medium per well, as recommended by the manufacturer. After 4 h at 37°C, the serum concentration was adjusted to 10%, and plates were further incubated at 37°C. For lytic and short-term infections, cells were infected with PV (strain Sabin 3, L2-2 or L2-2m) about 20 h post-transfection, at an m.o.i. of 0.01, 1 or 10 ID 50 per cell, for 30 min at 37°C. Total virus was recovered after 0, 2, 4, 6, 8 and 24 h at 37°C, clarified and titered on HEp-2 cells.

Persistently infected HEp/L2-2 cultures were transfected with siRNA as described above, except that they were washed three times before transfection, and 500 µl of OptiMEM was added to each well. One hundred µl was immediately withdrawn for titration (t 0 ). The siRNA-Lipofectamine mixture (concentration of each siRNA: 25nM) was added to cell monolayers, and cultures were further incubated at 37°C. This procedure is defined as a single transfection (ST). In double transfection (DT) assays, the cell supernatant was replaced after 2 h of incubation with 400 µl of OptiMEM and 100 µl of an identical siRNA-Lipofectamine mixture. After 4 h of incubation at 37°C, the serum concentration was adjusted to 10%. Samples of cell supernatant were frozen 4, 6, 8, 10 and 24 h after t 0 , and then weekly. Some cell cultures were transfected again 20 days after the first DT. The day before transfection, cultures of each well were split 1:2. Half of the cultures were again doubly transfected with siRNAs, as described above. These cultures are denoted QT for quadruple transfection.

Virus and viral RNA detection.

Infectious virus in supernatants of HEp/L2-2/6 cells was titered as described [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF].

When infectious virus was not detected in any of several undiluted samples of the same culture at various passages, its absence was verified by RT-nested PCR. RNA was extracted from confluent cell cultures and stabilized with the successive use of a RNeasy Protect Mini Kit (Qiagen) and QIAshredder Homogenizer (Qiagen), following the manufacturer's directions. For each cellular extract, the integrity of ribosomal RNAs was verified by electrophoresis in ethidium bromide-stained 1 % agarose gel. The sensitivity of the detection method was determined with known amounts of PV type 3 RNA (from 1 pg to 0.0001 fg) transcribed from plasmid pT7L/P1(L2-2) as described [START_REF] Duncan | Two amino acid substitutions in the type 3 poliovirus capsid contribute to the establishment of persistent infection in HEp-2c cells by modifying virus-receptor interactions[END_REF] and mixed with 1 µg of HEp-2 cellular RNA. The first RT-PCR step was the amplification of the 5'NC region, using primers (nt positions 164-184 and 959-976), RNasin (Promega) and the SuperScript One-step RT-PCR kit with Platinum Taq (Invitrogen). Nested PCR was performed with the same Platinum Taq DNA polymerase (Invitrogen) using a sense primer corresponding to nt positions 269-286 and an antisense primer to nt positions 775-795, following the same amplification protocol. PCR products were analyzed by electrophoresis in 1.5 % agarose gel.

Viral RNA extraction and sequencing.

cDNAs were prepared from viral RNA, obtained from 2 µl of medium of cultures with a titer > 10 6 ID 50 /ml, in the presence of RNasin (Promega) and 10 pmoles of antisense primer (nt positions 1169-86 and 7441-64). After 5 min denaturation at 80°C, primer and viral RNA were annealed for 5 min at 50°C (for the 5'NC region) or 45°C (for the 3Dpol region) and cDNA was synthesized in 20 µl of a mixture containing 1 mM of each of the dNTP, 2 µl dithiothreitol (Gibco) and 40 units of Superscript II RNase H -reverse transcriptase (Gibco) in the enzyme buffer. After incubation for 30 min at 50°C and denaturation for 5 min at 95°C, the cDNA regions between nt 614 and 1186 in the 5'NC region and nt 6536 and 7464 in the 3Dpol region were amplified [START_REF] Guillot | Natural genetic exchanges between vaccine and wild poliovirus strains in humans[END_REF], in 80 µl with 10 pmoles of primers and 2.6 units of Expand High Fidelity PCR System (Roche) according to the manufacturer's instructions. The amplified DNA fragments were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) on an ABI Prism DNA 377 Sequencer (Applied Biosystems). Nucleotide sequences were aligned with CLUSTAL W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF].

Northern blot analysis of RNA

Transfection with siRNAs and infection were performed as described above. Total RNA was extracted from persistently infected HEp/L2-2/6 cells and from HEp-2 cells 5 h after infection by PV strain Sabin 3, L2-2 or L2-2m, at a m.o.i. of 10 ID 50 per cell. TRIzol reagent (Life Technologies) was used as recommended by the manufacturer. After denaturation for 10 min at 85°C in 50% formamide, 2.2 M formaldehyde in running buffer as described [START_REF] Machado | Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter[END_REF], RNAs from 3 cell cultures in wells of 24-well plates were run on a 1.2% agarose -0.6 M formaldehyde gel. A full-length genomic PV transcript was used as a marker (7.4 kb). RNAs were transferred to a Nylon membrane (Hybond-N, Amersham) and fixed. To prepare the probes, the 5'NC-capsid region between nt 164 and 1186 of the PV Sabin 3 strain genomic template was amplified by PCR [START_REF] Guillot | Natural genetic exchanges between vaccine and wild poliovirus strains in humans[END_REF]. The human ribosomal 28S template was from Ambion. For each probe, 125 mCi of 32 P-UTP (Amersham) was used and 32 P-labeled riboprobes were produced with the MAXIscript kit (Ambion) and purified by following the manufacturer's instructions. Blots were successively hybridized with the PV and ribosomal 28S probes, as described [START_REF] Machado | Expression of a foreign gene by stable recombinant influenza viruses harboring a dicistronic genomic segment with an internal promoter[END_REF]. The intensity of radiolabeled bands was quantified with a STORM 820 phosporimager (Molecular Dynamics) and analyzed using the Image Quant program (Molecular Dynamics).

(

  see the Fig. in Supplementary Material). Therefore, a minimum of two transfections were performed. Preliminary experiments were performed with HEp/L2-2/6 cultures to compare the therapeutic effect of the Mix and Mixm. Two weeks post-transfection, the amount of viral RNA in HEp/L2-2/6 cultures doubly transfected with either the Mix or Mixm, was about 28 and 33%, respectively, of that in cultures doubly transfected with siRNA-IRR (data not shown). Three of the six cultures transfected twice with the Mix and two of the six transfected with the Mixm stopped producing virus 5 weeks post-transfection (data not shown). These data suggested that the Mix and Mixm had very similar efficiencies in HEp/L2-2/6 cultures.We studied the kinetics of virus production following a double transfection (DT) with the Mix. Infectious virus production remained high in the three cultures of HEp/L2-2/6 transfected with siRNA-IRR, whereas it dropped to undetectable levels 34 days after transfection with the Mix in one culture (Fig.5, upper panel, culture labeled Mix 1).

Legend to Figure 1 .Legend to Figure 2 .Legend to Figure 5 .Legend to Figure 7

 1257 Inhibition of virus growth by antiviral siRNAs. HEp-2 cells were infected with the Sabin 3 strain at a m.o.i. of 10 -2 ID 50 /cell, about 20 h after transfection with virus-specific siRNA-5'NC, siRNA-3Dpol, the mixture of both siRNAs, Mix, or with the irrelevant siRNA-IRR, or no siRNA. The results shown are means of 3-6 infected cultures and the SEM are indicated by vertical error scale bars. A diagram of the PV genome is given below, showing antiviral siRNAs targeting the 5'NC and 3Dpol regions. Yield of progeny virus produced by HEp-2 cells and Vero cells infected at a m.o.i. of 1 ID 50 /cell, 20 h after transfection by either antiviral or the irrelevant siRNA-IRR, or no siRNA. Samples were harvested 24 h pi and the results are expressed as a percentage of the virus yield from cells transfected by siRNA-IRR. Each bar represents the mean value ± SEM of at least 9 and 6 infected cultures of HEp-2 (A) and Vero cells (B), respectively. In HEp-2 cells, in the same conditions, the virus L2-2m/DT, produced by a HEp/L2-2/6 culture that was not cured despite two successive transfections with the Mix, was still susceptible to the same specific siRNAs (C). Decrease of virus production after transfection of persistently PV-infected cultures with a mixture of antiviral siRNAs. HEp-/L2-2/6 cells were treated 5 months pi. Two successive transfections were performed at a 2h-interval with either the Mix (siRNA-5'NC+siRNA-3Dpol) or the irrelevant siRNA-IRR. Cultures were split 20 days later and half of the cultures were again transfected twice with the same siRNAs at a 2h-interval. Cultures transfected twice and four times are denoted DT and QT, respectively. Extracellular virus progeny for each culture was titered at the times indicated after the first double transfection. Double transfections of siRNAs are indicated by arrows. ND: Infectious virus not detected. Numbers following the siRNA-IRR and the Mix indicate the culture number. Assay to detect PV genomes in HEp/L2-2/6 cultures after they stopped producing virus following transfections with antiviral siRNAs. Total RNAs were extracted and viral RNAs were reverse transcribed and amplified by nested PCR in the 5'NC region as described in Materials and Methods. Virus-producing HEp/L2-2/6 and uninfected HEp-2 cells were used as positive and negative controls, respectively. Amplification products corresponding to known amounts of PV type 3 RNA (1 pg to 0.0001 fg) are shown. ΦX174 DNA cleaved by Hae III was used as molecular weight markers. DT: double transfection; QT: quadruple transfection.

Figure 1

 1 Figure 1

ACKNOWLEDGEMENTS

We would like to thank C. Gouyette for siRNA synthesis and F. Riquet for providing PV RNA. We are grateful to A. Martin, S. Guillot, B. Blondel and N. Escriou for helpful discussions. This work was supported by grants from the Pasteur Institute (DVPI and Direction des PTR). A.S. was supported by a fellowship from the Ministère (MENESR).

Legend to Figure 3

Effect of central mismatches in siRNAs targeting the 5'NC region of PV on the multiplication of L2-2m in HEp-2 cells. True mismatches between the guide strand of siRNA (black) and the L2-2m genome (grey) are indicated; the wobble base pair is shown with a star. Experimental conditions were the same as those for Fig.