Karine Labadie

Isabelle Pelletier

Aure Saulnier

Javier Martin

Florence Colbère-Garapin

Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

Keywords: poliovirus, vaccine, enterovirus, picornavirus, persistent infection, enterocyte, intestine, Caco-2 cells, CD155

Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent

infections in human intestinal cells.

INTRODUCTION

Poliovirus (PV) is an enterovirus that causes paralytic poliomyelitis [START_REF] Landsteiner | Ubertragung der Poliomyelitis acuta auf Affen[END_REF][START_REF] Levaditi | Virus de la poliomyélite et culture des cellules in vitro[END_REF]. The PV genome, a single-stranded RNA molecule of positive polarity, is protected by an icosahedral capsid composed of 60 copies of polypeptides VP1 to VP4 [START_REF] Rossmann | Picornavirus structure overview[END_REF]. The cellular receptor common to all three PV serotypes is CD155, a glycoprotein member of the immunoglobulin superfamily [START_REF] Koike | The poliovirus receptor protein is produced both as membranebound and secreted forms[END_REF][START_REF] Mendelsohn | Cellular receptor for poliovirus : molecular cloning, nucleotide sequence and expression of a new member of the immunoglobulin superfamily[END_REF]. At the surface of susceptible cells, CD155 binds the virus and induces conformational transitions of the capsid that lead to viral uncoating at physiological temperature [START_REF] Hogle | Poliovirus receptors and cell entry[END_REF]. During these transitions, virions sedimenting at 160S externalize VP4 and the N-terminal part of VP1, thereby forming A particles sedimenting at 135S. Some A particles are released from cells at 37°C, while others remain cell-associated. The production of empty capsids sedimenting at 80S is believed to result from viral uncoating. CD155 is a major determinant of PV tissue tropism and it is present at the surface of human neurons [START_REF] Gromeier | Determinants of poliovirus pathogenesis[END_REF][START_REF] Pavio | Persistent poliovirus infection of human fetal brain cells[END_REF]. It is also present at the surface of human polarized enterocytes and M (microfold) cells, both on the apical face and more abundantly on the basolateral face [START_REF] Iwasaki | Immunofluorescence analysis of poliovirus receptor expression in Peyer's patches of humans, primates, and CD155 transgenic mice: implications for poliovirus infection[END_REF].

Poliomyelitis is efficiently prevented by either of two vaccines: the inactivated poliovaccine and the attenuated oral poliovaccine, OPV [START_REF] Minor | Poliovirus vaccines: molecular biology and immune response[END_REF]. The three Sabin strains of the OPV multiply to high titers in the human intestine, but normally do not invade the central nervous system. This vaccine is very safe. However, in immunodeficient persons with hereditary antibody deficiencies, Sabin PV strains of each of the three serotypes can continue replicating for long periods of times, more than 10 years, sometimes causing chronic poliomyelitis [START_REF] Bellmunt | Evolution of poliovirus type I during 5.5 years of prolonged enteral replication in an immunodeficient patient[END_REF][START_REF] Buttinelli | Nucleotide variation in Sabin type 2 poliovirus from an immunodeficient patient with poliomyelitis[END_REF][START_REF] Kew | Prolonged replication of a type 1 vaccine-derived poliovirus in an immunodeficient patient[END_REF][START_REF] Minor | Characteristics of poliovirus strains from long-term excretors with primary immunodeficiencies[END_REF][START_REF] Yoneyama | Neurovirulence of Sabin 1-derived polioviruses isolated from an immunodeficient patient with prolonged viral excretion[END_REF]. Prolonged virus multiplication in the intestine of patients causes long-term excretion of neurovirulent vaccine-derived PV (VDPV) in the stools.

Recently, Martin et al. characterized Sabin 3-derived PV isolates, which were excreted by a hypogammaglobulinemic patient over a period of 637 days post-vaccination [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF]. Virus excretion appeared to cease spontaneously after this period. The isolates followed a main lineage of evolution, with a rate (2.78% per year) of nucleotide substitution at synonymous third-base codon positions that was very similar to that estimated for wildtype PV during person-to-person transmission [START_REF] Kew | Molecular epidemiology of polioviruses[END_REF]. Some mutations in the isolates are in regions of the capsid possibly involved in adaptation for growth in the human gut and virus persistence [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF].

PV persistence has never been studied in intestinal cells, although it has been studied in various cell culture models, including neuronal and epidermoid cells. The molecular mechanisms of PV persistence vary greatly between different cell types, but modification of either CD155 expression or of PV-CD155 interactions is common to many persistent PV infections [START_REF] Duncan | Two determinants in the capsid of a persistent type 3 poliovirus exert different effects on mutant virus uncoating[END_REF][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]Pelletier, Duncan, and Colbère-Garapin, 1998). CD155 expression is reduced in cervix carcinoma HeLa cells [START_REF] Kaplan | Down regulation of poliovirus receptor RNA in HeLa cells resistant to poliovirus infection[END_REF] and in some clones of larynx carcinoma HEp-2c cells [START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF] following persistent PV infection. In addition, PV mutants (PVpi) selected during persistent infection of neuroblastoma IMR-32 cells harbor capsid mutations in regions interacting with CD155 (Pelletier et al., 1998). The kinetics of adsorption of PVpi onto CD155-expressing cells and of elution, and the conformational modification of the capsid induced by CD155 at 37°C are often different from those of the parental lytic PV strains [START_REF] Duncan | Two determinants in the capsid of a persistent type 3 poliovirus exert different effects on mutant virus uncoating[END_REF]. We have recently shown that 147S forms are produced upon interaction of particular PV mutants with either CD155-expressing cells or the soluble receptor PVR-IgG2a, at 0°C [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][START_REF] Pelletier | Characterization of the poliovirus 147S particle: new insights into poliovirus uncoating[END_REF]. These 147S forms have the same composition as the virion and may be a conformational intermediate between 160S virions and 135S A particles [START_REF] Pelletier | Characterization of the poliovirus 147S particle: new insights into poliovirus uncoating[END_REF].

To develop a cell culture model relevant to chronic PV infection of the human intestine, we studied PV persistence in Caco-2 cells, derived from a human colon adenocarcinoma. Caco-2 cells grown on permeable filters express genes that are specifically expressed in villus absorptive enterocytes of the small intestine [START_REF] Hidalgo | Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability[END_REF][START_REF] Pinto | Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture[END_REF][START_REF] Van Beers | Lactase and sucrase-isomaltase gene expression during Caco-2 cell differentiation[END_REF]. Therefore, despite their colonic origin, Caco-2 cells differentiate into small intestine enterocyte-like cells (Costa de [START_REF] Costa De Beauregard | Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells[END_REF][START_REF] Pinto | Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture[END_REF]. We show here that several of the PV mutants excreted by an immunodeficient patient can establish persistent infection in human intestinal cells, and that there is a correlation between this capacity and reduced adsorption kinetics onto cells.

RESULTS

Characterization of the intestinal cell culture models.

To study the capacity of VDPV mutants to establish persistent infections in undifferentiated dividing intestinal cells and in polarized enterocyte-like cells, we first characterized our intestinal cell culture models. Caco-2 cells were seeded onto Transwell filters as described in Materials and Methods. The interactions of PV with CD155 are generally involved in persistent PV infections, and therefore we tested for CD155 expression at the cell surface, by indirect immunofluorescence with a monoclonal antibody specific for CD155 and secondary antibodies labeled with fluorescein isothiocyanate (FITC). As previously shown (Tucker et al., 1993a), CD155 was present on both faces of the Caco-2 cells, and more abundant on the basal face of both 3 day-and 14 day-old cultures (Fig. 1).

Two to 4 day-old Caco-2 cells were used to study VDPV-CD155 interactions (see below). We verified that the average expression of CD155 at the cell surface was constant over this period of time by immunofluorescence and analysis with a fluorescence activated cell sorter (not shown). The structural protein F-actin, that is associated with the cytoskeleton and present in the microvilli of the brush border of polarized enterocytes, was detected by its specific binding to phalloïdin-alexa. F-actin was principally expressed at the basal face of 3-day old Caco-2 cells (Fig. 1,M). In contrast, it was principally expressed on the apical face of 14-day old cells, consistent with the presence of F-actin in brush border microvilli of 14-day old Caco-2 cells (Fig. 1,N). The presence of the brush border on the apical face of 14-day old Caco-2 cells was confirmed by labeling terminal fucose residues of the brush border glycocalyx with the lectin UEA1 conjugated to FITC, and by transmission electron microscopy (data not shown), as previously shown for enterocyte-like cells obtained by differentiation of a clone of Caco-2 cells [START_REF] Ouzilou | Poliovirus transcytosis through M-like cells[END_REF]. The trans-epithelial resistance increased from about 400 to 750 ohms.cm 2 between days 3 and 14 of culture, indicating that 14 day-old cells formed tight monolayers, due to the presence of tight junctions. Thus, 14 day-old Caco-2 cells acquired characters of polarized enterocytes, in particular cell polarity, a brush border and tight junctions.

Persistent poliovirus infection in Caco-2 cells.

Seven isolates of type 3 VDPVs obtained from a hypogammaglobulinemic patient, chronically infected by Sabin 3-derived PV mutants over almost two years until the patient spontaneously stopped excreting virus, were characterized previously [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF].

The number in the name of isolates (H36, H136, H307, H391, H442, H480 and H637) indicates the day post-vaccination when the virus was collected. The virus excreted by the patient on any one day could have been a mixture, and thus clones were obtained by plaquepurification, twice, and named H36-11 to H637-11. The genomic region encoding capsid proteins, which harbors all the determinants required to confer to PV a persistent phenotype in HEp-2c cells [START_REF] Calvez | Identification of a region of the poliovirus genome involved in persistent infection of HEp-2 cells[END_REF][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], was sequenced for each of these viral clones. This genomic region in four of the clones (H36-11, H136-11, H442-11 and H637-11) contained no missense mutations with reference to the consensus sequence of the corresponding isolates. It thus seems probable that these viral clones correspond to the major virus populations in the respective isolates. These 4 clones correspond to the first and last isolates, and to two intermediate isolates, and therefore cover the entire period of the evolution of the VDPVs. The 3 other clones had either a supplementary mutation (VP1 30 Ala>Val in H307-11, and VP3 79 Ser>Met in H480-11) or lacked one missense mutation (at residue VP1 34 in H391-11). Other clones of the same isolates also differed from the respective consensus sequences. These clones may be quasispecies. Therefore only VDPV clones H36-11, H136-11, H442-11 and H637-11 were included in all experiments.

The capacity of the four viral clones H36-11, H136-11, H442-11 and H637-11 to establish persistent infections was tested in undifferentiated, 4 to 5 day-old Caco-2 cells, using as controls the parental Sabin 3 strain, that is lytic in HEp-2c cells, and a type 3 PVpi, L2-2, that establishes persistent infections in 100 % of HEp-2c cell cultures [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]. During the first three weeks post-infection, cytopathic effects were detected in all infected Caco-2 cultures, and one month post-infection, a few growing cell colonies appeared in cultures infected with either PVpi L2-2 or one of VDPV H136-11, H442-11 or H637-11. Caco-2 cells persistently infected by VDPV mutants could be grown for at least 6 months. However, the percentage of persistently infected cultures was determined two months post-infection (Fig. 2). PVpi L2-2 established persistent infections in 47+12% of Caco-2 cell cultures. The Sabin 3 strain and viral clone H36-11 -the first isolatewere entirely lytic (Fig. 2). H136-11, H442-11 and H637-11 established persistent infections in 8 to 17% of cultures (Fig. 2). Thus, only 3 of 4 VDPVs were persistent, implicating viral determinants in the establishment of persistent infections in Caco-2 cells. These determinants were acquired (or became dominant) in the patient after day 36 and were present in VDPVs from day 136 to day 637 post-vaccination.

In HEp-2c cells, H36-11 was lytic, and H136-11, H442-11 and H637-11 mutants established persistent infections in 61+16, 17+10 and 69+16% of HEp-2c cell cultures, respectively. Therefore, each VDPV mutant was either lytic or persistent in both HEp-2c and Caco-2 cells.

Interestingly, 20 to 80% of Caco-2 cell cultures persistently infected by the VDPV mutants stopped excreting infectious virus between 1 month and 1 year post-infection.

Persistent poliovirus infection in polarized enterocyte-like cells.

We investigated whether differentiation of Caco-2 cells affects the establishment of persistent infections. Caco-2 cell cultures were grown for two weeks on microporous membranes until they were differentiated into polarized enterocyte-like cells, and then they were infected with H36-11, H136-11, H442-11 and H637-11, the parental S3 strain and PVpi L2-2. Although the level of CD155 expression is higher on the basolateral than on the apical face of cells, the level on the apical face is sufficient to mediate efficient infection (Tucker et al., 1993a), and therefore cells were infected either by the apical or by the basolateral face (Fig. 3). Five days post-infection, apical and basolateral supernatants were collected and the virus titered: all cultures excreted virus, and the titers were above 10 5 infectious doses 50 per ml (ID 50 /ml) in the apical chamber. Cytopathic effects were detected in all infected cultures.

The percentage of persistently infected cultures was determined 4 weeks post-infection, because the filters were not appropriate to grow enterocyte-like cells for longer periods of time. The parental S3 strain and H36-11 were lytic, as in undifferentiated Caco-2 cells. PVpi L2-2 established persistent infections in more than 65% of infected cultures, and the clones H136-11, H442-11 and H637-11 in more than 75% of infected cultures, independently of the face of cells used for virus inoculation (Fig. 3). The percentage of persistently VDPV-infected cultures was higher in enterocyte-like cells than in 4-5 day-old Caco-2 cell cultures, one and two months post-infection, suggesting that cellular determinants involved in the differentiation of enterocytes also contribute to the establishment of persistent infections by the VDPV mutants. No spontaneous cure was observed in VDPV-infected enterocyte-like cells, even when these cells could be maintained for periods of time longer than one month.

One-step virus growth cycles in polarized enterocytes.

We tested whether multiplication of the persistent VDPV mutants (H136-11, H442-11, H637-11) was impaired in polarized enterocyte-like cells. One-step growth experiments were performed at a m.o.i. of 10 ID 50 per cell with two of the persistent mutants (H136-11 and H442-11) and with the parental lytic S3 strain. No significant difference was observed between the multiplication of the persistent VDPVs and that of the S3 strain: the exponential growth and the intracellular yields were comparable for the 3 viruses. Intracellular yields were high (about 10 9 ID 50 per ml and per culture of 1.5 x 10 6 cells) (Fig. 4, upper panel). Virus liberation was also similar for all three viruses, and about 1000-fold more efficient from the apical face than from the basolateral face of cells, 48 h post-infection (Fig. 4, lower panel), in agreement with the results previously published for the Mahoney PV strain (Tucker et al., 1993b).

Reduced adsorption of the persistent VDPVs onto Caco-2 cells.

There are numerous mutations in the capsid proteins of the persistent VDPVs: some in the canyon (VP2 139 , VP2 140 ), in or near antigenic site 3a (VP1 288 ), site 4 (VP3 79 , VP3 116 ) or at the interface of protomers (VP3 91 ) [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF]. These residues may affect binding of VDPVs to CD155 and the plasticity of the viral capsid, respectively. Therefore, the early steps of the viral cycle were studied in Caco-2 cells for the three persistent VDPVs, the lytic VDPV H36-11 and the lytic S3 strain. The adsorption of H36-11 was more efficient than that of S3 (Fig. 5A), and similar to that of the wild-type strain T7/Leon used as an internal control (not shown). In contrast, the three persistent VDPVs had a lower adsorption than S3 onto Caco-2 cells (Fig. 5A). VDPVs H442-11 and H637-11, excreted late, had the lowest binding efficiency, about half that of S3. Thus, reduced adsorption correlated with the persistent phenotype of mutants H136-11, H442-11 and H637-11.

Virus elution and conformational transitions of the capsid

Increased elution or modified conformational transitions may contribute to the mechanism of the persistent infection, as previously shown for PVpi in HEp-2c cells [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]Pelletier, Duncan, and Colbère-Garapin, 1998). Therefore, these steps were studied and to compare the proportion of eluted particles regardless of the number of attached virions, values are expressed as a percentage of the total cell-associated radioactivity (Fig. 5B). The H442-11 and H637-11 viruses eluted from cells at 37°C faster and more efficiently than the lytic VDPV H36-11 and the S3 strain. The enhanced elution of H442-11 and H637-11 may thus contribute to the persistent phenotype of these viruses. However, the kinetics of elution of VDPV H136-11 was similar, and even slightly slower than that of S3. The VDPV H36-11 had the lowest elution rate (Fig. 5B) and its profile of elution was similar to that of the wild-type T7/Leon strain (not shown).

The uncoating transitions from 160S virions to 135S A particles were then analyzed by ultracentrifugation in sucrose gradients. Sedimentation profiles of cell-associated particles are shown for a 0 or 20 min incubation period at 37°C (Fig. 6). Like S3, all of the mutants sedimented at 160S after binding to Caco-2 cells for 3 h at 0°C, and the corresponding peak decreased after incubation at 37°C, leading to the formation of 135S A particles. The alteration ratio (i.e. the ratio of the area under the 135S peak to the area under the 160S peak) was between 0.3 and 0.4 for VDPVs H36-11, H136-11 and H637-11, and 0.5 for the control strain, S3 (Fig. 6). VDPV H442-11 had an alteration ratio almost twice as high as that of S3, and after 20 min the uncoating process of this VDPV appeared more complete than that of the other viruses tested. Thus, the extent of the uncoating transitions in 20 min did not correlate with the capacity of the VDPVs to establish persistent infections.

DISCUSSION

Immunodeficient patients whose gut is chronically infected by VDPVs can excrete large amounts of virus for years. Unfortunately, there is no convenient small animal model to investigate how PV establishes chronic infections in the gut. We therefore tested whether it is possible to establish persistent PV infections in vitro in human intestinal cells. The expression of CD155 was first verified at the surface of Caco-2 cell-derived enterocytes, and the differentiation of cells was monitored by examining the distribution of F-actin in the cells. In addition, the formation of a typical brush border was observed by transmission electron microscopy, and the presence of fucose residues in the glycocalyx was verified.

We have previously shown that the P1 region of PV type 3, the region that encodes the capsid proteins, harbors the main determinants involved in the phenotype persistence in epithelial-like HEp-2c cells [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]. In this study, four sequential VDPV clones excreted by an immunodeficient individual and covering a 637day period [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF] were studied in terms of their ability to establish persistent infections in intestinal cell cultures. H36-11, the first isolate from the patient was, like the parental S3 strain, lytic in dividing 4 to 5 day-old Caco-2 cultures. Interestingly, H136-11 and two other mutants isolated later (H442-11 and H637-11) established persistent infections in 8 to 17% of Caco-2 cell cultures. The same VDPVs established persistent infections in HEp-2c cells. Thus, viral mutations acquired or selected between 36 and 136 days post-vaccination were responsible for the phenotype of persistence. Seven capsid residues in VP1 and VP2 differentiate H136-11, which is persistent, from H36-11 which is lytic (Table 1). They include mutations at residues VP2 14 and VP1 288 close to residues VP2 13 and VP1 290 respectively, involved in the capacity of a type 3 PVpi to establish persistent infections in HEp-2c cells [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]. Therefore, residues VP2 14 and VP1 288 may contribute to the capacity of H136-11, H442-11 and H637-11 to establish persistent infections in Caco-2 (and HEp-2c) cells. However, this remains to be confirmed, and the involvement of the other mutations cannot be excluded.

Viral determinants play a major role in the establishment of persistent infections both in Caco-2 and in HEp-2c cells. Spontaneous cures occurred in persistently VDPV-infected Caco-2 cells, as previously observed in persistently PVpi-infected HEp-2c cells [START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF]. Therefore, persistent infections in Caco-2 and HEp-2c cells seem to have several common features, that differ from those in cultured neuronal cells, in which viral determinants do not play the major role, and in which no spontaneous cure has ever been observed (Colbère-Garapin, Pelletier, and Ouzilou, 2002).

VDPV mutants H136-11, H442-11 and H637-11 established persistent infections in enterocyte-like cells as they did in young Caco-2 cell cultures, but with a higher frequency, i.e. in more than 75% of cultures. This suggests that the same viral determinants play a role in the establishment of persistent infections in both types of intestinal cell cultures, but that cellular determinants involved in the differentiation of enterocytes also contributed to persistence. No spontaneous cure was observed in VDPV-infected enterocytes, possibly because these cultures, unlike Caco-2 cells, could not be maintained for several months.

Alternatively, the absence of cure in VDPV-infected enterocytes suggests either that cell division plays a role in the spontaneous cure of Caco-2 cells, or that the expression of cellular genes involved in enterocyte differentiation is not favorable to the cure of cells. If the absence of spontaneous cure in enterocyte-like cells is relevant to infection of fully differentiated enterocytes in patients, it is probable that these cells are normally eliminated in vivo when they are replaced by newly differentiated enterocytes.

Presumably several mechanisms are involved in persistence. The restriction of PV replication sometimes contributes to the mechanism of PV persistence [START_REF] Borzakian | Persistent poliovirus infection: establishment and maintenance involve distinct mechanisms[END_REF][START_REF] Calvez | Cell clones cured of persistent poliovirus infection display selective permissivity to the wild-type poliovirus strain Mahoney and partial resistance to the attenuated Sabin 1 strain and Mahoney mutants[END_REF][START_REF] Girard | Restriction of poliovirus RNA replication in persistently infected nerve cells[END_REF]. However, the establishment of persistent infections in enterocyte-like cells was not due to poor replication of VDPVs in these cells: one-step growth cycles were similar for the lytic S3 strain and the persistent VDPVs H136-11 and H442-11, with high intracellular yields and efficient, polarized virus liberation by the apical face. The early steps of PVpi multiplication are generally modified in HEp-2c cells [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]Pelletier, Duncan, and Colbère-Garapin, 1998), and consequently we studied adsorption, elution and uncoating transitions of VDPVs. The mutant H36-11, like T7/Leon (not shown), had an adsorption rate significantly higher than that of S3 (Fig. 5A).

This may be related either to the reversion in H36-11 of an attenuating determinant, at residue VP3 91 , toward the T7/Leon genotype, or to H36-11 residues VP2 186 Met and VP1 275 Thr (Table 1), the latter residue being close to the footprint of CD155 on the PV3 capsid [START_REF] He | Complexes of poliovirus serotypes with their common cellular receptor, CD155[END_REF]. Interestingly, the adsorption rates of persistent VDPVs (H136-11, H442-11 and H637-11) were lower than those of the lytic strains (S3 and H36-11). The high elution rate of H442-11 and H637-11 may contribute to the phenotype of persistence of these viruses, as previously reported for a type 1 PVpi (Pelletier, Duncan, and Colbère-Garapin, 1998).

However, the elution rate of H136-11 was slightly slower than that of S3, and yet H136-11 established persistent infections efficiently in enterocyte-like cells. As in the case of elution, there was no strict correlation between the efficiency of uncoating transitions and the capacity of VDPVs to establish persistent infections. No 147S particle was detected following adsorption of any of the VDPV mutants onto Caco-2 cells, confirming that there is no strict correlation between the formation or accumulation of these particles and PV persistence [START_REF] Duncan | Two determinants in the capsid of a persistent type 3 poliovirus exert different effects on mutant virus uncoating[END_REF]Pelletier et al., 1998).

Therefore, adsorption was the only early step potentially involved in the mechanism of persistence of the three persistent VDPVs in intestinal cell cultures. Although this reduced adsorption did not prevent a high level of viral replication, as mentioned above, it is possible that reduced interactions between persistent VDPVs and CD155 correlates with a low level of apoptosis in infected cells. Indeed, PV induces apoptosis in Caco-2 cells [START_REF] Ammendolia | Poliovirus infection induces apoptosis in CaCo-2 cells[END_REF] and we have recently shown that the frequency of PV-induced apoptosis is reduced when PV-CD155 interactions are affected by a mutation in the N-terminal domain of CD155, which interacts with PV [START_REF] Gosselin | Poliovirus-induced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells[END_REF]. The establishment of a persistent VDPV infection may also depend on several viral factors. A mutation in the 3A coding region of a PV is associated with reduced Vero cell lysis following viral multiplication [START_REF] Lama | Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells[END_REF]. Moreover, our results strongly suggest that cellular factors involved in the differentiation of enterocytes may contribute to the establishment of persistent infections in human intestinal cells. It would be particularly interesting to identify these factors and discover their role in persistent VDPV infection.

MATERIALS AND METHODS

Cells and viruses

The human colon adenocarcinoma cell line Caco-2 (passages 10 to 40) was grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS).

Enterocyte-like cells were obtained by seeding Caco-2 cells in the upper chamber of polyester Transwell-Clear filters (Corning Costar) with pores of 0.4 µm or 3 µm diameter, in DMEM supplemented with glucose (1 g/l) and 1% non-essential aminoacids (NEAA) in a humidified 9% CO 2 incubator. We used the vaccinal attenuated P3/Leon 12a 1 b Sabin strain (Sabin 3 or S3) and a PV mutant (PVpi), L2-2, derived from wild type P3/Leon/37 after 2.5 months of persistent infection in human neuroblastoma cell line IMR-32, and persistent in HEp-2c cells [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]. The seven sequential Sabin 3-derived viruses (H36 to H637) were isolated from a hypogammaglobulinemic patient who had excreted virus for 637 days [START_REF] Martin | Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion[END_REF]. We cloned each VDPV isolate twice by plaque purification on HEp-2c cells under an agarose (0.8%) overlay. All viral stocks were prepared on HEp-2c cells and virus infectivity was titered on these cells by an endpoint micromethod. The infectious titers are expressed per milliliter as the log 10 of the highest dilution for which cytopathic effects and cell lysis were observed in 50% of wells 7 days after infection at 37°C. Titers varied between 0.05 and 5 x 10 8 ID 50 /ml. The capsid protein-encoding region in each viral clone was sequenced.

Viral RNA sequencing

cDNA was synthesized exactly as previously described [START_REF] Oprisan | Natural genetic recombination between co-circulating heterotypic enteroviruses[END_REF] using 4 antisense primers (C1484, C2474, C2658, C3586; the number indicating primer position in viral genome). The synthesized cDNA was then amplified with 10 pmol genomic primers (G614, G1913, G2402) and 2.6 U Enzyme mix high fidelity (Roche). Amplification involved 29 cycles of denaturation at 94°C for 15 sec, annealing at 45°C for 30 sec and elongation at 68°C for 2 min, followed by a final cycle of denaturation at 94°C for 15 sec, annealing at 45°C for 30 sec and elongation at 68°C for 7 min. PCR products were analyzed by electrophoresis in ethidium bromide-stained 0.8% agarose gels and then purified using the QIAquick Spin Purification (Qiagen), according to the manufacturer's instructions. The amplified DNA fragments were sequenced using the Big-Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems) on the ABI Prism DNA 377 Sequencer (Perkin Elmer 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].

Indirect immunofluorescence

Caco-2 cells seeded on Transwell filters for 3 or 14 days were fixed for 15 min at 20°C in 4% paraformaldehyde and then treated for 10 min with NH 4 Cl (50 mM). Cells were incubated either with the anti-CD155 monoclonal antibody 404 (14 µg/ml) [START_REF] Lopez | Identification of a new class of Ig superfamily antigens expressed in hemopoiesis. In "Leucocyte typing VI, White Cell Differentiation Antigens[END_REF], or with a control immunoglobulin G1 for 60 min at 20°C, washed twice with phosphate-buffered saline (PBS) -Ca 2+ -Mg 2+ containing 2% filtered FCS and then incubated for 60 min with anti-mouse immunoglobulin conjugated to FITC (15 µg/ml). Cells were washed again, then permeabilized for 2 min with Triton X100 (0.5%), and were incubated with phalloïdin-alexa (0.4 U/ml) for 30 min to stain F-actin. Filters were washed again and mounted in Vectashield and cells were viewed under a Leica confocal microscope with a X63 objective. Fluorescence (green and red channels) from basolateral to apical face was quantified with MetaMorph Offline (Universal Imaging Corporation, version 4.6 r0).

Establishment of persistent infection in Caco-2 cells

Confluent 4 or 5-day-old Caco-2 cells, cultivated in 24-well plates, were washed with DMEM and then infected at a multiplicity of infection (m.o.i.) of about 10 -4 ID 50 /cell. Infected cells were grown at 37°C in a humidified 4% CO 2 incubator, in DMEM-2% FCS for the first week and then in DMEM-10% FCS for a minimum of 8 weeks, changing the medium once per week.

To induce Caco-2 cell differentiation into polarized enterocyte-like cells, Caco-2 cells were seeded at a density of 4.10 4 cells per cm 2 on tissue culture-treated polyester Transwell filters. Apical and basal media were replaced every 3 or 4 days for 14 days with DMEM-10% FCS until cells reached confluency and then with DMEM-2% FCS. Two weeks after seeding, cells were infected either from the apical or basolateral side as described above except that cells were grown in a humidified 9% CO 2 incubator for at least one month.

Single-cycle growth experiments

Enterocyte-like Caco-2 cells were infected, 2 weeks after seeding on filters by the apical face, at a m.o.i. of 10 ID 50 /cell in 200 µl of DMEM for 30 min at 37°C. Cells were washed three times and then incubated for 2, 4, 6, 12, 24 or 48 h at 37°C in 9% CO 2 atmosphere. For each time point, both apical and basolateral supernatants were collected.

Intracellular virus was recovered by freezing and thawing, and all samples were clarified before titering virus on Caco-2 cells.

Preparation of radiolabeled virions

Confluent HEp-2c cells were infected at a m.o.i. of about 10 ID 50 /cell and agitated for 30 min at 37°C. Medium supplemented with 2% FCS was then added, and cells were incubated for 2 h at 37°C. The medium was then changed to methionine-and cysteine-free medium for 30 min and 600 µCi of 35 S-methionine and 35 S-cysteine (TRAN 35 S-LABEL; ICN) were added. Cells were lysed 24 to 48 h post-infection by freezing and thawing. Virus was clarified and purified from cytoplasmic extracts by CsCl density gradient centrifugation as previously described [START_REF] Blondel | Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions[END_REF][START_REF] Pelletier | Characterization of persistent poliovirus mutants selected in human neuroblastoma cells[END_REF]. Purified virus was desalted by gel filtration on a PD-10 column (Pharmacia Biotech) and then stored at -80°C. As the specific activities of S3, PVpi L2-2 and VDPV mutants varied between 0.7 and 18 x 10 -4 cpm/ID 50 , the same m.o.i. (140 or 200 ID 50 /cell) was used in experiments with radiolabeled viruses. The results of virus adsorption and elution are expressed as percentages of cellassociated radioactivity.

Virus adsorption onto Caco-2 cells

Virus adsorption was studied, as previously 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]Pelletier, Duncan, and Colbère-Garapin, 1998), using 2 to 4-day-old Caco-2 cells in suspension on ice. Cells (0.75 x 10 6 ) were infected with radiolabeled virions at a m.o.i. of 140 ID 50 /cell in 150 µl of DMEM-2%FCS. Infected cells were agitated on ice for 30, 60, 90, 120, 150, 180 and 210 min after which cells were washed twice with DMEM-2%FCS and once with PBS. Cell-associated radioactivity was determined by scintillation counting.

Particle elution from Caco-2 cells at 37°C

Undifferentiated Caco-2 cells (0.75 x 10 6 ) in suspension were infected, as previously 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]Pelletier, Duncan, and Colbère-Garapin, 1998), with radiolabeled virions at a m.o.i. of 140 ID 50 /cell in 150 µl of ice-cold DMEM-2%FCS. After 2.5 h of adsorption, infected cells were washed twice and incubated for various times (0, 15, 30, 60, 90, 120 and 150 min) in 3 ml of DMEM-2%FCS preheated to 37°C. Cells were then washed once with ice-cold PBS and cell-associated radioactivity was determined by scintillation counting.

Capsid conformational transitions

Undifferentiated Caco-2 cells (0.5 x 10 7 ) in suspension were infected with radiolabeled virions at a m.o.i. of 200 ID 50 /cell for 3h at 0°C and then treated exactly as previously described (Pelletier, Duncan, and Colbère-Garapin, 1998). Briefly, Caco-2 cells were incubated at 37°C for either 0 or 20 min and lysed. The 20 min time point was chosen because at this time, about 50% of the capsids of the reference S3 strain were transformed into uncoating intermediates. The lysates were layered onto 15 to 30% (w/v) sucrose gradients that were centrifuged in a Kontron TST 41.14 rotor for 2h at 40,000 rpm at 4°C.

Figure 1 .

 1 Figure 1.Immunofluorescence analysis of CD155 expression in 3-day-old Caco-2 cells and polarized enterocytes. Caco-2 cells were seeded and cultured on Transwell filters for 3 days (left-hand panels) or 14 days (right-hand panels). Fluorescence is shown both on basolateral (Bl) and apical (Ap) planes of Caco-2 cells. The Bl and Ap planes of 3-day-old Caco-2 cells are designated by reference to differentiated enterocytes (the basal face is adjacent to the filter). The PV receptor (PVR or CD155) was detected with the monoclonal antibody 404[START_REF] Lopez | Identification of a new class of Ig superfamily antigens expressed in hemopoiesis. In "Leucocyte typing VI, White Cell Differentiation Antigens[END_REF] and anti-mouse immunoglobulin conjugated to FITC (A-D). To visualize Caco-2 cell differentiation, F-actin was stained with phalloïdin-alexa (E-H). Simultaneous detection of CD155 and F-actin is presented (I-L). Images were captured by confocal microscopy through a 63X objective lens. Panels M and N show fluorescence intensity profiles (in arbitrary units) for green (FITC) and red (alexa) channels, from Bl to Ap planes of 3-and 14-day-old Caco-2 cells, respectively. Each value in M and N represents the average fluorescence intensity for 3 separate fields in the same plane. Vertical bars indicate the standard errors of the means (SEM) for 18 and 21 planes of 3-and 14-day old cultures, respectively. The distance between two planes is 1 µm and the thickness of each slide is 0.3 µm. Fluorescence was quantified with MetaMorph Offline (Universal Imaging Corporation, version 4.6 r0).

  Figure 2. Persistent poliovirus infection in Caco-2 cell cultures. Confluent 4-or 5-day-old Caco-2 cells were infected at a m.o.i. of 10 -4 ID 50 per cell with the Sabin 3 strain (S3), the mutant PVpi L2-2 and the 4 VDPV clones H36-11 to H637-11. The results are expressed as the percentage of persistently infected cultures 2 months post-infection. The mean of 3 experiments is presented, and the horizontal error bars represent the SEM.

  Figure2

Figure 3 .

 3 Figure 3. Persistent poliovirus infection in polarized enterocyte-like cells. Caco-2 cells seeded on Transwell filters were grown for 14 days to induce the differentiation of Caco-2 cells into enterocyte-like cells. Cells were then infected at a m.o.i. of 10 -4 ID 50 /cell with the Sabin 3 strain (S3), the PVpi L2-2 or the 4 VDPV clones H36-11 to H637-11, either from the apical face (dark gray) or from the basolateral face (light gray). At least 3 independent experiments were performed with each virus, and the percentage of persistently infected cultures one month post-infection is presented with SEM (horizontal error bars).

Figure 4 .

 4 Figure 4. One-step virus growth cycles in polarized enterocytes. Caco-2 cells were seeded on Transwell filters and were infected 2 weeks later from the apical side at a m.o.i. of 10 ID 50 /cell with the lytic strain S3, and the persistent VDPV clones H136-11 and H442-11. Intracellular virus (upper panel) and extracellular apical or basolateral virus (lower panel) were titered on Caco-2 cells. Averages of 4 cultures from 2 independent experiments are presented and the SEM are indicated by vertical bars (they are barely visible because of the log scale).

Acknowledgements. We would like to thank Dr. Marc Lopez for providing the anti-CD155 monoclonal antibody 404. We are grateful to B. Blondel and B. Baron for critical reading of the manuscript. We thank E. Perret (Centre d'Imagerie Dynamique) for help with confocal microscopy and C. Bronnert (Plate-Forme de Microscopie électronique) for help with electron microscopy. This work was supported by grants from the Pasteur Institute.


Hélène Mainet

The paradoxical place of small towns in sustainable development policies. What is beyond the images of "places where the living is easy"?

Keywords:

Sustainable development policies have become new objectives of local actions since the implementation of the notion in the 1990's. Small towns promote images of places where the living is easy and with sustainable amenities like "natural" living environment. But, beyond these perceptions, operational sustainable development policies are quite occasional. Taking a sample of French small towns as examples, in the Auvergne region, it is interesting to analyse this paradox of an interesting potential leading to rare effective actions. It seems that it is quite difficult for local stakeholders to implement really transversal and not sectoral policies and the lack of capacity and budget is an important issue for them as it takes time and money to develop really participative and holistic programs.

Over the years the concept of sustainable development has led to various definitions, understandings and instrumentations. Basic definition of sustainable development implies interrelation of three dimensionsenvironment, economy and society, meaning that each of them is as important as the others. Sustainable development became a political objective of the United Nations with several important summits and the Rio Conference in 1992. Some of the key sustainable development policy frameworks include comprehensive action plan Agenda 21 (created in 1992 and reaffirmed in 2002). Sustainable development remains a powerful concept and has been used extensively in town development since 1994 when several European capital cities signed the "Aalborg Commitments". These commitments became the foundation of sustainable development planning in cities. Municipalities that signed these Commitments vowed to establish principles of effective governance, protection and preservation of natural resources, wellbeing and health of citizens. Nowadays issue of quality of life tends to be more and more important for the attractiveness of spaces in a context of increasing commuting and social demand for a good living environment. Small towns are often described as places were "the living is easy" or "human sized towns" and local actors tend to underline local amenities such as a good environment, the proximity to nature or strong social ties to attract newcomers, tourists, even new investors [START_REF] Knox | Small Town Sustainability: Economic, Social, and Environmental Innovation[END_REF]. Furthermore, in the sustainable development philosophy, the notion of "Think Global, act local" tends to favour local actions and thus small localities where such experimentations can take place. But what is beyond the promotion of fashionable criteria and whether small towns can really implement sustainable development policies, not only focusing on environmental aspects but associating environment, economy, social and cultural issues? Taking French small towns as case studies, the paper is questioning the implementation of real and concrete policies and programs such as Local Agenda 21 or Healthy City affiliations in such towns. Apart from national data, a survey has been conducted in a sample of 12 small towns in the Auvergne Region (interviews with local authorities and inhabitants). This sample has been chosen to represent a range of situations (demographic and economic trends, geographical distance from large cities). It appears to be quite difficult (politically and financially) to plan "green and soft" transports modes, encourage local food systems or design eco-neighborhoods, when local budgets are often limited and economies of scales not always possible. Finally, the place of small towns in sustainable development policies is quite paradoxical as few of them are really involved in sustainable development programs despite their images of places "where the living is easy".

Small towns: are they places "where the living is easy"? In France, small towns are often portrayed in iconic terms as "towns in the countryside", close to nature. They are generally seen as more sustainable places to live in because of the absence of congesting forces, such as traffic, pollution and crime. Reduced transportation costs in terms of fuel and time along with higher division of labor are regarded as main advantages of small and medium towns. In city branding, environmental, social and heritage aspects are very much used in the promotion of small towns (table 1). A study of Internet sites of a sample of more than 80 small towns shows that the living environment (described as "natural" and "preserved") and quality of life attributes are quoted more often than economic aspects [START_REF] Mainet | Les petites villes françaises en quête d'identité : ambigüité du positionnement ou image tactiquement combinée ? MOTS, Les langages du politique[END_REF]. Sources: analysis of a selection of French small town websites (2010)

Those good images and the importance of environmental aspects are also noticeable in images and perceptions of inhabitants (table 2). A survey conducted in Auvergne small towns, on a sample of 328 dwellers, shows that the evaluation of their quality of life is good and very good, largely linked with the characteristics of the place and prized amenities, like the living environment qualities. Social aspects are also important and outdistance economic features like the existence of job opportunities or the proximity to work. Considering the three sustainable development pillars, economy seems to be less important in residential choices and mobility. But the reality is often quite divergent from mainstream images. Like other communities, small towns are facing myriad of challenges, ranging from job and population losses in many places to development pressures and changing landscapes in others. Access to jobs, services, and transportation options can be often limited. Is the living still easy when public services are closing (this is the case in many of French small towns, where courts and tribunals, police stations, post offices, hospitals are affected by the national policy of restructuration of public services), when jobs in industries are reduced and young people are leaving? Ageing in lots of small towns is also a real issue. The promoted quality of life seems to be more ambiguous than it might appear. In that context, what is the place of local policies specifically dedicated to the improvement of quality of life issues and, more generally speaking, of sustainable development?

A difficult commitment in operational sustainable development policies An interesting paradox is the difference between the will and the capacity of local stakeholders to implement sustainable development policies. Different schemes and frameworks exist to develop sustainable development policies (climate and energy plan, environmental charter…). The analysis of different transversal and holistic programs proves that the place of small towns is not obvious. It seems that implementing official sustainable development policies is quite difficult for small towns, compared to other towns, especially large and medium sized towns. The first example is the Local Agenda 21. It has been created as a local adaptation of the Rio 1992 precepts [START_REF] Dooris | Healthy Cities and Local Agenda 21: the UK experience -challenges for the new millennium[END_REF]. In France, a national policy aims at encouraging communes and local authorities to adopt a Local Agenda 21, through a national process of branding (which has to be revaluated regularly). In 2013, 470 Local Agendas 21 were certified by the State, when 950 others were implemented without official recognition. Looking at the total of the French Local Agendas 21, it is interesting to notice that small towns (with population between 2,000 and 20,000 inhabitants) are hardly represented (tab. 3). The reasons to implement sustainable development policies are linked with the promotion of the town and the recognition of policies already implemented as a way to formalize them. It is also important to take advantage of being part of a network and sharing experiences and "good practices". The reasons why not are very interesting to understand from the small town actors' point of view. Small cities are at a distinct disadvantage when trying to enact sustainability policies due to capacity (staff time and skills, local revenue). They often have limited local government service delivery and planning capacity. In many small places the limiting factor in terms of any policymaking, including sustainability programs, is often capacity. Professional management (both in the form of a city/town manager and the specific dedication of staff time to sustainability) increased the adoption of sustainability policies. It can be an explanation of the "better" position of medium sized towns. Furthermore, small towns depend more on citizen or political leadership. The role of local leaders is very important in the decision to apply for membership of these programs. The role of citizens is also important in small towns, and local governments that created an official citizen commission to oversee sustainability actions seem to adopted more policies. The example of Issoire is typical, "with Group 21" dealing with inhabitants and actors dialogues and "walking workshops" aiming in situ observations of local issues. Small towns need structures to provide technical assistance and support (at regional or national levels). For example in Auvergne, an eco-development network of small and medium towns has been created in 2013, with the support of the State for funding and engineering. Another example is the national program for eco-districts. In 2011, on a total of 394 applications submitted, 27% came from small towns (and rather the same was the share of the rural communes), that is from communes in need for funding and support.

Conclusion

Finally, the place of small towns in sustainable development policies is quite paradoxical as few of them are really involved in operational and global programs. We observe a paradox between their images of sustainable places and the fact that most of them do not (and cannot) implement sustainable development policies. Another issue is the importance of collaborative schemes, with neighboring communes or with regional or national partners. The diversity of small towns should be taken into account as the possibilities are different depending on demographic and economic trends (the issue of declining economy and depopulation). Their location from greater urban centers is also a major spatial aspect as small towns integrated in metropolitan areas are facing different problems and have different opportunities than more the remote ones, assuming a role of local centers for rural surroundings.

Table 1 -

 1 Examples of images and word used to describe small towns amenities

	Words and notions used	Number	%
	Total sample	83	100
	Of which : Living (« natural » and « preserved ») environment	20	24,1
	Quality of life	17	20,5
	History, heritage	15	18,1
	Dynamism of local economy	11	13,3

Table 2 -

 2 Perceptions of quality of life by small towns' inhabitants

	A-Evaluation of quality of life	Percentage
	Excellent	49.0
	Good	43.2
	Rather good	4.2
	Bad	1.2
	B-Components of quality of life	Quotations rank
	Place amenities (quietness, living environment...)	1
	Personal aspects (family life, well-being...)	2
	Social aspects (neighbourhood, sense of security ...)

Table 3 -

 3 French communes with Local Agenda 21 and Healthy City membership by size

Size of communes (number of inhabitants) Share of local authorities with Local Agenda 21 by the type of communes Share of Healthy City members by the type of communes Share of urban units in the category in the national total

  Another program was the adoption of Healthy City measures. This program has been launched in 1986 with the Ottawa Charter by the World Health Organization. The objectives are to promote services and programs dedicated at a healthy living environment, taking into account social and spatial factors. Elements of sustainable development are very important in the Ottawa Charter and the local scale is chosen as the best one to develop actions through a "local health plan"[START_REF] Le Goff | Les villes-santé et le développement durable : convergence, concurrence ou écran ?[END_REF]. In France, in 2013, 86 communes are Healthy Cities members, but only 13.5 % of them are small towns (when they represent 89 % of the total urban units). Medium sized towns authorities are more involved in this program 1 . Looking at the sample of Auvergne small towns, it is interesting to identify how and why local actors have been implementing or not sustainable development programs. From 12 small towns, five have already entered operational actions (tab. 4). It is worth to notice that they are part of collaborative plans with surrounding communes. Only six of them have clear mention of sustainable development in the structure of their municipal commissions. But sustainable development is seldom considered as a transversal commission, often associated with sectoral and quite different authorities (like housing, environment, planning). It seems that the definition of sustainable development, which, in theory, is supposed to associate environment, economy and society, is quite blurred.

		%	%	%
	Less than 2,000	0.1	x	x
	2,000 -20,000	1.6	13.5	89,0
	20,000 -100,000	17,0	51.8	9.4
	Over 100,000	57,0	34.7	1.6
	Source: from a national survey (http://encyclopedie-dd.org/encyclopedie/economie/4-3-
		territoires-et-amenagement/les-agendas-21-locaux-et-les.html)
				and http://www.villes-sante.com/

Table 4 -

 4 Place of sustainable development (SD) policies in municipal organization of Auvergne small towns (ranked according to the importance of SD)

	Small Town	SD program	Existence of SD commission in
				the commune structure
	Cournon	Territorial Climate plan	SD and local planning
		(with	Clermont-Ferrand
		metropolitan area)
	Brioude	With the community of	Urbanism, housing and DD
		communes
	Issoire	With the community of	« Living together » and SD
		communes
	Ambert	With the community of	-
		communes
	Saint-Flour	With the community of	-
		communes
	Commentry	A21 planned	Works, urbanism, housing and
				SD
	Saint-Pourçain-sur-Sioule A21 planned	Education, citizenship, ecology,
				SD and environment
	Thiers	-		Water, sanitation, transports, SD
				and works
	Yssingeaux	-		-
	Vic-le-Comte	-		-
	Billom	-		-
	Lapalisse	-		-
				Source: survey, 2012-2013

As comparison, in Poland, with 43 Healthy Cities in

2013, 23 % are small towns, 53.4 the medium ones and 23.6 large cities.