Orhan Agirdag

Mieke Van Houtte

Piet Van Avermaet

Keywords: national identification, sub-national identification, educational segregation, ethnic school composition, school diversity, Flanders national identification, sub-national identification, educational segregation, ethnic school composition, school diversity

published or not. The documents may come

Introduction

A growing concern about ethnic school segregation is evident in many European countries. Such separation is considered not only unfavourable for educational achievement but also an obstruction for the social integration of immigrant and minority pupils (Belgium: [START_REF] Jacobs | De sociale lift blijft steken[END_REF]Van Houtte and Stevens 2009; for England: [START_REF] Burgess | Parallel lives? Ethnic segregation in schools and neighbourhoods[END_REF]France: Felouzis 2005; Germany: Kristen 2005; the Netherlands: Westerbeek 1999; [START_REF] Karsten | Choosing Segregation or Integration? The Extent and Effects of Ethnic Segregation in Dutch Cities[END_REF]and Sweden: Szulkin and Jonsson 2006). For example, in the aftermath of the 2001 riots in the UK, the ever-growing absence of social cohesion was linked to educational and residential ethnic segregation. Policy makers argued that desegregation is a requisite for the cultivation of a sense of citizenship, in particular a stronger national identification, i.e.

Britishness, and sub-national identification, i.e. Englishness [START_REF] Cantle | Community Cohesion: A Report of the Independent Review Team[END_REF]Ouseley 2001;Maxwell 2009). Similar evolutions took place in France. Although the topic of social integration there is commonly linked to the issue of residential segregation (les banlieues), educational segregation is seen as one of the most essential obstacles in attaining a stronger French national identification (Payet 1998;[START_REF] Felouzis | Ethnic segregation and its effects in middle school in France[END_REF]. In other words, policy makers in different countries generally work towards the dispersal of minority/immigrant pupils (we call these groups non-natives), as they believe that the mixing of ethnicities in schools will enhance the academic achievement and national identification, particularly with respect to non-native pupils (Mahieu 2002; [START_REF] Burgess | Parallel lives? Ethnic segregation in schools and neighbourhoods[END_REF].

However, these policies lack a firm evidential foundation, since little empirical research has been conducted into the determinants of national identifications among children and young adolescents (e.g. [START_REF] Carrington | What Makes a Person British -Childrens Conceptions of Their National Culture and Identity[END_REF]Verkuyten 2001;[START_REF] Barrett | Children's views of Britain and Britishness in[END_REF]Reizábal, Valencia and Barrett 2004;[START_REF] Lam | African and Caribbean adolescents in Britain: ethnic identity and Britishness[END_REF]. Moreover, although theorists of identity insist that social identifications are strongly dependent on contextual factors (Oakes, Turner and Haslam 1994), the impact of school attendance in segregated or integrated contexts on the national identifications of native and nonnative pupils is obscure. While research has been primarily conducted into the effect of ethnic school segregation on educational achievement, interethnic friendship, and racial attitudes (for reviews Schofield 1991; [START_REF] Braddock | The effects of school desegregation[END_REF], studies linking school segregation to social identifications have focused mostly on ethnic identity and in-group/out-group evaluations [START_REF] Kinket | Intergroup evaluations and social context: a multilevel approach[END_REF]Umaña-Taylor 2004).

Hence, the relation between de facto school segregation and the national identifications of pupils remains generally unknown, and consequently, the abovestated political beliefs concerning the negative impact of ethnic school context on the development of national identifications are not based on empirical observations.

The main aim of this study is to address this lacuna. More specifically, we investigate the impact of ethnic school context-in terms of ethnic concentration and the ethnic heterogeneity/diversity of the student body-on national identifications of 2,845 pupils in sixty-eight elementary schools in Flanders. Flanders, which is the Dutch-speaking regional state of Belgium, is a unique context, since as an autonomous state it is responsible for almost every aspect of educational policy. Therefore, besides the Belgian national identification, we also investigate the determinants of subnational, that is, Flemish identification of pupils.

National identification

As with gender or religious identifications, national identification can be regarded as a part of the individual's social identity [START_REF] Barrett | Children's views of Britain and Britishness in[END_REF]. Tajfel (1981, p.255) defines -4-social identity as 'that part of an individual's self-concept which derives from his knowledge of his membership of a social group (or groups) together with the value and emotional significance attached to that membership'. Drawing upon this definition, national identification can be considered as the self-identification and emotive meaning that an individual derives from membership in a national group.

In contrast to the large number of studies on ethnic and gender identity, relatively little research has been conducted on the national identity of children and young adolescents. Early studies have examined the time span of the national identification process in children. These studies reveal that children begin to categorize themselves as members of a national group by ages five or six. At this moment, however, children's gender and age identities seem to be more salient than their national identity. Nevertheless, national identity tends to increase in importance until the age of eleven or twelve. By the age of ten, youngsters are able to describe the characteristics of their national group (for reviews see [START_REF] Barrett | Children's views of Britain and Britishness in[END_REF]Reizábal et. al 2004).

More recently, different authors have focused on the impact of national identification on children's in-group favouritism. For example, [START_REF] Barrett | Children's views of Britain and Britishness in[END_REF] reports that youngsters who categorized themselves as 'quite British' or 'very British' were more likely to link British people with positive adjectives, such as 'friendly', 'nice', 'clean'. Studies of children and young adolescents in the Netherlands [START_REF] Kinket | Intergroup evaluations and social context: a multilevel approach[END_REF] and in the Basque country (Reizábal et. al 2004) [START_REF] Carrington | What Makes a Person British -Childrens Conceptions of Their National Culture and Identity[END_REF]. These studies underline the importance of the national context in which national identities are formed and negotiated. For example, Philippou (2005) shows that the importance placed on national identity by Greek-Cypriot pupils was related to the highly politicized context of Cyprus, namely, the presence of a 'national enemy' on the island. In contrast, [START_REF] Howard | It's like we're a normal way and everyone else is different': Australian children's constructions of citizenship and national identity[END_REF] indicate that Australian children were mostly uninterested and unconcerned about being Australian.

National identification and ethnic minorities

When we take ethnic differences into account, this picture becomes more complex.

For years, the one-dimensional assimilation model was the dominant framework. The theories based on it suggested that national identification and ethnic identification are antipodal, implying that the strength of the ethnic identify of non-natives determines the degree of their national identity (Phinney 1990). By contrast, current scholars argue for a bi-dimensional model. They have pointed out that ethnic identification does not necessarily compete with national identification, since people can combine multiple identities (Hutnik 1991;Phinney, Horenczyk, Liebkind and Vedder 2001).

For example, a Turkish immigrant in Flanders may perceive himself as 'very Belgian'(national identification) and simultaneously as 'very Flemish' (sub-national identification) and 'very Turkish' (ethnic identification). This is the notion of the hyphenated or multiple identities (Geartner and Dovido 2000;Verkuyten 2004;Modood 2005). Previous empirical studies on national identifications of immigrants and ethnic minorities have generally shown that these non-native groups identify less with the national category than natives do (for studies of Belgium see Phalet and Swyngedouw 2002;Van Craen, Vancluysen and Ackaert 2007). Research on children and young adolescents reveals similar results [START_REF] Carrington | What Makes a Person British -Childrens Conceptions of Their National Culture and Identity[END_REF][START_REF] Barrett | Children's views of Britain and Britishness in[END_REF][START_REF] Lam | African and Caribbean adolescents in Britain: ethnic identity and Britishness[END_REF]. [START_REF] Barrett | Children's views of Britain and Britishness in[END_REF] indicates that white-English children have a higher level of both 'Englishness' and 'Britishness' than children from ethnic minority groups. A study conducted by [START_REF] Carrington | What Makes a Person British -Childrens Conceptions of Their National Culture and Identity[END_REF] with eight to eleven years-old children demonstrates that more than 90 per cent of native children answered 'British' when responding to the question 'Are you British or something else'. By contrast, only 38 per cent of non-native children said they were 'British', while a fifth claimed a specific ethnic identity, such as 'Chinese' or 'Jamaican'. In line with the idea of 'hyphenated identities', 25 per cent of non-natives described themselves as having both ethnic and national identity.

For the purpose of the present study, it is important to note that various authors have argued that national identification is partly determined by the social context. For instance, Vadher and Barrett (2009) show that the British identity of Indian and Pakistani is different in private and public spheres. These authors suggest that at public level (e.g. at school) the British identity is more dominant than it is at the private level (e.g. at home). In their discussion, [START_REF] Lam | African and Caribbean adolescents in Britain: ethnic identity and Britishness[END_REF] argue that the ethnic school context may be an important determinant of national identifications; but unfortunately, they do not provide empirical evidence to test this hypothesis. Children and young adolescents spend almost half of their waking hours in a school context. Most theories on identity, self-categorisation theory in particular, emphasize that social identities are strongly dependent on context (Tajfel 1981;Oakes et. al 1994), and empirical evidence supports this point [START_REF] Hopkins | The role of the 'other' in national identity: Exploring the context-dependence of the national ingroup stereotype[END_REF]. It is, then, surprising that the relation between the school context and the national identifications of pupils is widely neglected. Therefore, our primary goal in this paper is to examine the manner in which pupils' national (Belgian) and sub-national (Flemish) identifications are influenced by contextual school factors, especially the ethnic composition of the student body, as measured by the proportion of non-native pupils (i.e. ethnic minority concentration), and the ethnic diversity/heterogeneity at school (see Variables section).

Identification and ethnic school composition

Reference group theory

The reference group theory [START_REF] Kelley | Two functions of reference groups[END_REF]Merton 1968;Hyman and Singer 1968) is a suitable starting point to investigate the association between ethnic minority concentration and the national and sub-national identifications of pupils. According to this theory, a group, in a given context, might constitute either a normative reference or a comparative reference group for individuals. When the group functions as the former, it sets and enforces standards for individuals. This means that when the normative reference group tends to exhibit high national identifications, the individual will also be inclined to have a higher national identification. On the other hand, when the group functions as a comparative reference group, it does not function as the norm, but as a benchmark against which individuals compare and judge themselves. In this case, the contrast between the individual and the group is highlighted, which might enlarge the differences between them [START_REF] Kelley | Two functions of reference groups[END_REF]. According to Merton (1968), individuals initially tend to choose their own group as their reference group. However, individuals might also choose groups to which they do not belong (e.g. a non-native pupil considering the native group as the point of reference). Because the relative size of a group in a given context (school) may increase or decrease the probability that it is chosen as a reference group, the proportion of natives or non-natives at school will be related to the likelihood that such groups will be chosen as the point of reference by individuals. As such, when normative reference group processes prevail and the proportion of native pupils at school increases (lower ethnic minority concentration), native pupils are more likely to be the normative reference group for both native and non-native individuals at school. This implies that the standards of natives (i.e. high national identification, see above) will be the norm for non-native individuals and might increase the latter's low national identifications. However, we do not expect that native individuals will be affected by the native reference group, since they already have high national identifications. In sum, the normative reference group theory generates the following hypothesis:

H1: An increasing proportion of native pupils at school (lower ethnic minority concentration) will be related to higher levels of national and sub national identifications for non-native pupils.

However, as stated above, comparative reference group processes might prevail as well. In this case, when the proportion of natives increases (lower ethnic minority concentration), the native groups' high-national identification becomes a benchmark against which non-native individuals will compare themselves, which might further decrease their national identifications. On the other hand, an increasing ethnic minority concentration implies that the low national identifications of the non-native group will be the yardstick for native pupils. This contrast between the low identification of the non-native reference group and the high identifications of native individuals can be expected to further increase the national identifications of native pupils. Hence, drawing upon the comparative reference group framework following hypotheses can be formulated. Note that Hypothesis 2 is in conflict with abovementioned Hypothesis 1.

H2: An increasing proportion of native pupils at school (lower ethnic minority concentration) will be related to lower levels of national and sub national identifications for non-native pupils.

H3: An increasing proportion of non-native pupils at school (higher ethnic minority concentration) will be related to higher levels of national and sub national identifications for native pupils.

Constrict theory

The above stated reference group theory is related to ethnic minority concentration, that is, the proportion of non-native pupils at school. In the literature, minority concentration is often confused with ethnic diversity/heterogeneity. However, these are two distinct concepts and two distinct measurements of ethnic composition (see Putnam 2007;Van Houtte and Stevens 2009). Ethnic diversity or heterogeneity refers to the degree of ethnic difference in a given context. For example, a school in England with Pakistani children only is less diverse than a school in which all the children are from different ethnic groups. To conceptualise the impact of ethnic diversity/heterogeneity at school level, we consider the 'constrict theory' as described in Putnam (2007).

According to the constrict theory the degree of ethnic diversity in a given context triggers social anomie or social isolation. In ethnically diverse environments, both natives and non-natives will be less engaged in collective actions and will have fewer close friends. It should be noted that Putnam states that this tendency applies only to the short run; in the long term, wider benefits of ethnic diversity are evident.

However, in the short-term, ethnic diverse settings can be expected to be associated with a decrease in collectivity: 'People living in ethnically diverse settings appear to "hunker down"-that is, to pull in like a turtle' (Putnam 2007, p.149). The original constrict theory takes neighbourhoods as units of contextual analysis, while we will test this hypothesis in the school context. Similarly, the original constrict theory does not link ethnic diversity/heterogeneity directly to national identifications, but to collectivity. However, we consider pupils' national and sub-national identifications as an utterance of such collectivity. After all, both native and non-natives are expected to share these collective identities. Flanders, for instance, witnesses a growing tendency of addressing the non-natives as 'new-Belgians' or 'new-Flemings', whereas in the past, they were primarily called 'immigrants' or 'allochtones'. In sum, the following hypothesis can be formulated by drawing upon the constrict theory: H4: Increasing ethnic diversity at school level will be associated with a decrease in national and sub-national identification for both native and nonnative pupils.

Interethnic friendship as mediator

The school-effects literature underlines the importance of intermediate relations, that is, of mediator variables that clarify the relationship between structural school features and the dependent variable (for reviews see Teddlie and Reynolds 2000;Van Houtte 2005). Therefore, the impact of ethnic school composition on the identifications of pupils should not be understood as a mechanical one, but rather as a mediating process. With regard to social identification, the Common Ingroup Identity Model (CIIM; Geartner and Dovidio 2000) points at the importance of interethnic contact and friendships. According to the CIIM, when extended contact takes place between individuals from different social groups, these individuals will show a greater propensity to re-categorize their social identities towards those that connect both groups. For non-natives, this implies that increasing contact and friendship with natives might result in increasing national and sub-national identification (see Van Craen et. al 2007;Maxwell 2009). For natives, on the other hand, increasing contact and friendship with non-natives might temper their national and sub national identifications, this, to minimize the social distance between them.

For pupils, these interethnic friendships, in turn, are partly determined by the ethnic composition of the school, since several studies have shown that in schools with higher ethnic heterogeneity, pupils are more engage in interethnic friendships [START_REF] Goldsmith | Schools ' role in shaping race relations: Evidence on friendliness and conflict[END_REF]Van Houtte and Stevens 2009). Hence, a mediational model can be formulated, which states that given the expected association between ethnic school composition and interethnic friendship and the relationship between interethnic friendship and national identification, the following hypothesis emerges:

H5:

The impact of the ethnic school composition on pupils' national and subnational identifications (H1, H2, H3, and H4) will be mediated by the amount of their interethnic friendships.

Methods

Sample

We used data gathered in 2008-2009 from 2,845 pupils (mean age 11.61) in a sample of sixty-eight primary schools in Flanders as part of the Segregation in Primary Education in Flanders (SIPEF) project. Multistage sampling was conducted. In the first instance, in order to encompass the entire range of ethnic composition, we selected three cities in Flanders with ethnically diverse populations. Second, using data gathered from the Flemish Educational Department, 116 primary schools within these selected cities were asked to participate; this yielded a positive response of 54 per cent. This relatively high non-response of schools is because Flemish schools are commonly swamped with such requests from investigators, generally resulting in a 'first come, first served' outcome. As such, the participating schools did not differ from those that opted out in terms of school sector (i.e. private and public schools) or ethnic composition (as measured by the proportion of non-native pupils). The schools in this dataset encompass the entire range of ethnic composition, from schools having almost no non-native pupils to pure-minority-concentrated schools having 100 per cent non-native pupils. Within these schools, our research team asked all fifth-grade pupils present at school during our visit to fill out a written questionnaire. If the number of fifth-grade pupils was less than thirty, we surveyed all the sixth-grade pupils as well.

The pupils completed the questionnaires in class in the presence of one or two

Research design

Given that we are dealing with a clustered sample of pupils nested within schools and with data at different levels, namely, variables of ethnic school composition as the main determinants at the school level and national and sub-national identifications as outcome at pupil level, the use of hierarchical linear modelling is most appropriate (HLM6, Raudenbush and Bryk 2002). As is common in multilevel analyses, we start by estimating unconditional models to determine the amount of variance that occurs among schools regarding national and sub-national identifications. Then, we add the main determinants to the model, controlling systematically for school features (size and sector) and pupil features [gender, grade, socioeconomic status (SES)]. In the second model, we add ethnic heterogeneity/diversity at school level, and in the third model interethnic friendship at pupil level. HLM only provides unstandardized gamma coefficients; to evaluate the strength of the associations, we standardize these coefficients. We carry out each analysis separately for native and non-native pupils, since the association between ethnic school composition and national/sub-national identification is hypothesized to be different for both groups.

Variables

Outcomes

To asses national (Belgian) and sub-national (Flemish) identification, we used a scale based on five items from the Collective Self-Esteem Scale twice [START_REF] Luhtanen | A Collective Self-Esteem Scale -Self-Evaluation of Ones Social Identity[END_REF]. Similar items were successfully used with same aged children in previous studies in the Netherlands (Verkuyten 2001). The items are as follows: 'I consider myself as a Belgian/Fleming'; 'I often regret that I am Belgian/Flemish' (reverse scored); 'I am glad to be a Belgian/Fleming'; I often feel that Belgium/Flanders is worthless' (reverse scored); 'I feel good about Belgium/Flanders'. There are five answer categories, ranging from absolutely do not agree (1) to completely agree (5). Responses to these five items were averaged. An exploratory factor analysis revealed that there was one underlying identity dimension for both scales. For the Belgian identification scale, the lowest loading is .62, with an Eigenvalue of 2.76 (explaining 55.26 per cent of variance) and Cronbach's alpha of .80. For the Flemish identification scale, the lowest loading is .66, with an Eigenvalue of 2.95 (explaining 58.52 per cent of variance) and Cronbach's alpha of .82. Table 1 demonstrates that, on average, native pupils have a significantly stronger Belgian identification (M=4.38, SD=0.69) and Flemish identification (M=4.22, SD=0.83) than non-native pupils, respectively (M=3.18, SD=0.89, t=37.99, p<0.001) and (M=3.13, SD=0.92; t=31.10, p<0.001).

School-level variables

As stated above, the school ethnic composition is measured by two indicators. First, we consider ethnic minority concentration, that is, the proportion of non-native respondents in a school in our database. On average, the proportion of non-native pupils is 51.50 per cent (SD = 34.16; Table 1). The minority concentration ranges from 2.63 per cent to 100 per cent non-native pupils.

The second indicator of school's ethnic composition measures the ethnic diversity/heterogeneity within a school, expressed as the total number of different groups of non-natives, corrected by their size. We use an index of ethnic diversity, which is the Herfindahl index as used by Putnam (2007) multiplied by -1, since Putnam in fact calculated an index of homogeneity, whereas we are interested in heterogeneity. The used index is calculated as (p ethnic group 1 )² + (p ethnic group 2 )² + … + (p ethnic group n )². We included the eleven ethnic groups listed above. The index has a range of -1 till 0 and a value of -1 implies no diversity at all, that is, only one ethnic group is enrolled in the school. A value that approaches zero means total diversity: all pupils in school have a different ethnic origin. In our data, on average, schools have a value of -0.46 (SD = 0.20).

We determined the school size from the total number of pupils, using data gathered from the Flemish Educational Department. The number of pupils varies from ninety-one in the smallest school to 526 in the largest. The schools catered for an average of 223 pupils (SD=104.03; Table 1). Finally, the variable school sector distinguishes between thirty-three public schools (municipal schools and state schools; score 0) and thirty-five private schools (score 1). It should be noted that in the Flemish educational system, no distinction is made between public schools and private schools with respect to state support.

Individual-level independent variables

Four individual-level variables were included in the multilevel analysis. Our research concentrated on the fifth-and sixth-grade pupils. Therefore, most of the respondents were age eleven (about 49 per cent) or twelve (about 36 per cent) in 2009. It should be noted that psychological research points out that this period is decisive for developing a national identity [START_REF] Barrett | The development of national ingroup bias: English children's attributions of characteristics to English, American and German people[END_REF]. Given the high multicollinearity between age and grade (Cramer's V=0.64; p<0.000), we had to choose between one of these two variables to enter the model. We opted for grade because the sample was unbalanced for grade.

The sample was equally divided with respect to gender, with 51.2 per cent of the respondents being female (male=0, female=1). We measured the socioeconomic status (SES) of origin of the pupils by means of the occupational prestige of the father and mother [START_REF] Erikson | Intergenerational Class Mobility in 3 Western European Societies -England, France and Sweden[END_REF]; the higher of the two is used as an indicator of the SES of the family. The respondents have a mean SES of 4.44 (SD=2.22). On average, the nonnative pupils have a significantly lower SES (M=3.24, SD=1.92) than the native pupils (mean=5.44, SD=1.94; Table 1).

Finally, to measure pupils' interethnic friendship we asked native respondents to state how many of their friends at school had a non-Belgian origin, and we asked non-native pupils to state how many of their friends at school had a Belgian origin.

There were five possible answers: nobody (score 1), a few (score 2), half of them (score 3), most of them (score 4), and all of them (score 5). As can be seen from Table 1, on average native pupils have lower scores on interethnic friendship (M=1.95, SD=0.84) than non-native pupils do (M=2.66, SD=1.17, t=17.37, p<0.001).

[TABLE 1 ABOUT HERE]

Results

Unconditional Model

Does the school context matter with respect to pupils' identifications? To provide an answer to this question, Table 2 presents the variance components from the unconditional models. We are particularly interested in the explained variance at school-level, computed as the between-school variance component divided by the sum of within-school and between-school variance (τ 0 / σ² + τ 0 ). Consistent with other 2 indicates that significant amounts of the variances in pupils' identifications are explained at school level (p<0.001). It is also clear that the schoolcontext is more prevalent for non-native pupils than it is for native pupils' identifications: for non-natives around 12 per cent of the variance in national identification and 11 per cent variance in sub-national identification are among schools, whereas the figure for natives is respectively around 3 per cent and 6 per cent (Table 2).

[TABLE 2 ABOUT HERE]

National identification

With respect to national (Belgian) identifications of non-native pupils, Model 1b (Table 3) indicates that non-native pupils attending schools with a higher proportion of non-native pupils, tend to exhibit lower levels of national identity (γ = -3.20; p<0.001), controlling for school characteristics (size and sector) and pupil-level control variables (grade, gender and SES). The addition of ethnic heterogeneity in the Model 2b does not change this picture, and ethnic heterogeneity is initially not significantly associated with Belgian identification of non-native pupils (γ = -0.066; p = 0.158; Table 3). However, as we consider interethnic friendship in Model 3b, the relation between both the ethnic composition variables and national identification changes considerably. Firstly, entering interethnic friendship reduces the effect of minority concentration, which is no longer significant. Interethnic friendship, in turn, Secondly, ethnic diversity/heterogeneity becomes negatively related to national identifications of non-native pupils (Model 3b, Table 3). These findings are in line with the hypotheses which we derived from normative reference group theory (H1), constrict theory (H4) and Common Ingroup Identity Model (CIIM, H5) and disconfirm the hypothesis drawn from comparative reference group theory (H2): for non-native pupils ethnic minority concentration has a negative effect on their national identifications, which can be explained by the number of their interethnic friendships.

The negative impact of ethnic diversity/heterogeneity is suppressed by interethnic friendship.

Considering the effects on native pupils, ethnic school composition variables are initially not significantly associated with their national identifications (Model 1a-2a, Table 3). However, in Model 3a, it becomes clear that there is a suppression effect caused by interethnic friendship. After the addition of interethnic friendship, ethnic minority concentration became significantly positively related to the national identifications of native pupils, while interethnic friendship is negatively related to their national identifications (Model 3a, Table 3). This means that the increasing presence of non-native pupils at school positively impacts this group's national identification, while interethnic friendship tempers their national identification.

Secondly, when interethnic friendship is hold constant, ethnic diversity/heterogeneity is negatively related to native pupils' Belgian identification, even though this effect is borderline (γ = -0.119; p<0.065). These findings are in line with the hypotheses, which we derived from comparative reference group theory (H3), constrict theory (H4), and CIIM (H5). Finally, while the individual level influences are not the primary concern of this article, it is worth mentioning that SES is only positively related to native pupils' national identifications (a stronger Belgian identification for pupils with higher SES) and that non-native girls exhibit higher level of Belgian identity than non-native boys.

Grade is not associated to Belgian identification (Table 3). When we enter age instead of grade (not shown here), there was no substantial difference with respect to the results.

[TABLE 3 ABOUT HERE]

Sub-national identification

Considering sub-national (Flemish) identifications of non-native pupils, the results are similar to those of their Belgian identifications (Table 4; Model 1b-3b): the strong negative impact of minority concentration in the first two models disappears in the third model, after controlling for interethnic friendship, while a relatively small, but significant negative effect of ethnic heterogeneity appears after the addition of interethnic friendship (γ = -0.080; p<0.05). This means that non-native pupils tend to exhibit higher levels of both national and sub-national identity in schools with a lower proportion of non-native pupils, and this finding can be explained by the increasing opportunities of interethnic friendship in such schools. In addition, in schools with a higher ethnic diversity/heterogeneity, non-native pupils have lower Flemish identifications, when their interethnic friendships are held constant. Analogous to the effects on Belgian identification, non-native pupils tend to have higher Flemish identification when they have more native friends, and non-native girls are more likely to have higher sub-national identifications than non-native boys (Model 1b-3b; Table 4). Again, these findings are in line with H1, H4 and H5, while disconfirming H2.

With respect to native pupils' sub-national identifications, however, Table 4 indicates that there is no school-level variable significantly associated with native pupils' sub-national identifications. Hence, with respect to Flemish identification of native pupils, we could not find support for any of our hypotheses. The individual level effects were similar to those on Belgian identification: a higher SES is positively related to native pupils' Flemish identifications, and increasing inter-ethnic friendships is associated with decreasing Flemish identification. (Table 4; Model 1a-3a)

[TABLE 4 ABOUT HERE]

Discussion and conclusion

In different European countries, policy makers strive for the dispersal of non-native pupils, believing that the mixing of pupils of different ethnic groups will enhance the social integration and national identifications of youngsters. Given the lack of research supporting such policy practices and because theorists of identity emphasize the importance of contexts, the aim of this study was to explore the association between ethnic school composition and pupils' national and sub-national (Belgian and Flemish) identifications.

The results of this inquiry indeed indicate that the ethnic school composition is significantly related to national identifications and partly related to sub-national identifications of native and non-native pupils. Firstly, ethnic minority concentration, as measured by the proportion of non-native pupils at school, has a negative impact on non-native pupils' Belgian and Flemish identifications, while it has a positive impact on native pupils' Belgian identifications. In other words, normative and comparative reference group processes prevail respectively for non-natives and for natives. An explanation of this differential effect could be derived from the general reference group theory, as it insists that normative reference processes are more likely to occur when individuals are striving for acceptance by the reference group (Merton 1968).

Given the socially dominant position of natives within Flemish society, we assume that non-natives are striving to obtain recognition from the native group. This might explain why normative reference processes prevailed for non-natives. Furthermore, this study lends support to the prediction that we derived from the constrict theory: as ethnic heterogeneity/diversity rises at school level, the national identifications of native and non-native pupils' decrease.

Four remarks should be added to what has been stated above. First, the relation between ethnic school composition variables and pupils' national and sub-national identifications is not a mechanical one, but as expected, it is mediated by interethnic friendships. In general, interethnic friendship has a positive impact on non-native pupils' national and sub-national identifications, while it tempers the identifications of native pupils. Secondly, compared to the impact of ethnic minority concentration, the size of the effect of ethnic diversity/heterogeneity is much smaller. Thirdly, ethnic diversity/heterogeneity only decreases pupils' identifications when interethnic friendship is held constant. In other words, the negative impact of ethnic diversity/heterogeneity is suppressed by interethnic friendship. This means that ethnic diversity/heterogeneity as such does not affect Flemish and Belgian identifications; rather it impacts them only if pupils do not engage in interethnic friendship relations. Fourth, we did not find a significant association between any school-level variable and native pupils' Flemish identifications. This could be explained by the political situation in Flanders where Flemish identity is being used by far right-wing and racist politics. Therefore, native pupils' Flemish identifications are potentially expressed with a certain restraint to avert an association with racism. However, this post-hoc explanation should be examined in further research.

While the individual level influences were not the primary concern of this article, an interesting finding is that non-native girls have higher Belgian and Flemish identifications than non-native boys. Maxwell ( 2009) argues that higher level of perceived discrimination by the broader society contributes to lower levels of national identification and research in Belgium shows that non-native men report more perceived discrimination than non-native women (De Rycke and Swyngedouw 1997).

In other words, while we do not have a conclusive explanation for the gender gap with respect to national and sub-national identifications, we can hypothesize that perceived discrimination might explain this gap.

It is important to keep in mind a few limitations of this study. Firstly, because the focus of the research project is on the impact of school ethnic composition, we only considered the influence of school context variables. However, other contexts (e.g. neighbourhood, country) might also contribute to youth's identifications and the position of the school on the school market might play a role as well (see Verhoeven, 2002). Secondly, this investigation employs a limited measure of interethnic friendship. We asked all pupils to state how many of their friends were natives and how were many non-natives. A more elaborated technique would be to provide pupils a list with the names of all pupils at school and to ask to indicate their best friends and friends. However, such a technique was unsuitable not only because the survey would Thirdly, we made a raw distinction between native and non-native pupils. This distinction neglects the ethnic differences within the non-native group. However, a separate analysis for each ethnic minority group would have harmed the reliability of our analysis, because the individual groups in our data are too small. While considering non-natives as one category is consistent with most of the previous studies conducted in Belgium (e.g., Jacobs et al., 2009;[START_REF] Agirdag | Exploring bilingualism in a monolingual school system: insights from Turkish and native students from Belgian schools[END_REF], future research should, if possible, make a distinction within the non-native groups.

In terms of educational policy, this article finds considerable support for the concerns about the negative impact of ethnic school segregation, but most importantly, it does also challenges these policies. At the one hand, our findings indicate that schools with a high minority concentration and high ethnic heterogeneity might indeed have a negative impact on non-native pupils' national and sub-national identifications.

However, we demonstrate that these effects are mediated by their interethnic friendships. While the school is an important context in which pupils make friends, opportunities outside the school for interethnic friendships should not be neglected.

Hence, besides school desegregation, policy makers should consider stimulating interethnic contact and friendship opportunities, both inside and outside the school. At this point, we should also notice that cultivating national identification might not be considered as a means of assimilation. As noted in our introduction section, national identities do not necessarily compete with ethnic identities and most people have multiple or hyphenated identities, which are very important with respect to pupils' well-being and educational achievement (see Phinney et al. 2001;Modood 2005). In this study, however, it was not feasible to measure all different ethnic identities, due to the large scale design of the research. However, we strongly recommend further

  reveal similar results. Qualitatively oriented research has focused on the negotiation of national identity by children and young adolescents, concentrating on the meaning derived by belonging' to a nation-state and the construction of their national identifications through the education system (for Australia: Howard and Gill 2001; for Greek-Cyprus: Philippou 2005; for Ireland: Tormey 2006; for Scotland Carrington

  researchers and a teacher. The questionnaires were anonymous and were analyzed in complete confidentiality.

  Since all of the analyses are performed separately for native and immigrant pupils, it is important to explain how we have made the distinction between them. The principal criterion was the birthplace of the pupils' grandmothers. If these data were missing, we considered their mothers and fathers' birthplaces, as most non-native pupils in Flanders are second-or third-generation immigrants. We consider eleven broad ethnic groups: (1) Native Belgians (46.7 per cent), (2) West-Europeans including pupils of Dutch, French, or German origin (5.6 per cent), (3) South-Europeans, including pupils of Italian or Spanish origin (6.6 per cent), (4) Turks (13.0 per cent), (5) Moroccans (15.6 per cent), (6) Other North-Africans (1.0 per cent), (7) East-Europeans (5.8 per cent), (8) Sub-Sahara Africans (1.8 per cent), (9) Middle-Easterners (1.2 per cent), (10) South-East Asians (1.7 per cent), and (11) others (1.1 per cent). As is common practice, and in line with the official Flemish definition of non-native groups, only West European origins (groups 1 and 2) were considered as producing native descent (seeVan Houtte and Stevens 2009). As such, we created a dichotomous variable (0=native, 1=non-native).

  research (Teddlie and Reynolds 2000), most of the variation occurs within schools, between pupils. Nevertheless, justifying the need for a multilevel analysis, Table

  to non-native pupils' Belgian identifications (γ = 0.298; p<0.001).

  the intersections between the school context and pupils' ethnic and national identities and examine the mediating role of interethnic relationships. 2002 'Desegregatie in functie van integratie [Desegregation in function of integration]', in TIMMERMAN, C., HERMANS, P. & HOORNAERT, J. (eds) Allochtone jongeren in het onderwijs. Een multidisciplinair perspectief [Non-native youth in education. A multidisciplinary perspective]. Leuven: Garant. MAXWELL, RAHSAAN 2009 'Caribbean and South Asian identification with British society: the importance of perceived discrimination', Ethnic and Racial Studies, vol. 32, no. 8, pp. 1449 -1469. MERTON, ROBERT KING 1968 'Continuities in the theory of reference groups and social structure', in MERTON, R. K. (ed) Social theory and social structure. Glencoe, IL: Free Press. MODOOD, TARIQ 2005 Multicultural politics : racism, ethnicity, and Muslims in Britain. Minneapolis: University of Minnesota Press. OAKES, PENELOPE J, HASLAM, S. ALEXANDER and TURNER, JOHN C 1994 Stereotyping and social reality. Oxford: Blackwell. OUSELEY, HERMAN 2001 'Community Pride Not Prejudice: Making Diversity Work in Bradford'. Bradford: Bradford Vision. PAYET, JEAN-PAUL 1998 'La ségrégation scolaire: Une perspective sociologique sur la violence à l'école', Revue française de pédagogie, no. 123, pp. 21-34. PHALET, KAREN and SWYNGEDOUW, MARC 2002 'National identities and representations of citizenship: A comparison of Turks, Moroccans and working-class Belgians in Brussels', Ethnicities, vol. 2, no. 1,

F

Page 32 of 36 URL: http://mc.manuscriptcentral.com/rers ethnic@surrey.ac.uk Ethnic and Racial Studies F o r P e e r R e v i e w O n l y

  PHINNEY, JEAN S. 1990 'Ethnic-Identity in Adolescents and Adults -Review ofResearch', Psychological Bulletin, vol. 108, no. 3, pp. 499-514. PHINNEY, JEAN S., HORENCZYK, GABRIEL, LIEBKIND, KARMELA and VEDDER, PAUL 2001 'Ethnic Identity, Immigration, and Well-Being: An Interactional Perspective', Journal of Social Issues, vol. 57, pp. 493-510. PUTNAM, ROBERT D. 2007 'E Pluribus Unum: Diversity and Community in the Twenty-first Century The 2006 Johan Skytte Prize Lecture', Scandinavian Political Studies, vol. 30, no. 2, pp. 137-174. RAUDENBUSH, STEPHEN W. and BRYK, ANTHONY S. 2002 Hierarchical linear models : applications and data analysis methods, 2nd ed. Thousand Oaks: WESTERBEEK, KARIN 1999 'The colours of my classroom: a study into the effects of the ethnic composition of classrooms on the achievement of pupils from different ethnic backgrounds', PhD, European University Institute, Florence. ORHAN AGIRDAG is Researcher in Research Group CuDOS, Department of Sociology at Ghent University. ADDRESS: Korte Meer 5, 9000 Gent, Belgium. Email: orhan.agirdag@ugent.be MIEKE VAN HOUTTE is Lecturer in Research Group CuDOS, Department of Sociology at Ghent University. ADDRESS: Korte Meer 5, 9000 Gent, Belgium. Email: mieke.vanhoutte@ugent.be Table1. Descriptive Statistics for the Dependent and Independent Variables: Frequencies , Means, Standard Deviations and Results of t-test comparing native and non-native pupils.

		Total		Natives	Non-natives
	Variables	Mean	SD	Mean	SD	Mean	SD Mean difference
	School Level
	% School sector	51.47	50.35	53.23	50.30	51.47	50.35
	F o r F o r (private) School size F % Non-Native o r Ethnic diversity	(N=68) 222.91 (N=68) 51.50 (N=68) (N=68) -0.46	104.03 34.16 0.20	(N=62) 226.27 (N=62) 46.80 (N=62) (N=62) -0.44	107.68 32.06 0.18	(N=68) 222.91 (N=68) 51.50 (N=68) (N=68) -0.46	104.03 34.16 0.20
	P e e r Sage Publications. REIZABAL, LUIXA, VALENCIA, JOSE and BARRETT, MARTYN 2004 'National P e e r Pupil Level P % Grade (sixth) 29.98 (N=2845) 45.82 26.83 (N=1487) 44.32 (N=1358) 33.43 e % Gender (female) 50.53 (N=2827) 49.98 52.12 (N=1479) 49.97 50.89 (N=1348) e SES 4.44 2.22 5.44 1.94 3.24 r (N=2676) (N=1464) (N=1212)	47.19 50.01 1.92	2.20 t=29.40***
	R e v i e w identifications and attitudes to national ingroups and outgroups amongst children living in the Basque Country', Infant and Child Development, vol. 13, no. 1, pp. 1-20. A Review of the Literature', Review of Research in Education,, vol. 17, pp. w w SCHOFIELD, JENET WARD 1991 'School Desegregation and Intergroup Relations: R e v i e Inter-ethnic friendship 2.28 (N=2561) 1.07 1.95 (N=1371) 0.84 (N=1190) 2.66 National 3.82 0.98 4.38 0.69 3.18 R PIET VAN AVERMAET is Director of Centre for Diversity and Learning at Ghent identification (N=2621) (N=1409) (N=1212) e piet.vanavermaet@ugent.be *p≤0.05, **p≤0.01, ***p≤0.001 e i ADDRESS: Sint-Pietersnieuwstraat 49, 9000 Gent, Belgium. Email: v Sub-national 3.72 1.03 4.22 0.83 3.13 University. identification (N=2522) (N=1370) (N=1152)	1.17 0.89 0.92	0.71 t=17.37*** 1.20 t=37.99*** 1.09 t=31.10***
	O n l y SZULKIN, RYSZARD and JONSSON, O. JAN 2006 'Ethnic segregation and 335-409. y y educational outcomes in Swedish comprehensive schools: a multilevel O n l l n O -33-
	analysis.', NEP: New Economic Papers.
	TAJFEL, HENRI 1981 Human groups and social categories. Cambridge: Cambridge
	PHILIPPOU, STAVROULA 2005 'Constructing national and European identities: the University Press
	case of Greek-Cypriot pupils', Educational Studies, vol. 31, no. 3, pp. 293-TEDDLIE, CHARLES and REYNOLDS, DAVID 2000 The international handbook
	315. of school effectiveness research. London: Falmer Press.
			-30--32-

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Table 2 .

 2 Variance components for national (Belgian) and sub-national (Flemish) identification from the unconditional model.

			Ethnic and Racial Studies		Page 34 of 36
			Natives		Non-natives
	Belgian identification	Flemish identification	Belgian identification	Flemish identification
	Within-school variance τ0	0.458	0.660	0.712	0.769
	Between-school variance σ²	0.013	0.039	0.095	0.092
	% Variance between school	2.76%***	5.53%***	11.82%***	10.68%***
	*p≤0.05, **p≤0.01, ***p≤0.001

URL: http://mc.manuscriptcentral.com/rers ethnic@surrey.ac.uk

Table 3

 3 Influences on national (Belgian) identification: results of the multilevel analysis, standardized gamma coefficients with the standard errors in parentheses

	Natives	Non-natives

Table 4 .

 4 Influences on sub-national (Flemish) identification: results of the multilevel analysis, standardized gamma coefficients with the standard errors in parentheses

	Natives	Non-natives

-24-have needed a higher time investment but also because it was completely anonymous.

Acknowledgements

This article was made possible through funding from the the Research Foundation Flanders (FWO-project G.040908). The authors would like to thank two anonymous referees for their constructive comments. Special thanks to Paul Mahieu, Loes Vandenbroucke and Simon Boone for their helpful comments.

school history: the Irish case', British Journal of Sociology of Education, vol. 27, no. 3, UMAÑA-TAYLOR, ADRIANA J. 2004 'Ethnic identity and self-esteem: examining the role of social context', Journal of Adolescence, vol. 27, no. 2, pp. 139-146. VADHER, KIREN and BARRETT, MARTYN 2009 'Boundaries of Britishness in British Indian and Pakistani young adults', Journal of Community & Applied Social Psychology, vol. 19, no. 6,. VAN CRAEN, MAARTEN , VANCLUYSEN, KRIS and ACKAERT, JOHAN 2007 Voorbij wij en zij? De sociaal-culturele afstand tussen autochtonen en allochtonen tegen de meetlat [Beyond us and them? Social-cultural distance between natives and immigrants measured]. Brugge: Vandenbroele. VAN HOUTTE, MIEKE 2005 'Climate or culture? A plea for conceptual clarity in school effectiveness research', School Effectiveness and School Improvement, vol. 16, no. 1, pp. 71-89. VAN HOUTTE, MIEKE and STEVENS, PETER A. J. 2009 'School Ethnic Composition and Students' Integration Outside and Inside Schools in Belgium', Sociology of Education, vol. 82, pp. 217-239. VERHOEVEN, MARIE 2002 École et diversité culturelle : regards croisés sur l'expérience scolaire de jeunes issus de l'immigration. Louvain-la-Neuve: Academia-Bruylant. VERKUYTEN, MAYKEL 2001 'National identification and intergroup evaluations in Dutch children', British Journal of Developmental Psychology, vol. 19, pp. 559-571. ---2004 The social psychology of ethnic identity. London: Psychology Press. Page 31 of 36 URL: http://mc.manuscriptcentral.com/rers ethnic@surrey.ac.uk Ethnic and Racial Studies


I Michiels

G Desombere

P Leroux-Roels

F Lemey

M Nevens

Ranst

Jannick Verbeeck

Leen Kwanten

François D'heygere

Arne Beguin

Isabelle Desombere

Geert Leroux-Roels

Philippe Lemey

Frederik Nevens

AND Marc Van Ranst
email: vanranstmarc@gmail.com

HCV genotype distribution in Flanders and Brussels (Belgium)

Keywords: Belgium, epidemiology, HCV genotype 5a, HCV genotype distribution, hepatitis 1 C virus, phylogeny

HCV genotype distribution in Flanders and Brussels (Belgium): unravelling the spread of an uncommon

HCV genotype 5a cluster

genotype 3 in East-Flanders, Antwerp, Flemish-Brabant and Limburg. In Brussels, 13 HCV genotype 4 was the second most prevalent genotype. This observation is due to the 14 immigration of patients from the Middle East and Africa. Remarkably, a cluster of 15 HCV genotype 5a was found in West-Flanders, where it represents the second most 16 prevalent genotype accounting for 26.2% of HCV infections. We could not identify one 17 major transmission source explaining the whole HCV genotype 5a epidemic. Instead, 18 several smaller possible transmission chains were identified and confirmed 19 phylogenetically. 20

Conclusion: 21

Overall, the HCV genotype 5a epidemic in West-Flanders seems to be mainly associated 22 with blood transfusion and unsafe medical practices.

INTRODUCTION 1

The hepatitis C virus (HCV) is a leading cause of chronic liver disease and confronts many 2 regions of the world with a severe disease burden. Worldwide, more than 170 million people 3 are infected with this virus [1]. Since its discovery in 1989, there is a growing interest in 4 different aspects of HCV research, including HCV epidemiology, evolution, host interactions 5 and antiviral defence mechanisms. Despite considerable research efforts, several aspects are 6 still poorly understood. [START_REF] Van Damme | Epidemiology of hepatitis C in 21 Belgium: present and future[END_REF] Phylogenetic analyses have revealed that HCV can be classified in 6 different genotypes and 8 multiple subtypes [START_REF] Simmonds | 7 Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes[END_REF]. These six genotypes have different geographical distributions [START_REF] Cristina | Genetic diversity and evolution of hepatitis C virus in the Latin 10 American region[END_REF]. HCV 9 genotypes 1a, 1b and 3a are highly prevalent "epidemic" strains that are found globally. These 10 strains spread swiftly around the world during the twentieth century, most likely through 11 infected blood, blood products and injecting drug use, and have relatively low levels of 12 genetic variation. In contrast, other HCV strains are highly divergent but are found in 13 restricted geographic areas. These "endemic" strains reflect long-term transmission at low 14 levels in particular regions. Genotypes 1, 2 and 4 appear to be endemic to regions of West and 15

Central Africa and the Middle East, whereas divergent endemic strains of genotypes 3 and 6 16 strains are found in South-East Asia [START_REF] Pawlotsky | Hepatitis C virus genetic variability: pathogenic and clinical 12 implications[END_REF]. HCV genotype 5a infections are mainly found in the 17 northern part of South Africa, where 40% of the HCV patients carry this genotype [START_REF] Chamberlain | The 14 complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in 15 South Africa[END_REF]. In 18 other regions of the world, HCV genotype 5a infections are scarce, and therefore, little is 19 known about the geographical distribution, evolution and treatment response of this 20 uncommon genotype. [START_REF] Ray | Genetic epidemiology of 15 hepatitis C virus throughout egypt[END_REF] In Belgium, HCV prevalence has been estimated at about 0.8%, representing about 80.000 22 patients [START_REF] Beutels | Prevalence 18 of hepatitis A, B and C in the Flemish population[END_REF][START_REF] Van Damme | Epidemiology of hepatitis C in 21 Belgium: present and future[END_REF]. A study of the epidemiological profile of HCV patients in Belgium conducted 23 between 1992 and 2002 showed that HCV genotype 1 is the most prevalent genotype, 24 followed by HCV genotype 3. HCV genotype 2 and HCV genotype 4 are roughly equally 25 distributed, and HCV genotypes 5 and 6 are only encountered sporadically [START_REF] Gerard | Evolution over a 10 year period of the epidemiological profile of 1,726 24 newly diagnosed HCV patients in Belgium[END_REF]. In 2006 1 however, a remarkable cluster of at least 100 HCV genotype 5a infected patients was 2 identified in the West-Flanders province of Belgium [START_REF] Verbeeck | Investigating the origin and spread of 27 hepatitis C virus genotype 5a[END_REF]. 3

In this paper, we describe the general epidemiological profile of HCV genotypes in Flanders, 4

Belgium for the period between 2001 and 2009. Next, we focus on the uncommon HCV 5 genotype 5a cluster in West-Flanders. In order to find out how HCV genotype 5a was 6 introduced in the population and to study its local spread, we conducted face-to-face 7 interviews with the infected patients using a detailed questionnaire to learn more about the 8 medical history and the HCV infection of each individual patient. We performed phylogenetic 9 analyses to confirm the links between the patients that were found during thorough 10 investigation of the questionnaires. 11

MATERIALS AND METHODS 1

Study population 2

Between January 2001 and August 2009, blood samples of 2301 new patients with chronic 3 hepatitis C were received for HCV genotyping at the University Hospital of Leuven. Only 4 HCV carriers presenting for the first time were included in this study. The study population 5 consists of blood samples from patients attending the medical consultation at the hospital and 6 blood samples from outpatients from other hospitals in Flanders that do not have their own 7 HCV genotyping facilities. Patients with anti-HCV antibodies who are HCV RNA negative 8 are not included. The selected study population is a representative sample of the total HCV 9 population in Flanders since patients from all provinces are included. 10

Genotyping was performed using the INNO LiPA 1.0 or the INNO LiPA 2.0 (developed by 11 Innogenetics Inc., Ghent, Belgium and distributed by Siemens Healthcare Diagnostics, IL, 12 USA) according to the manufacturer's instructions. Demographic and geographic information 13 (age, gender, city of residence) were collected for all patients. 14 Patients infected with HCV genotype 5a were invited to participate in our study. In total, 67 15 patients were willing to participate. Informed consent was obtained from all patients. Face-to-16 face interviews based on a detailed questionnaire were conducted for all 67 patients in order 17 to explore possible transmission routes. The questionnaire investigated HCV diagnosis, HCV 18 treatment outcome, HCV family history, hospitalisation history, general medical care, blood 19 transfusion history, contact with blood products, blood donations, military history, tattooing, 20 piercing, acupuncture, IDU, visits to general doctors, visits to dentists, travelling to foreign 21 countries, and possible links with South Africa. For the female patients, additional questions 22 concerning pregnancy and childbirth were included. The study was approved by the ethical 23 committee of the University Hospital Leuven.

Phylogenetic analysis 1

In order to reconstruct the phylogenetic relationships between the HCV genotype 5a patients, 2 98 serum samples were collected from 61 different patients. Viral RNA was isolated from 140 3 µl serum using the QIAamp Viral RNA Mini kit (Qiagen Benelux B.V., Venlo, the 4 Netherlands) according to the manufacturer's instructions, and eluted in 60 µl elution buffer. 5

One-step reverse transcription PCRs were carried out using the OneStep RT-PCR kit (Qiagen) 6 to amplify a 584 bp fragment from the highly variable E1-E2 region (genome location: nt 554 7 to nt 1138 according to the numbering of the H77 strain) and a 573 bp fragment from the 8 more conserved NS3-NS4 region (genome location: nt 5058 to nt 5631 according to the 9 numbering of the H77 strain) as described previously [START_REF] Verbeeck | Investigating the origin and spread of 27 hepatitis C virus genotype 5a[END_REF]. Purified DNA fragments were 10 directly sequenced on both strands using the ABI Prism BigDye Terminator Cycle 11 Sequencing Reaction Kit (Applied Biosystems). 12 Multiple nucleotide sequence alignments were created for the E1-E2 region, NS3-NS4 region, 13 and the concatenated E1-NS4 region using the Clustal X program version 1.83 [START_REF] Higgins | CLUSTAL: a package for performing multiple 29 sequence alignment on a microcomputer[END_REF]. 14 Alignments were adjusted by hand. For the E1-E2 region, the hypervariable region was 15 excluded from the analysis, since it is impossible to unambiguously align this region. [START_REF] Bourliere | Epidemiological changes in hepatitis C virus genotypes in France: evidence in 48 intravenous drug users[END_REF] Molecular phylogenies were estimated for each alignment using a maximum-likelihood (ML) 17 approach implemented in PAUP* v4.0b [11]. Model selection for each sequence alignment 18 was performed with the Modeltest 3.7 software [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF] under the Aikake Information Criterion. [START_REF] Bochud | Genotype 3 is 10 associated with accelerated fibrosis progression in chronic hepatitis C[END_REF] For the E1-E2 region and the concatenated region, the general time reversible model with 20 gamma distributed rate heterogeneity among sites and invariable sites (GTR+I+G) was 21 selected. For the NS3-NS4 region, the transversional model with gamma distributed rate 22 heterogeneity among sites and invariable sites (TVM+I+G) had the smallest AIC score. [START_REF] Delwaide | HCV genotype 21 4 in Belgium: three distinct patterns among patients from European and African origin[END_REF] Substitution model parameters were estimated on a initial neighbor-joining tree and were 24 fixed throughout heuristic search optimization during which the NNI branch-swapping 25 algorithm was used. Bootstrap analysis was performed using 500 replicates to assess the 1 statistical robustness of the estimated phylogenies. 2 3

Statistical analysis 4

Statistical analysis was performed using SPSS 11.5 (SPSS Inc., Chicago, USA). P-values 5 ≤0.05 were considered statistically significant. The Chi-Square test was used for the detection 6 of significant differences between dichotomous variables. For continuous variables, the 7 Mann-Whitney test was used. 8

RESULTS 1

General HCV genotype distribution in Flanders, Belgium 2 In this study, the HCV genotype of 2301 HCV positive patients was determined. HCV 3 genotype 1 was the most prevalent genotype and was found in 1401 (60.9%) of patients. HCV 4 genotype 3 was the second most prevalent genotype with 466 (20.3%) infected patients, 5 followed by HCV genotype 4 (184 patients, 8.0%), HCV genotype 2 (145 patients, 6.3%) and 6

HCV genotype 5 (104 patients, 4.5%). Only one patient was infected with HCV genotype 6. 7

No mixed infections with different genotypes were detected. HCV subtype information was 8 obtained for 80.4% of samples (11.2% 1a, 42.6% 1b, 0.7% 1a/1b, 3.6% 2a/2c, 1.0% 2b, 9 18.1% 3a, 0.1% 3b, 0.3% 4a/4c/4d, 2.4% 4c/4d, 0.2% 4f and 0.2% 4h). 10

In total, 1392 (60.5%) patients were male, and 909 (39.5%) were female. For HCV genotypes 11 1 and 3, significantly more men were infected than women (Pearson Chi-Square tests, 12 P<0.001). For the other HCV genotypes, no significant differences according to gender could 13 be found. The mean age ± SD of the total study population was 51.5 ± 15.6 years (range: 5-14 94). Patients infected with HCV genotype 3 were significantly younger than patients infected 15 with one of the other genotypes (42.4 ± 10.3 years; range: 5-84; Mann-Whitney test, 16 P<0.001). 17 In each province, HCV genotype 1 is the most prevalent. In Antwerp, Limburg, East-Flanders 1 and Flemish-Brabant, HCV genotype 1 is followed by HCV genotypes 3, 4, 2 and 5a 2 respectively. In Brussels, HCV genotype 4 is more prevalent than HCV genotype 3. 3 Remarkably, HCV genotype 5a is a minor genotype in all provinces, except for West-4 Flanders, were it is the second most prevalent genotype. In this province, more than a quarter 5 of the patients (26.2%) is infected with the uncommon HCV genotype 5a. A Pearson Chi-6

Square test indicated that the number of HCV genotype 5a patients is significantly higher 7 compared to the number of HCV genotype 5a patients in other provinces (P<0.001). The 8 HCV genotype 5a patients are significantly older (mean age 62 ± 14.7; range: 13 -88; Mann-9 Whitney test: P<0.001). 10

The data on the treatment response of the HCV genotype 5a patients are not based on the 11 official medical records, but on the information given by the patients during the interview. 12 Therefore, we do not have the exact information on the dosage for each patient. In total, 49 13 patients received antiviral therapy. Of these, 59.2% (29/49) showed sustained virological 14 response (SVR) after completion of therapy, 14.3% (7/49) were non-responders, and 26.5% 15 (13/49) relapsed after therapy. [START_REF] Bourliere | Epidemiological changes in hepatitis C virus genotypes in France: evidence in 48 intravenous drug users[END_REF] 17 Epidemiological investigation of the HCV genotype 5a cluster in Flanders 18 In total, 104 patients were infected with HCV genotype 5a. Of these, 67 patients were willing [START_REF] Bochud | Genotype 3 is 10 associated with accelerated fibrosis progression in chronic hepatitis C[END_REF] to be interviewed at their home using a standardized questionnaire to identify potential risk 20 factors. All 67 patients were born and are living in Belgium. Forty-four patients (65.5%) are 21 living in West-Flanders, 4 (6.0%) in East-Flanders, 5 (7.5%) in Flemish-Brabant, 6 (9.0%) in 22 Antwerp, 6 (9.0%) in Limburg, and 2 (3.0%) in Brussels. 23

Among the HCV genotype 5a patients, 49 (73.1%) received blood transfusion and 24 (35.8%) 24 patients were blood donors before they were diagnosed with HCV. The questionnaire 25 contained several questions that addressed the possibility of transmission via needles or 1 syringes. None of the patients reported a history of IDU. Thirteen patients underwent 2 acupuncture treatment, most of them at the practice of the family doctor. Two patients have a 3 tattoo, and 22 patients have one or more piercings. Seventeen patients received multiple 4 injections from multi-dose vials, mainly for pain relief. Two patients reported that they 5 received several injections during their military service, and that hygienic procedures were not 6 followed correctly. Vertical transmission does not seem to occur often, only two "mother-7 child" couples were identified. The same holds for horizontal or familial transmission. We 8 identified only one "husband-wife" couple, one "father-son" couple and two sibling couples. 9

The patients were asked to give an overview of the family doctors and dentists that they 10 visited in the past. The names of three different family doctors appeared on top of the list. 11

These family doctors were mainly medically active between 1960 and 1980. Two of them are 12 also infected with HCV genotype 5a, and performed several deliveries. Patient-to-doctor or 13 doctor-to-patient transmission might have occurred since hygienic procedures were less strict 14 prior to the identification of HCV in 1989. No obvious links between HCV infection and 15 dentistry history were identified. Patients were also requested to provide us with an overview 16 of the surgical procedures they underwent and details on when and where these were 17 performed. Surgeries were executed in several hospitals, at different wards, and in a period 18 spanning more than 50 years. Therefore, it is very difficult to make any links between HCV 19 transmission and surgical history. [START_REF] Smith | 13 The origin of hepatitis C virus genotypes[END_REF] Since HCV genotype 5a is most frequently found in South Africa, we asked the patients if 21 they had ever visited that country and whether they had any direct contact with blood or blood 22 products during their stay in South Africa. Eight patients visited South Africa but none of 23 them had contact with blood products, which excludes the possibility that one of the patients 24 was infected in South Africa through blood products or surgery. Five patients had travelled to 25 Congo. Two of them were active in a medical setting and performed surgical procedures 1 during their stay. Two other patients were there on holiday and reported no contact with blood 2 products. One patient was active in a military setting in Congo, and had a knee surgery during 3 his stay between 1959 and 1961. [START_REF] Pawlotsky | Hepatitis C virus genetic variability: pathogenic and clinical 12 implications[END_REF] 5

Phylogenetic reconstruction 6

Serum samples from 61 HCV genotype 5a infected patients were collected for phylogenetic 7

analysis. Thirty-one of these patients completed the questionnaire, enabling us to investigate 8 possible transmission routes in detail for this subset of patients. Figure 2 shows the 9 phylogenetic tree for the concatenated dataset. Only one viral strain per patient was included 10 for clarity purpose. Each patient is identified by a unique identification number. Numbers in 11 bold represent patients of whom both a questionnaire and a serum sample were available. In 12 general, the phylogenetic signal of the dataset is rather low, since the main branches of the 13 tree are not supported by high bootstrap values. However, some smaller clusters with higher 14 bootstrap values can be recognized. Remarkably, for most of these clusters, possible common 15 infection sources were identified during detailed inspection of the questionnaires. The strains 16 indicated in green originate from patients that visited the same family doctor for several years. 17

The doctor himself is also infected with HCV genotype 5a, but unfortunately, we were not 18 able to collect a serum sample. This sub-cluster (indicated by the dashed rectangle) harbours 19 some additional strains from patients that live in the same rural area. We do not have a 20 questionnaire from these patients and therefore can not confirm whether these patients also 21 visited this family doctor. The strains indicated in orange correspond to a "mother-son" 22 couple where vertical transmission occurred. The two patients who received multiple 23 injections during their military service at the same military base, cluster together with a 24 bootstrap value of 99 (strains indicated in blue). Three patients that do not live in West-25

Flanders also cluster with a high bootstrap value. The corresponding strains are indicated in 1 red. They all received blood transfusion in the same year and in the same geographical region. 2

One of the patients was also a blood donor in this geographical region before he/she was 3 diagnosed with HCV, and delivered multiple blood donations during almost 10 consecutive 4 years. The strains shown in yellow are also associated with blood transfusion procedures. 5

Two of the three patients (BE03048920 and BE04458951) were blood donors in the same 6 geographic region and in the same year as the third patient (BE02218930) received a blood 7 transfusion. 8

The small clusters that can be observed for the concatenated gene region, were also present in 9 the phylogenetic trees of the separate E1-E2 region and NS3-NS4 region (data available on 10 request). 11

12

DISCUSSION 1
This study aimed at evaluating the HCV genotype distribution in Flanders and Brussels 2 (Belgium) and at investigating the mechanisms underlying the occurrence and spread of an 3 uncommon HCV genotype 5a cluster which was described in a previous study [START_REF] Verbeeck | Investigating the origin and spread of 27 hepatitis C virus genotype 5a[END_REF]. [START_REF] Pawlotsky | Hepatitis C virus genetic variability: pathogenic and clinical 12 implications[END_REF] The global genotype distribution in our patient group indicates that HCV genotype 1 is 5 dominant, followed by HCV genotype 3, HCV genotype 4, HCV genotype 2, HCV genotype 6 5 and HCV genotype 6. This distribution pattern largely corresponds to the results obtained 7 during a previous survey performed in drug users from Belgium in 2005 [START_REF] Mathei | Molecular epidemiology of hepatitis C among drug users in Flanders, Belgium: 36 association of genotype with clinical parameters and with sex-and drug-related risk 37 behaviours[END_REF]. Since the study 8 population in this study was limited to drug users, the reported prevalence of HCV genotype 3 9 was higher than in our study. Compared to the results reported by Gérard and colleagues [START_REF] Gerard | Evolution over a 10 year period of the epidemiological profile of 1,726 24 newly diagnosed HCV patients in Belgium[END_REF], 10 the proportion of HCV genotype 3 patients in Belgium seems to be growing over the last 11 decade. This phenomenon is also observed in other European countries [START_REF] Roman | Hepatitis C virus genotypes 40 distribution and transmission risk factors in Luxembourg from 1991 to 2006[END_REF][START_REF] Chlabicz | Changing HCV genotypes distribution in Poland--relation to source 44 and time of infection[END_REF][START_REF] Bourliere | Epidemiological changes in hepatitis C virus genotypes in France: evidence in 48 intravenous drug users[END_REF][START_REF] Christofidou | 1 Distribution of hepatitis C virus genotypes in viremic patients with beta-thalassemia[END_REF] and is 12 probably due to the increase of injecting drug use (IDU) contamination, which has become 13 the most common way of new HCV transmission since blood screening became available in 14 1991 [START_REF] Kleter | Hepatitis C virus 6 genotypes: epidemiological and clinical associations. Benelux Study Group on Treatment of 7 Chronic Hepatitis C[END_REF]. Patients infected with HCV genotype 3 were significantly younger than patients 15 infected with one of the other HCV genotypes in our dataset. This also fits in the IDU 16 scenario. Since HCV genotype 3 has recently been associated with accelerated fibrosis 17 progression [START_REF] Bochud | Genotype 3 is 10 associated with accelerated fibrosis progression in chronic hepatitis C[END_REF], the growing share of this genotype may have important consequences for 18 the management of patients infected with this genotype. [START_REF] Bochud | Genotype 3 is 10 associated with accelerated fibrosis progression in chronic hepatitis C[END_REF] In the Brussels capital region, HCV genotype 4 is the second most prevalent genotype. This 20 HCV genotype is most common in the Middle East and in Northern and Central Africa [START_REF] Smith | 13 The origin of hepatitis C virus genotypes[END_REF]. [START_REF] Ray | Genetic epidemiology of 15 hepatitis C virus throughout egypt[END_REF] Egypt has the highest HCV prevalence worldwide, and more than 90% of the patients are 22 infected with HCV genotype 4 [START_REF] Ray | Genetic epidemiology of 15 hepatitis C virus throughout egypt[END_REF]. Recent studies indicate that HCV genotype 4 prevalence 23 has increased in European countries due to the immigration of HCV genotype 4-infected 24 patients [START_REF] Ansaldi | Different seroprevalence and molecular epidemiology patterns of hepatitis C virus 18 infection in Italy[END_REF][START_REF] Delwaide | HCV genotype 21 4 in Belgium: three distinct patterns among patients from European and African origin[END_REF][START_REF] Payan | Changing of hepatitis C virus genotype patterns in France 31 at the beginning of the third millenium: The GEMHEP GenoCII Study[END_REF][START_REF] Schroter | 34 Epidemiological dynamics of hepatitis C virus among 747 German individuals: new subtypes 35 on the advance[END_REF][START_REF] Van Asten | Spread of hepatitis C virus among European 39 injection drug users infected with HIV: a phylogenetic analysis[END_REF][START_REF] Van De Laar | Changes in risk behavior and dynamics of hepatitis C virus 43 infections among young drug users in Amsterdam, the Netherlands[END_REF]. A study on the emergence of HCV genotype 4 in The Netherlands revealed 25 three epidemiological profiles, one associated with immigration from the Middle East and 1 Africa, a second profile linked to injecting drug use, and a third one consisting of men who 2 have sex with men (MSM) that are co-infected with HIV [START_REF] De Bruijne | Emergence of hepatitis C virus genotype 47 4: phylogenetic analysis reveals three distinct epidemiological profiles[END_REF]. Brussels is a geographical 3 region with a relatively high immigration rate, which could be a possible explanation for the 4 rise in HCV genotype 4 prevalence in this region. Moreover, HCV genotype 4 is increasing in 5 the IDU population in Belgium (personnel communication, Frederik Nevens). 6

Another remarkable finding is the fact that HCV genotype 5a is the second most prevalent 7 genotype in West-Flanders, whereas it is only found sporadically in other Flemish provinces . 8 In order to study the epidemiology of HCV genotype 5a in Flanders, a thorough investigation 9 of possible transmission routes and risk factors was conducted using a detailed questionnaire. 10

For the patients infected with non-genotype 5a strains, epidemiological information is 11 lacking. Therefore, we are unable to discuss risk factors associated with transmission for these 12

HCV genotypes, and we can not compare the HCV genotype distribution in different risk 13 groups which is a drawback of our study. A survey in HCV patients using a simple 14 questionnaire during the consultations as suggested by De Maeght and colleagues [START_REF] De Maeght | A pilot observational survey 2 of hepatitis C in Belgium[END_REF] can 15 provide information on the risk factors and transmission profiles of HCV in Belgium. [START_REF] Bourliere | Epidemiological changes in hepatitis C virus genotypes in France: evidence in 48 intravenous drug users[END_REF] The main transmission route for this HCV genotype seems to be blood transfusion, as 73.1% 17 of the HCV genotype 5a patients received one or more transfusions before they were 18 diagnosed with HCV. Moreover, 24 patients (35.8%) were blood donors before they were 19 diagnosed with HCV, and they may have unconsciously contributed to the spread of the HCV 20 genotype 5a virus. In our previous study addressing the origin and spread of HCV genotype 21 5a, we hypothesized that transmission of HCV genotype 5a in Belgium was mainly associated 22 with contaminated blood products, and that HCV has been circulating in Belgium since mid 23 1800 [START_REF] Verbeeck | Investigating the origin and spread of 27 hepatitis C virus genotype 5a[END_REF]. A study on the epidemiology of HCV genotype 5a in central France suggests that 24 this uncommon genotype was transmitted by iatrogenic route before 1972 in this region, and 25 later on via transfusion to the whole district [START_REF] Abergel | The epidemiology and virology of hepatitis 6 C virus genotype 5 in central France[END_REF]. In Syria, transmission via blood transfusion, 1 IDU and tattooing was identified in 30% of the HCV genotype 5a patients, while no obvious 2 transmission mechanism could explain the infection in the remaining portion of patients [START_REF] Antaki | The unexpected discovery of a focus of hepatitis C virus genotype 5 in a Syrian 11 province[END_REF]. 3

In southeast Spain, the origin of infection was unknown for 80% of the patients, 16% 4 acquired infection by blood transfusion and 4% of patients were IDUs [START_REF] Jover | Infection by genotype 5a of HCV in a district of southeast Spain[END_REF]. 5

In contrast to HCV genotypes 1, 3 and 4 for which epidemiological data are well established, 6 the epidemic spread of HCV genotype 5a remains unknown. Since HCV genotype 5a is the 7 predominant genotype in South Africa [START_REF] Chamberlain | The 14 complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in 15 South Africa[END_REF], it may well be that this genotype originated in that 8 part of the world and was subsequently introduced in Belgium. However, our study reveals no 9 direct epidemiological links with South Africa. Although HCV genotype 5a is not commonly 10 found in Congo, we hypothesized in a previous study that this region could harbour the 11 original source of this genotype [START_REF] Verbeeck | Investigating the origin and spread of 27 hepatitis C virus genotype 5a[END_REF]. One general surgeon and one urologist were 12 professionally active in Congo, so it is possible that they had contact with HCV infected 13 patients during their stay. 14 Since HCV genotype 5a infections are rare, they theoretically represent an excellent marker to 15 follow the epidemiological spread. Tracing the predominant transmission routes mainly 16 depends on information obtained from the patients themselves, and is therefore subjected to 17 the obvious disadvantages of this kind of studies, such as recall bias. However the results of 18 our phylogenetic analysis confirm some findings of the face-to-face interviews. Based on the 19 combined results of both the epidemiological and phylogenetic study, small and localized 20 clusters of transmission have been identified. When, where and how HCV genotype 5a 21 originated and started spreading in the human population, still remains an unsolved issue. Our 22 results suggest that several independent transmission events have led to the local spread of 23 this genotype, and that transfusion and unsafe medical practices are the main transmission 24 routes for HCV genotype 5a in Flanders, Belgium.

18

  Figure 1 displays the HCV genotype distribution for the northern part of Belgium (Flanders 20

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 25

26

 26

FIGURE LEGENDS 1 2 Fig 1 5 Fig 2 Figure 2

 21522 FIGURE LEGENDS 1

ACKNOWLEDGEMENTS 1

We would like to thank all HCV genotype 5a patients for their kind participation in our study. 2 PL was supported by a postdoctoral fellowship from the Fund for Scientific Research (FWO) 3 Flanders and FWO grant G.0513.06. 4 5 6 7