Molecular Characterization of Noroviruses and HBGA from Infected Quilombola Children in Espirito Santo State, Brazil

Noroviruses (NoV) are the main etiological agents of gastroenteritis outbreaks worldwide and susceptibility to NoV infection has been related to the histo-blood group antigen (HBGA). This study aimed to determine the prevalence of NoV strains and to evaluate the HBGA phenotype and genotype of children from semi-isolated Quilombola communities, descendents of black slaves in Brazil. A total of 397 children up to eleven years old, with and without diarrhea, from Quilombola Communities in the Espirito Santo State, Brazil, were investigated for the presence of NoV from August 2007 to September 2009. Feces were collected from all the children, and blood from the NoV positive children. NoV was screened by reverse transcription-PCR with primers for the RNA-dependent RNA polymerase region; genogroup was determined by PCR with primers for the C and D regions and genotyped by sequencing. HBGA phenotype was performed by gel-spinning and FUT2 and FUT3 were analyzed by PCR or sequencing analysis. NoV were detected in 9.2% (12/131) of diarrheic and 1.5% (4/266) of non-diarrheic children (p<0.05, Fisher’s exact test). GI and GII genogroups were present in 12.5% and 87.5% of the samples, respectively. The following genotypes were characterized: GII.4 (25%), GII.12 (25%), GII.6 (12.5%) and GI.1 (6.3%), GI.3 (12.5%) and GI.4 (6.3%). Children infected with NoV showed the A (n = 6), O (n = 6), and B (n = 2) HBGA phenotypes, and 13 of them were classified as secretors (Se) and one as a non secretor (se). Mutations of Se 40, 171,216,357,428,739,960 were found for the FUT2 gene and mutations of Le 59, 202, 314 for the FUT3 gene. The only se child was infected by NoV GI, whereas the Se children were indiscriminately infected by GI or GII. This study showed rates of NoV infection in symptomatic and asymptomatic Quilombola children consistent with other studies. However, children under 12 months were seven times more affected than those between 1 and 5 years old. GII.12 was as frequent as GII.4 and GI.1 and GI.4 were described for the first time in Brazil. Owing to the small number of cases studied, no clear pattern of susceptibility and/or HBGA resistance could be inferred.


Introduction
Gastroenteritis of infectious etiology is still an important cause of morbidity in the human population worldwide [1]. Diarrhea and vomiting associated with a lack of access to primary health care and supportive treatment for dehydration, can lead to serious clinical consequences, particularly in developing countries [2,3]. Noroviruses (NoVs) are the main viral agents in acute diarrhea outbreaks and sporadic cases for all age groups worldwide, except for rotaviruses in children under three years old, leading to more than 1 million hospitalizations annually [1,3]. However, this scenario is changing in countries, including Brazil [4] that have adopted the use of rotavirus vaccines tending to pass all age groups be headed by NoV. In addition, asymptomatic excretion is reported in healthy individuals, favoring virus transmission [1,2,3,5].
Human NoVs belong to the Caliciviridae family, Norovirus genus and are classified into three genogroups: GI, GII, and GIV. Differences in the sequence of the major viral capsid proteins (VP1) allow further classification into eight GI and twenty-one GII genotypes and one GIV genotype [6]. GII.4 is considered the most prevalent genotype worldwide [1,7].
At present, there is no in vitro replication system available for human NoVs. Nevertheless, the expression of recombinant VP1 allows the in vitro reconstruction of VLPs (virus-like particles), morphologically and antigenically similar to the wild virions [8] that show bind to molecules characterized as histo-blood group antigens (HBGA) [9,10,11,12,13,14]. HBGAs are oligosaccharides synthesized by the stepwise addition of monosaccharides onto glycan precursors via the glycosyltransferases FUT2, FUT3 and A/B of the ABO and Lewis blood group systems. The FUT2 enzyme adds a fucose, in the a1,2 linkage, onto a galactose of the precursor, generating the H antigens. The FUT3 enzyme adds a fucose residue, in the a1,4 (or 1,3) position, onto the Nacetylglucosamine of the precursor, generating the Lewis a (Le a ) or the Lewis b (Le b ) antigens when combined with the a1,2 fucose residue. The A or B enzymes catalyze the addition of an Nacetylgalactosamine or a galactose onto the H antigens, giving rise to the A and B antigens, respectively. The FUT2, FUT3 and ABO genes possess functional alleles encoding the FUT2, FUT3, A and B enzymes respectively. These FUT2, FUT3 and ABO genes also possess null alleles which are unable to generate active enzymes. Individuals who have the H antigen in their epithelial tissues and secretions are called secretors. In contrast, individuals who inherited two FUT2 null alleles are devoid of H, Le b , A and B antigens and are called non-secretors. Similarly, FUT3 null homozygotes lack the Le a and Le b antigens and homozygotes for O alleles (null alleles of the ABO gene), are devoid of either A and B antigens and are therefore of the blood group O [15].
Approximately 80% of the human population has a secretor phenotype (Se) and is considered to be susceptible to NoV infections by the majority of strains with a variation depending on the ABO phenotype and on the ability of strains to recognize the A, B, H or Le b antigens. The remaining 20% of the population with the non secretor phenotype (se) is considered to be naturally resistant to most NoV strains [16,17,18,19]. However, VLPs from some GI and GII strains have been reported to bind to carbohydrates such as Le a or Le x mainly present in non-secretors. Accordingly, non-secretor individuals are occasionally infected by NoVs [14,20,21,22,23].
Although HBGA polymorphisms representing populations from the five continents have already been characterized, the populations studied hitherto in South America have been restricted to the Colombian Amerindians, Maya, Suruí, Karitiana and Pima Indians [24]. This is the first study involving a black population from Southeastern Brazil, and moreover, infected with NoV. This population consists of descendants of former African slaves, who live in semi-isolated communities, called ''Quilombola Communities'' and the people living there are known as ''quilombolas'' [25]. Even today, in the 21 st century, they are still underserved communities with poor sanitary conditions, conducive to gastrointestinal diseases.
The aims of this study were: i) to determine the prevalence and genotypes of NoV among children up to 11 years old, with and without diarrhea, who are residents in the Quilombola Communities in the Espírito Santo State, Southeastern Brazil and; ii) to characterize the HBGA phenotypes and genotypes of the infected children to elucidate their susceptibility to NoV infection.

Study Design and Site Description
This is a descriptive study with Quilombola children up to 11 years old, who are descendants of slaves and who live in Quilombola Communities located in the semi-isolated rural areas, known as North Sapê, in the North of Espírito Santo State, Southeastern Brazil. Today there are 30 communities in the North Sapê region with an estimated population of more than 3,600 people, 950 residences and numerous farms dedicated to the cassava plant. They are between 6.21 mi (10 km) and 18.64 mi (30 km) from urban centers and from each other, and are connected by dirt roads of very difficult access.
This study was approved by the Ethics Research Committee of the Centro de Ciências da Saúde, Universidade Federal do Espírito Santo (002A/08) and Statement of Consent was obtained from the guardians and from the children themselves, whenever appropriate.

Fecal and Blood Specimens
A total of 397 fecal specimens were obtained, 131 (33%) from children with diarrhea (symptomatic) and the remaining 266 (67%) from children without diarrhea (asymptomatic), matched by community, between August 2007 and September 2009. To carry out the HBGA studies, blood samples (3 ml) were collected by venipuncture in tubes containing ethylenediamine tetraacetic acid (EDTA) as anticoagulant from children who were positive for NoV. Blood samples were immediately analyzed for phenotype as described below; leukocytes were obtained by centrifugation at 2,0006g for 15 min and stored at 220uC for further analysis. [26]. Feces and peripheral blood leukocytes were frozen at 220uC for nucleic acid extraction.

Noroviruses Detection and Genogroup and Genotype
Viral nucleic acid was extracted from a 10% fecal suspension in Tris-calcium by using a guanidine isothiocyanate and silica method, as previously described [27]. Complementary DNA (cDNA) was obtained in a reverse transcription reaction using the 20 mU random primer pd(N) 6 TM (Amersham Bioscience, UK) and the SuperscriptII TM reverse transcriptase (Invitrogen, USA) [28].
NoVs genotyping was determined by partial sequencing of PCR amplicons obtained with primers for ORF-2 (G1SKF/G1SKR and G2SKF/G2SKR for the C region and Cap A, B1, B2/Cap C, D1, D3 for the D region of the VP1 gene) [31,32] (Table 1). Amplicon purification was performed using the QIAquickH kit (Qiagen) and sequencing using the commercial kit BigDyeH Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). Sequences were aligned using the BioEdit SequenceH Alignment Editor version 7.0.9.0, deposited in the GenBank database and then compared with sequences of Brazilian and reference samples of each genotype. A phylogenetic tree was constructed by the Neighbor-joining method using the MEGA program version 5.10, and the genetic distance was calculated with the Kimura 2 parameter model, using 2,000 pseudo-replicates.
Nucleotide sequences obtained in this study were submitted to the National Center for Biotechnology Information (Gen Bank, http://www.ncbi.nlm.nih.gov/) and received the following acces-

Histo-Blood Group Antigen
Phenotypic analysis. The characterization of the blood groups was carried out with the peripheral blood samples from the NoV positive children by: (i) the tube agglutination technique with anti-A, anti-B, anti-AB and anti-D (DiaClon TM , Diamed, Br, BIO RAD) monoclonal antibodies (MAbs), and (ii) the Lewis antigens detection system and the secretor status categorization by gelcentrifugation was performed with capture MAbs anti-Le a or anti-Le b (DiaClon TM , Diamed, Br, BIO RAD) in two different reactions, according to the manufacturer's instructions.
Genotypic Analysis of the FUT2 gene (secretor). Genotype analysis was carried out with DNA extracted from peripheral blood leukocytes by treatment with 1.6 M sucrose and 10 mg/ml proteinase K [33]. Inactivating mutations of FUT2 gene were screened by: (i) PCR for the most common mutation G428A with specific primers for the mutation Se antisense (as) combined with Se 1s sense (s) for the wild-type allele and PCR with primers for the mutant gene se with Se2s, as previously described [26] (Table 2) and; (ii) gene sequencing of exon 2, using a set of primers designed for this study from the reference sequence available in GenBank (NCBI). The primers were targeted to amplify the entire exon, from the position -61F, up to +98 R, as follows: A 50 ml final reaction mixture contained 0.2 mM dNTP, 1.5 mM MgCl 2 , 0.2 mM of each primer, 1 unit of Platinum Taq DNA polymerase (InvitrogenH, Carlsbad, CA, USA), 16 PCR buffer and 1 ml of DNA under the following conditions: initial denaturation at 95uC for 2 min followed by 25 cycles at 95uC for 45 s, 60uC for 45 s and 72uC for 75 s, and a final extension at 72uC for 5 min. The gene for human growth hormone (HGH) was used as the internal control of the reaction [34].
For sequencing, the PCR products were purified with ExoSAP-IT TM (GE/USB). The thermal cycle was 37uC and 80uC for 15 min. each. Sequencing was performed with the BigDyeH Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). Sequences were aligned using the BioEdit Sequence Alignment Editor version 7.0.9.0.
Genotypic Analysis of the FUT3 gene (Lewis). The presence of four major human FUT3 mutations T59G, T202C, C314T and T1067A was investigated [26]. Two PCRs were performed for each mutation with a set of primers containing III-55 antisense and (i) the initiator for the wild-type allele and (ii) the initiator for the mutant allele (Table 2). One microliter of DNA extracted was added to a 50 ml final reaction mixture containing 1.5 mM MgCl 2 , 200 mM of each dNTP, 0.2 mM of each primer and 1 unit of GoTaqH DNA polymerase (Promega, Madison, USA). Amplification was performed under the following conditions: initial denaturation at 95uC for 2 min followed by 25 cycles at 95uC for 45 sec, 60uC for 45 s and 72uC for 75 s, and final extension at 72uC for 5 min. Similarly to the previous item, an internal control reaction was performed.

Statistical Analyses
The two sided Fisher's exact test was performed for comparisons between groups using the GraphPad program version 5.0a, and p values below 0.05 were considered significant.

Detection and Molecular Characterization of Noroviruses
Noroviruses were detected in 4% (16/397) of the fecal specimens obtained from Quilombola children, of whom 131 (33%) were diarrheic (symptomatic) and 266 (67%) were without  Twelve strains were genotyped by means of partial sequence analysis of ORF2 (regions C and D), evidencing GII.4 (n = 4), GII.12 (n = 4), GII.6 (n = 2), and GI.3 (n = 2) genotypes ( Table 3, Figures 1 and 2). GII.6 was detected exclusively among asymptomatic children, while the other genotypes were found only among symptomatic cases. The four strains that could not be genotyped were three GIIs (two from symptomatic cases and one from an asymptomatic case), and one GI (from an asymptomatic case). NoV GI.1, GI.4 and GII.6 (Figures 1 and 2) were also detected in an urban community, consisting of predominantly black people, near the traditional Quilombola Communities (unpublished results).
HBGA was determined for 14 out of the 16 children infected with NoV, six belonged to blood group A, six to O and two to B (Table 3); 13 children were characterized as Se and one as se. The Se children were infected by GI and GII NoV strains, while the only se was infected by a GI strain. The frequency of se individuals in this population is close to 0.2 (data not shown) and individuals of this phenotype are under-represented among infected children (1/ 14). All three children infected by GI belonged to the O blood group, while those infected by GII were equally distributed between A and O blood groups. Here again, this difference did not reach statistical significance owing to the small number of cases.

Characterization of the Polymorphisms of the FUT2 and FUT3 Genes
The full coding sequence of the FUT2 gene revealed several mutations in the black population studied, at the 40, 171, 216, 357, 428, 739 and 960 positions ( Table 4). The inactivating mutation G428A was in a homozygous state in the only child with se phenotype.
For the FUT3 gene, five children were homozygotes for the wild-type alleles. The mutations Le 59 (n = 5) and Le 202,314 (n = 4) were always detected in the heterozygous state, both in those phenotypically characterized as Lewis positive and those characterized as negative, suggesting that other mutations could account for the Le (a-b-) phenotype in this population (Table 3).

Discussion
NoVs were found in the Quilombola children at rates consistent with other studies addressing community infection by NoVs [35,36,37]. Despite this, the rate of NoVs among the symptomatic and asymptomatic children was relatively high, since the studied communities are rural and sparsely populated, which could compromise the transmission of virus particles. Although the positive NoVs cases were concentrated between March and May 2009, the great diversity of the genotypes coupled with the small number of cases in each community at each collection date does not allow this result to be characterized as an outbreak.
NoV infection is known to occur in individuals of all ages due to its vast diversity of genotypes, its infectivity, the high rates of mutation and recombination which together lead to antigenic varieties and escape from the host immune system [29,38,39,40,41]. However, in our study, most cases of symptomatic NoV infection occurred among children up to 12 months old. Although this age group corresponded to only 9.6% of the total of samples collected, it had a higher prevalence compared to the prevalence found in the 1 to 5 year-old children, suggesting there was either a greater susceptibility of very young children or early immunization in this type of community. Ferreira et al. (2012) [42] also showed a higher prevalence of NoVs in the youngest children, but in children older than those in this present study.
This study of NoVs in 30 Quilombola communities of Southeastern Brazil was conducted in parallel to the recently published study carried out in a similar community in the North of Brazil [25]. These regions are approximately 2,300 km apart from each other. In contrast to our results, the study in the North showed twice the number of NoV cases in children with diarrhea but did not find the virus in any of the asymptomatic children [25]. This study complements earlier studies with hospitalized patients or outpatients at emergency rooms that involved patients with a more severe clinical profile [3]. Ferreira et al. (2012) [42] in a 15 year retrospective study of outbreaks or sporadic cases of gastroenteritis, found NoVs in 28.8% of all cases.
Most of the NoV strains characterized in this study belonged to GII, in agreement with other studies that show GII as the most prevalent in the world, whereas GI is sporadically present at high prevalence [1,7,35]. However, a third the cases here consisted of GI, a rate that can be considered relatively high for Brazil since previous studies have described lower frequencies [3,42,43] as well as the Quilombola study in the North [25], although Soares et al. (2007) [44] described a GI prevalence of 47.6% in Brazil.
This study shows the first description for GI.1 and GI.4 in Brazil among the three different GI genotypes detected (GI.1, GI.3 and GI.4), which represents a relatively high diversity. Studies in others countries have also shown high diversity of GI types, albeit with a low frequency for each [43,45]. Generally, the diversity observed for GI and GII was interesting because they were found in semiisolated communities. A broad genetic diversity was also observed in the Quilombola children in the North of Brazil [25].
The GII.12 strains align very closely to those referred to as ''post-2009'', when the rise of a new NoV GII.12 recombinant in the U.S. winter 2009/2010 was described [40]. Interestingly, stools of the present study were collected six months before the North American samples (March 2009), suggesting that the new GII.12 recombinant probably circulated in Brazil concomitantly Figure 1. Phylogenetic tree of C region. Phylogenetic tree constructed by the Neighbor-joining method based on the partial nucleotide sequence of the norovirus VP1 capsid protein gene C region. Highlighted strains identified by ''Sapê'', originated in fecal samples from ''Quilombola'' children and were analyzed along with the prototype norovirus genotypes of GI, GII and GIV taken from the ''GenBank.'' All strains were identified by genotype/accession number/year/origin. Sapovirus/AB630067.2/2008/JP was added as a reference group. Values of ''Bootstrap (2,000 replicates) are shown at the junction of the branches. N Samples Q51, Q151 and Q220 belong to another study site near the Quilombola communities. ¤ Samples of this study. doi:10.1371/journal.pone.0069348.g001 to the U.S. Nevertheless, one should take into account that the sequence alignment was made on the basis of the C and D regions of VP1 gene, requiring the sequence of B region and its interface with C region to prove a recombinant phenomenon, as recently described by Fumian et al. (2012) [39].
In the last decade the most prevalent genotype, in people of all ages worldwide has been the GII.4 [1,43]. The GII.4 strains observed were similar to the 2006b variant, shown in Figure 1 by the prototype GQ246799.1/Dijon/2009/FRA. This variant has been detected in at least seven other states in Brazil [43]. In terms of its epidemiological impact, worldwide since 1995, this genotype has been widely studied in order to understand its mechanisms of viral evolution and escape from the immune system as well as its ability to recognize HBGAs [38,46,47,48,49]. In the present study, we observed that it was not as overwhelmingly represented as previously reported but was similar in prevalence to GII.12. This suggests that GII.4 may be in the process of being replaced by the new GII.12 recombinant as the dominant circulating strain.
To assess if there was any parasite-host relationship typical for Brazil, we characterized the HBGA phenotype and genotype of the Quilombola children infected with NoVs, as representative of people of African descendant in Brazil. They correspond to 50% of the population, according to the Brazilian Institute of Geography and Statistics (IBGE 2011).
Here, no significant association between infection and ABO, secretor, or Lewis phenotypes was observed. Nevertheless, all the patients infected by a GII strain, for whom HBGAs typing was performed, were all Se, apart from a single se, who was found infected, suggesting a lower likelihood of se becoming infected. GI strains belonging to the Lewis-binder group have been described and the single se patient was Lewis positive, making it plausible that the GI strain involved in that case belonged to this group. Unfortunately, it was not possible to genotype and amplify the VP1 coding sequence of that GI strain in order to assay its HBGA specificity. Previous studies showed an association with infection and the Se phenotype [16]. Nevertheless cases of infected se individuals have been described [23]. Consistent with this, several strains belonging to either GI or GII were found capable of binding to carbohydrates present in the se individuals, such as the Le a antigen [14,23].
Since the frequency of se individuals in this population is close to 0.2 (data not shown), individuals of this phenotype are underrepresented among infected children (1/14 ) that have been previously described (BGMUT/NCBI) [50]. There are descriptions of many FUT2 alleles in the human population, with 19 alleles containing a single substitution (SNP -single nucleotide polymorphisms). Among these, G428A is the main inactivating mutation of FUT2 responsible for the se phenotype since it generates a stop codon at position 143 (Trp-X) [51]. This mutation is present in European, African and Iranian populations [52]. In this study, the Se phenotype was found in the majority (14/15) of individuals tested. The se 428 was homozygous in the single individual with a se phenotype.
In conclusion, this work revealed that, despite the peculiar characteristics of the Quilombola population under study, rates of NoVs infection in symptomatic and asymptomatic children were consistent with other studies and describes for the first time the circulation of GI.1 and GI.4 in Brazil. Moreover, there was a greater frequency of younger children (,1 year) among the NoV infected children. The preliminary analysis of the genetic diversity of the HBGAs of the Quilombola population revealed a surprising number of similarities with other populations. Although the limited number of positive samples jeopardizes an appropriate analysis, the results showed diverse genotypes of NoV infecting African descendents belonging to different ABO, secretor and Lewis blood groups.