Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Norovirus Diversity in Diarrheic Children from an African-Descendant Settlement in Belém, Northern Brazil

Norovirus Diversity in Diarrheic Children from an African-Descendant Settlement in Belém, Northern Brazil

  • Glicélia Cruz Aragão, 
  • Joana D'Arc Pereira Mascarenhas, 
  • Jane Haruko Lima Kaiano, 
  • Maria Silvia Sousa de Lucena, 
  • Jones Anderson Monteiro Siqueira, 
  • Túlio Machado Fumian, 
  • Juliana das Mercês Hernandez, 
  • Consuelo Silva de Oliveira, 
  • Darleise de Souza Oliveira, 
  • Eliete da Cunha Araújo


Norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) are viral pathogens that are associated with outbreaks and sporadic cases of gastroenteritis. However, little is known about the occurrence of these pathogens in relatively isolated communities, such as the remnants of African-descendant villages (“Quilombola”). The objective of this study was the frequency determination of these viruses in children under 10 years, with and without gastroenteritis, from a “Quilombola” Community, Northern Brazil. A total of 159 stool samples were obtained from April/2008 to July/2010 and tested by an enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) to detect NoV, SaV and HAstV, and further molecular characterization was performed. These viruses were detected only in the diarrheic group. NoV was the most frequent viral agent detected (19.7%-16/81), followed by SaV (2.5%-2/81) and HAstV (1.2%-1/81). Of the 16 NoV-positive samples, 14 were sequenced with primers targeting the B region of the polymerase (ORF1) and the D region of the capsid (ORF2). The results showed a broad genetic diversity of NoV, with 12 strains being classified as GII-4 (5–41.7%), GII-6 (3–25%), GII-7 (2–16.7%), GII-17 (1–8.3%) and GI-2 (1–8.3%), as based on the polymerase region; 12 samples were classified, based on the capsid region, as GII-4 (6–50%, being 3–2006b variant and 3–2010 variant), GII-6 (3–25%), GII-17 (2–16.7%) and GII-20 (1–8.3%). One NoV-strain showed dual genotype specificity, based on the polymerase and capsid region (GII-7/GII-20). This study provides, for the first time, epidemiological and molecular information on the circulation of NoV, SaV and HAstV in African-descendant communities in Northern Brazil and identifies NoV genotypes that were different from those detected previously in studies conducted in the urban area of Belém. It remains to be determined why a broader NoV diversity was observed in such a semi-isolated community.


Acute gastroenteritis (AGE) is a common childhood disease that causes high rates of hospitalization and mortality, particularly in less-developed regions of the world. Each year, AGE is responsible for about 1.4 million deaths, occurring primarily in low and middle income countries [1], [2]. Although different groups of pathogens are involved in the etiology of AGE, enteric viruses play a key role as causative agents. The most relevant of these enteric viruses are group A rotavirus (RV-A), norovirus (NoV), adenovirus types 40/41 (AdV-40/41), human astrovirus (HAstV) and sapovirus (SaV) [3]. In settings where most of the population lives under conditions of poor hygiene, including poor quality water, food and sanitation, these viruses are common causes of AGE, which occurs most often in the first year of life [4], [5].

NoV and SaV, also known as human caliciviruses (HuCV), are classified in the Caliciviridae family. These viruses are responsible for causing outbreaks and sporadic cases of acute viral gastroenteritis in humans. NoV is recognized as the major cause of extensive AGE outbreaks that involve different settings, such as restaurants, nursing homes, schools and cruise ships [6], [7], [8], [9]. Recent studies have emphasized the role of NoV as a cause of AGE in hospitalized children [10], [11], [12], [13]. NoV strains are classified into five genogroups (GI-GV) on the basis of sequence similarity, of which the GI, GII and GIV genogroups are associated with human infections [14]. These genogroups are further divided into at least 32 genotypes; however, genotype GII-4 has emerged as the dominant strain worldwide, responsible for 70–80% of NoV outbreaks during the past 20 years [15], [16], [17], [18].

Enteric viruses are transmitted by the fecal-oral route, primarily through contaminated food and water, as well as through person-to-person spread [9]. Contaminated food is a high-risk source for the occurrence of outbreaks, especially those caused by NoV [19], [20].

Studies on the molecular epidemiology of enteric viral infections in hospitals, outpatient health units and the community have already been conducted [3], [5], [7]; nevertheless, studies on the epidemiology of infection in semi-closed communities such as Indian villages and African-descendant settlements are lacking.

The African-descendant settlements, known as “Quilombola” communities in Brazil, are rural communities that are composed of the descendants of African slaves. Members of these communities primarily survive through subsistence agricultural activity, with cultural traditions that preserve a strong link with the past. These social groups show ethnic and cultural identities that are distinct from the rest of the society [21].

This study aimed to assess the frequency of NoV, SaV and HAstV in cases of AGE among children living in a “Quilombola” community in the State of Pará, Brazil. This is the first study in the Amazon region, Northern Brazil that aims to detect enteric viruses in African-descendant communities.

Patients, Materials and Methods

Study population

This study was conducted in the semi-closed Abacatal “Quilombola” Community, located in the outskirts of Ananindeua and Marituba municipalities, which belong to the metropolitan area of Belém, Pará State, Amazon region, Brazil. This community is located 8.2 km from downtown Ananindeua (Figure 1).

Figure 1. Map showing the location of the Abacatal “Quilombola” Community, in the metropolitan region of Belém city, Pará State, Amazon region, Brazil.

The community occupies an area of 308 hectares (01°25′18″S e 48°20′58″W) and includes 84 families with a population of about 400 persons, including about 120 children aged less than 10 years. An official stratified data involving this subgroup is not available, but those individuals from whom fecal samples (n = 159) were obtained, were distributed as follows: 0–1 (14.5%), >1–5 (55.3%) and >5–10 (30.2%) years.

The socioeconomic level is very low, and the economy is based on agricultural subsistence activity (vegetables and fruits). These products are commercialized once a week in a free market in the Ananindeua municipality. Most of the families live in wooden or clay houses and have close contact with domestic animals, such as dogs, cats, chickens, ducks and pigs. Sewage infrastructure is not available, and access to potable water is lacking. Medical assistance is provided by health units located in downtown Ananindeua. Accessing this community is difficult, as it is accessible only through one land road that is difficult to access during the rainy season. The “Quilombola” have contact with outside urban communities during the sale of their products and through young people who attend high school in Ananindeua.

Patients and clinical specimens

This study was conducted from April/2008 to July/2010. Fecal samples were obtained during twice-a-week visits to the village by two pediatricians, who provided treatment to children with diarrhea. Samples were also obtained during active surveillance for diarrheic cases (three or more liquid or semi-liquid evacuations in a 24 hours period), also carried out twice-a-week in the community. For each diarrheic case collected, one normal fecal specimen (asymptomatic case) from an individual of similar age was obtained to be used as a control. The samples were refrigerated in iceboxes and brought to the Virology Section, Evandro Chagas Institute, and kept frozen at −20°C until virus analysis.


The Ethics Committee on Human Research of the Evandro Chagas Institute (IEC-CEPH) granted ethical approval to our study under number 0024.0.072.000-06. The ethical consent form was applied to the subjects of this research. Initially, the study team held meetings with community members, such as community leaders, health visitors and school directors, in order to obtain a better understanding of the study area and to inform them about the research objectives. Written informed consent was signed by parents or guardians of the children during the fecal specimen collection.

NoV antigen detection

The detection of NoV was initially performed using a commercial enzyme immunoassay (EIA) (Ridascreen®Norovirus 3rd Generation, R-Biopharm AG, Darmstadt Germany), following the manufacturer's instructions. The kit is a sandwich EIA whereby specific monoclonal antibodies against NoV antigens GI and GII came adhered to the micro-wells. Briefly, 10% (wt/vol) fecal suspensions were prepared with the diluent solution (NaCl buffer) and added into the wells together with an enzyme-labeled polyclonal antibody. Following incubation, a horseradish peroxidase conjugate was used for detection. The results were visually read and confirmed by absorbance measurements [22].

Viral RNA extraction and reverse transcription (RT)

Viral RNA was extracted from a 10% fecal suspension (the same as used in the EIA test) using a guanidine isothiocyanate/silica method [23], followed by cDNA synthesis performed using a pd(N)6™ random primer (Amersham Biosciences, UK) and the Superscript™ II RNAse H Reverse (Invitrogen, USA).

Molecular detection and characterization

The viral genome was detected by polymerase chain reaction (PCR) using the following primers: p289/p290 to detect HuCV (NoV and SaV) [24]; Mon 431–434 to detect NoV GI and GII [25]; and Mon 269/270 to detect HAstV [26]. PCR was carried out using the Taq DNA polymerase (Biotools, Spain). NoV-positive samples by EIA or PCR were also tested by another PCR using a set of primers that targeted the capsid region [27].

The PCR amplicons obtained from PCR-positive samples were purified using the commercial kits QIAquick® Gel Extraction Kit or QIAquick® PCR Purification Kit (QIAGEN, CA, USA), as recommended by the manufacturers. The purified DNA was subjected to a sequencing reaction, in both directions, using a Big Dye Terminator Cycle Sequencing Ready Reaction Kit® (v.3.1) (Applied Biosystems) and an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA). The chromatograms were analyzed, and sequences were edited using the BioEdit Sequence Alignment Editor (v. software. A dendogram was constructed by the neighbor-joining method using a matrix of genetic distances established under the Kimura-two parameter model using MEGA 5.05 [28], [29]. The robustness of each node was assessed by bootstrap analysis using 2,000 pseudo-replicates. All sequenced strains were also compared with sequences available in the GenBank database, including reference strains. A nucleotide sequence Basic Local Alignment Tool (BLAST) search was performed for each NoV strain detected, and strains were also analyzed using the automated typing-tool available on line (, which is recommended for genotyping nomenclature harmonization [30].

The nucleotide sequences obtained in this study were deposited into the National Center for Biotechnology Information (GenBank: under the accession number JX047011-JX047023.

Statistical analysis

Statistical analyses were performed using the software BioEstat 5.0 [31]. The G test (G) was applied to analysis correlating the ages of children to NoV detection. Odds Ratio (OR) was used to compare the prevalence between the Brazilian school-vacation months (January, February and July) and the other months of the year; p-values≤0.05 were considered to be statistically significant.


Of the 159 samples collected, 81 and 78 came from 50 diarrheic and 57 non-diarrheic children, respectively. In the diarrheic group, one, two, three, and four stool samples were collected from 30, 12, 5, and 3 children, respectively. All samples from non-diarrheic children were negative for all enteric viruses tested. NoV, SaV and HAstV were detected in 23.5% (19/81) of fecal samples obtained from diarrheic children. NoV was the most frequently detected pathogen (19.7%-16/81), followed by SaV (2.5%-2/81) and HAstV (1.2%-1/81).

Of the samples that were positive for NoV, eight (50%) were detected by both EIA and PCR (with primers specific for the polymerase and capsid region); seven (43.7%), only by PCR; and one (6.3%), by EIA only (Table 1).

Table 1. Detection of norovirus in fecal specimens from diarrheic children from the Abacatal “Quilombola” Community, according to the methodology used. April/2008 to July/2010.

NoV genotype characterization showed a broad genetic diversity and a significant circulation of genotypes other than the GII-4. Partial nucleotide sequencing of the polymerase gene using the primers Mon 431–433 classified 12 strains as GII-4 (5–41.7%), GII-6 (3–25%), GII-7 (2–16.7%), and GII-17 (1–8.3%) and one strain as GI-2 (1–8.3%). Using primers for the capsid region (partial VP1 gene), 12 samples were genetically characterized as follows: six (50%) as GII-4 [variants 2006b (n = 3) and 2010 (n = 3)], three (25%) as GII-6, two (16.7%) as GII-17, and one (8.3%) as GII-20. Ten of these positive samples were characterized by both polymerase and capsid genes (ORF1/ORF2 partial nucleotide sequencing). Nine strains were classified as the same genotype by both analyzed regions; however, one strain grouped in a different genotype: GII-7/GII-20 (Figure 2a and 2b). The two SaV-positive samples were sequenced and classified as genotypes GI-1 and GII-2, and the HAstV strain as genotype 3.

Figure 2. Dendograms constructed using the partial region of the norovirus RNA polymerase sequence (ORF1-B) (A) and capsid region (ORF2-D) (B) amplified from 14 strains from diarrheic children of Abacatal “Quilombola” Community, metropolitan region of Belém, Pará State, Brazil.

April/2008 to July/2010. Prototype strains are presented together with strains from other locations. The number above each branch corresponds to the bootstrap value (2,000 replicates). The scale bar is proportional to the genetic distance. Study samples were marked (♦): PA-local/Qui-code in the study+Fx-Number of the stool/country/month+year of collection.

NoV was detected primarily in fecal samples from children in the 0–1 year (23.5%) and >1–5 years (20%) age groups, while SaV and HAstV were detected in the >1–5 years age group. No statistically significant difference was observed when comparing the NoV frequencies among each age group (G = 0.0094; p = 0.9228) (Table 2).

Table 2. Distribution by age group of positive samples for norovirus, sapovirus and astrovirus detected in children from the Abacatal “Quilombola” Community.

The monthly distribution of NoV-positive samples denoted a higher prevalence in the months of August/2008, January/2009 and January-February/2010; SaV was detected in the months of February and April/2010, and HAstV was detected in February/2010 (Figure 3). A statistically significant difference was observed when the positivity for NoV in the school-vacation months (January, February and July) was compared with the positivity in other months of the year (OR = 11.86; p<0.0004).

Figure 3. Monthly distribution of the positive cases of norovirus, sapovirus and astrovirus (including their genotypes) detected in stool specimens from diarrheic children of the Abacatal “Quilombola” Community, Pará State, Brazil.

April/2008 to July/2010.

It was observed that samples classified as NoV GII-6 were all detected in August/2008 and GII-17 in January/2009. Most of the GII-4 strains circulate in January and February/2010.


Our study showed that NoV circulates at high rates and found a broad genetic diversity among children with gastroenteritis disease in a semi-closed “Quilombola” community. NoV, SaV and HAstV were only detected in diarrheic individuals, with a positivity of 19.7%, 2.5% and 1.2%, respectively. Rotavirus showed a positivity of 4.7% [32], lower than the rates observed for NoV. Epidemiological studies involving the detection of viral gastroenteritis pathogens in the “Quilombola” population are still rare, as most of the studies have been conducted in hospitals and health units in urban settings.

The results obtained in this study for NoV (19.7%) were higher than the partial results demonstrated in another study that is still under way in different “Quilombola” communities in Espírito Santo State, Southeastern Brazil. In these communities, NoV was detected in both symptomatic (8.5%) and asymptomatic groups (2.3%) [33]. A study conducted in Belém involving hospitalized and non-hospitalized children with AGE in 2003 demonstrated a NoV detection rate of 12.5%, lower than the results obtained in the Abacatal community [10]. In a seroepidemiological survey involving eight isolated Indian tribes in the Amazon region, six located in Pará State, one in Amazonas State and one in Venezuela, the NoV positivity ranged from 39% to 100% [34].

The broad diversity of NoV genotypes found in the present study is in contrast with results obtained previously in other studies conducted in Belém, where GII-4 was the dominant circulating genotype, aside from a low circulation of other genotypes [10], [22]. On the other hand, our results agree with those from previous recent studies where NoV GII-4, albeit predominant, co-circulates with other genotypes [35], [36]. However, most of these studies included either hospitalized children or outbreaks of AGE [10], [37], [38], [39].

The genetic diversity of NoV was recently described in two studies: one that tested fecal samples from several Brazilian states and one that examined specimens from Rio de Janeiro State [40], [41]. Those authors observed a predominance of GII-4 (78% and 80.7%, respectively), which co-circulated with other genotypes. Some of these genotypes (GII-6, GII-7, and GII-17) were also detected in the Abacatal community, in addition to genotype GII-20. One NoV strain (QUI 38 F1) showed different genotype specificities for its polymerase and capsid regions (GII-7/GII-20), revealing a recombination event by partial sequencing of the ORF1/2 junction region and SimPlot analysis [42].

The GII-4 variants (2006b and 2010) detected in this study have a worldwide distribution and are mainly related to outbreaks and epidemics of AGE [43], [44]. The GII-4 2010 variant showed a 99% similarity with the nucleotide sequences of samples described in 2009, such as GII-4 New Orleans, which is also associated with AGE outbreaks [45].

One child developed two NoV infections: one by genotype GII-17, in August/2008, and a subsequent infection by GII-6, in January/2009. This finding highlights the concept that naturally induced cross-protection does not occur in NoV infections. Interestingly, both genotypes had been previously detected in Brazil in the states of Acre and Rio de Janeiro, in 2005 and 2007, respectively [40], [46]. The genotype GII-6 has also been detected in studies conducted in China (2008–2009), Japan and India (2007–2009) [47], [48], [49].

One isolate of NoV GI-2 was also found. This genotype is detected less often compared to NoV GII. In a surveillance study conducted in Belém city [10], only one sample was found to be GI-4, while in Rio de Janeiro, two GI-2 samples were among hospitalized children during a period of four years (2005 to 2008) [37].

Most of the NoV positive cases were detected during the Brazilian school vacation months (January, February and July) or just thereafter (August). It is likely that, in this period, children and several families resident in the “Quilombola” community are more likely to travel outside the village, providing more exposure to infection and a greater possibility for introducing NoV to the community. The OR value demonstrated that the probability of NoV infection in the community is highest in the vacation months, with individuals almost 12 times more likely to be affected by the disease. However, during 2009, fecal specimens were not collected in certain months as a result of heavy rainfall, which posed some logistical problems for gaining access to the community. The lack of complete information prevented us from properly assessing seasonality. The fact that some diarrheic cases were missed also represents a limitation of the study.

The search for SaV is still limited in Brazil, with few studies conducted to date. In the “Quilombola” community, SaV accounted for 2.5% of the pathogens detected in the diarrheic cases. This result is lower than those previously observed in Belém (3%, 4.9%, and 8.9%) and higher than rates observed in Salvador (0.7%) [10], [50], [51], [52].

The two cases that were positive for SaV were detected in the same child during diarrheic episodes, in two separate months (February and April) and with two different genotypes (GI-1 and GII-2). Thus, the first infection may not have elicited immunity that was sufficient to prevent reinfection.

A single sample was HAstV-3-positive. This genotype has been detected only rarely in Belém: in a study conducted for 18 years in Belém, this type was detected in 4.2% of the positive samples, while genotype 1 was detected in 60.6% of cases [53].

Our study provided evidence of the co-circulation of major enteric viral pathogens in a semi-closed African-descendant community, underscoring the need for a continuous surveillance, especially considering the relatively recent introduction of rotavirus vaccination in Brazil. The NoV-related findings (high rate, broad diversity, reinfection) are relevant and, as a consequence, a challenge for current efforts toward the development of a vaccine.


The authors would like to thank the valuable technical support provided by Dielle Monteiro, Ian Lima, Antônia Mendonça, Ariane Mendonça, Vanessa Pereira, Lorena Martins and João Lima. They are also grateful to the Geoprocessing Laboratory (LABGEO), especially to the technician Fabricio Dias and to the Library staff, both of Evandro Chagas Institute. They also thank the infants, their families, health workers and community leaders of the Abacatal “Quilombola” Community. Comments on the manuscript from Dr. Pedro Vasconcelos were gratefully acknowledged.

Author Contributions

Care and collection of clinical data of children: CSO ECA. Conceived and designed the experiments: JDPM YBG. Performed the experiments: GCA MSSL JAMS TMF JMH DSO LSS JHLK. Analyzed the data: GCA JAMS ACL YBG. Contributed reagents/materials/analysis tools: JDPM YBG. Wrote the paper: GCA JDPM JAMS TMF ACL YBG.


  1. 1. Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, et al. (2010) Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet 375: 1969–1987.
  2. 2. Wardlaw T, Salama P, Brocklehurst C, Chopra M, Mason E (2010) Diarrhoea: why children are still dying and what can be done. Lancet 375: 870–872.
  3. 3. Levidiotou S, Gartzonika C, Papaventsis D, Christaki C, Priavali E, et al. (2009) Viral agents of acute gastroenteritis in hospitalized children in Greece. Clin Microbiol Infect 15: 596–598.
  4. 4. Glass RI, Bresee B, Jiang J, Gentsch T, Ando R, et al. (2001) Gastroenteritis viruses: an overview. Novartis Found Symp 238: 5–25.
  5. 5. Vernacchio L, Vezina RM, Mitchell AA, Lesko SM, Plaut AG, et al. (2006) Diarrhea in American infants and young children in the community setting: incidence, clinical presentation and microbiology. Pediatr Infect Dis J 25: 2–7.
  6. 6. Blanton LH, Adams SM, Beard RS, Wei G, Bulens SN, et al. (2006) Molecular and epidemiologic trends of caliciviruses associated with outbreaks of acute gastroenteritis in the United States, 2000–2004. J Infect Dis 193: 413–421.
  7. 7. Svraka S, Duizer E, Vennema H, Bruin E, Van der Veer B, et al. (2007) Etiological role of viruses in outbreaks of acute gastroenteritis in the Netherlands from 1994 through 2005. J Clin Microbiol 45: 1389–1394.
  8. 8. Bruggink L, Sameer R, Marshall J (2010) Molecular and epidemiological characteristics of norovirus associated with community-based sporadic gastroenteritis incidents and norovirus outbreaks in Victoria, Australia, 2002–2007. Intervirol 53: 167–72.
  9. 9. Karst SM (2010) Pathogenesis of noroviruses, emerging RNA viruses. Viruses 2: 748–781.
  10. 10. Aragão GC, Oliveira DS, Santos MC, Mascarenhas JDP, Oliveira CS, et al. (2010) Molecular characterization of norovirus, sapovirus and astrovirus in children with acute gastroenteritis from Belém, Pará, Brazil. Rev Pan-Amaz Saúde 1: 149–158.
  11. 11. Gonzalez-Galan V, Sánchez-Fauqier A, Obando I, Montero V, Fernandez M, et al. (2011) High prevalence of community-acquired norovirus gastroenteritis among hospitalized children: a prospective study. Clin Microbiol Infect 17: 1895–1899.
  12. 12. Lopman BA, Hall AJ, Curns AT, Parashar UD (2011) Increasing rates of gastroenteritis hospital discharges in US adults and the contribution of norovirus, 1996–2007. Clin Infect Dis 52: 466–474.
  13. 13. Wiegering V, Kaiser J, Tappe D, Weissbrich B, Morbach H, et al. (2011) Gastroenteritis in childhood: a retrospective study of 650 hospitalized pediatric patients. Int J Infect Dis 15: 401–407.
  14. 14. Zheng DP, Ando T, Fankhauser RL, Beard RS, Monroe SS (2006) Norovirus classification and proposed strain nomenclature. Virol 346: 312–323.
  15. 15. Green KY (2007) Caliciviridae: the noroviruses. In: Knipe DM, Howley PM, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins. pp. 949–978.
  16. 16. Siebenga JJ, Vennema H, Zheng DP, Vinjé J, Lee BE, et al. (2009) Norovirus illness is a global problem: emergence and spread of Norovirus GII.4 Variants, 2001–2007. J Infect Dis 200: 802–812.
  17. 17. Donaldson EF, Lindesmith LC, Lobue AD, Baric RS (2010) Viral shape-shifting: norovirus evasion of the human immune system. Nat Rev Microbiol 8: 231–241.
  18. 18. Zheng DP, Widdowson MA, Glass RI, Vinjé J (2010) Molecular epidemiology of genogroup II-genotype 4 noroviruses in the United States between 1994 and 2006. J Clin Microbiol 48: 168–177.
  19. 19. Lysén M, Thorhagen M, Brytting M, Hjertgvist M, Anderson Y, et al. (2009) Genetic diversity among food-borne and waterborne norovirus strains causing outbreaks in Sweden. J Clin Microbiol 47: 2411–2418.
  20. 20. Bae JY, Lee JS, Shin MH, Lee SH, Hwang IG (2011) Effect of wash treatments on reducing human norovirus on iceberg lettuce and perilla leaf. J Food Prot 74: 1908–1911.
  21. 21. Silva JAN (2007) Condições Sanitárias e de Saúde em Caiana dos Crioulos, uma Comunidade Quilombola do Estado da Paraíba. Saúde Soc 16: 111–124.
  22. 22. Siqueira JAM, Linhares AC, Oliveira DS, Lucena MSS, Wanzeller AL, et al. (2011) Evaluation of third-generation RIDASCREEN enzyme immunoassay for the detection of norovirus antigens in stool samples of hospitalized children in Belém, Pará, Brazil. Diagn Microbiol Infect Dis 71: 391–395.
  23. 23. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-Van Dillen PM, et al. (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28: 495–503.
  24. 24. Jiang X, Huang PW, Zhong WM, Farkas T, Cubitt DW, et al. (1999) Design and evaluation of a primer pair that detects both Norwalk and Sapporo-like caliciviruses by RT-PCR. J Virol Methods 83: 145–154.
  25. 25. Anderson AD, Garret VB, Sobel J, Monroe SS, Fankhauser RL, et al. (2001) Multistate outbreak of Norwalk-like virus gastroenteritis associated with a common caterer. Am J Epidemiol 154: 1013–1019.
  26. 26. Noel JS, Lee TW, Kurtz JB, Glass RI, Monroe SS (1995) Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing. J Clin Microbiol 33: 797–801.
  27. 27. Vinjé J, Hamidjaja RA, Sobsey MD (2004) Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses. J Virol Met 116: 109–117.
  28. 28. Felsenstein J (1995) PHYLIP (Phylogeny Inference Package) Version 3.57c. Available: Accessed: 5 Nov 2012.
  29. 29. Tamura K, Petreson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likehood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2736–2739.
  30. 30. Kroneman A, Vennema H, Deforche K, Avoort HV, Peñaranda S, et al. (2011) An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol 51: 121–125.
  31. 31. Ayres M, Ayres Júnior M, Ayres DL, Santos AAS (2007) BioEstat 5.0: Aplicações estatísticas nas áreas das ciências biológicas e médicas. Belém-PA: Mamirauá Press. 364 p.
  32. 32. Kayano JHL, Aragão GC, Moura A, Araujo EC, Rodrigues IRC, et al. (2010) Ocorrência de rotavírus e parasitas intestinais em crianças com diarreia aguda na Comunidade Quilombola do Abacatal, Ananindeua, Pará. In: XVI Congresso Brasileiro de Infectologia Pediátrica.
  33. 33. Vicentini F, Gomes YM, Barreira DM, Costa MS, Denadai W, et al. (2011) Molecular characterization of noroviruses from children in the “quilombola” communities in the north of Espirito Santo State, Brazil. In: XXII National Meeting of Virology and VI Mercosur Meeting of Virology. Virus Reviews & Research 16: 159–159.
  34. 34. Gabbay YB, Glass RI, Monroe SS, Carcamo C, Estes MK, et al. (1994) Prevalence of antibodies to Norwalk Virus among Ameridians in isoleted Amazonia Communities. Am J Epidemiol 139: 728–733.
  35. 35. Pang XL, Preiksaitis JK, Wong S, Li V, Lee BE (2010) Influence of novel norovirus GII.4 variants on gastroenteritis outbreak dynamics in Alberta and the Northern Territories, Canada between 2000 and 2008. PloS One 5: e11599.
  36. 36. Siebenga JJ, Lemey P, Pond S, Rambout A, Vennema H, et al. (2010) Phylodynamic reconstruction reveals norovirus GII.4 epidemic expansions and their molecular determinants. PLoS Pathog 6: e1000884.
  37. 37. Ferreira MSR, Xavier MPTP, Fumian TM, Victoria M, Oliveira SA, et al. (2008) Acute gastroenteritis cases associated with Noroviruses infection in the state of Rio de Janeiro. J Med Virol 80: 338–344.
  38. 38. Mathijs E, Denayer S, Palmeira L, Botteldoorn N, Scipioni A, et al. (2011) Novel norovirus recombinants and of GII.4 sub-lineages associated with outbreaks between 2006 and 2010 in Belgium. Virol J 8 10.1186/1743-422X-8-310.
  39. 39. Zeng M, Xu X, Zhu C, Chen J, Zhu Q, et al. (2012) Clinical and Molecular Epidemiology of Norovirus Infection in Childhood Diarrhea in China. J Med Virol 84: 145–151.
  40. 40. Fioretti JM, Ferreira MSR, Victoria M, Vieira CB, Xavier MPTP, et al. (2011) Genetic diversity of noroviruses in Brazil. Mem Inst Oswaldo Cruz 106: 942–947.
  41. 41. Ferreira MSR, Victoria M, Carvalho-Costa FA, Vieira CB, Xavier MPTP, et al. (2010) Surveillance of Norovirus Infections in the State of Rio De Janeiro, Brazil 2005–2008. J Med Virol 82: 1442–1448.
  42. 42. Fumian TM, Aragão GC, Mascarenhas JDP, Kaiano JH, Siqueira JAM, et al. (2012) Detection of a novel recombinant strain of norovirus in an African-descendant community from the Amazon region of Brazil in 2008. Arch Virol 157: 2389–2392.
  43. 43. Eden JS, Bull RA, Tu E, McIver CJ, Lyon MJ, et al. (2010) Norovirus GII.4 variant 2006b caused epidemics of acute gastroenteritis in Australia during 2007 and 2008. J Clin Virol 49: 265–271.
  44. 44. Zhou X, Li X, Sun L, Mo Y, Chen S, et al. (2012) Epidemiological and molecular analysis of a waterborne outbreak of norovirus GII.4. Epidemiol Infect 8: 1–8.
  45. 45. Vega E, Barclay L, Gregoricus N, Williams K, Lee D, et al. (2011) Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks, United States. Emerg Infect Dis 17: 1389–1395.
  46. 46. Barreira DM, Ferreira MS, Fumian TM, Checon R, Sadovsky AD, et al. (2010) Viral load and genotypes of noroviruses in symptomatic and asymptomatic children in southeastern Brazil. J Clin Virol 47: 60–64.
  47. 47. Chan-It W, Thongprachum A, Okitsu S, Nishimura S, Kikuta H, et al. (2011) Detection and genetic characterization of norovirus infections in children with acute gastroenteritis in Japan, 2007–2009. Clin Lab 57: 213–220.
  48. 48. Nataraju SM, Pativada M, Chatterjee D, Nayak MK, Ganesh B, et al. (2011) Molecular epidemiology of norovirus infections in children and adults: sequence analysis of region C indicates genetic diversity of NVGII strains in Kolkata, India. Epidemiol Infect 139: 910–918.
  49. 49. Zeng M, Gong Z, Zhang Y, Zhu Q, Wang X (2011) Prevalence and genetic diversity of norovirus in outpatient children with acute diarrhea in Shanghai, China. Jpn J Infect Dis 64: 417–422.
  50. 50. Nakamura LS, Oliveira DS, Silva PF, Lucena MS, Mascarenhas JDP, et al. (2006) Molecular characterization of calicivirus in feces of children with acute diarrhea, attending a public hospital, in Belém, Pará. In: XVII National Meeting of Virology. Virus Reviews & Research 11: 95–95.
  51. 51. Siqueira JAM, Nascimento IS, Oliveira DS, Soares LS, Aragão GC, et al. (2010) Caracterização molecular de calicivírus humano em amostras fecais de crianças atendidas em um posto de saúde de Belém, Pará entre os anos de 1998 e 2000. In: 62a Reunião Anual da Sociedade Brasileira para o Progresso da Ciência.
  52. 52. Xavier MPTP, Oliveira SA, Ferreira MSR, Victoria M, Miranda V, et al. (2009) Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil. Braz J Med Biol Res 42: 438–444.
  53. 53. Gabbay YB, Leite JPG, Oliveira DS, Nakamura LS, Nunes MRT, et al. (2007) Molecular epidemiology of astrovirus type 1 in Belém, Brazil, as an agent of infantile gastroenteritis, over a period of 18 years (1982–2000): Identification of two possible new lineages. Virus Res 129: 166–174.