Noroviruses associated with outbreaks of acute gastroenteritis in the State of Rio Grande do Sul, Brazil, 2004–2011

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Journal of Clinical Virology 61 (2014) 345–352

Contents lists available at ScienceDirect

Journal of Clinical Virology

jou rn al hom epage: www.elsev ier .com/ locate / j cv

oroviruses associated with outbreaks of acute gastroenteritis in thetate of Rio Grande do Sul, Brazil, 2004–2011

uliana da Silva Ribeiro de Andradea,∗, Monica Simões Rochaa,elipe Aníbal Carvalho-Costaa, Julia Monassa Fioretti a,aria da Penha Trindade Pinheiro Xaviera, Zenaida Maria Alves Nunesb,

eanice Cardosoc, Alexandre Madi Fialhoa, José Paulo Gagliardi Leitea,arize Pereira Miagostovicha

Laboratory of Comparative and Environmental Virology – Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Ministry of Health, Rio de Janeiro, RJ, BrazilCentral Laboratory of State of Rio Grande do Sul, State Foundation of Production and Health Research, Porto Alegre, RS, BrazilDepartment of Epidemiologic Surveillance, State Department of Health, Porto Alegre, RS, Brazil

r t i c l e i n f o

rticle history:eceived 31 March 2014eceived in revised form 19 August 2014ccepted 26 August 2014

eywords:cute gastroenteritis outbreaksorovirusesenotypesII variantsouthern Brazil

a b s t r a c t

Background: Acute gastroenteritis norovirus (NoV) in a country of continental dimensions like Brazil hasresulted in under-reporting of the number of outbreaks, as well as the genotypes associated.Objectives: To demonstrate the role of NoV in outbreaks occurring in the State of Rio Grande do Sul,Southern Brazil, we determined its prevalence, as well as the genotypes associated, and evaluated clinicaland epidemiological aspects.Study design: NoV investigation was carried out in rotavirus group A negative stool samples from 2265patients from 741 outbreaks that occurred in the State of Rio Grande do Sul, Brazil, during a period ofeight years (2004–2011). NoV detection and nucleotide sequencing for genotype characterization wascarried by using sets of primers targeting a conservative Rd-Rp polymerase genome region and the viralcapsid gene, respectively.Results: NoVs were detected in 817 stool samples (36.1%) and associated with 327 outbreaks (44.1%). NoVGII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21; and GI.1 and GI.3 were characterized.GII.4 was the most frequently detected (72.3%), with five variants identified (Asia 2003, Hunter 2004,

Yerseke 2006a, Den Haag 2006b, New Orleans 2009). This study describes the first detection of GI.1 andGII.13 and GII.15 in Brazil and demonstrates NoV winter-spring seasonality in this region of the country.Conclusions: NoVs were responsible for almost 50% of outbreaks, with about 70% of them resulting fromgenotype GII.4 and its variants. The seasonality observed could help health authorities to establish asystem of active surveillance in order to reduce NoV impact especially in congregate settings.

. Background

Noroviruses (NoVs) (genus Norovirus) belong to the family Cali-iviridae and possess a single-stranded positive-sense RNA genomehat encodes three open-reading frames (ORF 1-3) [1]. Genetically

Abbreviations: AG, acute gastroenteritis; NoVs, noroviruses; ORF, open-readingrame; RVA, rotavirus A; RS, Rio Grande do Sul; EIA, enzyme immunosorbent assay;AGE, polyacrylamide gel electrophoresis; nt, nucleotide.∗ Corresponding author at: Avenida Brasil, 4365, Manguinhos, CEP 21040-360 Rioe Janeiro, RJ, Brazil. Tel.: +55 21 2562 1875; fax: +55 21 2562 1851.

E-mail addresses: [email protected], juliana [email protected]. de Andrade).

ttp://dx.doi.org/10.1016/j.jcv.2014.08.024386-6532/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

diverse, NoVs are divided into five genogroups (GI–GV), of whichGI, GII, and GIV have been shown to infect humans [2]. Thesegenogroups are divided into at least 35 genotypes and the geneticclassification is based on phylogenetic analysis of the capsid region(ORF-2) [2].

The major impact of NoV infections in public health has beendemonstrated by their global distribution, which are primarilyresponsible for water- and food-borne outbreaks of acute gastroen-teritis (AG) worldwide [3–5]. NoVs are transmitted by the fecal–oralroute and most AG outbreaks are described in confined places and

large gatherings, such as cruises, resorts and nursing homes [6–8].

The emergence of new variants of NoV GII.4 is associated withepidemics of large public health impact. The rapid evolution andspread of these viruses require frequent studies to determine

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46 J.d.S.R. de Andrade et al. / Journal

revalence, molecular epidemiology, and geographical distribu-ion, in order to establish measures to prevent AG outbreaks androvide data that could contribute to the development of an effec-ive vaccine for the control of AG [9–11].

In Brazil, despite studies conducted in different regions demon-trating the high prevalence and diversity of NoVs circulatingn the country, there is still no active laboratory surveillanceetwork for the diagnosis of these viruses [11–14]. In South-rn Brazil, only one study has reported NoV GII in Porto Alegreity [15], despite the several AG outbreaks with no etiologicefined whose initial investigation has focused on group A rotavirusRVA).

. Objectives

This study aimed to associate NoV infection to AG outbreakshat occurred in the state of Rio Grande do Sul (RS), Southernrazil, over eight years (2004–2011), providing epidemiologicalnd molecular characterization of genotypes and variants respon-ible for those outbreaks. Clinical characteristics of the AG casesere also assessed.

. Study design

.1. Study area

The State of Rio Grande do Sul is the coldest Brazilian state,ocated between latitudes N – 27◦04′ and S – 33◦45′. The climates subtropical and the four seasons are well marked. The meanemperature varies from 14 to 17 ◦C (winter) and 23 to 25 ◦C (sum-

er) and the rainy season includes the months of September toovember. It has about 11 million inhabitants and covers a landrea of 268,781,896 km [2], comprising 496 municipalities groupednto seven mesoregions [16].

able 1rimers used in the polymerase chain reaction for amplification of the genome of human

Primers Sequence (5′-3′)a Orientati

Primers to detection NoVb

Mon431f TGG ACI AGR GGI CCY AAY CA +

Mon432e TGG ACI CGY GGI CCY AAY CA +

Mon433f GAA YCT CAT CCA YCT GAA CAT −

Mon434 e GAA SCG CAT CCA RCG GAA CAT −

Primers to Classification of Genogroupsd

Cap Ae GGC WGT TCC CAC AGG CTT −

Cap B2 e TAT GTI GAY CCW GAC AC +

Cap B1e TAT GTT GAC CCT GAT AC +

Cap Cf CCT TYC CAK WTC CCA YGG −

Cap D3 f TGY CTY ITI CCH CAR GAA TGG +

Cap D1f TGT CTR STC CCC CAG GAA TG +

Primers to Classification of Genogroups g

G1SKFe CTGCCCGAATTYGTAAATGA +

G1SKRe CCAACCCARCCATTRTACA −

G2SKFf CNTGGGAGGGCGATCGCAA +

G2SKR f CCRCCNGCATRHCCRTTRTACAT −

Primers to classification of NoV GII.h

EVP2Ff GTR CCR CCH ACA GTT GAR TCA +

EVP2Rf CCG GGC ATA GTR GAY CTR AAG AA −

a IUPAC code to indicate degenerate positions: I, iosine; R, purine (A/G); Y, pyrimidine

b Reference: [19].c Pol, Polimerase.d Reference: [20].e Genogroup I.f Genogroup II.g Reference: [21].h Reference: [22].i Primer positions based on Norwalk (M87661) for NoV GI.j Primer positions based on Lordsdale (X86557) for NoV GII.

ical Virology 61 (2014) 345–352

3.2. Clinical specimens

Stool samples were obtained in the context of AG outbreak mon-itoring under the Unified Health System (SUS-Brazil) comprisinga hierarchical network in which samples are provided by spon-taneous demand from outpatients assisted in different MunicipalHealth Centers. These samples, through the Central Laboratory ofthe State (LACEN-RS), are forwarded to the Regional Rotavirus Ref-erence Laboratory, Laboratory of Comparative and EnvironmentalVirology (RRRC-LVCA) for investigating RVA presence by polyacryl-amide gel electrophoresis (PAGE) [17] and enzyme immune-assay(EIA) (Premier Rotaclone, Meridian Bioscience, Inc.; Ridascreen, R-Biopharm).

3.3. Case definition and inclusion criteria

For this study, one stool sample of 2265 patients with a neg-ative diagnosis for RVA from 741 AG outbreaks that occurred inthe period 2004–2011, in 97 out of 496 municipalities of the state,were selected for NoV investigation. AG outbreaks were charac-terized by the Surveillance Epidemiologic Service as an increasein the number of cases above the expected range for the popula-tion involved in that specific period following the definition of theBrazilian Ministry of Health [18].

To characterize NoV genotypes, 112 samples were selected on ageographic and temporal distribution, in which 50% of the munic-ipalities of each mesoregion of State of Rio Grande do Sul wereselected by random drawing in Excel 2010. One sample of eachoutbreak per year was selected afterwards.

3.4. Fecal suspension, RNA extraction, and reverse-transcription

Fecal suspensions were prepared in 10% Tris/HCl/Ca2+ (0.01 M,pH 7.2). The extraction of viral RNA was performed using themethodology of the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia,CA, USA) according to the manufacturer’s instructions.

norovirus.

on Localization Region Position

RNA Polc B 5093–5112i

RNA Pol B 5093–5112i

RNA Pol B 5285–5305i

RNA Pol B 5285–5305i

VP1 D 6897–6914i

VP1 D 6738–6754i

VP1 D 6738–6754i

VP1 D 6667–6684j

VP1 D 6432–6452j

VP1 D 6342–6451j

VP1 C 5671–5689i

VP1 C 5058–5076i

VP1 C 5401–5423j

VP1 C 5401–5423j

VP1 P2 6381–6403j

VP1 P2 6381–6403j

(C/T); S, C/G; I, A/T/C/G; W, A/T; K, G/T; H, A/T/C.

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Complementary DNA (cDNA) was synthesized using the Highapacity cDNA Reverse Transcription kit (Applied Biosystems, Fos-er City, CA, USA) according to the manufacturer’s instructions.

.5. Detection and molecular characterization

Table 1 resume the list of primers used for NoV detection andharacterization showing their sequences and genome positionsccording the region target and the genogroup.

For NoV diagnosis, a set of primers for amplifying the partialegion of the ORF-1 that encodes RNA-dependent RNA polymeraseRp-Rd) known as Region B was used [19].

For NoV genotype characterization by nucleotide sequencing,ositive stool samples were processed initially by using a set ofrimers specific for GI and GII targeting the ORF-2 (region D) [20].amples that did not amplify in the D region were processed withhe amplification of the C region (ORF-2) using primers for GInd GII [21]. Samples characterized as GII.4 were processed withrimers for amplification of the region P2 (hypervariable region ofP1) [22]. Amplicon purification, nucleotide sequencing, edition,lignment, and phylogenetic analysis of nucleotide sequences wereerformed according to a previous study [11]. The statistical signif-

cance of phylogenetic trees obtained was estimated using 2000ootstrap replications.

The nucleotide sequence data reported in this study were sub-itted to GenBank under the numbers: KJ179659–KJ179807 and

J195431–KJ195435.

.6. Statistical analysis

Statistical analyses were performed using Epi Info 7 [23]. Ratesf NoV positivity and association between the frequency of symp-oms and seasonality were compared using the chi-square testithout correction, and when the number of samples was less than

0, the Fisher exact test was used. For the analysis of the results byge group, the chi-square test for linear trend was used. A statisticalignificance was established at p ≤ 0.05.

. Results

.1. Diagnostic and molecular characterization

NoVs were detected in 36.1% (817/2265) of the samples andere associated with 44.1% (327/741) of AG outbreaks, with 33.3%

109/327) of them being reported in 2006 when a higher (Table 2)etection rate (57.8%; 380/658) of positive cases was observed.

Twelve genotypes of NoV were characterized circulating inhe State of RS during the studied period. GII.4 (81/112; 72.3%)as the most frequent, followed by GII.6 (n = 11; 9.8%), GII.3

n = 6; 5.4%), GII.17 (n = 3; 2.7%), GII.12 (n = 2; 1.8%), GII.15 (n = 2;.8%), GII.2 (n = 2; 1.8%), GII.13 (n = 1; 0.9%), GII.21 (n = 1; 0.9%),nd GII.14 (n = 1; 0.9%). Two samples were genotyped as GI.1nd GI.3 (Figs. 2 and 3). The characterization of variants of the6 GII.4 samples revealed the predominance of Den Haag 2006bariant, detected in 47.8% (22/46) of samples, then the variantew Orleans 2009 was detected in 41.3% (19/46), Yerseke 2006a.3% (2/46), Hunter 2004 4.3% (2/46), and Asia 2003 2.2% (1/46).

GII.4 was found in all years of the studied period, with higheretection in 2006 (37%; 30/81), and in this year occurred theo-circulation of two variants (Table 2). In the other years, a replace-ent of GII.4 variants was observed. The genotypes cocirculation of

oV GII was observed in all years, especially in the years 2007, 2008,nd 2010, when five different genotypes were detected (Table 2).

A total of 112 out of 327 outbreaks were characterized with2.3% (81/112) of them being associated with the GII.4 genotype Ta

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Fig. 1. Distribution of monthly per year positive cases and frequency of norovirus that occurred in the state of Rio Grande do Sul from 2004 to 2011.

Table 3Distribution of norovirus infections according to the seasons over a period of 8 years(2004–2011).

Season No. of positive samples/studied (%)

Summer 135/504 (26.8)Fall 86/357 (24.1)Winter 307/714 (43)Spring 289/690 (41.9)

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Table 4Frequency detection of norovirus according to the age group, 2004–2011.

Age group in years No. of positive samples/studied (%)

≤1 157/514 (30.5)>1 ≤ 2 153/421(36.3)>2 ≤ 5 104/344 (30.2)>5 ≤ 10 50/166 (30.1)>10 ≤ 20 52/143 (36.4)>20 ≤ 50 197/472 (41.7)>50 94/172 (54.6)No informations 10/33 (30.3)

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tatistically significant differences between Summer and Spring: p ≤ 0.05; Summernd Winter: p ≤ 0.05; Fall and Spring: p ≤ 0.05 and Fall and Winter: p ≤ 0.05. p value:hi-square test without correction.

data not shown). Table 2 presents the temporal distribution ofhese outbreaks showing also GII.4 variants identified.

.2. Epidemiological and clinical aspects

Monthly distribution of NoV infection revealed a greater num-er of cases during the colder months of the year (Fig. 1) and thenalysis over the years has shown a seasonal pattern (p ≤ 0.05) with

significantly higher detection in spring and winter (Table 3).

able 5linical signs and symptoms in 932 patients infected and not infected with norovirus (No

Clinical features ≤1 year >1 year ≤ 10

NoV(+)a (n = 47) NoV(−)b (n = 71) NoV(+)a (n = 1

Presence of mucus in feces 11 (23.4) 25 (35.2) 19 (13.1)

Presence of blood in feces 1 (2.1) 6 (8.4) 4 (2.7)

Fever (≥37.5 ◦C) 20 (42.5) 33 (46.5) 60 (41.4)

Vomit 35(74.5) 38 (53.5) 104(71.7)

Abdominal pain 20 (42.5) 39 (54.9) 102(70.3)

Coryza 14 (29.8) 25 (35.2) 34 (23.4)

Cough 12 (25.5) 25 (35.2) 36 (24.8)

Dehydration 1 (2.1) 13 (18.3) 19 (13.1)

oV, noroviruses.oldface indicates a p value ≤0.05, Chi-square or Fisher’s exact test, as appropriate.amples percentages (%) are indicated in brackets.

a Norovirus positive samples.b Norovirus negative samples.

Total 817/2265 (36.1)

Chi-square for linear trend, p ≤ 0.05.

Regarding age groups affected, adults older than 20 years hada higher rate of NoV infection and the detection rate of NoVsrose significantly with increasing age (p ≤ 0.05) (Table 4). In all

age groups, vomiting was significantly more frequent among NoV-positive subjects (p ≤ 0.05). Additionally, presence of blood andmucus in feces and cough was significantly more frequent in NoV-negative patients (Table 5).

V), according to age group.

years >10 years Total

45) NoV(−)b (n = 225) NoV(+)a (n = 253) NoV(−)b(n = 191)

47(20.9) 39 (15.4) 28 (14.6) 16912 (5.3) 1 (0.4) 14(7.3) 3893 (41.3) 52 (20.5) 50 (26.2) 308

112 (49.8) 149(58.9) 85 (44.5) 523128 (56.9) 167 (66.0) 114 (59.7) 570

59 (26.2) 22 (8.7) 22 (11.5) 17645 (20.0) 15 (5.9) 24(12.6) 15737 (16.4) 49 (19.4) 38 (19.9) 157

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Fig. 2. Phylogenetic tree analysis based on the partial region (D) of the gene encod-ing the VP1 protein of 57 samples belonging to Norovirus genogroup II (GII).Phylogenetic tree based on the partial nucleotide sequence (214 bp) of the codingregion for the protein VP1 norovirus GII reconstructed from the Neighbor-Joining

ical Virology 61 (2014) 345–352 349

5. Discussion

Several studies conducted in Brazil have demonstrated theimpact of NoV infections in cases of GA from outbreaks and sporadiccases, including hospitalized children [13,24,25]. A retrospectivestudy conducted in a nursery in the state of Rio de Janeiro, alsoevaluating a long period of time, also made clear the importanceof NoV infections in children up to 4 months old kept indoors [26].However, this study performed in a period of 8 years shows for thefirst time in Brazil the importance of NoV outbreaks in the pop-ulation as a whole in a temperate region, as well as the impactof the introduction of new variants in a given region. The associ-ation of NoV to almost half of the AG outbreaks emphasizes thepublic health importance of those viruses in the country and theneed to establish a NoV diagnosis network in laboratories aroundthe country as observed in other countries from Europe or UnitedStates, where this high proportion is also observed [27]. Unfortu-nately, the fragility of the notification system still does not allowthe proper identification of the source of each outbreak so it was notpossible to identify if there were outbreaks of food- or water-borneor person–person origins.

The diversity of NoVs circulating in the state corroborated theimportance of permanently conducting molecular epidemiologicalstudies. Here, NoVs GII.13, GII.15, and GI.1 were first described inLatin America. NoV GII.13, with few reports in the literature, hasbeen associated with outbreaks and hospitalization of children indifferent countries [28–30]. The GII.15 genotype has been responsi-ble for outbreaks in Japan, Turkey, and China [31–33], and GI.1 wasassociated with outbreaks and sporadic cases in countries such asSouth Korea, India, and Italy [34–36]. Due to the larger number ofoutbreaks, a strategy for NoV molecular characterization used inthis study selected one sample/outbreak, not allowing the evalua-tion of occurrence of different genotypes/outbreaks.

The impact of the circulation of GII.4 in the state of Rio Grande doSul was evident as observed in other Brazilian studies [13,26,37,38],and worldwide since the mid-1990s [9,10,39]. The associationof five variants of GII.4 confirms the epidemiological impact ofintroducing new NoV variants into a geographic region [9,40].

The variants Asia 2003 and Hunter 2004, detected in 2004,2005,and 2006, showed a pattern of movement similar to thatwhich occurred in other countries [41,42], although they have beendetected at low frequency. However, variants of GII.4 characterizedin 2006 (Yerseke 2006a and Den Haah 2006b) accounted for 37%of all GII.4. These variants have been identified in many studiesas the cause of outbreaks around the world and are recognizedas pandemic variants [39,43,44]. In this study, Den Haag 2006bvariant was the most frequent, being detected during four years(2006–2009). Its introductions into the state may explain the highrate of detection of NoV as well the prevalence of GII.4 in 2006. Thevariant Yerseke 2006a was detected only in 2009, in contrast tosome studies that report the Yerseke 2006a variant as prevalent insome countries during the period 2005–2006, and then replaced bythe Den Haag 2006b variant in 2006–2007 and remained in circu-lation until the period 2008–2009 [9,43,45]. In 2010 and 2011, theNew Orleans 2009 variant was the only variant detected in the stateof RS. In the United States, this variant was first detected in the win-ter of 2009–2010, and involved a large number of outbreaks caused

by GII.4 in 2010 [22]. The substitution of GII.4 variants observed inNoV outbreaks exposes the population susceptible to a virus forwhich it has poor immunity, explaining the frequent involvement

method with the model Kimura 2-parameter and 2000 bootstrap replicates. Thesamples are marked with a black circle. The bootstrap values are indicated in thephylogenetic tree, values less than 70% are not represented. The bar at the bottomof the figure is proportional to the genetic distance.

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Fig. 3. Phylogenetic tree analysis based on the partial region (P2) of the gene encoding the VP1 protein of 45 samples belonging to Norovirus genogroup II genotype 4 (GII.4).Phylogenetic tree based on partial nucleotide sequence (629 bp) of the coding region of the P2 subdomain of VP1 protein of norovirus GII.4, rebuilt from the Neighbor-Joiningmethod with Kimura 2-model parameters and 2000 bootstrap replicates. The samples are marked with a black circle. Bootstrap values are shown in the phylogenetic tree,valore less than 70% are not represented. The bar at the bottom of the figure is proportional to the genetic distances.

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f this genotype in AG outbreaks and its high prevalence worldwide39,43].

In this study, genotype GII.6 was the second-most frequentlyetected, corroborating data of its prevalence in the country11–13], and GII.12, 17, and 21 have also been described recentlyn the country [11,26]. Outbreaks caused by other genotypes ofoV GII are also described although in lower frequency [28,29].he low frequency of NoVs belonging to GI corroborates previ-us studies that also found a low prevalence of this genogroup11,26,37]. Nonetheless, other studies have shown that GI NoVsould be more involved in outbreaks when compared to GII7,46].

In addition to an extensive characterization of the virus, thistudy over 8 years has demonstrated the seasonality of NoV in aemperate climate region. Although the seasonal pattern of NoVs its still controversial [47,48], detection peaks in the driest monthsave been described in other Brazilian studies [13,37] including inhe city of Porto Alegre with a higher NoV detection occurring inpring [15].

Concerning age groups affected, there was a greater detection ofoV in older age groups, but it is important to remember that this

tudy was conducted using samples previously negative for RVA.he diversity of the etiologic profile of AG in infants including aigher rate of infection by other viral agents, such as enteric ade-oviruses, as well as emerging viruses such as human bocaviruses,ichi viruses, and parasitic and bacterial etiologies infections couldxplain the lower rate of detection in younger age groups whenompared to adults [49–51].

Vomiting was the most significant clinical manifestationbserved in individuals infected with NoV, which corroborates pre-ious research showing that vomiting is particularly associatedith NoV infections [13,27]. Symptoms such as abdominal pain can

e difficult to detect in children less than 1 year of age, which mayxplain the lower frequency in this age group.

It was demonstrated that bloody and mucous diarrhea was lessrequent in the NoV-positive patients, confirming previous studiesnd suggesting the presence of enteropathogenic bacteria amongoV-negative patients, although the diagnosis for these pathogensas not been available [21,52].

The global analysis results obtained in this 8-year study thatncluded clinical, epidemiological, and molecular aspects of NoVemonstrates the role of these viruses in cases of an outbreak ofG, contributing to local information necessary for evaluation inase of adoption of norovirus vaccine currently available. The greativersity of NoV, as well as the emergence of new GII.4 variantst regular intervals, emphasizes the need of continuous molec-lar epidemiological surveillance of these viruses in the countrynd the strengthening the surveillance network of the Braziliantates and neighboring countries. Due to the high cost of diagno-is, usually done on a molecular basis, few studies have focusedn the detection and molecular characterization of NoV in Latinmerica [53,54]. Recently, the presence and diversity of NoV wasemonstrated in Paraguayan children [53] with strain similarityanging from 77% to 99% for the genotypes detected when com-ared to nucleotide sequences obtained in this study (data nothown). Additionally, strategies for characterization using differ-nt regions of the genome do not allow comparative analysis.n conclusion, there is a need to establish a low-cost diagnostic

ethod that can be used in a network of active surveillance in theegion.

unding

This study was supported by the PROEX/CAPES, CNPq, andOC/Fiocruz.

[

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Competing interests

No conflicts of interest have been identified.

Ethical approval

This study is part of a project that covers diagnosis, surveillance,and molecular epidemiology of viruses that cause GA approved bythe Ethics Committee of Fiocruz (CEP: 311/06).

Acknowledgements

We thank the Central Laboratory of State of Rio Grande do Sul(LACEN-RS) staff, Marcelle Figueira, Tatiana Rose and Tulio Fumianfor generous contributions. M.P. Miagostovich and J.P.G. Leite arefellows of the Brazilian National Council for Scientific and Techno-logical Development (CNPq).

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