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Journal of Clinical Virology 58 (2013) 378– 384

Contents lists available at ScienceDirect

Journal of Clinical Virology

jo u r n al hom epage: www.elsev ier .com/ locate / j cv

olecular epidemiology of norovirus strains in Paraguayan childrenuring 2004–2005: Description of a possible new GII.4 cluster

aria Eugenia Galeanoa,b, Magaly Martinezb, Alberto A. Amarillab,1,raciela Russomandob, Marize Pereira Miagostovicha, Gabriel I. Parrab,∗,

osé Paulo Leitea

Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Ministry of Health, Rio de Janeiro, RJ, BrazilMolecular Biology and Genetics Department, Health Sciences Research Institute, National University of Asunción, Asunción, Paraguay

r t i c l e i n f o

rticle history:eceived 20 March 2013eceived in revised form 28 June 2013ccepted 12 July 2013

eywords:oroviruscute gastroenteritisaraguayan childrenII.4 norovirus

a b s t r a c t

Background: Noroviruses (NoV) have been shown to be an important cause of morbidity and mortalityin children worldwide, only second after Group A rotaviruses (RVA). In Paraguay, acute gastroenteritis(AGE) is the third cause of mortality in children ≤5 years old.Objectives: To analyze the presence and diversity of NoV in Paraguayan children ≤5 years old presentingAGE.Study design: Three hundred seventy eight fecal samples, negative for pathogenic bacteria and RVA, werecollected from children admitted as ambulatory and hospitalized patients in a large private hospitalfrom Asuncion, Paraguay from 2004 to 2005. The presence and diversity of NoV was determined by twodifferent RT-PCR strategies and nucleotide sequencing.Results: One hundred and sixty one samples were positive for NoV by partial amplification of the viralpolymerase gene (RdRp). No seasonality or differences in the viral prevalence for the different age-groupswere detected. GII and GI NoVs were associated to 58% and 42% of the infections, respectively. The geno-

type was determined in 18% (29/161) NoV-positive samples. The genotypes detected were: GII.4 (18%),GII.17 (18%), GII.6 (14%), GII.7 (14%), GII.3 (10%), GII.5 (3%), GII.8 (3%), GII.16 (3%), GI.3 (14%) and GI.8 (3%).Amplification of the ORF2 from the GII.4 strains showed the presence of a new GII.4 variant.Conclusions: The results showed a continuous circulation of NoV in children throughout the two years ofstudy and an extensive diversity of genotypes co-circulating, highlighting the need for better surveillanceof NoV in Paraguayan children.

© 2013 Elsevier B.V. All rights reserved.

. Background

Acute gastroenteritis (AGE), both epidemic and sporadic, areommon causes of morbidity and mortality in people of all ages1]. According to Black et al., 1.336 million of deaths occur annu-lly in children ≤5 years old due to diarrheic infections worldwide

2]. Noroviruses (NoV) are considered the most common cause ofGE outbreaks in adults worldwide and the second most frequentiral agent causing AGE in young children after group A rotavirus

∗ Corresponding author. Present address: Caliciviruses Section, Laboratory ofnfectious Diseases, NIAID, National Institute of Health, Bethesda, MD, USA.el.: +1 301 402 1258; fax: +1 301 480 5031.

E-mail addresses: gabriel parra@hotmail.com, parrag@niaid.nih.gov,arrag@niaid.nih (G.I. Parra).1 Present address: Departamento de Análises Clínicas, Toxicológicas e Broma-

ológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirãoreto, SP, Brazil.

386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jcv.2013.07.008

(RVA) [3,4]. In developing countries, NoV have been estimated tocause around 1.1 million hospitalizations and up to 200,000 chil-dren deaths annually [5].

NoV are small (27–38 nm in diameter), non-enveloped virusesthat belong to the Caliciviridae family. The single-stranded,positive-sense genome is organized in three open reading frames(ORFs): ORF1 encodes for 6 nonstructural (NS) proteins, includingthe RNA-dependent RNA polymerase (RdRp); ORF2 encodes themajor capsid protein (VP1); and ORF3 encodes the minor capsidprotein (VP2) [6]. The marked genetic diversity presented by NoVprompted the development of multiple protocols for detection andmolecular characterization, which target different regions of theORF1 and ORF2 [7–11].

Based on sequence differences on the VP1, NoV have been classi-

fied into six genogroups (GI–GVI), and multiple genotypes [11,12].NoV from GI, GII, and GIV have been shown to infect humans, beingthe GII.4 strains the most prevalent. Although GII.4 strains havebeen shown to be the main cause of AGE outbreaks worldwide,

Clinical Virology 58 (2013) 378– 384 379

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M.E. Galeano et al. / Journal of

ther NoV strains (e.g. GII.3) have been shown at high incidence inporadic cases of diarrhea in children ≤5 years old [13].

In Paraguay, AGE is considered the third cause of mortality inhildren ≤5 years old [14]. Since 1998, epidemiological studiesave been carried out to determine the role of RVA in the etiologyf non-bacterial AGE infections in Paraguayan children, indicatinghat is responsible for approximately 25% of all AGE infections inhat population [15,16].

. Objectives

To analyze the presence and diversity of NoV in children ≤5ears old with AGE, previously determined negative for RVA andnteric pathogenic bacteria.

. Study design

.1. Sample collection

A total of 927 fecal samples were collected from children ≤5ears old with non-bacterial AGE who were admitted as ambu-atory and hospitalized patients in a large private hospital fromsuncion, Paraguay between January 2004 and December 2005.ll samples were screened for RVA [17]. Approximately 50% of theVA-negative samples from each month were randomly selected

or NoV screening (378 out of 701 RVA-negative samples; 161 col-ected during 2004 and 217 during 2005). In months were a lowumber of samples was originally collected, at least 10 samplesere selected if enough material was available. In only two months

March and May, 2004) less than 10 samples were selected (6 and, respectively) for NoV screening. No data on whether the patientsere linked to outbreaks or serious cases of gastroenteritis was

ollected.

.2. RNA extraction and PCR amplification

Viral RNA was extracted from 10% fecal suspension inris–HCl–Ca++ [0.01 M] (pH: 7.2) buffer using guanidine isoth-ocyanate/silica method, as described by Boom et al. [18].everse transcription was carried out using random primersInvitrogen®, Carlsbad, CA, USA). For NoV detection, genericrimers (Mon431/Mon432 and Mon433/Mon434) that target theegion B of the ORF1 (RdRp gene) [7], were used according tohe conditions described by Beuret et al. [19]. The molecularharacterization was performed by using degenerated primersapA/CapB1/CapB2 and CapC/CapD1/CapD3 that target the VP1ene (region D) as described by Vinjé et al. [9]; and the primersV12Y, JV12i, Ni-R and G1 that target the RdRp gene as describedreviously by Boxman et al. [10]. PCR products were resolved on 2%garose gel electrophoresis, followed by ethidium bromide stain-ng, and the images were obtained using the Kodak Gel Logic 212mage capture system (Carestream Health Inc., Upland, CA, USA).

.3. Nucleotide sequencing and bioinformatic analyzes

PCR products from the region D were purified directly fromhe agarose gels using the QIAquick® Gel extraction kit (QIAGEN®,alencia, CA, USA) in accordance with manufacturer’s instructions.ucleotide sequencing was carried out using both primers andig Dye Terminators v3.0 and an automated sequencer ABI PRISM730 Genetic Analyzer (Applied Biosystems®, Foster City, CA, USA).

he GII.4 strain characterization was performed by sequencinghe PCR product of the entire ORF2 with GII.4-specific primershat target the 5′ and 3′ end of the ORF2. Internal primers wereesign to cover nearly the full ORF2 (primers available upon

Fig. 1. Frequency of norovirus infections in children with acute gastroenteritis inAsuncion, Paraguay by age-group.

request). All sequencing reactions were repeated at least twiceto verify the accuracy of the differences. The sequences obtainedwere analyzed with BioEdit v.7.0.9.0 software, using Clustal W forsequence alignment [20]. The phylogenetic trees were constructedusing the Neighbor-Joining method and Kimura 2-parameter asa model of nucleotide substitution using the MEGA v4.0 softwarepackage [21]. The statistical significance was estimated by boot-strap method (1000 pseudo-replicates). The nucleotide sequences(≥200 pb) reported in this study are available at GenBank databaseunder the accession numbers: KC736563–KC736586. The X-raysolved structure of the VP1 from TCH0 5 (Protein Data Bank [PDB]accession number: 3SKB) was used to identify the antigenic sitesand differences among GII.4 strains. Figure was rendered usingMacPyMol (DeLano Scientific LLC).

4. Results

4.1. Viral detection

NoV genome was detected in 161 (43%) of 378 analyzed sam-ples. All age groups presented similar frequencies of NoV infection(Fig. 1). NoV infection was detected throughout the year, withpeaks distributed across different months (Fig. 2). Climatic datalike mean temperature, relative humidity and rainfall was obtainedfrom the Climate Department of the Meteorology and HydrologyService from the Ministry of Defense (Paraguay); and no relation-ship between NoV infections and those factors was observed in thisstudy (data not shown).

4.2. Noroviruses characterization

NoV genogroup was determined in 84 out of 161 positive sam-ples using region B and/or region D amplification. GII strains weredetected in 54.7% (46/84) of the samples, GI in 40.5% (34/84),and GI/GII mixed infections were observed in 4 samples (4.7%).Partial sequence of the ORF2 was obtained from 29 samples(Table 1). Paraguayan GI strains were classified into two geno-types: GI.3 and GI.8 (Fig. 3). Norovirus GI.3 strains were groupedin two phylogenetic clusters: GI.3a and GI.3b [22]. Three out ofthe four GI.3 Paraguayan strains grouped with the prototype strainHu/Osaka/010314/2001/JP (EF547393) within the cluster GI.3b[23]; while the strain Py830SR05 was related to Brazilian GI.3asamples detected during 2006 and 2007 [24,25] and the prototypestrain Hu/DesertShield395/1993/US (U04469), described in USA

soldiers in Saudi Arabia in the 1990s [26]. The strain Py698SR04,was related to the Boxer strain responsible for viral AGE outbreakson USA Navy Ships in 2001, and was, therefore, classified as geno-type GI.8.

380 M.E. Galeano et al. / Journal of Clinical Virology 58 (2013) 378– 384

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Fig. 2. Monthly distribution of norovirus infections in children with acute gastroenteritis in Asuncion, Paraguay. The number of analyzed samples (gray bars), norovirus-positive samples (black bars) and percentage of norovirus infections (line) are shown for each month.

Hu/GI.3/DesertShield395/1990/US/U04469

Hu/GI.3/13484/2007/RJ/BRA/GU132470

Hu/GI.3/13440/2007/RJ/BRA/GU132469

Hu/GI.3/2232/2006/BRA/DQ496216

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GI.3a

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Hu/GI.3/Py837SR05/2005/PY

Hu/GI.3/Py1348SR05/2005/PY

Hu/GI.3/Py398SR04/2004/PY

Hu/GI.3/Osaka/010314/2001/JP/EF547393

Hu/GI.3/Potsdam196/2000/DE/AF439267

Hu/GI.3/2525/2006/BRA/DQ496217

Hu/GI.3/VA98115/1998/US/AY038598

Hu/GI.3/LittleRock/316/1994/US/F414405

Hu/GI.3/Honolulu/219/1992/US/AF414403

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Hu/GI.6/Hesse3/1997/DE/AF093797

Hu/GI.1/Norwalk/1968/US/M87661

HU/GI.7/Winchester/1994/UK/AJ277609

Hu/GI.4/Chiba407/1987/JP/AB042808

HU/GI.5/Musgrove/1989/UK/AJ277614

HU/GI.2/Whiterose/1996/UK/AJ277610

Hu/GI.2/Southampton/1991/UK/L07418

Hu/GII.4/Bristol/1993/UK/X76716

Hu/GII.4/Lordsdale/1993/UK/X86557

Hu/GIV/Alphatron/98-2/1998/NET/AF195847

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0.001

Fig. 3. Phylogenetic tree of the partial gene from major capsid protein of GI norovirus strains detected in Paraguayan children during 2004–2005. The tree was constructedusing Kimura 2-parameter as a nucleotide substitution model and Neighbor-Joining for tree reconstruction. The statistical significance was estimated by bootstrap method(1000 pseudo-replicates). Bootstrap values are shown at the branch nodes (values <70% not shown). The Paraguayan strains are marked with a filled circle. For each strain(where available), name, country of origin, year of isolation and GenBank accession numbers are shown.

M.E. Galeano et al. / Journal of Clinical Virology 58 (2013) 378– 384 381

Table 1Norovirus (NoV) genotypes circulating in Asuncion, Paraguay during 2004–2005.

NoV genotypes No. of samples

2004 2005 Total (%)

GI.3 1 3 4 (14)GI.8 1 0 1 (3)GII.3 3 0 3 (10)GII.4 2 3 5 (18)GII.5 0 1 1 (3)GII.6 3 1 4 (14)GII.7 0 4 4 (14)GII.8 1 0 1 (3)GII.16 0 1 1 (3)

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Fig. 4. Phylogenetic tree of the partial gene from major capsid protein of GIInorovirus strains detected in Paraguayan children during 2004–2005. Tree recon-

GII.17 2 3 5 (18)Total 13 16 29

Eight genotypes from GII were detected in 24 Paraguayan sam-les: GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.16 and GII.17 (Table 1,ig. 4). GII.4, GII.6, GII.8 and GII.17 were found in both years, whileII.3 strains were found in samples collected in 2004, and GII.5nd GII.7 in samples collected in 2005 (Table 1). To further char-cterize the Paraguayan GII.4 strains, the ORF2 (nt 5130–6688;ased on Hunter504D [GB: DQ078814]) was sequenced and com-ared with representative GII.4 strains from each of the describedlusters. The phylogenetic analysis grouped the GII.4 Paraguayantrains between the Hunter and the Yerseke clusters (Fig. 5a).mportantly, when the nucleotide sequence was submitted to theorovirus Genotyping Tool v1.0 [27], the GII.4 cluster could note assigned. Thus, although nucleotide and amino acid differenceshowed high similarity of the Paraguayan strains with Hunter-likend Yerseke strains (Fig. 5b); analyzes of the amino acids involvedn important antigenic sites [28–30], showed three differences inntigenic sites A and E (Fig. 5c) when compared with Hunter-likend Yerseke strains. Therefore, the phylogenetic clustering and themino acid analyzes suggest that these strains belong to a newII.4 cluster. Because the phylogenetic analyzes of the region Dlustered together Paraguayan and Brazilian GII.4 strains (data nothown) we tentatively named “South-American cluster” to thisewly described GII.4 variant.

. Discussion

In this study, we sought to determine the presence and molecu-ar diversity of NoV associated to AGE cases in Paraguayan children5 years old. Forty three percent (161/378) of the analyzed sam-les were positive for NoV. This incidence may be higher thanreviously reported [31–33], due that only samples negative foracteria and RVA were selected for this study; however, severaleports have shown similar incidences among RVA-negative sam-les [13,34]. NoV are usually referred as “stomach flu viruses”ecause of their high incidence in outbreaks of AGE during theinter season [31,33,35,36]; however, in Paraguay NoV-positive

amples were observed over the two years of study with no sea-onal pattern. This lack of seasonality is consistent with previouseports for this geographic region [24,37].

NoV genetic diversity has lead to the development of sev-ral strategies for detection and molecular characterization, whicharget different regions for genome amplification. Consequently,ombined molecular methods were recommended for accurateoV identification and characterization [9,38,39]. Following thispproach, two different amplification protocols targeting RdRpene (region B) and the major capsid protein gene (region D) were

pplied in this study [9,10]. The identification of the genogroup wasnly possible in approximately 50% of the NoV-positive samples.ifferences in the primer-binding site of the Paraguayan strainsould explain the low success of molecular characterization. GI and

struction methods as described in Fig. 3 and Study Design section. The Paraguayanstrains are marked with a filled circle. For each strain (where available), name,country of origin, year of isolation and GenBank accession numbers are shown.

382 M.E. Galeano et al. / Journal of Clinical Virology 58 (2013) 378– 384

Fig. 5. Characterization of the GII.4 strains detected in Paraguay. (a) Phylogenetic tree of the VP1 region from GII.4 noroviruses. Representative strains from each GII.4 clusterwere used to compare with the Paraguayan GII.4 strains (marked with a circle). For each strain the name/year of isolation/country/GenBank accession number was provided.(b) Percentage of nucleotide (NT) and amino acid (AA) differences from the Paraguayan GII.4 strain (Py735SR04) and each of the strains described in the phylogenetic tree. (c)Amino acid differences at the antigenic sites (A–E) from Paraguayan GII.4 strains and representative GII.4 strains. Differences between the Paraguayan strain (Py735SR04) andthe Hunter284E and Yerseke38 strains are highlighted in blue. Same amino acid differences were detected with the Paraguayan strain Py794SR05. The antigenic sites (A–E)are indicated in light green in the dimeric structure from the TCHO5 strain (PDB: 3SKB). Top and side view of the dimer are shown as indicated. The dashed line indicates theline of symmetry between the two monomers. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)

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II strains were detected at similar frequencies, being 40.5% and4.7%, respectively. The remaining samples (4.7%) showed GI/GIIixed infections, which was not surprising considering the high

requencies of both GI and GII. Several reports have shown theredominance of GII strains over GI, at rates of 90% and 10%, respec-ively; in both outbreaks and sporadic AGE cases [5,25,40–43].owever, Soares et al. have shown similar incidences of GI/GII

trains in children <10 years old from Rio de Janeiro, Brazil [24],hich might reflect a particular pattern of circulation in South-merican countries and non-epidemic settings. More accurateharacterization of the samples is needed to confirm these findings.

In this study, the NoV genotype was determined in 29 sam-les. Ten different genotypes were identified, and all of themresented similar frequencies (3–18%); suggesting that multiplesenotypes were circulating in Paraguayan children with AGE dur-ng 2004–2005. This is in contrast with previous studies that havehown the large predominance of one or two strains (in mostases GII.4 and GII.3) in both outbreaks and sporadic cases of AGE39,40,44,45]. Even though a small number of strains were char-cterized, this study shows that multiple NoV genotypes, ratherhan single strain, could be responsible for AGE in Paraguayan chil-ren. Interestingly, GII.5 strains are being reported for the first time

n South America, which expands the diversity of NoV reported forhis geographic area. GII.3 strains, the second most prevalent geno-ype in the world, were described in the 1990s and early 2000sn Argentina [46] and Brazil [25,47], respectively. In Paraguayanhildren, GII.3 strains were observed in three samples collected ineptember and October 2004, and were phylogenetically related tohe Brazilian strains that circulated in 2003. GII.17 strains were ini-ially described in the USA [48], and later in Argentina [41], Brazil49] and Nicaragua [33]. In Paraguay, GII.17 strains were found inanuary 2004 and mid-2005, suggesting that this genotype haveeen circulating in the Americas for over a decade.

GII.4 strains have been shown to be the predominant NoV strainsorldwide [4,6,30,42]. The chronological emergence of new phy-

ogenetic clusters and changes in one of the five antigenic sitesA–E) described in the VP1 from GII.4 strains has shown to corre-ate with large local and global epidemics [28–30,50]. A new GII.4ariant that has been circulating in South American children since003 it is being described here. Strains from this cluster showedigh similarity with prototype strains from two GII.4 clusters, butifferences on the antigenic sites A and E as well as phylogeneticnalyses confirms the new variant. Interestingly, these GII.4 strainsrom the South-American cluster have been detected in childrenith sporadic cases of AGE, but not associated to outbreaks so far.

In conclusion, the findings reported in this study, i.e. the con-inuous circulation of NoV throughout the year and the extensiveiversity of genotypes detected, highlight the need for betterurveillance of NoV in Paraguayan children population and otheratin American countries. This is particularly important becausehe RVA vaccine was introduced in the National Immunization Pro-ram from many countries in Latin America [51], and the NoV couldecome the major viral pathogen of diarrhea in children under ≤5ears old.

unding

MEG was a scholarship recipient from the National Council forcientific and Technological Development (CNPq), and the Brazilianederal Agency for Support and Evaluation of Graduate EducationCAPES), through the Exchange Student Post-graduation Program

PEC-PG). This study was partially supported by the National Uni-ersity of Asuncion, Oswaldo Cruz Institute/Fiocruz, PROEP/CNPq,arlos Chagas Filho Foundation for Research Support of the State ofio de Janeiro (FAPERJ), CAPES and CNPq.

[

[

l Virology 58 (2013) 378– 384 383

Competing interests

None.

Ethical approval

This study was approved by the Ethics Review Committee(P39/08) of the Health Sciences Research Institute, National Uni-versity of Asunción.

Acknowledgements

We would like to thank Dr. Eduardo de Mello Volotão and Dr.Filipe Carvalho-Costa for their critical reading of the initial versionsof the manuscript. We also thank to the Climate Department of theMeteorology and Hydrology Service from the Ministry of Defense(Paraguay) for contributing with climatologic data and to personalfrom San Roque Private Hospital for providing samples.

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