Norovirus infection in children with acute gastroenteritis, Madagascar, 2004-2005.Of 237 children with acute gastroenteritis gastroenteritis: see enteritis. gastroenteritis Acute infectious syndrome of the stomach lining and intestines. Symptoms include diarrhea, vomiting, and abdominal cramps. in Antananarivo, Madagascar, during May 2004-May 2005, 14 ([approximately equal to] 6%) were infected with norovirus. Seasonality (November-December peak) was detected. Reverse transcription-PCR identified GII GII Global Information Infrastructure GII Getty Information Institute GII Gasherbrum II (26,360 ft. mountain near Pakistan-China) GII Government Information Infrastructure GII Ghana Integrity Initiative as the most common genogroup. GIs belonged to GI.1, GI.3, and GI.4. Noroviruses in Madagascar show extensive genetic diversity. ********** Gastroenteritis is a major public health issue worldwide. Noroviruses are now considered emerging pathogens (1) and are recognized as the leading cause of nonbacterial, acute gastroenteritis in humans (2). The 2 genera, Norovirus and Sapovirus, are members of the family Caliciviridae and have a positive-sense, single-stranded RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic genome [approximately equal to] 7.5 kb long. Because no readily available cell culture system exists, characterization and classification of noroviruses are based on reverse transcription reverse transcription n. The process by which DNA is synthesized from an RNA template. (RT)-PCR, genomic sequencing genomic sequencing The sequencing of the entire genome of an organism. A Closer Look The technique that allows researchers to read and decipher the genetic information found in the DNA of anything from bacteria to plants to animals is , and phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. analysis (2-5). According to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the latest scheme for norovirus nomenclature, the 29 genetic norovirus clusters or genotypes are classified into 5 genogroups (GI-V) (6). GI and II infect humans, and GII.4 has been the most highly prevalent genotype worldwide during the past decade (2,5,7). The saying "Madagascar is not an island, but an archipelago" captures an important aspect of the country, whose geography and history have combined to produce a society of considerable diversity and uniqueness (8). In recent years, many studies have investigated the role of human noroviruses in childhood diarrhea and found worldwide distribution (4,7,9,10). However, no studies have reported the prevalence and molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, of noroviruses in Madagascar, which we report here. The Study From May 2004 to May 2005, a study of acute gastroenteritis in children [less than or equal to] 16 years of age was undertaken by the Institut Pasteur, Antananarivo, Madagascar. Children with a diagnosis of acute dehydrating watery diarrhea who were seen at the rehydration rehydration /re·hy·dra·tion/ (-hi-dra´shun) the restoration of water or fluid content to a patient or to a substance that has become dehydrated. re·hy·dra·tion n. 1. clinics and hospitals of Antananarivo were eligible for the study. Antananarivo, the capital city of Madagascar, has a population of [approximately equal to] 4 million. The study was approved by the Ethical Review Board of the Institut Pasteur, Antananarivo, Madagascar. Fecal samples were collected from the children and stored at -80[degrees]C until analysis was undertaken at the University of Liverpool The University of Liverpool is a university in the city of Liverpool, England. History The University was established in 1881 as University College Liverpool, admitting its first students in 1882. , UK. There, viral RNA was extracted from 150 [micro]L of 10%-20% fecal suspensions in phosphate-buffered saline by using a guanidine guanidine /gua·ni·dine/ (gwah´ni-den) the compound NHdbondC(NH2)2, a strong base found in the urine as a result of protein metabolism and used in the laboratory as a protein denaturant. and silica method (11). Sapoviruses were not sought in the initial screening. RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. was performed in a 50-[micro]L reaction mixture with primers that targeted the capsid capsid /cap·sid/ (kap´sid) the shell of protein that protects the nucleic acid of a virus; it is composed of structural units, or capsomers. cap·sid n. N terminus/shell gene. For GI, forward primer G1SKF SKF Svenska Kullagerfabriken SKF Svenska Klätterförbundet (Sweden) SKF Smithsonian Kite Festival SKF San Antonio Kelly Field Annex (Lackland AFB, Texas) (5'-CTGCCCGAATTYGTAAATGA-3') and reverse primer G1SKR SKR South Korea SKR Sveriges Kvinnojourers Riksförbund (Swedish Association of Women’s Shelters) SKR Swedish Kroner (unit of currency) SKR Serial Killin Records SKR Safe Keeping Receipt SKR Sally K. (5'-CCAACCCARCCATTRTACA-3') were used, yielding a PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) product of 330 bp. For GII, the primers used were forward primer G2SKF (5'-CNTGGGAGGGCGATCGCAA-3') and reverse primers G2SKR (5'-CCRCCNGCATRHCCRTTRTACAT-3') and G2ALSKR (5'-CCACCAGCATATGAATTGTACAT-3'), yielding a 344-bp product (12). The PCR was performed with an initial denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. at 94[degrees]C, followed by 40 cycles of 60-sec denaturation at 94[degrees]C, 60-s primer annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. at 50[degrees]C, an extension for 2 min at 72[degrees]C, followed by a final extension stage of 15 min at 72[degrees]C. Amplification products were examined under ultraviolet light Ultraviolet light A portion of the light spectrum not visible to the eye. Two bands of the UV spectrum, UVA and UVB, are used to treat psoriasis and other skin diseases. after electrophoresis through a 2% agarose agarose more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. gel with ethidium bromide Ethidium bromide (sometimes abbreviated as EtBr) is an intercalating agent commonly used as a nucleic acid stain in molecular biology laboratories for techniques such as agarose gel electrophoresis. staining. Products were extracted by using the QIAquick PCR purification kit (QIAGEN, Basingstoke, UK) and were sequenced by Macrogen Inc. (Seoul, South Korea). Phylogenetic relationships were examined by aligning sequences with the ClustalW multiple alignment program (European Molecular Biology Laboratory The European Molecular Biology Laboratory (EMBL) is a molecular biology research institution supported by 19 countries comprising nearly all of western Europe and Israel. , Heidelberg, Germany). A phylogenetic tree phylogenetic tree Diagram showing the evolutionary interrelations of a group of organisms that usually originated from a shared ancestral form. The ancestor is in the tree trunk; organisms that have arisen from it are placed at the ends of tree branches. was constructed according to the neighbor-joining method by using ClustalX (version 1.83) and the alignment file obtained by analysis with ClustalW. Bootstrap See boot. (operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen. values on a scale from 1 to 1,000 were also calculated. An unrooted phylogram of norovirus isolates from the present study and prototype strains was plotted in the PHYLIP PHYLIP Phylogeny Inference Package (genetics software) format (http://evolution.genetics.washington. edu/phylip.html) output by using TreeView software, version 3.1 (http://taxonomy.zoology zoology, branch of biology concerned with the study of animal life. From earliest times animals have been vitally important to man; cave art demonstrates the practical and mystical significance animals held for prehistoric man. .gla.ac.uk/rod/treeview. html). Assignment of norovirus to genotype was made according to the scheme proposed by Zheng et al. (6). The nucleotide sequences of the Malagasy strains have been deposited at GenBank (accession nos. EF213624-EF213635, EF213638, and EF213640.) Norovirus-positive samples were screened for other viruses by negative-stain electron microsopy and by RT-PCR for rotavirus rotavirus /ro·ta·vi·rus/ (ro´tah-vi?rus) any member of the genus Rotavirus. ro´taviral Rotavirus /Ro·ta·vi·rus/ (ro´tah-vi?rus and astrovirus (13,14). During this 12-month study, 258 children with acute gastroenteritis in Madagascar were screened for norovirus infection. Because 21 samples contained insufficient stool, 237 samples were analyzed (142 from boys and 95 from girls). Overall, 85% of children were <3 years of age, 77% were <2 years, 43% were <1 year, and 3% were newborns. The median age of the study population was 20 months (range 1 day to 16 years). Fourteen noroviruses (5.9%) were detected in 237 children (Table 1). Noroviruses were found in all age groups. Infection rates did not differ between boys and girls boys and girls mercurialisannua. (6.3% and 5.2%, respectively, p>0.1). No coinfections with other viruses (rotavirus, astrovirus, and adenovirus adenovirus Any of a group of spheroidal viruses, made up of DNA wrapped in a protein coat, that cause sore throat and fever in humans, hepatitis in dogs, and several diseases in fowl, mice, cattle, pigs, and monkeys. ) were detected. Ten (71%) of the noroviruses detected were identified as belonging to genogroup GII; the remaining 4 (29%), to GI. Most GII noroviruses belonged to a potentially novel cluster (Figure 1). GI noroviruses were further classified into 3 genotypes: GI.1 (1 isolate), GI.4 (1 isolate), and GI.3 (2 isolates). [FIGURE 1 OMITTED] The median age of children with norovirus infection was 18 months (range 3-51 months). Most infections ([approximately equal to] 86%) occurred in children <36 months of age. No infections were recorded in newborns or children >5 years of age. All infections with genogroup GI noroviruses were found in children <24 months of age (Table 2). Noroviruses were detected throughout the year; however, infections peaked during the wet season in Madagascar. November and December were the months of major norovirus prevalence (35.7% each month). GI noroviruses preceded GII infections by a few days (Figure 2). [FIGURE 2 OMITTED] Conclusions To our knowledge, ours is the first study that has used molecular detection methods to investigate the role of noroviruses in pediatric pediatric /pe·di·at·ric/ (pe?de-at´rik) pertaining to the health of children. pe·di·at·ric adj. Of or relating to pediatrics. gastroenteritis in Madagascar. We showed that infections with GI and GII noroviruses are relatively common. In a 1-year collection of stool samples, we detected noroviruses by RT-PCR in [approximately equal to] 6% of children with acute gastroenteritis in Antananarivo. This rate establishes norovirus as the second most commonly detected enteric virus enteric virus n. See enterovirus. in this population, behind rotavirus (38%) and followed by astrovirus (2.5%) (data not shown). These findings are consistent with those of studies elsewhere (7,10). The median age of children with norovirus infection (18 months) was higher than previously reported (7) and higher than that of the rotavirus-infected group (median 10 months, range 1 day to 48 months) and that of the astrovirus-infected group (median 10 months, range 5-20 months) (data not shown). Noroviruses were detected throughout the year, but the number peaked in November and December. Such seasonality in a tropical country is not really expected, as year-round circulation has been previously documented (15). Our findings confirm the finding of previous studies that GII is the predominant norovirus genogroup circulating in communities worldwide. Considerable genetic diversity was observed among the norovirus GII isolates, and some were identified as belonging to a potentially novel cluster. The closest reference strain to the potentially novel cluster was the recombinant Hu/NoV/GII.1/Hawaii/1971/ US. In contrast, norovirus GI isolates were clustered with prototype strains; Hu/NoV/GI.3/DSV395/1990/SA (Desert Shield) was predominant (2 strains), followed by Hu/NoV/ GI.1/Norwalk/1968/US (Norwalk) and Hu/NoV/GI.4/Chiba407/1987/JP (1 each). This study has several limitations. First, it is a preliminary study. The sample size was small, and we examined samples collected over a 13-month period. Longer, longitudinal studies longitudinal studies, n.pl the epidemiologic studies that record data from a respresentative sample at repeated intervals over an extended span of time rather than at a single or limited number over a short period. are required to address issues such as norovirus seasonality and temporal genetic variability. In addition, we restricted our analysis to specimens collected from patients at rehydration clinics and hospitals, so prevalence of norovirus infections in the general population may have been underestimated. Furthermore, the use of short conserved sequences, although successful for diagnosis of norovirus infection, should be used with caution for classification and phylogenetic analyses. Further analysis by full capsid sequencing might be required. Nevertheless, continued norovirus surveillance is needed to monitor the spread and persistence of the various genotypes infecting children in Madagascar. Acknowledgments We are grateful to P. Mauclere, for providing the opportunity to work with these samples. The study was funded internally by the University of Liverpool. References (1.) Widdowson MA, Monroe SS, Glass RI. Are noroviruses emerging? Emerg Infect Dis. 2005;11:735-7. (2.) Radford AD, Gaskell RM, Hart CA. Human norovirus infection and the lessons from animal caliciviruses. Curr Opin Infect Dis. 2004;17:471-8. (3.) Ando T, Noel JS, Fankhauser RL. Genetic classification of "Norwalk-like viruses." J Infect Dis. 2000; 181 (Suppl2):S336-48. (4.) Zintz C, Bok K, Parada E, Barnes-Eley M, Berke T, Staat MA, et al. Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States. Infect Genet genet: see civet. Evol. 2005;5:281-90. (5.) Kageyama T, Shinohara M, Uchida K, Fukushi S, Hoshino FB, Kojima S, et al. Coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to norovirus in Japan. J Clin Microbiol. 2004;42:2988-95. (6.) Zheng DP, Ando T, Fankhauser RL, Beard RS, Glass RI, Monroe SS. Norovirus classification and proposed strain nomenclature. Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression . 2006;346:312-23. (7.) Dove W, Cunliffe NA, Gondwe JS, Broadhead RL, Molyncux ME, Nakagomi O, et al. Detection and characterization of human calici-viruses in hospitalized children with acute gastroenteritis in Blantyre, Malawi. J Med Virol. 2005;77:522-7. (8.) Covell M. Geographical and historical background. In: Madagascar: politics, economics and society. London: Pinter Pub Ltd; 1987. p. 8. (9.) Hansman GS, Katayama K, Maneekam N, Peerakome S, Khamrin P, Tonusin S, et al. Genetic diversity of norovirus and sapovirus in hospitalized infants with sporadic cases of acute gastroenteritis in Chiang Mai, Thailand. J Clin Microbiol. 2004;42:1305-7. (10.) Bereciartu A, Bok K, Gomez J. Identification of viral agents causing gastroenteritis among children in Buenos Aires, Argentina. J Clin Virol. 2002;25:197-203. (11.) Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-Van Dillen PM, Van Der Noordaa J. Rapid and simple method for purification of nucleic acids Nucleic acids The cellular molecules DNA and RNA that act as coded instructions for the production of proteins and are copied for transmission of inherited traits. . J Clin Microbiol. 1990;28:495-503. (12.) Kojima S, Kageyama T, Fukushi S, Hoshimo FB, Shinohara M, Uchida K, et al. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J Virol Methods. 2002; 100:107-14. (13.) Cunliffe NA, Dove W, Bunn JE, Ben Ramadan M, Nyangao JW, Riveron RL, et al. Expanding global distribution of rotavirus serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon. se·ro·type n. See serovar. v. G9: detection in Libya, Kenya, and Cuba. Emerg Infect Dis. 2001;7:890-2. (14.) Cunliffe NA, Dove W, Gondwe JS, Thindwa BD, Greensill J, Holmes JL, et al. Detection and characterization of human astroviruses in children with acute gastroenteritis in Malawi. J Med Virol. 2002;67:563-6. (15.) O'Ryan ML, Mamani N, Gaggero A, Avendano LF, Prieto S, Pena A, et al. Human caliciviruses are a significant pathogen of acute sporadic diarrhea in children of Santiago, Chile. J Infect Dis. 2000;182:1519-22. Dimitrios C. Papaventsis, * Winifred Dove,* Nigel A. Cunliffe, * Osamu Nakagomi, * ([dagger]) Patrice Combe combe Noun same as coomb , ([double dagger]) Pierre Grosjean, ([double dagger]) and C. Anthony Hart * * University of Liverpool, Liverpool, United Kingdom; ([dagger]) Nagasaki University, Nagasaki, Japan; and ([double dagger]) Institute Pasteur, Antananarivo, Madagascar Dr Papaventsis is an honorary clinical lecturer in medical microbiology and genitourinary medicine, Department of Host Defense and Infection, Faculty of Medicine, University of Liverpool, UK. His main research interests include the molecular epidemiology of enteric viruses. Address for correspondence: C. Anthony Hart, Department of Medical Microbiology and Genito-Urinary Medicine, University of Liverpool, Duncan Building, Daulby St, Liverpool L69 3GA, UK; email: c.a.hart@ liverpool.ac.uk
Table 1. Characteristics of viruses from children with acute
gastroenteritis in Madagascar *
GenBank Sample
Identification no. accession no. ([dagger]) date Age, mo
DG6004-Madag04 EF213640 May 2004 14
DC2022-Madag04 EF213626 July 2004 14
DG6003-Madag04 EF213634 Oct 2004 44
DR0011-Madag04 EF213628 Nov 2004 12
DR0025-Madag04 EF213625 Nov 2004 17
DC2048-Madag04 EF213624 Nov 2004 3
DR0023-Madag04 EF213638 Nov 2004 9
DR0045-Madag04 EF213627 Nov 2004 19
DC2054-Madag04 EF213629 Dec 2004 16
DG6020-Madag04 EF213630 Dec 2004 30
DR0046-Madag04 EF213631 Dec 2004 14
DT1020-Madag04 EF213632 Dec 2004 12
DT1032-Madag04 EF213633 Dec 2004 8
DM4025-Madag05 EF213635 Mar 2005 51
Identification no. Sex Norovirus PCR
DG6004-Madag04 M GI pos
DC2022-Madag04 F GII pos
DG6003-Madag04 M GII pos
DR0011-Madag04 M GII pos
DR0025-Madag04 M GI pos
DC2048-Madag04 M GI pos
DR0023-Madag04 F GI pos
DR0045-Madag04 F GII pos
DC2054-Madag04 M GII pos
DG6020-Madag04 M GII pos
DR0046-Madag04 F GII pos
DT1020-Madag04 F GII pos
DT1032-Madag04 M GII pos
DM4025-Madag05 M GII pos
EM for
Norovirus NoV, RV, AV
Identification no. sequencing (double dagger])
DG6004-Madag04 GI-1 Neg
DC2022-Madag04 GII novel Neg
DG6003-Madag04 GII novel Neg
DR0011-Madag04 GII novel Neg
DR0025-Madag04 GI-4 Neg
DC2048-Madag04 GI-3 Neg
DR0023-Madag04 GI-3 Neg
DR0045-Madag04 GII novel Neg
DC2054-Madag04 GII novel Pos
(putative NoV)
DG6020-Madag04 GII novel Neg
DR0046-Madag04 GII novel Neg
DT1020-Madag04 GII novel Neg
DT1032-Madag04 GII novel Neg
DM4025-Madag05 GII novel Neg
* EM, electron microscopy, NoV, norovirus; RV, rotavirus; AV,
astrovirus; pos, positive; neg, negative.
([dagger]) Fecal samples.
([double dagger]) All norovirus-positive samples were negative
for AV and RV when tested by reverse transcription-PCR.
Table 2. Distribution of viruses and norovirus genogroups in
children with acute gastroenteritis, by age, Madagascar, May
2004-May 2005
Age group, mo
Virus 0-12 13-24 25-36 >36
Other than 96 75 18 34
norovirus
Norovirus GI 2 2 0 0
Norovirus GII 3 4 1 2
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