Abstract
From 2010-2016, a total of 251 stool samples were screened for norovirus using next-generation sequencing (NGS) followed by phylogenetic analysis to investigate the genotypic diversity of noroviruses in rural and low-income urban areas in northern Brazil. Norovirus infection was detected in 19.9% (50/251) of the samples. Eight different genotypes were identified: GII.4_Sydney[P31] (64%, 32/50), GII.6[P7] (14%, 7/50), GII.17[P17] (6%, 3/50), GII.1[P33] (6%, 3/50), GII.3[P16] (4%, 2/50), GII.2[P16] (2%, 1/50), GII.2[P2] (2%, 1/50), and GII.4_New Orleans[P4] (2%, 1/50). Distinct GII.6[P7] variants were recognized, indicating the presence of different co-circulating strains. Elucidating norovirus genetic diversity will improve our understanding of their potential health burden, in particular for the GII.4_Sydney[P31] variant.
References
Atmar RL (2010) Noroviruses—state of the art. Food Environ Virol 2(3):117–126. https://doi.org/10.1007/s12560-010-9038-1
Glass PJ, White LJ, Ball JM et al (2000) Norwalk virus open reading frame 3 encodes a minor structural protein. J Virol 74(14):6581–6591. https://doi.org/10.1128/jvi.74.14.6581-6591.2000
Kroneman A, Vennema H, Deforche K et al (2011) An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol 51(2):121–125. https://doi.org/10.1016/j.jcv.2011.03.006
Chhabra P, de Graaf M, Parra GI et al (2019) Updated classification of norovirus genogroups and genotypes. J Gen Virol 100(10):1393–1406. https://doi.org/10.1099/jgv.0.001318. Corrigendum: Updated classification of norovirus genogroups and genotypes. J Gen Virol 2020;101(8):893. https://doi.org/10.1099/jgv.0.001475
Matsushima Y, Ishikawa M, Shimizu T et al (2015) Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region. Euro Surveill 20(26):21173. https://doi.org/10.2807/1560-7917.es2015.20.26.21173
Siqueira JAM, Bandeira RDS, Oliveira DS et al (2017) Genotype diversity and molecular evolution of noroviruses: A 30-year (1982–2011) comprehensive study with children from Northern Brazil. PLoS ONE 12(6):e0178909. https://doi.org/10.1371/journal.pone.0178909
Parra GI, Green KY (2015) Genome of emerging norovirus GII.17, United States, 2014. Emerg Infect Dis 21(8):1477–1479. https://doi.org/10.3201/eid2108.150652
Eden JS, Hewitt J, Lim KL et al (2014) The emergence and evolution of the novel epidemic norovirus GII.4 variant Sydney 2012. Virology 450–451:106–113. https://doi.org/10.1016/j.virol.2013.12.005
Hoa Tran TN, Trainor E, Nakagomi T et al (2013) Molecular epidemiology of noroviruses associated with acute sporadic gastroenteritis in children: global distribution of genogroups, genotypes and GII.4 variants. J Clin Virol 56(3):185–193. https://doi.org/10.1016/j.jcv.2012.11.011
Parra GI, Squires RB, Karangwa CK et al (2017) Static and evolving norovirus genotypes: implications for epidemiology and immunity. PLoS Pathog. 13(1):e1006136. https://doi.org/10.1371/journal.ppat.1006136
van Beek J, Ambert-Balay K, Botteldoorn N et al (2013) Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012. Euro Surveill 18(1):8–9
Lu J, Sun L, Fang L et al (2015) Gastroenteritis outbreaks caused by norovirus GII.17, Guangdong Province, China, 2014–2015. Emerg Infect Dis 21(7):1240–1242. https://doi.org/10.3201/eid2107.150226
Andrade JSR, Fumian TM, Leite JPG et al (2017) Detection and molecular characterization of emergent GII.P17/GII.17 Norovirus in Brazil, 2015. Infect Genet Evol 51:28–32. https://doi.org/10.1016/j.meegid.2017.03.011
Lun JH, Hewitt J, Sitabkhan A et al (2018) Emerging recombinant noroviruses identified by clinical and waste water screening. Emerg Microbes Infect 7(1):50. https://doi.org/10.1038/s41426-018-0047-8
Medici MC, Tummolo F, Calderaro A et al (2015) Identification of the novel Kawasaki 2014 GII.17 human norovirus strain in Italy, 2015. Euro Surveill 20(35):30010. https://doi.org/10.2807/1560-7917.ES.2015.20.35.30010
Mattison CP, Cardemil CV, Hall AJ (2018) Progress on norovirus vaccine research: public health considerations and future directions. Expert Rev Vaccines 17(9):773–784. https://doi.org/10.1080/14760584.2018.1510327
Barreira DMPG, Fumian TM, Tonini MAL et al (2017) Detection and molecular characterization of the novel recombinant norovirus GII.P16-GII.4 Sydney in southeastern Brazil in 2016. PLoS ONE 12(12):e0189504. https://doi.org/10.1371/journal.pone.0189504
Fioretti JM, Fumian TM, Rocha MS et al (2018) Surveillance of noroviruses in Rio De Janeiro, Brazil: occurrence of new GIV genotype in clinical and wastewater samples. Food Environ Virol 10(1):1–6. https://doi.org/10.1007/s12560-017-9308-2
Hernandez JM, Silva LD, Junior ECS et al (2018) Molecular epidemiology and temporal evolution of norovirus associated with acute gastroenteritis in Amazonas state, Brazil. BMC Infect Dis. 18(1):147. https://doi.org/10.1186/s12879-018-3068-y
Gondim RDG, Pankov RC, Prata MMG et al (2018) Genetic diversity of norovirus infections, coinfections, and undernutrition in children from Brazilian Semiarid Region. J Pediatr Gastroenterol Nutr 67(6):e117–e122. https://doi.org/10.1097/MPG.0000000000002085
Cantelli CP, da Silva MFM, Fumian TM et al (2019) High genetic diversity of noroviruses in children from a community-based study in Rio de Janeiro, Brazil, 2014–2018. Arch Virol. 164(5):1427–1432. https://doi.org/10.1007/s00705-019-04195-z
Dábilla N, Almeida TNV, Franco FC et al (2019) Recombinant noroviruses detected in Mid-West region of Brazil in two different periods 2009–2011 and 2014–2015: atypical breakpoints of recombination and detection of distinct GII.P7-GII.6 lineages. Infect Genet Evol 68:47–53. https://doi.org/10.1016/j.meegid.2018.12.007
Tahmasebi R, Luchs A, Tardy K, Hefford PM, Tinker RJ, Eilami O et al (2020) Viral gastroenteritis in Tocantins, Brazil: characterizing the diversity of human adenovirus F through next-generation sequencing and bioinformatics. J Gen Virol. https://doi.org/10.1099/jgv.0.001500 ((epub ahead of print))
Watanabe ASA, Luchs A, Leal É et al (2018) Complete genome sequences of six human bocavirus strains from patients with acute gastroenteritis in the North Region of Brazil. Genome Announc 6(17):e00235-e318. https://doi.org/10.1128/genomeA.00235-18
Cilli A, Luchs A, Leal E et al (2019) Human sapovirus GI2 and GI3 from children with acute gastroenteritis in northern Brazil. Mem Inst Oswaldo Cruz 114:e180574. https://doi.org/10.1590/0074-02760180574
Ribeiro GO, Luchs A, Milagres FAP et al (2018) Detection and characterization of enterovirus B73 from a child in Brazil. Viruses 11(1):16. https://doi.org/10.3390/v11010016
Luchs A, Leal E, Tardy K et al (2019) The rare enterovirus c99 and echovirus 29 strains in Brazil: potential risks associated to silent circulation. Mem Inst Oswaldo Cruz 114:e190160. https://doi.org/10.1590/0074-02760190160
Rosa UA, Ribeiro GO, Villanova F et al (2019) First identification of mammalian orthoreovirus type 3 by gut virome analysis in diarrheic child in Brazil. Sci Rep 9(1):18599. https://doi.org/10.1038/s41598-019-55216-5
da Costa AC, Luchs A, Milagres FAP et al (2018) Near full length genome of a recombinant (E/D) cosavirus strain from a rural area in the central region of Brazil. Sci Rep 8(1):12304. https://doi.org/10.1038/s41598-018-30214-1
da Costa AC, Luchs A, Milagres FAP et al (2018) Recombination located over 2A–2B junction ribosome frameshifting region of saffold cardiovirus. Viruses 10(10):520. https://doi.org/10.3390/v10100520
Luchs A, Leal E, Komninakis SV et al (2018) Wuhan large pig roundworm virus identified in human feces in Brazil. Virus Genes 54(3):470–473. https://doi.org/10.1007/s11262-018-1557-0
Leal É, Luchs A, Milagres FAP et al (2019) Recombinant strains of human parechovirus in rural areas in the North of Brazil. Viruses 11(6):488. https://doi.org/10.3390/v11060488
da Costa AC, Leal E, Gill D et al (2019) Discovery of Cucumis melo endornavirus by deep sequencing of human stool samples in Brazil. Virus Genes 55(3):332–338. https://doi.org/10.1007/s11262-019-01648-0
Tahmasebi R, Costa ACD, Tardy K et al (2020) Genomic analyses of potential novel recombinant human adenovirus C in Brazil. Viruses 12(5):508. https://doi.org/10.3390/v12050508
Li L, Deng X, Mee ET et al (2015) Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent. J Virol Methods 213:139–146. https://doi.org/10.1016/j.jviromet.2014.12.002
Charlys da Costa A, Thézé J, Komninakis SCV et al (2017) Spread of Chikungunya Virus East/Central/South African Genotype in Northeast Brazil. Emerg Infect Dis 23(10):1742–1744. https://doi.org/10.3201/eid2310.170307
Deng X, Naccache SN, Ng T et al (2015) An ensemble strategy that significantly improves de novo assembly of microbial genomes from metagenomic next-generation sequencing data. Nucleic Acids Res 43(7):e46. https://doi.org/10.1093/nar/gkv002
Altan E, Delaney MA, Colegrove KM et al (2020) Complex virome in a mesenteric lymph node from a Californian Sea Lion (Zalophus californianus) with polyserositis and steatitis. Viruses 12(8):E793. https://doi.org/10.3390/v12080793
Ng TF, Marine R, Wang C et al (2012) High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. J Virol 86(22):12161–12175. https://doi.org/10.1128/JVI.00869-12
Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. https://doi.org/10.1093/bioinformatics/btm404
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054
Fu JG, Ai J, Zhang J et al (2016) Molecular epidemiology of genogroup II norovirus infection among hospitalized children with acute gastroenteritis in Suzhou (Jiangsu, China) from 2010 to 2013. J Med Virol 88(6):954–960. https://doi.org/10.1002/jmv.24429
Xue Y, Pan H, Hu J et al (2015) Epidemiology of norovirus infections among diarrhea outpatients in a diarrhea surveillance system in Shanghai, China: a cross-sectional study. BMC Infect Dis 15:183. https://doi.org/10.1186/s12879-015-0922-z
Kim YE, Song M, Lee J et al (2018) Phylogenetic characterization of norovirus strains detected from sporadic gastroenteritis in Seoul during 2014–2016. Gut Pathog 10:36. https://doi.org/10.1186/s13099-018-0263-8
Ouédraogo N, Kaplon J, Bonkoungou IJ et al (2016) Prevalence and genetic diversity of enteric viruses in children with diarrhea in Ouagadougou, Burkina Faso. PLoS ONE 11(4):e0153652. https://doi.org/10.1371/journal.pone.0153652
Diez-Valcarce M, Lopez MR, Lopez B et al (2019) Prevalence and genetic diversity of viral gastroenteritis viruses in children younger than 5 years of age in Guatemala, 2014–2015. J Clin Virol 114:6–11. https://doi.org/10.1016/j.jcv.2019.03.006
Gupta S, Krishnan A, Sharma S et al (2018) Changing pattern of prevalence, genetic diversity, and mixed infections of viruses associated with acute gastroenteritis in pediatric patients in New Delhi, India. J Med Virol 90(3):469–476. https://doi.org/10.1002/jmv.24980
Reymão TKA, Fumian TM, Justino MCA et al (2018) Norovirus RNA in serum associated with increased fecal viral load in children: Detection, quantification and molecular analysis. PLoS ONE 13(7):e0199763. https://doi.org/10.1371/journal.pone.0199763
Paula FL, Sardi SI, Tigre DM et al (2018) Acute gastroenteritis associated with norovirus GII.4 variants. Arq Gastroenterol 55(3):264–266. https://doi.org/10.1590/S0004-2803.201800000-67
Gray JJ, Kohli E, Ruggeri FM et al (2007) European multicenter evaluation of commercial enzyme immunoassays for detecting norovirus antigen in fecal samples. Clin Vaccine Immunol 14(10):1349–1355. https://doi.org/10.1128/CVI.00214-07
Moyo S, Hanevik K, Blomberg B et al (2014) Genetic diversity of norovirus in hospitalised diarrhoeic children and asymptomatic controls in Dar es Salaam, Tanzania. Infect Genet Evol 26:340–347. https://doi.org/10.1016/j.meegid.2014.06.013
Mesquita JR, Nascimento MS (2014) Norovirus GII.4 antibodies in the Portuguese population. J Infect Dev Ctries 8(9):1201–1204. https://doi.org/10.3855/jidc.4616
Abugalia M, Cuevas L, Kirby A et al (2011) Clinical features and molecular epidemiology of rotavirus and norovirus infections in Libyan children. J Med Virol 83(10):1849–1856. https://doi.org/10.1002/jmv.22141
Mathew S, Alansari K, Smatti M et al (2019) Epidemiological, molecular, and clinical features of norovirus infections among pediatric patients in Qatar. Viruses 11(5):400. https://doi.org/10.3390/v11050400
Eftim SE, Hong T, Soller J et al (2017) Occurrence of norovirus in raw sewage—a systematic literature review and meta-analysis. Water Res 111:366–374. https://doi.org/10.1016/j.watres.2017.01.017
Fumian TM, Fioretti JM, Lun JH et al (2019) Detection of norovirus epidemic genotypes in raw sewage using next generation sequencing. Environ Int 123:282–291. https://doi.org/10.1016/j.envint.2018.11.054
Mans J, Murray TY, Taylor MB (2014) Novel norovirus recombinants detected in South Africa. Virol J 11:168. https://doi.org/10.1186/1743-422X-11-168
Bruggink LD, Moselen JM, Marshall JA (2016) The comparative molecular epidemiology of GII.P7_GII.6 and GII.P7_GII.7 norovirus outbreaks in Victoria, Australia, 2012–2014. Intervirology 59(1):60–65. https://doi.org/10.1159/000448100
Utsumi T, Lusida MI, Dinana Z et al (2017) Occurrence of norovirus infection in an asymptomatic population in Indonesia. Infect Genet Evol 55:1–7. https://doi.org/10.1016/j.meegid.2017.08.020
Volpini LPB, Barreira DMPG, Almeida PLDS et al (2020) An outbreak due to a norovirus GII.Pe-GII.4 Sydney_2012 recombinant in neonatal and pediatric intensive care units. J Infect Public Health 13(1):89–93. https://doi.org/10.1016/j.jiph.2019.06.012
Hernandez JDM, Silva LDD, Sousa Junior EC et al (2016) Analysis of uncommon norovirus recombinants from Manaus, Amazon region, Brazil: GII.P22/GII.5, GII.P7/GII.6 and GII.Pg/GII.1. Infect Genet Evol 39:365–371. https://doi.org/10.1016/j.meegid.2016.02.007
Cai H, Yu Y, Jin M et al (2017) Cloning, sequencing and characterization of the genome of a recombinant norovirus of the rare genotype GII.P7/GII.6 in China. Arch Virol 162(7):2053–2059. https://doi.org/10.1007/s00705-017-3325-1
Dong X, Qin M, Wang ZE et al (2019) Should we pay attention to recombinant norovirus strain GII.P7/GII.6? J Infect Public Health 12(3):403–409. https://doi.org/10.1016/j.jiph.2018.12.007
Fajardo Á, Tort FL, Victoria M et al (2014) Phylogenetic analyses of Norovirus strains detected in Uruguay reveal the circulation of the novel GII.P7/GII.6 recombinant variant. Infect Genet Evol 28:328–332. https://doi.org/10.1016/j.meegid.2014.10.026
Puustinen L, Blazevic V, Salminen M et al (2011) Noroviruses as a major cause of acute gastroenteritis in children in Finland, 2009–2010. Scand J Infect Dis 43(10):804–808. https://doi.org/10.3109/00365548.2011.588610
Fumian TM, Andrade JSR, Leite JP et al (2016) Norovirus recombinant strains isolated from gastroenteritis outbreaks in Southern Brazil, 2004–2011. PLoS ONE 11(4):e0145391. https://doi.org/10.1371/journal.pone.0145391
Chan MCW, Hu Y, Chen H et al (2017) Global spread of Norovirus GII.17 Kawasaki 308, 2014–2016. Emerg Infect Dis 23(8):1359–1354. https://doi.org/10.3201/eid2308.161138
Degiuseppe JI, Gomes KA, Hadad MF et al (2017) Detection of novel GII.17 norovirus in Argentina, 2015. Infect Genet Evol 47:121–124. https://doi.org/10.1016/j.meegid.2016.11.026
Silva LD, Bandeira RD, Junior EC et al (2017) Detection and genetic characterization of the emergent GII.17_2014 norovirus genotype among children with gastroenteritis from Northern Brazil. Infect Genet Evol 48:1–3. https://doi.org/10.1016/j.meegid.2016.11.027
Medici MC, Tummolo F, Martella V et al (2014) Novel recombinant GII.P16_GII.13 and GII.P16_GII.3 norovirus strains in Italy. Virus Res 188:142–145. https://doi.org/10.1016/j.virusres.2014.04.005
Medici MC, Tummolo F, Martella V et al (2018) Emergence of novel recombinant GII.P16_GII.2 and GII.P16_GII.4 Sydney 2012 norovirus strains in Italy, winter 2016/2017. New Microbiol 41(1):71–72
Pabbaraju K, Wong AA, Tipples GA et al (2019) Emergence of a novel recombinant norovirus GII.P16-GII.12 strain causing gastroenteritis, Alberta, Canada. Emerg Infect Dis 25(8):1556–1559. https://doi.org/10.3201/eid2508.190059
Hoffmann D, Mauroy A, Seebach J et al (2013) New norovirus classified as a recombinant GII.g/GII.1 causes an extended foodborne outbreak at a university hospital in Munich. J Clin Virol 58(1):24–30. https://doi.org/10.1016/j.jcv.2013.06.018
Nahar S, Afrad MH, Begum N et al (2013) High prevalence of noroviruses among hospitalized diarrheal patients in Bangladesh, 2011. J Infect Dev Ctries 7(11):892–896. https://doi.org/10.3855/jidc.2944
Arana A, Cilla G, Montes M et al (2014) Genotypes, recombinant forms, and variants of norovirus GII.4 in Gipuzkoa (Basque Country, Spain), 2009–2012. PLoS ONE 9(6):e98875. https://doi.org/10.1371/journal.pone.0098875
Iritani N, Kaida A, Abe N et al (2012) Increase of GII.2 norovirus infections during the 2009–2010 season in Osaka City, Japan. J Med Virol. 84(3):517–525. https://doi.org/10.1002/jmv.23211
Wang YH, Zhou DJ, Zhou X et al (2012) Molecular epidemiology of noroviruses in children and adults with acute gastroenteritis in Wuhan, China, 2007–2010. Arch Virol. 157(12):2417–2424. https://doi.org/10.1007/s00705-012-1437-1
Bidalot M, Théry L, Kaplon J et al (2017) Emergence of new recombinant noroviruses GII.p16-GII.4 and GII.p16-GII.2, France, winter 2016 to 2017. Euro Surveill 22(15):30508. https://doi.org/10.2807/1560-7917.ES.2017.22.15.30508
Ao Y, Xie X, Dong X et al (2019) Genetic analysis of an emerging GII.P2-GII.2 norovirus associated with a 2016 outbreak of acute gastroenteritis in China. Virol Sin 34(1):111–114. https://doi.org/10.1007/s12250-019-00084-6
Liu LT, Kuo TY, Wu CY et al (2017) Recombinant GII.P16-GII.2 Norovirus, Taiwan, 2016. Emerg Infect Dis 23(7):1180–1183. https://doi.org/10.3201/eid2307.170212
Niendorf S, Jacobsen S, Faber M et al (2017) Steep rise in norovirus cases and emergence of a new recombinant strain GII.P16-GII.2, Germany, winter 2016. Euro Surveill 22(4):30447. https://doi.org/10.2807/1560-7917.ES.2017.22.4.30447
Cheung SKC, Kwok K, Zhang LY et al (2019) Higher viral load of emerging norovirus GII.P16-GII.2 than pandemic GII.4 and epidemic GII.17, Hong Kong, China. Emerg Infect Dis 25(1):119–122. https://doi.org/10.3201/eid2501.180395
Mizukoshi F, Nagasawa K, Doan YH et al (2017) Molecular evolution of the RNA-dependent RNA polymerase and capsid genes of human norovirus genotype GII.2 in Japan during 2004–2015. Front Microbiol 8:705. https://doi.org/10.3389/fmicb.2017.00705
Silva-Sales M, Leal E, Milagres FAP, Brustulin R, Morais VDS, Marcatti R, Araújo ELL, Witkin SS, Deng X, Sabino EC, Delwart E, Luchs A, Costa ACD (2020 Dec) Genomic constellation of human Rotavirus A strains identified in Northern Brazil: a 6-year follow-up (2010–2016). Rev Inst Med Trop Sao Paulo. 18(62):e98. https://doi.org/10.1590/S1678-9946202062098
Acknowledgements
We thank Luciano Monteiro da Silva for administrative support. Our thanks also to the Coordenação Geral de Laboratórios de Saúde Pública do Departamento de Articulação Estratégica da Secretaria de Vigilância em Saúde do Ministério da Saúde (CGLAB/DAEVS/SVS-MS), MP Biomedicals Inc., and Zymo Research Corporation, for the donation of reagents.
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This study was partially supported by FAPESP #2016/01735-2 and CNPq #400354/2016-0. Antônio Charlys da Costa is funded by FAPESP #2017/00021-9, Adriana Luchs is funded by FAPESP #2015/12944-9, Vanessa S. Morais is funded by FAPESP #2019/21706-5, and Elcio Leal is funded by CNPq #302677/2019-4.
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RJT, ACC, RT, ED, ECS, EL and AL conceived the study; RJT, ACC, EL and AL designed the study protocol; FAPM, RB and MART participated in the conduct of the study, collection and screening of the specimens; ACC, RT and VSM performed the deep-sequencing assays; ACC, XD, ED and EL analyzed the big data; RJT, ACC, RT, RPP, AJA, MSC, ELLA, MMG, ECS, EL and AL analyzed and interpreted the data; ED, ECS, EL and AL supervised the study; RJT, ACC, MSC, MMG, EL and AL drafted the manuscript; all authors critically revised the manuscript for intellectual content and approved the final version. ACC and AL are guarantors of the paper.
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Tinker, R.J., da Costa, A.C., Tahmasebi, R. et al. Norovirus strains in patients with acute gastroenteritis in rural and low-income urban areas in northern Brazil. Arch Virol 166, 905–913 (2021). https://doi.org/10.1007/s00705-020-04944-5
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DOI: https://doi.org/10.1007/s00705-020-04944-5