Os norovírus (NV) são uma importante causa de hospitalização infantil. Crianças internadas por gastroenterite por NV (GENV) são consideradas portadoras de diarreia grave. O objetivo desse estudo, realizado na cidade do Rio de Janeiro, Brasil, é descrever as características clínicas e a frequência da diarreia por NV em crianças hospitalizadas, comparando as taxas de detecção de NV em crianças vacinadas e não vacinadas contra rotavírus (Rotarix). Foram coletadas 659 amostras de fezes de igual número de crianças e encaminhadas para análise pela reação em cadeia pela polimerase, precedida de transcrição reversa no período de janeiro de 2004 a dezembro de 2009. O percentual de amostras positivas para os NV foi de 27,3% nesse período. Das 180 amostras positivas para NV, 55% tiveram origem na comunidade (aqCo) e 45% foram de aquisição nosocomial (aqNo). O percentual de GENV nos dois anos anteriores (2004 e 2005) à introdução da vacina Rotarix foi de 28,3%, sendo 11,3% o percentual de amostras aqCo. Nos dois anos posteriores (2008 e 2009), a GENV significou 24,4%, e as amostras aqCo foram 14,9% (p<0,05). Em 647 crianças, 494 não receberam a vacina Rotarix, enquanto 151 crianças receberam, pelo menos, uma dose. O percentual de GENV foi de 23,8% e 39,7%, respectivamente (p<0,05). Apesar do comportamento sazonal dos casos de GENV aqCo, esse fato não teve significância estatística. Das 180 crianças, 61,6% tinham peso &#8804; p10 do NCHS, 82,2% tinham idade &#8804; 5anos. As crianças com idade &#8804; 2 anos foram mais acometidas nos casos de aqCo do que àquelas de aqNo (p<0,05). Foram observados em 82 crianças: vômitos (73,2%), febre (54,9%), tosse (20,7%), coriza (2,2%), sangue nas fezes (8,5%), erupção cutânea (4,9%) e broncoespasmo (7,3%). Houve significância estatística com relação à frequência maior de febre, coriza, tosse e broncoespasmo nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). De 69 crianças, 73,9% apresentaram desidratação e, dessas, 76,5% necessitaram de hidratação venosa. Esses dados tiveram significância estatística, representada por maiores percentuais nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). Esse estudo demonstra que os NV foram um importante agente etiológico nos casos de gastroenterites em crianças hospitalizadas e responsável por altas taxas de infecções nosocomiais. Estatisticamente, não foi comprovada uma tendência de aumento dos casos de GENV no período do estudo, como também do aumento da frequência de GENV nos anos posteriores em relação aos anos anteriores à introdução da vacina Rotarix no Brasil em 2006. No entanto, houve significância estatística quando foi avaliado o percentual de GENV em crianças hospitalizadas vacinadas e não vacinadas contra RV. Um aumento dos casos de GENV em crianças poderá vir a acontecer nos próximos anos, quando é esperado que um número maior de crianças será vacinado contra RV. Tosse, coriza e broncoespasmo são sintomas que devem ser mais detalhadamente investigados. Estratégias de prevenção contra a disseminação dos NV são condutas importantes em unidades de internação. Uma vacina eficaz contra norovírus pode ser um benefício significativo para reduzir o percentual de crianças hospitalizadas por diarreia. / Noroviruses (NV) are a major cause of infant hospitalization. Children hospitalized for gastroenteritis NV (NVGE) are considered to have severe diarrhea. The purpose of this study, conducted in the city of Rio de Janeiro, Brazil, is to describe the clinical characteristics and frequency of norovirus diarrhea in hospitalized children, comparing the rates of detection of NV in vaccinated and unvaccinated children against rotavirus (RV). We collected 659 fecal samples from equal numbers of children and sent for analysis by polymerase chain reaction, reverse transcription from January 2004 to December 2009. The percentage of samples positive for NV was 27.3% in this period. Of the 180 samples positive for NV, 55% meant source community (Coaq) and 45% of nosocomial acquisition (Noaq). The percentage of NVGE the previous two years (2004 and 2005) the introduction of the vaccine against RV was 28.3% and 11.3% represented the percentage of samples Coaq. In the two subsequent years (2008 and 2009), NVGE meant to 24.4%, and the samples were Coaq 14.9% (p <0.05). In 647 children, 494 received no vaccine against RV while 151 children received at least one dose. The percentage of NVGE was 23.8% and 39.7%, respectively (p <0.05). Despite the seasonal behavior of NVGE aqCo cases, this did not reach statistical significance. Of the 180 children, 61.6% had weight &#8804; p10 NCHS, 82.2% were aged &#8804; 5 years old. Children aged &#8804; 2 years were most affected in cases of Coaq than those of Noaq (p <0.05). Were observed in 82 children: vomiting (73.2%), fever (54.9%), cough (20.7%), coryza (2.2%), blood in stools (8.5%), rash (4.9%) and bronchospasm (7.3%). There was statistical significance with respect to the higher frequency of fever, coryza, coughing and wheezing in children with NVGE of Coaq than those of Noaq (p <0.05). Of 69 children, 73.9% had dehydration and of these, 76.5% required intravenous hydration. These data were statistically significant, represented by the highest percentage of children with NVGE of Coaq than those of Noaq (p <0.05). This study demonstrates that the NV were an important etiologic agent in cases of gastroenteritis in hospitalized children and responsible for high rates of nosocomial infections. Statistically, , it was not shown a tendency to increase in cases of NVGE during the study period, as well as the increased frequency NVGE in later years relative to years prior to the introduction of RV vaccine in Brazil. However, statistical significance was assessed as the percentage of NVGE in hospitalized children vaccinated and unvaccinated against RV. An increase in cases of children in NVGE could happen in the next few years, when it is expected that a greater number of children will be vaccinated against RV. Cough, coryza and wheezing are symptoms that should be further investigated. Strategies for preventing the spread of NV are important conduits in inpatient units. An effective vaccine against norovirus can be a significant benefit to reduce the percentage of children hospitalized for diarrhea.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T13:51:59Z No. of bitstreams: 1 vivianhonoratobarletta.pdf: 2632443 bytes, checksum: c92921b16b55178bf7a44b922bd843ef (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:10:07Z (GMT) No. of bitstreams: 1 vivianhonoratobarletta.pdf: 2632443 bytes, checksum: c92921b16b55178bf7a44b922bd843ef (MD5) / Made available in DSpace on 2017-08-07T19:10:07Z (GMT). No. of bitstreams: 1 vivianhonoratobarletta.pdf: 2632443 bytes, checksum: c92921b16b55178bf7a44b922bd843ef (MD5) Previous issue date: 2011-02-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os norovírus (NoV) são importantes agentes etiológicos, responsáveis por surtos e casos esporádicos de doença diarréica aguda, que acometem indivíduos de todas as idades. A partícula viral não apresenta envelope e o material genético é constituído por uma molécula de RNA de fita simples, de polaridade positiva. Pertencem à família Caliciviridae, gênero Norovirus e estão classificados em cinco genogrupos (GI–V), sendo que os NoV humanos estão agrupados nos genogrupos I, II e IV e destes, os NoV do genogrupo II e genótipo 4 (GII.4) são os mais comumente encontrados, em todo o mundo. Apesar da associação destes vírus com a doença diarréica aguda estar bem documentada na literatura mundial, no Brasil, os trabalhos são escassos e restritos aos grandes centros e adjacências. Assim, considerando-se o pouco conhecimento sobre os norovírus e a inexistência de dados epidemiológicos na cidade de Juiz de Fora, MG, foi realizado o presente estudo, cujos objetivos foram a detecção e caracterização molecular de amostras de NoV, associadas a casos esporádicos de doença diarréica aguda infantil, bem como a avaliação da influência de fatores climáticos e demográficos na ocorrência destas infecções. De janeiro de 2008 a dezembro de 2009, 218 espécimes fecais foram analisados para a presença de NoV, por RT-PCR convencional, todos obtidos de crianças de 0 a 12 anos de idade, proveniente de atendimentos ambulatoriais (89,45%) e hospitalizados (10,55%). Foram detectadas 20 (9,17%) amostras positivas e observou-se uma tendência de sazonalidade das infecções no período da estação seca, no ano de 2008, fato que não se repetiu em 2009. A maioria das amostras positivas foi detectada em crianças na faixa de 0 a 36 meses e não houve correlação, estatisticamente significante, entre a ocorrência das infecções e o sexo. Das 20 amostras detectadas, 19 foram caracterizadas como NoV GII e 1 como NoV GI. O sequenciamento parcial do genoma e a análise filogenética das amostras selecionadas, revelou a presença de NoV dos genótipos GII.4 e GII.6, que cocircularam nos dois anos do estudo. As amostras NoV GII.4, detectadas em Juiz de Fora, apresentaram maior similaridade de nucleotídeos e de aminoácidos com aquelas que circularam no estado do Rio de Janeiro nos anos de 2006, 2007 e 2008. A análise filogenética das amostras NoV GII.6 detectadas em Juiz de Fora, associada à alta similaridade das sequências de nucleotídeos e aminoácidos, mostrou que estas foram mais proximamente relacionadas com a amostra NoV GII.6 (GU132461/2007), detectada no estado do Rio de Janeiro em 2007, fatos que, aliados à proximidade geográfica de ambas as cidades, sugerem uma possível linhagem comum entre as mesmas. Este levantamento epidemiológico permitiu constatar a presença e circulação de NoV na população infantil de Juiz de Fora, MG, demonstrando sua importante participação como agente etiológico das diarreias agudas, também nesta comunidade / Noroviruses (NoV) are important etiological agents responsible for outbreaks and sporadic cases of acute diarrhea in individuals of all ages. The viral particles are nonenveloped with and the genome is composed of a positive single-stranded RNA. Norovirus belongs to the Caliciviridae family, Norovirus genus and are classified into five genogroups (GI-V), with GI, GII and GIV being found in human and among them, the NoV GII genotype 4 (GII.4) are the most commonly found worldwide. In Brazil, norovirus surveys are realized mainly in research institutes, carried out in the biggest centers and surroundings. Thus, considering the little knowledge about these viruses and the lack of epidemiological data on this viral infection in the Juiz de Fora city, MG state, it was performed this study, which aimed to detect and characterize the NoV samples, associated with sporadic cases of acute infantile diarrhea, as well as asses the influence of climatic and demographic factors in the occurrence of these infections. Between January 2008 to December 2009, 218 fecal specimens were analyzed for the presence of NoV by conventional RT-PCR, all obtained from children 0-12 years of age, from outpatient (89.45%) and inpatients (10.55%). We detected 20 (9.17%) positive samples and there was a tendency for seasonal infections during the dry season in 2008, a fact which was not repeated in 2009. The biggest number of positive samples were detected in children aged 0 to 24 months and there was no statistically significant correlation between the occurrence of infections and sex. Of the 20 samples detected, 19 were characterized as NoV GII and 1 as NoV GI. The partial genome sequencing and phylogenetic analysis of selected samples revealed the presence of NoV genotypes GII.4 and GII.6, which co-circulated in the two years of study. Samples NoV GII.4 detected in Juiz de Fora, showed greater similarity of nucleotides and aminoacids with those that circulated in the state of Rio de Janeiro during 2006, 2007 and 2008. Phylogenetic analysis of the samples GII.6 NoV, detected in Juiz de Fora, associated with the high similarity of nucleotide and amino acid sequences showed that they were most closely related to the sample GII.6 NoV (GU132461/2007) detected in the state of Rio de Janeiro in 2007. This fact associated with the geographical proximity of both cities, suggesting a possible common lineage between them. This epidemiological survey revealed the presence and circulation of NoV in the infantile population of Juiz de Fora, MG, demonstrating its important role as an etiologic agent of acute diarrhea, also in this community.

MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human norovirus (NoV) is the etiological agent associated with acute gastroenteritis (AGE) in both children and adults worldwide. Children of <5 years of age, the elderly and individuals suffering from chronic diseases are potentially at high risk of NoV-associated illness. High morbidity and mortality rate associated with NoV have been reported worldwide. In children under the age of 5 years about 1.8 million death cases have been reported in developing countries alone. Despite the fact that the virus is affecting people of all age groups, there is lack of data to elucidate the importance and the role of NoV in children of the age above 5 years and adults. OBJECTIVE: To characterize human norovirus in patients with diarrhoea in rural communities of Vhembe district, Limpopo province. MATERIALS AND METHODS : From August 2017 to October 2018, outpatient between 5 and 68 years of age from rural communities of Vhembe district, Limpopo province were recruited for this study. A total of n=80 stool samples were collected from patients with diarrhoea and were kept at 4˚C throughout the transportation to the laboratory and refrigerated at - 20˚C prior to RNA extraction. Stool samples were tested for norovirus using the RIDA©GENE NOROVIRUS I & II real-time RT-PCR. The RNA extracts tested positive for norovirus were subjected to RT-PCR amplification. The RT-PCR products of the amplified fragments were sequenced, and phylogenetic trees were constructed by the neighbor-joining method using MEGA 7 software. RESULTS: NoV was detected in 13(16%) out of 80 stool samples collected, of which 6 (46%) strains belonged to norovirus GII and 7 (54%) strains to norovirus GI. A total of 5 genotypes were detected (GII.Pg, GII.1, GII.2, GII.4 Sydney 2012). The phylogenetic analysis revealed circulation of NoV genotypes with considerable diversity. CONCLUSION: This study illustrates NoV prevalence and substantial genetic diversity in patients above 5 years of age living in rural communities of Vhembe district, Limpopo province. Continued systematic surveillance to evaluate norovirus association with diarrhoea is needed to have a full picture on the epidemiology and disease burden in people of all the age groups. / NRF

<p>Vi&scaron;e od 100 vrsta virusa ljudi i životinja se izlučuje u spolja&scaron;nju sredinu. Prisustvo ovih virusa u povr&scaron;inskim vodama reflektuje fekalnu kontaminaciju i ukazuje na<br />opasnost za zdravlje ljudi i životinja. Na području Srbije se ne prati prisustvo patogenih virusa u povr&scaron;inskim vodama, pa&nbsp; čak ni u vodama za piće, a nije uspostavljena&nbsp; ni&nbsp; metodologija ovih ispitivanja. Shodno tome, cilj disertacije je da se utvrdi i analizira prisustvo animalnih i humanih virusa u povr&scaron;inskim vodama primenom najsavremenijih metoda koncentrovanja i detekcije virusa. U okviru disertacije ispitano je prisustvo sledećih virusa u povr&scaron;inskim vodama na teritoriji Vojvodine: humanih adenovirusa (HAdV); norovirusa (NoV) i hepatitis A virusa (HAV), adenovirusa svinja (PAdV),&nbsp; poliomavirusa goveda (BPyV) i hepatitis E virus (HEV).</p><p>Ispitano je ukupno 108 uzoraka povr&scaron;inskih i otpadnih voda koji su prikupljani od oktobra 2012. godine do juna 2014. godine. U radu su primenjene najsavremenije metode koncentrovanja i detekcije virusa u vodi, koje se u Srbiji nisu koristile za ovu namenu. Sprovedenim ispitivanjima dokazano je da su animalni i humani virusi prisutni u povr&scaron;inskim vodama na području Vojvodine. Najče&scaron;će detektovan&nbsp; virus u povr&scaron;inskim vodama je humani adenovirus (42,4%), a potom norovirusi GII i GI (40,4% i 15,2%), adenovirus svinja (11,1%), poliomavirus goveda (7,1%) i hepatitis E virus (3,0%). U ukupno 9 testiranih uzoraka gradske kanalizacione vode najče&scaron;će je detektovan HAdV (44,4%), NoV GII i GI&nbsp; (66,7% i 22,2%), BPyV je detektovan u samo jednom od 9 uzoraka, a niti u jednom nisu detektovani PAdV i HEV. Hepatitis A virus nije detektovan u uzorcima, a eksperimentalno je potvrđeno da su metode primenljive i za detekciju ovog virusa. Na osnovu rezultata prinosa procesne kontrole i utvrđenog prisustva virusa u uzorcima,&nbsp; zaključeno&nbsp; je da se ove metode mogu veoma uspe&scaron;no koristiti za detekciju virusne kontaminacije&nbsp; povr&scaron;inskih voda. Izvr&scaron;ena je igenotipizacija virusa iz odabranih uzoraka metodom sekvenciranja dela virusnog genoma. Indirektno je potvrđeno da su infekcije&nbsp;&nbsp; detektovanim virusima prisutne u populaciji životinja i ljudi. Prisustvo virusa u&nbsp;&nbsp; povr&scaron;inskim vodama i uzorcima gradske kanalizacije odražava infektivni status stanovni&scaron;tva, ali predstavlja i značajan rizik za zdravlje životinja i ljudi na području koje gravitira ispitanim vodama.&nbsp;</p> / <p>Over 100 types of pathogenic viruses are excreted in human and animal wastes. The presence of human and animal pathogenic enteric viruses in water environments reflects fecal contamination and indicates a risk to public health.&nbsp; Republic of Serbia does not implement surveillance for the presence of pathogenic human and animal viruses in surface waters and even in drinking water, neither is the established methodology of these studies in any institution in Serbia.&nbsp; Accordingly, the aim of the study was to determine and analyze the presence of human and animal viruses in surface water,&nbsp; using the latest methods&nbsp; of&nbsp; concentration and detection of the viruses.&nbsp; Within the dissertation examined the presence of the following viruses in surface waters in Vojvodina:&nbsp; Human adenoviruses&nbsp; (HAdV), noroviruses (NoV)&nbsp; and hepatitis A virus), Porcine adenovirus (PAdV) and Bovine polyomavirus (BPyV)&nbsp; and&nbsp; Hepatitis E virus (HEV).<br />A total of 108 samples of surface water and waste water were collected from October 2012 to June 2014. The paper are applied the most advanced methods and the concentration of virus detection in water, which in Serbia are not used for this purpose. The conducted tests have proven that the animal and human viruses present in surface waters in Vojvodina. The most commonly detected virus in surface water was human adenovirus (42.4%), followed by Norovirus GI&nbsp; and GII (40.4% and 15.2%),&nbsp; Porcine adenovirus&nbsp; (11,1%),&nbsp; Bovine polyomavirus&nbsp; (7.07%) and hepatitis E virus (3,0%).<br />In total of&nbsp; nine analysed sewage samples human adenovirus was detected in 44,4%&nbsp; of&nbsp; samples. The prevalence of norovirus GII and GI in sewage&nbsp; samples was&nbsp; 66,7%&nbsp; and 22,2%. Bovine&nbsp; polyomavirus was detected in one of nine samples while porcine adenovirus and hepatitis E virus were not detected in any of analyzed samples.&nbsp; Hepatitis A virus was not detected in samples, but&nbsp; it has been experimentally confirmed that the methods applicable for detection of the virus. Based on the results of process control and yield determined the presence of virus insamples, it was found that these methods can be successfully used to detect viral contamination of surface waters. Also, in these study was performed genotyping of viruses from selected samples by sequencing a part of the viral genome. Indirectly it is confirmed that the infection detected viruses present in a population of animals and humans. The presence of virus in samples of surface water and urban sewage reflects the infectious status of the population, but also constitutes a significant risk to the health of animals and people in the area that gravitates with tested waters.</p><p>&nbsp;</p>

<p>Human noroviruses (HuNoVs) are the leading cause of an acute form of non-bacterial gastroenteritis, where strains belonging to genogroup (G) II are dominant. Upon expression with the baculovirus culture system, virus-like particles (VLPs) of HuNoVs are expected to assemble into T = 3 icosahedral capsid particles resembling the structure of the infectious virion particles. However, some strains were found to assemble into either T = 1 or T = 4 capsids, or a combination of two different capsid forms. In this study, we showed that VLPs of the Virginia 1997 387 (VA387) GII.4 outbreak strain assembled into T = 1, T = 3, and T = 4 capsids upon expression in insect cell culture, the first case for a naturally occurring HuNoV strain to assemble into all three capsid states. TEM analysis revealed that T = 1 icosahedral particles were the most abundant in purified samples, which contrasts previous findings where either T = 3 or T = 4 were the most abundant. We resolved the cryo-EM structures of the T = 1 shell (S) domain, T = 3, and T = 4 particles to 2.24, 2.44, and 3.43 Å, respectively, making them the most resolved norovirus (NoV) structures to date. Single particle cryo-EM 3D analysis showed that the protruding (P) domain of T = 1 and T = 4 VLPs are highly dynamic. Additionally, we showed that T = 3 VLPs are resistant while T = 1 and T = 4 VLPs are sensitive to digestion in the presence of trypsin. This suggested that T = 1 and T = 4 capsids are less stable among the VLPs, which is consistent with the highly dynamic P domain inferred from our cryo-EM 3D analysis. During infection, HuNoVs travel through the gastrointestinal (GI) tract where they encounter a broad range of variable conditions that include pH, ionic strength, and host defenses (e.g., proteases). Our analyses suggest that virions are T = 3 particles as they can survive the GI tract upon exiting the host. We determined the first cryo-EM structure of T = 3 VLPs in complex with the known HuNoV host cell receptor, histo-blood group antigen (HBGA), to a resolution of 2.51 Å, demonstrating that NoV VLPs can serve as a platform in the structural characterization of small ligand molecules. Lastly, we identified a histidine residue retained in the S domain of all identified caliciviruses critical in the assembly of capsids. Our structures and their characterization will contribute to the development of therapeutic agents to combat noroviruses. </p>

MSc (Microbiology) / Department of Microbiology / See the attached abstract below