Hajj is the annual gathering of Islam practitioners in Mecca, Saudi Arabia. During the event, gastrointestinal infections are usually experienced and outbreaks have always been a concern; nevertheless, a deep and integrative study of the etiological agents has never been carried out. Here, I describe for the first time the epidemiology of pathogenic enteric viruses during Hajj 2011, 2012 and 2013. The focus of this study was the common enteric viruses Astrovirus, Norovirus, Rotavirus and Adenovirus. An enzyme Immunoassay established their presence in 14.9%, 15.0% and 6.6% of the reported cases of acute diarrhea for 2011, 2012 and 2013, respectively. For the three years of study, Astrovirus accounted for the majority of the viral infections. To our knowledge, this is the first time an epidemiological study depicts Astrovirus as the main viral agent of gastroenteritis in a mass gathering event. Hajj is rich in strains of Astrovirus, Norovirus and Rotavirus. A first screening by RT-PCR resulted in ten different genotypes. Strains HAstV 2, HAstV 1 and HAstV 5 were identified for Astrovirus. GI.6, GII.3, GII.4 and GII.1 were described for Norovirus and G1P[8], G4P[8] and G3P[8] were found for Rotavirus. The majority of the Astrovirus isolates could not be genotyped suggesting the presence of a new variant(s). Cases like this encourage the use of metagenomics (and nextgeneration sequencing) as a state-of-the-art technology in clinical diagnosis. A sample containing Adenovirus particles is being used to standardize a process for detection directly from stool samples and results will be obtained in the near future. The overall findings of the present study support the concept of Hajj as a unique mass gathering event that potentiates the transmission of infectious diseases. The finding of Norovirus GII.4 Sydney, a variant originated from Australia, suggests that Hajj is a receptor of infectious diseases worldwide. This work is part of the Hajj project, a collaborative effort with the Ministry of Health of the Kingdom of Saudi Arabia in order to describe entirely the epidemiology of gastrointestinal diseases in Hajj. It is expected that the results of this study will serve in the refinement of public health policies.

No description available.

Humane Astroviren (HAstV) stellen wichtige Erreger kindlicher Gastroenteritiden dar, die in ihrer Bedeutung lange Zeit unterschätzt wurden. Sie werden in acht Serotypen klassifiziert, die nach dem bisherigen Kenntnisstand mit den Genotypen korrespondieren. Ziel dieser Arbeit war es, Einsicht in die molekulare Epidemiologie der in Deutschland zirkulierenden Astroviren zu vermitteln. Eine HAstV-spezifische RT-PCR bildete die Grundlage für die phylogenetische Analyse eines Genomabschnitts, der für die Kapsidproteine des Virus kodiert. Dazu wurden 16 deutsche Astrovirusisolate aus den Jahren 1997-1999 unter Einbeziehung bereits publizierter Sequenzdaten der Serotypen 1-8 untersucht. In Anlehnung an ein in der vorliegenden Literatur verwendetes Klassifizierungsschema erfolgte die Einteilung der deutschen Isolate in unterschiedliche Genotypen. Hierbei bildete eine Konvergenz von mindestens 85% das Kriterium für die Zuordnung differenter Isolate zum gleichen Genotyp. Es konnte gezeigt werden, dass in Deutschland wenigstens vier HAstV-Genotypen (1-4) kozirkulieren. Über dem betrachteten Genomabschnitt stimmten einige Isolate aus unterschiedlichen geografischen Regionen in ihrer Sequenz überein. Im Rahmen dieser Arbeit wurde erstmals die Totalsequenz eines humanen Astrovirus vom Serotyp 3 ermittelt. Während in der phylogenetischen Analyse des betreffenden Isolats nur ein Genomabschnitt betrachtet wurde, ließ sich an seiner Totalsequenz demonstrieren, dass die Einordnung in den Geno- bzw. Serotyp 3 auch in anderen Genomregionen Gültigkeit besitzt. Analog zu den bisher bekannten Gesamtgenomsequenzen der Serotypen 1, 2 und 8 lassen sich drei überlappende offene Leseraster (ORFs) identifizieren. In den beiden am 5'-Ende gelegenen ORFs 1a und 1b erweisen sich die putativen Motive der Protease und der RNA-Polymerase zwischen den vier Serotypen als hochkonserviert, ebenso wie vier potentielle Transmembrandomänen, ein ribosomales frameshift-Signal und ein nukleäres Lokalisationssignal. In dem am 3'-Ende gelegenen ORF 2 befinden sich drei hochkonservierte potentielle N-Glykosylierungs-Sites sowie ein hochkonserviertes Glykosaminoglykan-Attachment-Site. Ein wesentlicher Befund im Zusammenhang mit der Totalsequenz ist der Nachweis einer 45 Nukleotide umfassenden Deletion im ORF1a im Originalmaterial (Stuhl). Diese wurde bisher nur bei Astroviren gefunden, die in Zellen kultiviert wurden. Von Interesse und weiteren Untersuchungen vorbehalten ist ihre Nähe zur nukleären Lokalisationssequenz, die für die Beeinflussung des Zielzell-Tropismus von Astroviren verantwortlich sein könnte. / Human astroviruses (HastV) are an important cause of infantile gastroenteritis. To date, there are eight recognized serotypes which correlate with genotypes. The aim of this study was to investigate the molecular epidemiology of astroviruses circulating in Germany. Based on a HAstV-specific RT-PCR, phylogenetic analysis of a segment of the capsid protein gene was performed. The examination included sequence data of 16 German astrovirus isolates from the years 1997-1999 as well as published sequence information of the serotypes 1-8. Molecular typing was carried out following published classification strategies. The criterion for classification of isolates into one genotype was sequence identity of at least 85%. Astroviruses of at least four different genotypes (1-4) were found to cocirculate in Germany. The nucleotide sequences of several isolates from different geographical regions were identical. As part of this study, the complete genomic sequence of a type 3 human astrovirus was determined. The classification of the virus as a genotype 3 astrovirus as suggested by phylogenetic analysis over a limited genome section was supported by sequence comparison over two different genomic regions. Similar to the known total sequences of serotypes 1,2 and 8, three overlapping open reading frames (ORFs) were identified. The 5' end ORFs 1a and 1b contain the putative protease and polymerase motifs, which are highly conserved between the four serotypes. A high degree of sequence identity was also found for four potential transmembrane domains, a ribosomal frameshift signal and a nuclear localisation signal. The 3' end ORF 2 encodes three almost totally conserved potential N-glycosylation sites and one highly conserved putative glycosaminoglycan attachment site. As an outstanding feature, the virus, which was isolated and sequenced directly from diarrheal feces, presents a 45-nucleotide deletion in ORF 1 a. This deletion has previously only been found in cell cultured astroviruses. Further studies are needed to determine whether all viral genomes within the quasispecies carry the deletion.

<p>Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalo&scaron;ću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiolo&scaron;ka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, &scaron;to se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemiolo&scaron;kih mera da se spreči nastanak epidemija i njihovo &scaron;irenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvr&scaron;i genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodi&scaron;njeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vr&scaron;ena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodi&scaron;njem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najče&scaron;će su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najče&scaron;će rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su če&scaron;će hospitalizovana i imala su statistički značajno vi&scaron;u temperaturu. Pored vi&scaron;e temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno če&scaron;će kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno če&scaron;će su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno če&scaron;će bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njače&scaron;će je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najvi&scaron;e obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodi&scaron;njem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najče&scaron;će kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najče&scaron;će kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i vi&scaron;a je nego &scaron;to je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno kori&scaron;ćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, &scaron;to je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja.</p> / <p>Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease&mdash;accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion&mdash;leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high&mdash;higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.</p>

O objetivo deste trabalho foi caracterizar astrovírus em amostras fecais coletadas de crianças com e sem diarréia, em São Paulo, Brasil, e divididas em dois grupos, EPM e HU, de acordo com a origem. A detecção foi realizada utilizando-se RT-PCR, com primers específicos. Os resultados para as amostras EPM mostram que 66/234 (28,2%) foram positivas para astrovírus. Para as amostras HU, 18/187 (9,6%) foram positivas. A genotipagem foi realizada com a técnica de nested/RT-PCR. De 66 amostras positivas (EPM), 19 (28,7%) foram caracterizadas como HAstV-1, 4 (6,0%) como HAstV-2, 2 (3,0%) como HAstV-3, 1 (1,5%) como HAstV-5 e 3 (4,5%) como HAstV-8. Das 18 positivas do HU, 1 (5,5%) amostra foi caracterizada como HAstV-1, 7 (38,8%) como HAstV-2 e 1 (5,5%) como HAstV-8. As amostras genotipadas em ambos os grupos foram submetidas ao seqüenciamento de nucleotídeos para confirmação dos resultados. Detecção e genotipagem de astrovírus em casos de diarréias pediátricas são técnicas são importantes e descrevem como esse vírus está circulando em São Paulo, Brasil. / The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo city, Brazil, and grouped into two distinct groups, EPM and HU. Detection was carried out using RT-PCR with specific primers. Results for EPM set showed that 66/234 (28,2%) were positive. In the HU set of samples, 18/187 (9,6%) were positive for astrovirus. Genotyping was carried out with nested/RT-PCR. Out of 66 astrovirus positive EPM samples, 19 (28,7%) were characterized as HAstV-1, 4 (6,0%) as HAstV-2, 2 (3,0%) as HAstV-3, 1 (1,5%) as HAstV-5 and 3 (4,5%) as HAstV-8. Among 18 astrovirus positive HU samples, 1 (5,5%) was characterized as HAstV-1, 7 (38,8%) as HAstV-2 and 1 (5,5%) as HAstV-8. Genotyped samples were confirmed by nucleotide sequencing. Detection and genotyping of astrovirus in pediatric diarrhea are important and describes how this virus is circulating in São Paulo, Brazil.

As doenças entéricas associadas com diarreia, desidratação e perda de peso constituem um dos principais problemas da bovinocultura mundial, contribuindo para expressivos índices de morbidade e mortalidade, principalmente entre neonatos. Nesse contexto, exigências para o aprimoramento do diagnóstico laboratorial de doenças entéricas de natureza infectocontagiosa, tornaram-se constantes, face às perdas econômicas vinculadas. Entre os principais enteropatógenos virais, de distribuição mundial e história recente, destaca-se o astrovírus bovino (BoAstV), cuja primeira identificação data de 1978. BoAstV entérico induz diarreia principalmente entre neonatos e imunocomprometidos, tendo uma prevalência acima de 60% nas 5 primeiras semanas de vida dos animais. Devido à carência de dados a respeito da prevalência desse vírus no Brasil, o presente estudo foi realizado objetivando a detecção e caracterização moleculares de cepas de BoAstV em amostras fecais de bovinos com e sem diarreia de diferentes idades. O estudo foi conduzido a partir de 272 animais em diferentes estados do Brasil, obtendo-se 14,3% de positividade através de RT-PCR, sendo que 11 amostras, provenientes dos estados de São Paulo, Minas Gerais e Rio Grande do Sul, foram submetidas ao sequenciamento nucleotídico. A similaridade entre as sequências deduzidas de aminoácidos das amostras obtidas foi maior do que 86,8%, quando comparadas umas com as outras, e situou-se entre 86,2 a 94,8% quando comparadas com sequências de outros BoAstV descritos. Nas reconstruções filogenéticas, 9 amostras agruparam-se conjuntamente em clado distinto de outros BoAstV, uma amostra (BoAstV-155-BRA) formou ramo parafilético a clado que agrupa tanto outros BoAstV como astrovírus de cervídeos e a amostra restante (BoAstV-267-BRA) formou ramo basal aos astrovírus bovinos e suínos. No entanto, o posicionamento das 2 amostras divergentes (BoAstV-155-BRA e BoAstV-267-BRA) não derivou de episódios de seleção positiva ou recombinação. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de variantes de BoAstV entérico em rebanhos de estados da região centro-sul do Brasil. / Enteric diseases associated with diarrhea, dehydration and weight loss is one of the major problems of cattle worldwide, contributing to significant morbidity and mortality, especially among newborns. In this context, demands for improving the laboratory diagnosis of infectious enteric diseases, became constant, given the economic losses involved. Among the major viral enteropathogens of worldwide distribution and recent history, highlight the bovine astrovirus (BoAstV), whose first mention dates from 1978. Enteric BoAstV induce diarrhea especially among newborns and immunocompromised animals, having a prevalence above 60% in the first 5 weeks of life. Due to lack of data concerning the occurrence of this virus in Brazil, the present study was aimed at molecular detection and characterization of BoAstV strains in fecal samples from cattle with and without diarrhea of different ages. The study was conducted on 272 animals from different states of Brazil, obtaining positivity of 14.3% by RT-PCR, and 11 samples from the states of São Paulo, Minas Gerais and Rio Grande do Sul were subjected to nucleotide sequencing. The similarity between the deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and was between 86.2 to 94.8% compared with sequences of other BoAstV described. In phylogenetic reconstructions, 9 samples were grouped together in a separate clade from other BoAstV, a sample (BoAstV-155-BRA) formed a paraphyletic branch to a clade that groups both other BoAstV and deer astrovirus and the remaining sample (267-BRA-BoAstV ) formed a basal branch to bovine and porcine astrovirus. However, the positioning of the two different samples (BoAstV-155-BRA and BoAstV-267-BRA) did not derive from episodes of positive selection or recombination. In summary, the results indicate, in an unprecedented manner, the circulation of enteric BoAstV variants in herds of cattle form states of the south-central region of Brazil.

As bactérias patogênicas em moluscos bivalves podem ser agentes causadores de doenças como a gastroenterite e responsáveis por vários surtos de origem alimentar, representado um risco para os consumidores. Os vírus entéricos são a causa mais comum de surtos de gastroenterites não bacteriana em humanos no mundo e podem ser encontrados nas águas utilizadas no cultivo de moluscos bivalves. Este estudo teve como objetivo avaliar a contaminação de mexilhões (Mytella falcata) e ostras (Crassostrea brasiliana) provenientes do Complexo Estuarino Lagunar de Cananéia-Iguape, Estado de São Paulo, por bactérias (coliformes totais, coliformes termotolerantes, patotipos de Escherichia coli), por astrovírus e norovírus humanos. Um total de 150 amostras de moluscos bivalves (75 ostras e 75 mexilhões) foram coletadas de junho de 2016 a fevereiro de 2017. A estimativa de coliformes totais nos tecidos das ostras variou de 14,1 a 154,5 número mais provável (NMP)/g e de coliformes termotolerantes de 3,0 a 48,6 NMP/g, enquanto que para as amostras de mexilhões, os coliformes totais variaram de 97,4 a 1300 NMP/g e coliformes termotolerantes de 3,6 a 927 NMP/g. E. coli foi detectada em 24 amostras (16%), em concentrações variando entre &lt;3 e &gt;927 NMP/g. Quatro amostras (17%) foram identificadas com Escherichia coli enteropatogênica (EPEC), apresentando o gene eae por PCR (Reação em Cadeia da Polimerase) e RFLP (Polimorfismo no Comprimento de Fragmentos de Restrição), e os amplicons positivos foram sequenciados. As porcentagens de similaridade relativas ao gene phoA de E. coli, para as cinco amostragens realizadas no estudo, apresentaram valores iguais ou superiores a 88,6%. As sequências de EPEC agruparam-se em diferentes clados com outras sequências do Brasil, Suíça e Uruguai, exibindo similaridade de 57,7 e 97,1% quando comparadas umas as outras. Quando comparadas a outras sequências de referência depositadas no GenBank, a similaridade variou entre 56,2 e 95,4%. Estes resultados são os primeiros a indicar a presença de EPEC em moluscos bivalves no Brasil. Astrovírus não foram identificados nas amostras de moluscos analisadas neste estudo. Norovírus (NoV) foi identificado em 21 (14%) das amostras, sendo 38% de mexilhões e 62% de ostras. As amostras de NoV genogrupo II (GII) foram agrupadas num clado único, juntamente com outras sequências de NoV GII, sendo mais próximas filogeneticamente de sequências originárias do Brasil, Japão e México, com similaridade de 93,8 a 96,6% do que com as outras sequências homólogas. A triagem de moluscos bivalves para coliformes, E. coli e presença de vírus entéricos significativos para a saúde pode ajudar na prevenção de surtos entre os consumidores e contribuir para a melhoria do ambiente estuarino. / The pathogenic bacteria in bivalve molluscs are causative agents of diseases such as gastroenteritis and responsible for several food-borne outbreaks, representing a risk to consumers. Enteric viruses are the most common cause of outbreaks of non-bacterial gastroenteritis in humans in the world and can be found in waters used in the cultivation of bivalve molluscs. The objective of this study was to evaluate the contamination of mussels (Mytella falcate) and oysters (Crassostrea brasiliana) from the estuarine complex Lagunar of Cananéia-Iguape, State of São Paulo, by bacteria (total coliforms, thermotolerant coliforms, Escherichia coli) and by human astroviruses and noroviruses. A total of 150 samples of bivalve molluscs (75 oysters and 75 mussels) were collected from June 2016 to February 2017. The total coliform estimate in oyster tissues varied from 14.1 to 154.5 most probable number (MPN)/g and thermotolerant coliforms from 3.0 to 48.6 MPN/g, whereas for mussel samples, total coliforms ranged from 97.4 to 1300 MPN/g and thermotolerant coliforms from 3.6 to 927 MPN/g. E. coli was detected in 24 samples (16%) at concentrations ranging from &lt;3 to &gt;927 NMP/g. Four (17%) were identified with enteropathogenic Escherichia coli (EPEC), presenting the gene eae by PCR (Polymerase Chain Reaction) and RFLP (Restriction fragment length polymorphism), and the positive amplicons were sequenced. The percentages of similarity relative to the phoA gene of E. coli, for the five samplings carried out in the study, presented values equal or superior to 88.6%. The EPEC sequences were grouped in different clades with other sequences from Brazil, Switzerland and Uruguay, exhibiting similarity of 57.7 and 97.1% when compared to each other. When compared to other reference sequences deposited in GenBank, the similarity ranged from 56.2 to 95.4%. These results are the first to indicate the presence of EPEC in bivalve molluscs in Brazil. Astroviruses were not identified in the mollusk samples analyzed in this study. Norovirus (NoV) was identified in 21 (14%) of the samples, representing 38% of mussels and 62% of oysters. NoV genogroup II (GII) samples were clustered in a single clade, along with other NoV GII sequences, keeping phylogenetically closest to sequences originating in Brazil, Japan and Mexico, with similarity of 93.8 to 96.6% than with the other homologous sequences. The screening of bivalve molluscs for coliforms, E. coli and the presence of enteric viruses significant to health can help preventing outbreaks among consumers and contribute to the improvement of the estuarine environment.

As doenças entéricas associadas com diarreia, desidratação e perda de peso constituem um dos principais problemas da bovinocultura mundial, contribuindo para expressivos índices de morbidade e mortalidade, principalmente entre neonatos. Nesse contexto, exigências para o aprimoramento do diagnóstico laboratorial de doenças entéricas de natureza infectocontagiosa, tornaram-se constantes, face às perdas econômicas vinculadas. Entre os principais enteropatógenos virais, de distribuição mundial e história recente, destaca-se o astrovírus bovino (BoAstV), cuja primeira identificação data de 1978. BoAstV entérico induz diarreia principalmente entre neonatos e imunocomprometidos, tendo uma prevalência acima de 60% nas 5 primeiras semanas de vida dos animais. Devido à carência de dados a respeito da prevalência desse vírus no Brasil, o presente estudo foi realizado objetivando a detecção e caracterização moleculares de cepas de BoAstV em amostras fecais de bovinos com e sem diarreia de diferentes idades. O estudo foi conduzido a partir de 272 animais em diferentes estados do Brasil, obtendo-se 14,3% de positividade através de RT-PCR, sendo que 11 amostras, provenientes dos estados de São Paulo, Minas Gerais e Rio Grande do Sul, foram submetidas ao sequenciamento nucleotídico. A similaridade entre as sequências deduzidas de aminoácidos das amostras obtidas foi maior do que 86,8%, quando comparadas umas com as outras, e situou-se entre 86,2 a 94,8% quando comparadas com sequências de outros BoAstV descritos. Nas reconstruções filogenéticas, 9 amostras agruparam-se conjuntamente em clado distinto de outros BoAstV, uma amostra (BoAstV-155-BRA) formou ramo parafilético a clado que agrupa tanto outros BoAstV como astrovírus de cervídeos e a amostra restante (BoAstV-267-BRA) formou ramo basal aos astrovírus bovinos e suínos. No entanto, o posicionamento das 2 amostras divergentes (BoAstV-155-BRA e BoAstV-267-BRA) não derivou de episódios de seleção positiva ou recombinação. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de variantes de BoAstV entérico em rebanhos de estados da região centro-sul do Brasil. / Enteric diseases associated with diarrhea, dehydration and weight loss is one of the major problems of cattle worldwide, contributing to significant morbidity and mortality, especially among newborns. In this context, demands for improving the laboratory diagnosis of infectious enteric diseases, became constant, given the economic losses involved. Among the major viral enteropathogens of worldwide distribution and recent history, highlight the bovine astrovirus (BoAstV), whose first mention dates from 1978. Enteric BoAstV induce diarrhea especially among newborns and immunocompromised animals, having a prevalence above 60% in the first 5 weeks of life. Due to lack of data concerning the occurrence of this virus in Brazil, the present study was aimed at molecular detection and characterization of BoAstV strains in fecal samples from cattle with and without diarrhea of different ages. The study was conducted on 272 animals from different states of Brazil, obtaining positivity of 14.3% by RT-PCR, and 11 samples from the states of São Paulo, Minas Gerais and Rio Grande do Sul were subjected to nucleotide sequencing. The similarity between the deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and was between 86.2 to 94.8% compared with sequences of other BoAstV described. In phylogenetic reconstructions, 9 samples were grouped together in a separate clade from other BoAstV, a sample (BoAstV-155-BRA) formed a paraphyletic branch to a clade that groups both other BoAstV and deer astrovirus and the remaining sample (267-BRA-BoAstV ) formed a basal branch to bovine and porcine astrovirus. However, the positioning of the two different samples (BoAstV-155-BRA and BoAstV-267-BRA) did not derive from episodes of positive selection or recombination. In summary, the results indicate, in an unprecedented manner, the circulation of enteric BoAstV variants in herds of cattle form states of the south-central region of Brazil.

Made available in DSpace on 2014-12-05T18:34:09Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 101.pdf: 12703986 bytes, checksum: b3d8bcb5c201aa863f8d9d57acdab347 (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A utilização e a manutenção dos recursos hídricos são temas que apresentam uma importância crescente nas discussões mundiais, devido à preocupação com a futura escassez de água dentro dos padrões de qualidade aceitáveis para o consumo. Entre os maiores problemas na utilização da água, destaca-se a sua poluição por resíduos biológicos e químicos, tornando as águas contaminadas um importante meio de transmissão de várias doenças, entre estas, as gastroenterites de etiologia viral. Este estudo teve como objetivo implementar e analisar uma metodologia para concentração de astrovirus humano (HAstV) em águas ambientais, associada a métodos moleculares de detecção, caracterização e quantificação. Foi realizado um estudo experimental com amostras de diferentes matrizes aquáticas, incluindo água de rio, mar, consumo e esgoto (efluente e afluente) para avaliação da metodologia empregada e dois estudos de campo com um total de 100 amostras ambientais (48 provenientes de uma Estação de Tratamento de Esgoto [ETE] e 52 de água de rio). Foi utilizado o método de concentração por adsorção/eluição com membrana carregada negativamente seguido de ultrafiltração associada à detecção por reação em cadeia pela polimerase precedida da transcrição reversa (RT-PCR) e a quantificação pela PCR em tempo real quantitativa (qPCR). A caracterização molecular do vírus detectado foi realizada por sequenciamento parcial do genoma da segunda fase aberta de leitura. Experimentalmente, a taxa de recuperação variou de 2,4 por cento a 65 por cento entre os diferentes tipos de amostras de água. No estudo de campo, foram detectados 16 por cento de HAstV no total de amostras ambientais, 17,3 por cento em amostras de águas de igarapés e 16,7 por cento na ETE. Uma amostra de rio foi caracterizada como HAstV-1 [...] / The use and maintenance of water resources are issues that present increasing significance in world debates, due to concern with the future scarcity of water acceptable for consuming according to quality standards. Among the major problems of water use, the more important is the pollution by biological and chemical wastes, which makes contaminated water a significant transmission route of several diseases, as gastroenteritis of viral etiology. This study aimed to implement and analyze a methodology for human astrovirus (HAstV) concentration from environmental waters, associated with molecular methods of detection, characterization and quantification. An experimental research was performed with different aquatic sources samples, including river water, seawater, potable water and sewage (inflow and outflow) to evaluate the methodology used, and two field’s research with a total of 100 environmental samples (48 from a Sewage Treatment Plant [STP] and 52 from river water). A method of concentration by adsorption/elution into an electronegative membrane followed by ultrafiltration combined to reverse transcriptase – polymerase chain reaction (RT-PCR) and real time PCR (qPCR) quantification was utilized. The molecular characterization of the detected virus was performed by genome partial sequencing of the second open reading frame (ORF-2). Experimentally, the recovery rate ranged from 2.4% to 65% among the different matrices of water samples. In field research, 16% of HAstV were detected of the total environmental samples, 17,3% from river water samples, and 16,7% from STP. One river water sample was characterized as HAstV-1. The qPCR method exhibited an 18 copies/reaction sensibility and the comparative analysis of the results showed a low detection (2%) in comparison with RT-PCR (15%). The association of the methods used proved the presence of HAstV in environmental waters, being an important tool for water quality assessment, for viral particle removal efficiency assessment in different treatment water methods and to elucidate the epidemiology HAstV waterborne outbreaks

O objetivo deste trabalho foi caracterizar astrovírus em amostras fecais coletadas de crianças com e sem diarréia, em São Paulo, Brasil, e divididas em dois grupos, EPM e HU, de acordo com a origem. A detecção foi realizada utilizando-se RT-PCR, com primers específicos. Os resultados para as amostras EPM mostram que 66/234 (28,2%) foram positivas para astrovírus. Para as amostras HU, 18/187 (9,6%) foram positivas. A genotipagem foi realizada com a técnica de nested/RT-PCR. De 66 amostras positivas (EPM), 19 (28,7%) foram caracterizadas como HAstV-1, 4 (6,0%) como HAstV-2, 2 (3,0%) como HAstV-3, 1 (1,5%) como HAstV-5 e 3 (4,5%) como HAstV-8. Das 18 positivas do HU, 1 (5,5%) amostra foi caracterizada como HAstV-1, 7 (38,8%) como HAstV-2 e 1 (5,5%) como HAstV-8. As amostras genotipadas em ambos os grupos foram submetidas ao seqüenciamento de nucleotídeos para confirmação dos resultados. Detecção e genotipagem de astrovírus em casos de diarréias pediátricas são técnicas são importantes e descrevem como esse vírus está circulando em São Paulo, Brasil. / The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo city, Brazil, and grouped into two distinct groups, EPM and HU. Detection was carried out using RT-PCR with specific primers. Results for EPM set showed that 66/234 (28,2%) were positive. In the HU set of samples, 18/187 (9,6%) were positive for astrovirus. Genotyping was carried out with nested/RT-PCR. Out of 66 astrovirus positive EPM samples, 19 (28,7%) were characterized as HAstV-1, 4 (6,0%) as HAstV-2, 2 (3,0%) as HAstV-3, 1 (1,5%) as HAstV-5 and 3 (4,5%) as HAstV-8. Among 18 astrovirus positive HU samples, 1 (5,5%) was characterized as HAstV-1, 7 (38,8%) as HAstV-2 and 1 (5,5%) as HAstV-8. Genotyped samples were confirmed by nucleotide sequencing. Detection and genotyping of astrovirus in pediatric diarrhea are important and describes how this virus is circulating in São Paulo, Brazil.