G1P[8] Rotavirus in children with severe diarrhea in the post-vaccine introduction era in Brazil: Evidence of reassortments and structural modifications of the antigenic VP7 and VP4 regions.

Worldwide rotaviruses A (RVA) are responsible for approximately 215,000 deaths annually among children aged <5 years. RVA G1P[8] remains associated with >50% of gastroenteritis cases in this age group. The aim of this study was to assess the genetic variability of G1P[8] strains detected in children with severe diarrhea in Belém, Pará, Brazil, during the post-rotavirus vaccine introduction era. Phylogenetic analysis clustered the VP4 and VP7 genes of 40 samples selected between 2009 and 2011 into lineages found to be different from the Rotarix® vaccine strain. A detailed investigation of their complete genotype constellations identified 2 reassortant viruses (5%), resulting from reassortments between the genogroups Wa-like and DS-1-like (G1-P[8]-I1-R2-C1-M1-A1-N1-T2-E1-H1) and Wa-like and AU-1-like (G1-P[8]-I1-R3-C1-M1-A1-N1-T1-E1-H1) genotype constellations. A comparison of the amino acid residues presents in the antigenic epitopes of VP7 and VP4, showed differences in the electrostatic charges distribution, between wild type Brazilian strains and the Rotarix® and RotaTeq® vaccine strains. These findings reflect the structural analyses of the antigenic regions of VP7 and VP4 of the RVA G1P[8] in children with gastroenteritis in Northern Brazil raising the hypothesis that structural modifications at these sites over time may account for the emergence of new strains that could possibly pose a challenge to current vaccines.

Rotavirus analyses by SYBR Green real-time PCR and microbiological contamination in bivalves cultivated in coastal water of Amazonian Brazil.

The contamination of mussels and oysters by viruses and bacteria is often associated with water contamination and gastroenteritis in humans. The present study evaluated viral and bacterial contamination in 380 samples, from nine mollusk-producing regions in coastal water north of the Brazilian Amazon. Rotavirus contamination was studied for groups A to H, using a two-step SYBR Green RT-qPCR (quantitative reverse transcription polymerase chain reaction), and bacterial families Enterobacteriaceae, Vibrionaceae, and Aeromonadaceae by classical and molecular methods. From the 19 pools analyzed, 26.3% (5/19) were positive for group A Rotavirus, I2 genotype for VP6 region, without amplifications for groups B-H. Bacteriological analysis identified Escherichia coli isolates in 89.5% (17/19) with identification of atypical enteropathogenic E. coli aEPEC in 10.5% (2/19), Salmonella (Groups C1 and G) (10.5%, 2/19), Vibrio alginolyticus (57.9%, 11/19) V. parahaemolyticus (63.2%, 12/19), V. fluvialis (42.1%, 8/19), V. vulnificus (10.5%, 2/19), V. cholerae non-O1, non O139(10.5%, 2/19) and Aeromonas salmonicida (52.6%, 10/19). All the samples investigated presented some level of contamination by enterobacteria, rotavirus, or both, and these results may reflect the level of contamination in the Northern Amazon Region, due to the natural maintenance of some of these agents or by the proximity with human populations and their sewer.

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.(AU)

Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Brazilian Amazon region, 2005.

BACKGROUND: An epidemic of acute gastroenteritis occurred in Rio Branco City, Acre State, in Brazil's Amazon region in 2005. An investigation was conducted to confirm the etiology and identify possible risk factors for death. METHODS: Rio Branco municipality surveillance data for the period May to October 2005 were reviewed. In a case-control study, children who died following acute gastroenteritis were compared to age-matched controls with acute gastroenteritis who survived. Rotavirus A (RV-A) was investigated in 799 stool samples and genotyped by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cumulative incidence of diarrhea in children aged <5 years was 21%. A fatal outcome was significantly associated with uncovered household water storage containers. RV-A was identified in 88% of samples and G9 was the prevalent genotype (71%). CONCLUSIONS: Oral rehydration solution and boiling or chlorinating drinking water likely limited mortality. This epidemic was caused by RV-A genotype G9. After the outbreak, a rotavirus vaccine was introduced into the official childhood immunization schedule in Brazil.

Tetravalent rhesus-human rotavirus vaccine (RRV-TV) in Belém, Brazil: efficacy against prevailing P and G genotypes.

To determine the efficacy of a reassortant rhesus-human tetravalent rotavirus vaccine (RRV-TV) (4 x 10(4) pfu/dose) against P and G rotavirus genotypes, 90 positive samples were tested using reverse transcription-polymerase chain reaction. The efficacy of the RRV-TV vaccine against P[8] and G1 individually or in binary combination P[8], G1 was 72 per cent (p < 0.005) 61 per cent (p < 0.013), and 70 per cent (p < 0.009), respectively, only for the first year of follow-up. In the second year, as well as after 2 years of follow-up, no efficacy was observed to these genotypes. These data indicate that further studies with rotavirus vaccines should focus on the molecular characterization of rotaviruses genotypes, in order to see whether or not cross-protection among different G and P genotypes may occur as a result of common bearing of VP4 specificities.

Detection and characterization of rotavirus G and P types from children participating in a rotavirus vaccine trial in Belém, Brazil.

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P], G genotypes were determined in 93.3%, 95.9%, 93.3%, 73.3%, 95.5% and 92.2% of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9%, 30% and 4.4% of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4% of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8 + 6] were detected in 54.5%, 26.7%, 12.2%, and 2.2% of the cases, respectively. The predominant genotypes were P[8], G1 and P[4], G2 with 53% and 26.6% of the infections, respectively. Unusual strains accounted for 20.5% including P[4], G1, P[6], G1, P[6], G4, P[6], G5, P[8], G2, P[8], G5. Mixed infections involving P[8 + 6], G2 and P[8 + 6], G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil.

Detection and characterization of Rotavirus G and P types from children participating in a Rotavirus vaccine trial in Belém, Brazil

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P],G genotypes were determined in 93.3 percent, 95.9 percent, 93.3 percent, 73.3 percent, 95.5 percent and 92.2 percent of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9 percent, 30 percent and 4.4 percent of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4 percent of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8+6] were detected in 54.5 percent, 26.7 percent, 12.2 percent, and 2.2 percent of the cases, respectively. The predominant genotypes were P[8],G1 and P[4],G2 with 53 percent and 26.6 percent of the infections, respectively. Unusual strains accounted for 20.5 percent including P[4],G1, P[6],G1, P[6],G4, P[6],G5, P[8],G2, P[8],G5. Mixed infections involving P[8+6],G2 and P[8+6],G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil

Inmunogenicidad, inocuidad y eficacia de una vacuna tetravalente obtenida por recombinación genética de rotavirus aislados de monos rhesus y seres humanos en Belém, Brasil / Immunogenicity, safety and efficacy of tetravalent rhesus-human, reassortant rotavirus vaccine in Belém, Brazil

Se evaluó la inocuidad, inmunogenicidad y eficacia de una vacuna tetravalente obtenida por recombinación genética de rotavirus aislados de monos rhesus y seres humanos (RRV-TV) (4 x 104 unidades formadoras de placas por dosis) en un ensayo prospectivo, aleatorio, a doble ciego y controlado con placebo que se efectuó con 540 lactantes brasileños. Se administraron dosis de vacuna o de placebo a la edad de 1, 3 y 5 meses. No se observaron diferencias significativas en la frecuencia de diarrea o vómito en los bebés de ninguno de los dos grupos después de administrar la dosis correspondiente. De 2 a 3% de los vacunados tuvieron fiebre baja los días tercero a quinto después de recibir la primera dosis, pero no después de las dosis segunda o tercera. Se observó una respuesta de anticuerpos del tipo IgA al rotavirus aislado de monos rhesus (RRV) en 58% de los vacunados y en 33% de quienes recibieron placebo. La respuesta de anticuerpos neutralizantes a cada serotipo no pasó de 20% cuando se determinó con la prueba de reducción de focos de fluorescencia, pero fue superior a 40% al medirse con la prueba de neutralización a base de reducción de placas. Se presentaron 91 casos de diarrea causada por rotavirus entre los niños que recibieron las tres dosis (de vacuna o de placebo) durante un seguimiento de 2 años, 36 de ellos en los niños vacunados. La eficacia general de la vacuna fue de 8% (P = 0,005) contra toda clase de diarrea y de 35% (P = 0,03) contra la diarrea causada por rotavirus. La protección durante el primer año de seguimiento, cuando predominó el rotavirus G del serotipo 1, fue de 57% (P = 0,008), pero se redujo a 12% en el segundo año. Se obtuvieron resultados similares al restringir el análisis a episodios en que el rotavirus fue el único agente patógeno identificado. Se observó en la vacuna una mayor tendencia a proteger contra casos de enfermedad con un promedio de seis o más deposiciones diarias. Estos resultados son lo suficientemente...

A tetravalent rhesus-human reassortant rotavirus (RRV-TV) vaccine (4 x 104 plaque-forming units/dose) was evaluated for safety, immunogenicity and efficacy in a prospective, randomized, double-blind, placebo-controlled trial involving 540 Brazilian infants. Doses of vaccine or placebo were given at ages, 1, 3 and 5 months. No significant differences were noted in the occurrence of diarrhoea or vomiting in vaccine and placebo recipients following each dose. Low-grade fever occurred on days 3­5 in 2­3% of vaccinees after the first dose, but not after the second or third doses of vaccine. An IgA antibody response to rhesus rotavirus (RRV) occurred in 58% of vaccinees and 33% of placebo recipients. Neutralizing antibody responses to individual serotypes did not exceed 20% when measured by fluorescent focus reduction, but exceeded 40% when assayed by plaque reduction neutralization. There were 91 cases of rotavirus diarrhoea among the 3-dose (vaccine or placebo) recipients during two years of follow-up, 36 of them among children given the vaccine. Overall vaccine efficacy was 8% (P = 0.005) against any diarrhoea and 35% (P = 0.03) against any rotavirus diarrhoea. Protection during the first year of follow-up, when G serotype 1 rotavirus predominated, was 57% (P = 0.008), but fell to 12% in the second year. Similar results were obtained when analysis was restricted to episodes in which rotavirus was the only identified pathogen. There was a tendency for enhanced protection by vaccine against illness associated with an average of 6 or more stools per day. These results are sufficiently encouraging to warrant further studies of this vaccine in developing countries using a higher dosage in an attempt to improve its immunogenicity and efficacy.

Concomitant rotavirus serotypes 1 and 4 infections in a diarrhoeic from Belem, Brazil

Infeccoes simultaneas por sorotipos 1 e 4 de rotavirus foram observadas em uma crianca de 15 meses de idade, do sexo feminino, internada com diarreia aguda contraida na comunidade que perdurou por 7 dias, evoluindo com desidratacao moderada. As evidencias dessas infeccoes foram inferidas baseadas em testes tais como: a) ensaio imunoenzimatico (ELISA), evidenciando-se reacao positiva para os sorotipos 1 e 4; e b) migracoes extras de segmentos de ARN visualizados a eletroforese em gel de poliacrilamida (EGPA). Esses resultados sugerem que as condicoes precarias de higiene e saneamento em que vivia essa crianca propiciam a infeccao macica por esses agentes virais. Alem disso, a co-circulacao de diferentes sorotipos no mesmo ambiente sustenta a necessidade de utilizar-se, no futuro, uma vacina polivalente, que proteja as criancas contra os quatro sorotipos G, epidemiologicamente importantes

Incidencia das diarreias infantis agudas em um bairro periferico de Belem, Para: fase preparatoria de um futuro estudo de campo com uma vacina contra rotavirus / Incidence of the diarrhea infantile acute in one perifheric district of Belem, Para: preparatory phase of one future study of field with one vaccine against rotaviruses

No periodo de fevereiro a setembro de 1988 foram recruta-das e submetidas a acompanhamento sistematico, 78 criancas habitantes de um bairro periferico de Belem, Brasil. Observou-se que cinco (6,4%) desses individuos foram excluidos no decurso do estudo, uma ves que passaram a morar em outra areada cidade. Visitas domiciliares, duas vezes por semana, foram empreendidas por agentes de campo, sendo os cuidados medicos proporcionados por dois profissionaisde nosso grupo. Foram registrados 67 episodios de diarreia, obtendo-se especimesfecais para exames em 62 (92.5%) deles. Rotavirus foram detectados em 4 (6.4%) dos episodios analisados laboratorialmente, todos classificados como subgrupo II(perfil eletroforetico de ARN longo). De 60 especies fecais processados quanto apresenca de enteropatogenos bacterianos, logrou-se obter 7(11,7%) isolamentos deEscherichia coli enteropatogenicas, 3 (5,0%) de Sjigella sonnei e 1 (1,7%) deSalmonella grupo C1. Detectou-se, ainda, Campilobacter sp em 2 (4,9%) dos 41 es-pecimes fecais cultivados com o proposito especifico de detectar-se esse agente Cryptosporidium sp. foi observado em apenas 1 (2.7%) dos 37 especimes fecais analisados. A maior incidencia de diarreia, 24,5%, ocorreu em abril, enquanto que amenor, 9,3%, em marco. Das 78 criancas recrutadas, 52 (66.7%) apresentaram diarreia. A taxa de ataque clinico especifica por idade nao exibiu diferencas niti-das nos intervalos de 0 a 3 e 5 a 8 meses, porem, configurou-se um pico aos 4. Ataxa de diarreia cumulativa atingiu a cifra de 1,4 episodio por crianca, aos oi-to meses. As frequencias de vomitod (75%), febre (50%) e desidartacao (50%) revelaram-se superiores aos mesmos parametros avaliados no grupo de diarreias relacionadas a outros agentes etiologicos.