Genómica de rotavirus. Impacto en salud pública / Rotavirus genomics. Public health impact

Resumen: Con la introducción de las vacunas de rotavirus Rotarix (RV1) o RotaTeq (RV5) en programas nacionales de vacunación de diversos países, surgió la preocupación de que la presión inmune generada condujera al aumento en la prevalencia de genotipos virales no incluidos en las vacunas, o bien del surgimiento de nuevas cepas que pudieran escapar a la respuesta inmune protectora inducida por la vacunación. La variación natural de los rotavirus ha hecho que sea muy difícil distinguir si el cambio en las cepas circulantes se debe a la presión selectiva impuesta por las vacunas o bien a la fluctuación natural de las cepas. Si acaso ha habido una presión selectiva, ésta ha sido hasta ahora baja. Sin embargo, es importante mantener la vigilancia epidemiólogica y poner atención al surgimiento de cepas resistentes a la inmunidad, en particular en países en desarrollo en los que se ha descrito una mayor diversidad viral.

Abstract: With the introduction of rotavirus vaccines Rotarix (RV1) or RotaTeq (RV5) in the immunization programs of an increasing number of countries, there is concern that the immune selection pressure induced will cause an increase in the prevalence of virus genotypes not included in the vaccine formulation, or to the appearance of novel rotavirus strains that could evade the protective immune response. The natural fluctuation of rotaviruses makes it difficult to distinguish if the change in the circulating strains is due to the vaccine selective pressure or to the natural diversity fluctuation of viruses. If there has been a selective pressure, it has been low so far. However, it is important to keep an epidemiological surveillance and pay attention to the emergence of strains that are resistant to the vaccine, in particular in those countries where the viral diversity has been shown to be higher.

Conocimiento vaccinal en madres con hijos menores de 5 años / Vaccinal knowledge, in mothers with children under 5 years old

La inmunización, es un mecanismo de defensa que asegura la inmunidad humoral para prevenir enfermedades infantiles graves, y es responsabilidad de los padres. Objetivo: Se plantea indagar el conocimiento vaccinal, en madres con hijos menores de 5 años. Métodos: el estudio se orienta bajo el paradigma cualitativo, enfoque fenomenológico; Los participantes del estudio fueron madres que vacunaron a sus hijos en el centro de salud 20 de febrero, distrito 12D03 Quevedo-Mocache, Ecuador. Para el análisis de los datos se utilizó el soware Atlas ti, vinculando 18 preguntas de reflexión. Resultados: Las categorías emergentes fueron: 1) Las vacunas son muy importantes y necesarias. 2. Vacunaron porque sus hijos crecen sanos y fuertes. 3. para algunas el vacunatorio es agradable y para otras no. 4. Algunas reciben buen trato y atención, otras no. 5. Es insuficiente la información sobre las vacunas. 6. Piden cambios de enfermeras y horarios. Conclusión: el conocimiento vaccinal de las madres, esdeterminante para la protección y la no propagación de enfermedades en sus hijos(AU)

Immunization is a defense mechanism that ensures humoral immunity to prevent serious childhood diseases, and is the responsibility of the parents. Objective: to investigate the vaccine knowledge in mothers with children under 5 years. Methods: the study is oriented under the qualitative paradigm, hermeneutic approach; e study participants were mothers who vaccinated their children in the health center February 20, district 12D03 Quevedo-Mocache, Ecuador. The Atlas ti soware was used to analyze the data, linking 18 reflection questions. Results: The emerging categories were: 1) Vaccines are very important and necessary. 2. They vaccinated because their children grow up healthy and strong. 3. For some the vaccine is nice and for others it is not. 4. Some receive good treatment and attention, others do not. 5. Insufficient information about vaccines. 6. Ask for changes of nurses and schedules. Conclusion: the vaccinal knowledge of the mothers, is decisive for the protection and non-propagation of diseases in their children(AU)

Diversity of group A rotavirus genes detected in the Triângulo Mineiro region, Minas Gerais, Brazil

ABSTRACT Group A rotaviruses are the main causative agent of infantile gastroenteritis. The segmented nature of the viral genome allows reassortment of genome segments, which can generate genetic variants. In this study, we characterized the diversity of the VP7, VP4 (VP8*), VP6, NSP4, and NSP5 genes of the rotaviruses that circulated from 2005 to 2011 in the Triângulo Mineiro (TM) region of Brazil. Samples with genotypes G2 (sublineages IVa-1 and IVa-3), G1 (sublineage I-A), G9 (lineage III), G12 (lineages II and III), G8 (lineage II), G3 (lineage III), P[4] (sublineages IVa and IVb), P[8] (sublineages P[8]-3.6, P[8]-3.3, and P[8]-3.1), I2 (lineage VII), E2 (lineages VI, XII, and X), and H2 (lineage III) were identified. The associations found in the samples were G1, G9, or G12 with P[8]-I1-E1-H1; G2 or G8 with P[4]-I2-E2-H2; G12 with I3-E3-H6; and G3 with P[4]-I2-E3-H3 (previously unreported combination). Reassortment events in G2P[4] strains and an apparent pattern of temporal segregation within the lineages were observed. Five TM samples contained genes that exhibited high nucleotide and amino acid identities with strains of animal origin. The present study includes a period of pre- and post-introduction of rotavirus vaccination in all Brazilian territories, thereby serving as a basis for monitoring changes in the genetic constitution of rotaviruses. The results also contribute to the understanding of the diversity and evolution of rotaviruses in a global context.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Detection of Rotavirus Genotypes in Korea 5 Years after the Introduction of Rotavirus Vaccines

Rotavirus (RV) is one of the most important viral etiologic agents of acute gastroenteritis (AGE) in children. Although effective RV vaccines (RVVs) are now used worldwide, novel genotypes and outbreaks resulting from rare genotype combinations have emerged. This study documented RV genotypes in a Korean population of children with AGE 5 yr after the introduction of RVV and assessed potential genotype differences based on vaccination status or vaccine type. Children less than 5-yr-old diagnosed with AGE between October 2012 and September 2013 admitted to 9 medical institutions from 8 provinces in Korea were prospectively enrolled. Stool samples were tested for RV by enzyme immunoassay and genotyped by multiplex reverse-transcription polymerase chain reaction. In 346 patients, 114 (32.9%) were RV-positive. Among them, 87 (76.3%) patients were infected with RV alone. Eighty-six of 114 RV-positive stool samples were successfully genotyped, and their combinations of genotypes were G1P[8] (36, 41.9%), G2P[4] (12, 14.0%), and G3P[8] (6, 7.0%). RV was detected in 27.8% of patients in the vaccinated group and 39.8% in the unvaccinated group (P=0.035). Vaccination history was available for 67 of 86 cases with successfully genotyped RV-positive stool samples; RotaTeq (20, 29.9%), Rotarix (7, 10.4%), unvaccinated (40, 59.7%). The incidence of RV AGE is lower in the RV-vaccinated group compared to the unvaccinated group with no evidence of substitution with unusual genotype combinations.

Anticorpos IgY específicos para rotavírus do grupo A. Uma abordagem terapêutica para rotavirose em Macaca fascicularis / Specific IgY antibodies to rotavirus group A. A therapeutic approach for rotavirus disease in macaca fasciculares

A produção de anticorpos em aves imunizadas seguida da extração desses anticorpos da gema dos ovos (IgY), tem atraído o interesse da comunidade científica, como pode ser demonstrado pelo aumento significativo da literatura sobre a IgY. [...] Destaca-se que a tecnologia da IgY oferece novas possibilidades de aplicação em imunoterapia e métodos de diagnóstico, tanto para aplicação humana quanto veterinária, incluindo estratégias de tratamentode doenças intestinais graves em crianças, particularmente em países pobres. Neste presente estudo, objetivou-se avaliar a eficiência terapêutica da IgY utilizando macacos cynomolgus (Macaca fascicularis) jovens desafiados com o rotavírus do grupo A (RVA) humano, a maior causa de morbidade e mortalidade de crianças em todo o mundo, especialmente em países em desenvolvimento. Para esta proposta, anticorpos IgY específicos contra o RVA foram produzidos em aves, purificados por polietileno glicol, caracterizados por eletroforese em gel depoliacrilamida, western blotting e um teste de neutralização em cultura de células (MA-104). Este experimento preliminar rendeu uma suspensão altamente concentrada de IgY específica antirotavírus(IgY anti-RVA) (média de 37 mg/mL). O macaco cynomolgus foi estabelecido como modelo de infecção experimental após uma única administração de suspensão de rotavírus humano (3,1x106FFU/mL) por sonda gástrica. Os animais foram acompanhados durante onze dias, sendo observadas as manifestações clínicas, cargas virais sérica e fecal, hematologia e dosagem de eletrólitos séricos...

The production of antibodies in chickens and the extraction of specific antibody suspensions fromegg yolk (IgY) are increasingly attracting the interest of the scientific community, asdemonstrated by the significant growth of the IgY literature. [...] Of note, the IgY-technology offersnew possibilities for application in human and veterinary diagnostics and therapeutics, includingstrategies for the treatment of severe intestinal diseases in children, particularly in poor countries.In this study, we aimed to evaluate the therapeutic efficacy of the IgY by using youngcynomolgus monkeys (Macaca fascicularis) challenged with human rotavirus group A (RVA), amajor cause of morbidity and mortality in children worldwide, especially in developing countries.For this purpose, specific IgY antibodies against RVA were produced in hens, purified bypolyethylene glycol, characterized by polyacrylamide gel electrophoresis, western blotting and aneutralization assay in a cell culture system (MA-104). This preliminary experiment has yielded ahigh concentrated suspension of anti-rotavirus specific IgY (anti-RVA IgY) (average 37 mg/ml).The cynomolgus experimental infection model was established after a single administration of ahuman rotavirus suspension (3.1x106FFU/ml) by oral gavage. The confined animals werefollowed during a period of eleven days, observed for clinical signs, measurement of serum andfaecal viral load, and evaluation of hematology and serum electrolytes. The main clinical sign(observed in two of the seven inoculated monkeys) was diarrhea associated with a decrease inserum potassium during three days, followed by recovery...

Changes in epidemiology of rotavirus in the Triângulo Mineiro region of Brazil: lack of two consecutive rotavirus seasons

Rotaviruses are the main cause of infantile acute diarrhea, and a monovalent (G1P[8]) vaccine against the virus was introduced into the Brazilian National Immunization Program for all infants in March 2006. The objectives of this study were to determine the rate and genotype distribution of rotavirus causing infantile diarrhea in the Triângulo Mineiro region of Brazil during 2011-2012 and to assess the impact of local vaccination. Fecal specimens were analyzed for detection and characterization of rotavirus using polyacrylamide gel electrophoresis, reverse transcription followed by polymerase chain reaction (PCR), and PCR-genotyping assays. Overall, rotavirus was diagnosed in 1.7% (6/348) of cases. Rotavirus positivity rates decreased 88% [95% confidence intervals (CI)=15.2, 98.3%; P=0.026] in 2011 and 78% (95%CI=30.6, 93.0%; P=0.007) in 2012 when compared with available data for baseline years (2005/2006) in Uberaba. In Uberlândia, reductions of 95.3% (95%CI=66.0, 99.4%; P=0.002) in 2011, and 94.2% (95%CI=56.4, 99.2%; P=0.004) in 2012 were also observed compared with data for 2008. The circulation of rotavirus G2P[4] strains decreased during the period under study, and strains related to the P[8] genotype reemerged in the region. This study showed a marked and sustained reduction of rotavirus-related cases, with a lack of rotavirus in the 2011 and 2012 seasons, suggesting a positive impact of the vaccination program.

Leite de mulheres brasileiras apresenta anticorpos IgA secretores (SIgA) que neutralizam o rotavírus G9P[5] / Milk from Brazilian women presents secretory IgA antibodies and neutralizes rotavirus G9P[5]

OBJETIVO: Verificar a presença de SIgA anti-rotavírus sorotipo G9P[5] e a capacidade de neutralização do vírus de amostras de leite de mulheres brasileiras. MÉTODOS: Foram determinados os níveis de anticorpos SIgA reativos contra rotavírus G9 em 30 amostras de leite materno por ELISA usando suspensões purificadas do vírus. A capacidade das amostras de neutralizarem o rotavírus G9P[5] foi analisada em ensaio de de Neutralização utilizando células MA-104. RESULTADOS: Foram observadas grandes variações individuais referentes aos níveis de SIgA e títulos de neutralização, mas todas as amostras mostraram certa capacidade de neutralizar o G9P[5]. Verificamos uma correlação positiva altamente significativa entre os níveis de anticorpos e os títulos de neutralização. CONCLUSÕES: A alta correlação entre níveis de anticorpos anti-rotavírus e a capacidade neutralizante das amostras de leite sugere um possível papel protetor desses anticorpos contra a infecção. Esses resultados também apoiam o incentivo à prática do aleitamento materno.

OBJECTIVE: To verify the presence of anti-rotavirus serotype G9P[5] SIgA and the virus neutralization capacity of milk samples from Brazilian women. METHODS: SIgA antibody levels reactive to rotavirus G9 were determined in 30 maternal milk samples by enzyme-linked immunosorbent assay (ELISA) using purified virus suspensions. The samples' capacity to neutralize rotavirus G9P[5] was analyzed using the MA-104 cells neutralization assay. RESULTS: Great individual variations were observed regarding the SIgA levels and neutralization titers, but all samples showed some G9P[5] neutralizing ability. A highly significant positive correlation was observed between antibody levels and neutralization titers. CONCLUSIONS: The high correlation between anti-rotavirus antibody levels and neutralizing capacity of the milk samples suggests a possible protective role of these antibodies against infection. These results also support the encouragement of the breast-feeding practice.

Caracterização molecular dos genótipos do rotavírus em pacientes pediátricos imunossuprimidos e não imunossuprimidos / Molecular characterization of rotavirus genotypes in immunosuppressed and non-immunosuppressed pediatric patients

OBJETIVO: Descrever a variabilidade genotípica do rotavírus grupo A (RVA) encontrado em pacientes pediátricos imunocompetentes e imunocomprometidos tratados no Hospital de Clínicas/Universidade Federal do Paraná (HC/UFPR), Curitiba, Paraná. MÉTODOS: Foi realizado um estudo transversal com 1.140 amostras de fezes coletadas, de abril de 2001 a dezembro de 2008, em pacientes ambulatoriais e pacientes hospitalizados com gastroenterite aguda encaminhados ao hospital. As técnicas usadas foram o método da aglutinação do látex e imunoensaio enzimático para diagnóstico de RVA. Foi realizada transcrição reversa, seguida por PCR multiplex semi-nested e sequência de nucleotídeos para caracterização do genótipo. Foram relatados dados de combinações de genótipos, clínicos, epidemiológicos, laboratoriais e sobre a presença de infecções hospitalares. RESULTADOS: Foi analisado um total de 80 amostras de fezes positivas para rotavírus. As associações mais frequentes entre os genótipos G e P foram: G4 P[8] (38,9%), G1 P[8] (30,5%), G9 P[8] (13,9%), G2 P[4] (6.9 %) e G3 P[8] 1,4%). O genótipo prevalente foi G2 P[4] depois da implementação da vacina nos anos de 2006 e 2008. Verificou-se que um total de 62,5% das crianças com idade abaixo de 12 meses estavam infectadas. Destas, 55,6% tinham grave desidratação, e 26,7% precisaram de cuidados intensivos. Encontrou-se uma frequência de 12,5% de infecções hospitalares. Não se observou correlação entre o genótipo e a gravidade da infecção nos pacientes estudados. CONCLUSÃO: As infecções por RVA podem associar-se a manifestações clínicas graves e é crucial a vigilância da variabilidade genotípica desse vírus para monitorizar a emergência de novas cepas e o impacto da imunização nesses pacientes.

OBJECTIVE: To describe the genotypic variability of group A rotavirus (RVA) found in immunosuppressed and non-immunosuppressed pediatric patients treated at the Hospital de Clínicas da Universidade Federal do Paraná (HC-UFPR), Curitiba, Paraná. METHODS: A cross-sectional study was conducted with 1,140 stool samples collected from April, 2001 to December, 2008 in outpatients and hospitalized patients with acute gastroenteritis referred to the hospital. RVA diagnosis was performed through the latex agglutination method and enzyme immunoassay. Reverse transcription followed by multiplex hemi-nested polymerase chain reaction (PCR) and nucleotide sequencing were used for genotype characterization. Genotype combinations, clinical data, epidemiological data, laboratory data, and presence of hospital-acquired infections were reported. RESULTS: A total of 80 rotavirus-positive stool samples were analyzed. The most frequent associations between genotypes G and P were: G4 P[8] (38.9%), G1 P[8] (30.5%), G9 P[8] (13.9%), G2 P[4] (6.9%), and G3 P[8] (1.4%). G2 P[4] was the most prevalent genotype after the vaccine implementation in the years 2006 and 2008. A total of 62.5% of children aged less than 12 months were found to be infected. Of these, 55.6% had severe dehydration and 26.7% needed intensive care. A frequency of 12.5% of nosocomial infections was found. No correlation was observed between genotype and severity of infection in the study patients. CONCLUSION: RVA infections can be associated with severe clinical manifestations, and the surveillance of genotypic variability of this virus is crucial to monitor the emergence of new strains and the impact of the immunization in these patients.

Diversidad genotípica de rotavirus grupo A: correlación entre el tipo G3 y severidad de la infección. Valencia, Venezuela / Genetic diversity of rotavirus group A: correlation between G3 type and severity of the infection. Valencia, Venezuela

La variabilidad genética y antigénica de los rotavirus (RV) parece tener implicaciones en la severidad de la infección, pero los estudios no son concluyentes. Por este motivo, en el presente trabajo se compararon las medias de severidad entre los episodios de diarrea causados por RV tipo G1 y G3, durante el período 2001-2005, en la Ciudad Hospitalaria “Dr. Enrique Tejera” de Valencia, Venezuela. RV se detectó por ELISA, los tipos G y P por RT-PCR. La severidad de la infección se estimó utilizando el sistema de Ruuska-Vesikari, las medias de severidad se compararon mediante la prueba t de Student (2 colas, 95%IC). RV se detectó en 24,5% (3.193/13.026) de los pacientes. G3 fue más frecuente (50,3%), seguido por G1 (39,2%), G9 (6,2%), G2 (0,6%), G4 (0,6%) y 3,1% mixtos (G1+G3). El 87,3% de las muestras resultaron P[8], 10,9% P[4] y 1,8% P[6]. Al comparar los episodios G1 y G3, no se observaron diferencias significativas (P>0,05) entre los grupos etarios, frecuencia de desnutridos, deshidratación y lactancia materna. Sin embargo, el grupo G3 se caracterizó por presencia significativa (P< 0,05) de fiebre, episodios con una duración ≥ 6 días, 6 o más evacuaciones en 24 horas y 3 o más días con vómitos. La media de severidad para los episodios G3 (11,1) fue mayor significativamente (P<0,05) a la G1 (7,8). Estos resultados muestran la asociación de G3 con diarreas severas y apoyan la importancia de conocer la variabilidad y frecuencia de los tipos virales para medir el impacto de las vacunas antirotavirus.

Genetic and antigenic rotavirus (RV) variabilities may have implications in the severity of the infection caused by these agents; however the studies are not conclusive. For that purpose, the mean severity scores of diarrhea episodes caused by RV types G1 and G3 were compared, at Ciudad Hospitalaria “Dr. Enrique Tejera” in Valencia, Venezuela, between 2001- 2005. RV were identified by ELISA, G and P types by RT-PCR. The severity of infection was determined using the Ruuska-Vesikari system and the mean severity values were compared using the Student’s t-test (two-tailed, 95%CI). RV were detected in 24.5% of patients (3193/13026), being G3 the most common (50.3%), followed by G1 (39.2%), G9 (6.2%), G2 (0.6%), G4 (0.6%) and of mixed infection 3.1% (G1+G3). Type P[8] was present in 87.3% of samples, 10.9% P[4] and 1.8% P[6]. There were not statistically significant differences (P≥0.05) observed between the episodes caused by G1 and G3 when age, breast feeding, and degrees of malnutrition and dehydration were considered. Nevertheless, in the G3 positive group, fever, episodes of more than 6 days, 6 or more evacuations in 24 hours and 3 or more days with vomit, were observed. The mean severity score for the G3 episodes (11.1) was significantly higher (P< 0.05) than for G1 (7.8). These results show that G3 was associated with severe diarrhea, supporting the hypothesis that the knowledge of the variability and frequency of viral types is essential to measure the impact of an anti-rotavirus vaccine.

An unusual case of concomitant infection with chicken astrovirus and group A avian rotavirus in broilers with a history of severe clinical signs

A molecular study of intestinal samples from 21 broiler flocks with a history of enteritis revealed that 23.8% and 14.3% were positive for chicken astrovirus (CAstV) and avian rotavirus (ARV), respectively. CAstV and group A ARV were simultaneously detected in only one broiler flock. Birds in this group developed the significant intestinal lesions characterized by frothy contents, paleness, and thin intestinal walls. In this report we present an unusual case of runting stunting syndrome (RSS) with a history of high mortality and growth retardation in broiler chickens. We also make the first identification of CAstV and group A ARV in broiler chickens in Korea.

Rotavirus genotyping in gastroenteritis cases of an infantile population from Western Brazilian Amazonia / Genotipagem de rotavírus em casos de gastrenterites de uma população infantil do oeste da Amazônia brasileira

INTRODUCTION: During the period from 2000 to 2002, 79 rotavirus-positive stool samples were collected from children presenting diarrhea in the Western Brazilian Amazon. METHODS: Molecular characterization of the G and P genotypes was performed using RT-PCR and electropherotyping analysis by polyacrylamide gel electrophoresis. RESULTS: A total of 59 samples were confirmed as group A rotavirus. A long electrophoretic profile was exhibited by the G1P[8], G3P[8], and G4P[8] genotypes. The G1P[8] genotype was found in greater proportion. The short electropherotype was exhibited only by G2 genotype strains. CONCLUSIONS: The proportion of the rotavirus genotypes observed was not different from that in other areas of Brazil. This study is the first genotyping of rotavirus in the Western Brazilian Amazon.

INTRODUÇÃO: Entre 2000 e 2002, 79 amostras positivas para rotavírus foram coletadas de crianças com diarreia na Amazônia ocidental brasileira. MÉTODOS: Para a caracterização molecular dos genótipos G e P foram realizadas as reações de RT-PCR e a análise dos eletroferotipos por eletroforese em gel de poliacrilamida (PAGE). RESULTADOS: 59 amostras foram confirmadas como pertencentes ao rotavírus grupo A. Os genótipos G1P[8], G3P[8] e G4P[8] apresentaram perfis eletroforéticos longos. O genótipo G1P[8] foi encontrado em maior proporção. O eletroferotipo curto ocorreu apenas em genótipos G2. CONCLUSÕES: A proporção dos genótipos de rotavírus observada não foi diferente de outras áreas do Brasil. Este estudo é a primeira genotipagem de rotavírus na Amazônia ocidental brasileira.

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.

Prevalência de rotavírus em crianças atendidas na rede pública de saúde do estado de Pernambuco / Rotavirus prevalence in infants and children in the public healthcare system of the state of Pernambuco

INTRODUÇÃO: Os rotavírus são considerados, importantes agentes etiológicos de gastroenterite aguda e ainda causa comum de hospitalização de crianças na faixa etária de zero a quatro anos de idade. No Brasil, a incidência de rotavírus nas crianças com gastrenterite é de 12 a 42 por cento, e a distribuição da infecção esta relacionada à sazonalidade que aparentemente ocorre em diferentes períodos e intensidade de acordo com a região de ocorrência. O estudo pesquisou rotavírus do grupo A em amostras fecais de crianças com caso suspeito atendidas na rede pública de saúde do Estado de Pernambuco. MÉTODOS: O diagnóstico foi realizado através de ensaios imunoenzimático ELISA e teste imunoquímico de aglutinação em látex. RESULTADOS: Foram estudadas 171 amostras. Destas, 33 (19,3 por cento) apresentaram positividade. Das amostras positivas 24 (72,7 por cento) eram do sexo masculino e 09 (27,3 por cento) do sexo feminino. Dentro da amostragem positiva 15,2 por cento eram vacinadas. Quando comparamos os resultados obtidos entre o teste Elisa e aglutinação pelo Látex, houve 100 por cento de concordância entre a positividade pelo Látex e o ELISA. CONCLUSÕES: A alta incidência desta infecção reforça necessidade de monitoramento desse vírus, definindo políticas de saúde relacionadas ao diagnóstico, profilaxia, melhores condições sócio-econômicas e aprimoramento da vacina.

INTRODUCTION: Rotaviruses are considered important etiological agents of acute gastroenteritis and a common cause of the hospitalization of children aged zero to four years-old. In Brazil, the incidence of rotavirus gastroenteritis in children is 12 to 42 percent and the distribution of infection is related to seasonality, which apparently occurs in different periods and intensity according to each region. The study investigated group A rotavirus in fecal samples of suspected children attended by the public health system of the State of Pernambuco. METHODS: Diagnosis was achieved by ELISA and the Latex agglutination test. RESULTS: Of the 171 samples studied, 33 (19.3 percent) presented positivity for Rotavirus A. Among positive samples, 72.7 percent belonged to male patients and 27.3 percent to female. Among the positive casuistic, 15.2 percent were vaccinated. When comparing the results obtained for the ELISA and Latex agglutination tests, 100 percent agreement between positivity by Latex agglutination and ELISA was verified. CONCLUSIONS: The high incidence of this infection reinforces the need to monitor this virus and define health policies concerning its diagnosis, prophylaxis, improvement in socioeconomic conditions and the refinement of the vaccine.

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7 percent nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8 percent identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8 percent. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

Molecular characterization of G1 human rotaviruses detected in children from Belém, Pará, Brazil / Caracterização molecular de rotavírus humanos do tipo G1 detectados em crianças de Belém, Pará, Brasil

Rotavirus is responsible for 40 percent of gastroenteritis infections worldwide, resulting in 611 thousand deaths annually among infants and young children. The aim of the present study was to perform molecular characterization of strains of the most common circulating rotavirus genotype (G1), which was obtained from children participating in studies previously conducted in Belém, northern Brazil over a 21-year period (1982 to 2003). G1 type rotavirus was detected by polyacrylamide gel electrophoresis, enzyme immunoassay and by reverse transcription polymerase chain reaction for the VP7 and VP4 genes. Of 798 specimens that were found to be positive for rotavirus, 330 (41 percent) had G1-specificity by EIA using monoclonal antibodies. A total of 148 G1 strains were analyzed by reverse transcription polymerase chain reaction. Electropherotypes and P genotypes characterization of G1 rotavirus occurred at frequencies of 78 percent and 88 percent, respectively. Three long electropherotype varieties were identified, with the L1 variety the most frequently found (79 percent). The G1P[8] combination was the most frequent, responsible for 64 percent of cases. Mixed infections of G1P[6]+P[8], G1P[4]+P[8], G1P[4]+P[6] and G1P[4]+P[6]+P[8] were found in 11 (7 percent), 11 (7 percent), 3 (2 percent) and 1 (0.6 percent) samples, respectively. One sample displaying a mixed G1+G4 infection was found. To our knowledge, this is the first study to focus on G1 rotavirus molecular characterization in Brazil. Our findings provide information that will allow a better understanding of the molecular diversity of G1 rotavirus infections in our region.

Os rotavírus são responsáveis por 40 por cento das ocorrências de gastroenterites infantil no mundo, resultando em 611 mil mortes anualmente, e o rotavírus do tipo G1 representa o seu genótipo circulante mais comum. O objetivo do presente artigo foi realizar a caracterização molecular das amostras de rotavírus do tipo G1 obtidas de crianças que participaram de estudos anteriormente conduzidos na Cidade de Belém, norte do Brasil, por um período de 21 anos (1982 a 2003). O rotavírus do tipo G1 foi detectado por meio de eletroforese em gel de poliacrilamida, ensaio imunoenzimático (EIA) e reação em cadeia da polimerase precedida da transcrição reversa para os genes VP7 e VP4. Dos 798 espécimes positivos para rotavírus, 330 (41 por cento) apresentavam especificidade G1 por EIA, usando anticorpos monoclonais. Um total de 148 amostras do tipo G1 foram analisadas por meio da reação em cadeia da polimerase precedida da transcrição reversa. A caracterização dos eletroferotipos e genótipos P dos rotavírus do tipo G1 ocorreu em frequências de 78 por cento e 88 por cento, respectivamente. Três variedades de eletroferotipos longos foram identificados, sendo L1 a predominante (79 por cento). A combinação G1P[8] foi a mais frequente, responsável por 64 por cento dos casos. As infecções mistas G1P[6]+P[8], G1P[4]+P[8], G1P[4]+P[6] e G1P[4]+P[6]+P[8] foram encontradas em 11 (7 por cento ), 11 (7 por cent), 3 (2 percent) e 1 (0,6 por cento) amostras, respectivamente. Uma amostra apresentando infecção mista G1+G4 foi identificada. Ressalte-se que este é o primeiro estudo a abordar a caracterização molecular de rotavírus do tipo G1 no Brasil. Nossos achados permitirão melhor compreensão a respeito da diversidade molecular associada às infecções por rotavírus do tipo G1 em nossa região.

Caracterização molecular dos genes de VP1, VP2, VP3, VP4 e VP7 de amostras de rotavírus A genótipo G5P(8) circulando no Brasil entre 1986 e 2005 / Molecular characterization of genes VP1, VP2, VP3, VP4 and VP7 of rotavirus samples of genotype A G5P (8) circulating in Brazil between 1986 and 2005

Nas décadas de 80 e início de 90 os rotavírus A (RV-A) de genótipo G5, comum em suínos, equinos e bovinos eram detectados com frequência em amostras fecais de crianças brasileiras. Após 1996, deixou de circular em caráter endêmico, tornando-se apenas esporadicamente detectado, enquanto o genótipo G9 começou a ser detectado com frequência. Esta situação leva a crer que houve a substituição do genótipo G5 pelo G9. Tendo em vista a escassez de dados moleculares a respeito de amostras de genótipo G5 de RV-A, no presente estudo foi realizada a análise filogenética para os genes que codificam para as proteínas VP1, VP2, VP3, VP4 e VP7 de vinte e oito amostras de RV-A humano de genótipo G5P(8), coletadas em diferentes estados brasileiros entre 1986 e 2005. A análise filogenética do gene que codifica para a proteína VP7 demonstrou que as mesmas agrupam juntamente com amostras humanas brasileiras de genótipo G5 (IAL28, Br 1054 e Br H8), no entanto a análise do gene que codifica para a proteína VP4 demonstrou que circularam três linhagens do genótipo P(8) (P(8)-1, P(8)-2 e P(8)-3) no Brasil entre 1986 e 2005 em associação com G5. As análises filogenéticas para os genes que codificam para VP1, VP2 e VP3 demonstraram que os mesmos pertencem ao genogrupo Wa-Like, comum a humanos, o que sugere que estas amostras possam ter se originado de uma amostra de RV-A humano. As análises filogenéticas dos genes de VP1, VP2 e VP3 revelaram que todas as amostras foram classificadas dentro dos genótipos R1, M1 e C1, respectivamente. Os resultados do presente estudo enfatizam a importância do monitoramento contínuo e caracterização molecular das amostras de RV-A circulantes, principalmente para prever a possível emergência e/ou re-emergência de genótipos após a introdução de uma vacina contra RV-A nos diferentes continentes do mundo e para se melhor entender a dinâmica e o padrão de evolução dos RV-A de genótipo G5.

Group A rotavirus genotypes and the ongoing Brazilian experience: a review

Brazil was the first Latin American country to introduce universal group A rotavirus (RV-A) vaccination in March 2006, resulting in a unique epidemiological scenario. Since RV-A first identification in Brazil, 2,691 RV-A-positive stool samples, collected between 1982- 2007, were typed by independent research groups throughout the country. In the pre-vaccination era, 2,492 RV-A-positive samples collected from 1982-2005 were successfully typed, while 199 samples were analyzed from 2006-2007. According to the reviewed studies, there were two important times in the pre-vaccination era: (i) the period from 1982-1995, during which the detection of G5P[8] RV-A, in addition to the classical genotypes G1-4, challenged vaccine development programs; and (ii) the period from 1996-2005, during which genotype G9P[8] emerged, following a global trend. The rate of G2P[4] RV-A detection decreased from 26 percent (173/653) during 1982-1995 to 2 percent (43/1,839) during 1996-2005. The overall detection rate of RV-A genotypes from 1982-2005 was as follows: 43 percent (n = 1,079) G1P[8]/G1P[not typed (NT)]; 20 percent (n = 488) G9P[8]/G9P[NT]; 9 percent (n = 216) G2P[4]/G2P[NT]; 6 percent (n = 151) G3P[8]/G3P[NT]; 4 percent (n = 103) G4P[8]/G4P[NT]; and 4 percent (n = 94) G5P[8]/G5P[NT]. Mixed infections accounted for 189 (7 percent) of the positive samples, while atypical G/P combinations or other genotypes, including G6, G8, G10 and G12, were identified in 172 (7 percent) samples. The initial surveillance studies carried out in several Brazilian states with RV-A-positive samples collected in 2006 and 2007 show a predominance of G2P[4] strains (148/199 or 74 percent). Herein, we review RV-A typing studies carried out since the 1980s in Brazil, highlighting the dynamics of RV-A strain circulation profiles before and early after universal use of RV-A vaccine in Brazil.

Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil

Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2 percent) sequences clustered with NSP4 genotype B, and 12 sequences (7.8 percent) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.

Rotavírus A em crianças de até três anos de idade, hospitalizadas com gastroenterite aguda em Campo Grande, Estado do Mato Grosso do Sul / Rotavirus A among hospitalized infants, up to three years of age, with acute gastroenteritis in Campo Grande, State of Mato Grosso do Sul

Através da eletroforese em gel de poliacrilamida e do ensaio imunenzimático combinado para rotavírus e adenovirus, foram analisadas 380 amostras fecais de crianças com até 3 anos, hospitalizadas com diarréia aguda, entre maio de 2000 e janeiro de 2004, em Campo Grande, MS. Do total de amostras, 88 (23,2 por cento) foram positivas para Rotavirus A. Dentre essas, 81 (92 por cento) tiveram padrão eletroferotípico definido, sendo 77 (87,5 por cento) de padrão longo e quatro (4,5 por cento) de padrão curto. A caracterização genotípica G e P foi feita por RT-Nested-PCR para 85 amostras, sendo 56 (65,9 por cento) genotipáveis para genótipo G. Dentre essas, 49 (87,5 por cento) foram G1, cinco (8,9 por cento) G4, uma (1,8 por cento) G3 e uma (1,8 por cento) G9. Considerando a genotipagem P, 37 (43,5 por cento) foram genotipáveis e todas eram P[8]. A associação G e P mais observada foi G1P[8], 33 (89,2 por cento) amostras; seguida de G4P[8], duas (5,4 por cento) amostras; G3P[8], uma (2,7 por cento) amostra; e G9P[8], uma (2,7 por cento) amostra.

Polyacrylamide gel electrophoresis and combined immunoenzyme assay for rotavirus and adenovirus were used to analyze 380 fecal samples from children up to three years of age who were hospitalized with acute diarrhea in Campo Grande, State of Mato Grosso do Sul, between May 2000 and January 2004. Among all the samples, 88 (23. 2 percent) were positive for Rotavirus A. Out of these, 81 (92 percent) had a defined electrophoretic pattern: 77 (87. 5 percent) with a long pattern and four (4. 5 percent) with a short pattern. Genotype G and P characterization was done by nested RT-PCR for 85 samples, of which 56 (65. 9 percent) were genotyped as type G. Among these, 49 (87. 5 percent) were G1, five (8. 9 percent) were G4, one (1. 8 percent) was G3 and one (1. 8 percent) was G9. The genotype was found to be type P in 37 samples (43. 5 percent) and all of these were P[8]. The G and P association most observed was G1P[8], with 33 samples (89. 2 percent), followed by G4P[8], two samples (5. 4 percent); G3P[8], one sample (2. 7 percent); and G9P[8], one sample (2. 7 percent).