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.

Projecting the effectiveness of RotaTeq® against rotavirus-related hospitalisations in Brazil

RotaTeq® (Merck & Company, Inc, Whitehouse Station, NJ, USA) is an oral pentavalent rotavirus vaccine (RV5) that has shown high and consistent efficacy in preventing rotavirus gastroenteritis (RGE) in randomised clinical trials previously conducted in industrialised countries with high medical care resources. To date, the efficacy and effectiveness data for RV5 are available in some Latin American countries, but not Brazil. In this analysis, we projected the effectiveness of RV5 in terms of the percentage reduction in RGE-related hospitalisations among children less than five years of age in four regions of Brazil, using a previously validated mathematical model. The model inputs included hospital-based rotavirus surveillance data from Goiânia, Porto Alegre, Salvador and São Paulo from 2005-2006, which provided the proportions of rotavirus attributable to serotypes G1, G2, G3, G4 and G9, and published rotavirus serotype-specific efficacy from the Rotavirus Efficacy and Safety Trial. The model projected an overall percentage reduction of 93 percent in RGE-related hospitalisations, with an estimated annual reduction in RGE-related hospitalisations between 42,991-77,383 in the four combined regions of Brazil. These results suggest that RV5 could substantially prevent RGE-related hospitalisations in Brazil.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9 percent) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6 percent) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

Molecular characterization of astrovirus in stool samples from children in São Paulo, Brazil

The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo, Brazil, grouped into two sets: EPM and HU. Detection and genotyping were carried out using reverse transcription nested polymerase chain reaction (RT-PCR) with specific primers directed towards the genome open reading frame 2 (ORF2). Results for EPM set showed that 66/234 (28.2 percent) were positive: 28/94 (29.7 percent) from children with acute diarrhea, 14/45 (31.1 percent) with persistent diarrhea, and 9/55 (16.3 percent) from control individuals. No data was available for 15/40 (37.5 percent) of samples. Mixed infections with other viruses were found in 33 samples. In the HU, 18/187 (9.6 percent) were positive: 12/158 (7.6 percent) from individuals with acute diarrhea and 6/29 (20.7 percent) from control children. Four samples were mixed with other viruses. Out of 66 astrovirus positive EPM samples, 18 (27.2 percent) were characterized as human astrovirus type-1 (HAstV-1), two (3.0 percent) as HAstV-2, two (3.0 percent) as HAstV-3, and three (4.5 percent) as HAstV-8. Among 18 astrovirus positive HU samples, one (5.5 percent) was characterized as HAstV-1, six (33.3 percent) as HAstV-2, and one (5.5 percent) as HAstV-8. Two HAstV-8 genotyped samples were further confirmed by nucleotide sequencing. Our results shows that astroviruses are circulating in a constant manner in the population, with multiple serotypes, in higher frequency than it was described for other Brazilian regions. For the first time in Sao Paulo, Brazil, it was shown that astroviruses play an important role in children gastroenteritis, as described for most locations where they were detected.

Serological and molecular diversity of human rotavirus in São Paulo, Brazil

From a total of 187 fecal samples from children with ages between 0 and 5 years, collected in the Hospital Universitário -USP, Brazil, from 1994 to 1996, 54 (28.9 percent) were positive for rotavirus. Positive samples were characterized by electropherotyping, subgrouping, G serotype and genotype and P genotype. Rotavirus electropherotypes were characterized in four different long genome patterns (38.9 percent), one short genome pattern (34.0 percent) and 18.0 percent were characterized as an unusual pattern. Subgroup I was found in 38.9 percent strains, subgroup II in 50.0 percent and 7.7 percent was subgroup nonI-nonII. For G serotypes, G2 was found in 59.3 percent, G1 was identified in 33.3 percent of strains, two samples showed mixtures of G1+G2 and one sample was G1+G3. Ten samples characterized as serotype G2 showed a long eletropherotype. Genotype G2 was the most frequently and was found in 37 (44.0 percent) samples (23 samples as a single genotype and 14 as mixtures of genotypes). G1 was found in 15 samples. G3 and G4 was detected mainly in mixtures of genotypes and G5, G6 and G9 were identified only in mixtures. A total of 20 (38.5 percent) samples were characterized as G genotype mixtures and P mixtures were found in 16 (29.6 percent) samples. P[4] was found in 55.6 percent of samples, P[8] in 51.9 percent and P[6-M37 like] in 22.3 percent of cases. P[6-Gottfried like] and P[11] were detected only in mixtures. One sample with G6 specificity, mixed with a G2 rotavirus and a P[11] strain, mixed with P[4] and P[8]strain was described for the first time in Latin America.

De um total de 187 amostras fecais de crianças com idades entre 0 e 5 anos, coletadas no Hospital Universitário -USP, Brasil, de 1994 a 1996, 54 (28.9 por cento) foram positivas para rotavírus. As amostras positivas foram caracterizadas quanto ao eletroferótipo, subgrupo, sorotipo G e genotipo G e P. Foram identificados quatro diferentes eletroferótipo longos em 38.9 por cento das amostras, um eletroferótipo curto (34,0 por cento) e 18,0 por cento foram caracterizadas como um eletroferótipo não usual. O subgrupo I foi encontrado em 38,9 por cento amostras, o subgrupo II em 50,0 por cento e nãoI-nãoII em 7,7 por cento. O sorotipo G2 foi encontrado em 59,3 por cento e G1 em 33,3 por cento. Duas amostras apresentaram misturas de G1+G2 e outra amostra G1+G3. Dez amostras caracterizadas como sorotipo G2 mostraram perfil eletroferótico longo. O genotipo G2 foi o mais freqüente, encontrado em 37 amostras (23 como único genotipo e 14 associados a outro genotipo). G1 foi encontrado em 15 amostras; G3 e G4 foram detectados principalmente em misturas e G5, G6 e G9, identificados somente em misturas. Um total de 20 (38,5 por cento) amostras foram identificadas como misturas de genotipo G e foram encontradas 16 (29,6 por cento) amostras com misturas de genotipo P. P[4] foi encontrado em 55,6 por cento das amostras, P[8] em 51,9 por cento e P[6-M37 like], em 5,5 por cento das amostras. P[6-Gottfried like] e P[11] foram detectados somente em misturas. Uma amostra com especificidade G6, associada ao genotipo G2 e outra P[11] misturada com P[4] e de P[8] foram identificadas pela primeira vez na América Latina.

G and P rotavirus genotypes in stool samples from children in Teresina, State of Piauí

A total of 123 stool specimens collected in Teresina, Piauí between 1994 and 1996, from 0 to 2-year-old children with diarrhea, were used for this study. Molecular characterization of the G and P rotavirus genotypes was performed using the reverse transcriptase polymerase chain reaction. The following results were obtained for the P genotypes: P[8] (17. 1 percent), P[1] (4. 9 percent), P[4] (3. 3 percent), P[6, M37] (2. 4 percent) and mixtures (27. 6 percent). The P[1]+P[8] mixture was found in 19. 5 percent of the samples. For the G genotypes, the results were: G1 (25. 2 percent), G5 (13. 8 percent), G2 (2. 5 percent), G4 (2. 5 percent), G9 (0. 8 percent) and mixtures (41. 5 percent). G1+G5 was the mixture most frequently found (12. 1 percent). Our results showed unusual combinations such as P[1]G5 and P[1]+P[8]G5. The high percentage of mixtures and unusual combinations containing mixtures of human and animal rotavirus genotypes strongly suggests the possibility of gene reassortment and interspecies transmission.

Um total de 123 amostras fecais de crianças de 0 a 2 anos com diarréia, coletadas em Teresina, Piauí, entre 1994 e 1996 foi utilizada neste estudo. Para a caracterização molecular dos genótipos G e P de rotavírus, foram realizadas as reações de transcriptase reversa e reação em cadeia pela polimerase. Os seguintes resultados foram obtidos para o genótipo P: P[8] (17,1 por cento), P[1] (4,9 por cento), P[4] (3,3 por cento), P[6, M37] (2,4 por cento) e misturas (27,6 por cento). A mistura P[1]+P[8] foi encontrada em 19,5 por cento das amostras. Para o genótipo G os resultados foram: G1 (25,2 por cento), G5 (13,8 por cento), G2 (2,5 por cento), G4 (2,5 por cento), G9 (0,8 por cento) e misturas (41,5 por cento). A mistura G1+G5 foi a mais freqüentemente encontrada (12,1 por cento). Nossos resultados mostram combinações não usuais como P[1]G5 e P[1]+P[8]G5. A alta porcentagem de misturas e as combinações não usuais contendo misturas de genótipos de rotavirus humanos e animais sugerem fortemente a possibilidade de rearranjo genético e transmissão interspecies.

Detection of rotavirus in dogs with diarrhea in Brazil

Rotavirus was detected by the enzyme-linked immunosorbent assay (ELISA) in the faeces of a diarrheic dog. Virus particles with morphology typical of rotavirus were visualized by direct electron microscopy. This sample was subsequently tested for the four main human serotypes (G1-G4), by ELISA with monoclonal antibodies. G genotyping was attempted by RT-PCR using G1-G6 and G8-G11 primers but no positive results could be yielded. Also using RT-PCR it was possible to characterize this canine strain as belonging to P[ 3] genotype. This is the first canine rotavirus detected in Brazil.

Epidemiological features of rotavirus infection in Goiânia, Goiás, Brazil, from 1986 to 2000

A total of 2,605 faecal specimens from children up to 10 years old with or without diarrhoea were collected. Samples were obtained from 1986 to 2000 in hospitals, outpatient clinics and day-care centers in Goiânia, Goiás. Two methodologies for viral detection were utilized: a combined enzyme immunoassay for rotavirus and adenovirus and polyacrylamide gel electrophoresis. Results showed 374 (14.4 percent) faecal specimens positive for Rotavirus A, most of them collected from hospitalized children. A significant detection rate of rotavirus during the period from April to August, dry season in Goiânia, and different frequencies of viral detection throughout the years of study were also observed. Rotavirus was significantly related to hospitalization and to diarrhoeal illness in children up to 24 months old. This study reinforces the importance of rotavirus as a cause of diarrhoea in children and may be important in regards to the implementation of rotavirus vaccination strategies in our country

Characterization of mixed infections with different strains of bovine rotavirus in an outbreak of diarrhea in dairy herds in Goiás, Brazil

Ten faecal samples of bovine rotavirus from calves less than 30 days old from an outbreak of diarrhea in Hidrolândia, Goiás, Brazil were submitted to serological and molecular characterization, using enzyme immonuassay for subgrouping and serotyping, PAGE for determination of electropherotypes and PCR for genome typing. Nine samples belonged to group A/subgroup I rotavirus one samples was group A / soubgroup non-I/non-II. Four samples were characterized as G10P[11] (B223-like), four samples showed a mixture of two rotavirus strains (G6G10 and P[5]P[11]), one sample was characterized as G6P[11] and one sample was characterized only by G serotyping/genotyping, and did not react with any primer used. Two electropherotypes were detected and both were present in the same animal. This study demonstrates that two different electropherotypes and/or serotypes of bovine rotavirus can circulate in the same outbreak.

Identification, propagation and subgroup characterization of an equine rotavirus isolated in Säo Paulo, Brazil

Duas amostras de fezes de equinos (EQ/28 e EQ/29) foram recebidas para diagnóstico no Instituto Biológico. As amostras eram provenientes de Orlândia, Säo Paulo, de dois animais com diarréia. A identificaçäo de rotavírus foi feita através de ensaio imunoenzimático (EIARA/FIOCRUZ) e por eletroforese em gel de poliacrilamida (PAGE). As amostras foram positivas para rotavírus pelas duas técnicas, com eletroferótipo característico de rotavírus equino (segmentos 3 e 4 bem próximos) e segmentos 7, 8 e 9 separados). O rotavírus foi isolado em linhagem celular de rim fetal de macaco Rhesus (MA104), com a adiçäo de tripsina, em equipamento do tipo "roller", chegando a sexta passagem, com efeito citopítico característico. Os lisados de cada passagem em culturas celulares foram positivos para rotavírus por EIARA e PAGE, com eletroferótipo semelhante ao da amostra original, em relaçäo aos segmentos 1 a 6, e com algumas diferenças na migraçäo dos segmentos 7 a 11. As amostras originais e lisados das passagens em culturas de células foram testados para determinaçäo de subgrupo, com anticorpos monoclonais (MAb) específicos para grupo A, subgrupo I e subgrupo II, através de um teste imunoenzimático. Tanto as amostras originais quanto os lisados reagiram com o MAb específico para o grupo A e näo reagiram com os anticorpos antisubgrupos I e II, da mesma forma que a amostra H-2 e outros rotavírus equinos. Este é o primeiro isolamento de um rotavírus equino no Brasil, tornando-se importante elucidar sua prevalência em animais normais e com diarréia