Molecular analysis of NSP4 coding gene of porcine rotavirus in Brazil / Análise molecular do gene codificador da NSP4 de rotavírus suínos no Brasil

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas(AU)

A proteína não estrutural 4 (NSP4) desempenha diferentes funções na replicação e na morfogênese dos rotavírus, apresentando, ainda, uma atividade de enterotoxina, causando diarreia do tipo secretória. Um total de 11 sequências parciais de nucleotídeos do gene codificador da NSP4 de rotavírus suínos de criações brasileiras foram definidas como pertencentes ao grupo A. Comparando-se as sequências virais da área do peptídeo toxigênico, que compreende a porção entre os aminoácidos de 114 a 135, constatou-se uma única mutação pontual no aminoácido 135, sendo que duas amostras apresentaram alanina, e as demais, valina. A análise filogenética do gene demonstrou que todas as amostras pertencem ao genotipo E1, e que a identidade nucleotídica das amostras brasileiras variou de 92,4% a 100%, enquanto que a identidade de aminoácidos, de 95,8% a 100%. Apenas um resíduo (aa 138) sofreu seleção positiva enquanto que pelo menos outros 119 apresentam seleção negativa. Assim, esses dados mostram a ocorrência de um genotipo comum da NSP4 já descrito anteriormente em suínos, com uma baixa diversidade entre as amostras encontradas(AU)

MOLECULAR ANALYSIS OF NSP4 CODING GENE OF PORCINE ROTAVIRUS IN BRAZIL

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas.

Molecular analysis of NSP4 coding gene of porcine rotavirus in Brazil / Análise molecular do gene codificador da NSP4 de rotavírus suínos no Brasil

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas

A proteína não estrutural 4 (NSP4) desempenha diferentes funções na replicação e na morfogênese dos rotavírus, apresentando, ainda, uma atividade de enterotoxina, causando diarreia do tipo secretória. Um total de 11 sequências parciais de nucleotídeos do gene codificador da NSP4 de rotavírus suínos de criações brasileiras foram definidas como pertencentes ao grupo A. Comparando-se as sequências virais da área do peptídeo toxigênico, que compreende a porção entre os aminoácidos de 114 a 135, constatou-se uma única mutação pontual no aminoácido 135, sendo que duas amostras apresentaram alanina, e as demais, valina. A análise filogenética do gene demonstrou que todas as amostras pertencem ao genotipo E1, e que a identidade nucleotídica das amostras brasileiras variou de 92,4% a 100%, enquanto que a identidade de aminoácidos, de 95,8% a 100%. Apenas um resíduo (aa 138) sofreu seleção positiva enquanto que pelo menos outros 119 apresentam seleção negativa. Assim, esses dados mostram a ocorrência de um genotipo comum da NSP4 já descrito anteriormente em suínos, com uma baixa diversidade entre as amostras encontradas

Molecular analysis of nsp4 coding gene of porcine rotavirus in brazil

The non-structural protein 4 (NSP4) has different roles in rotaviral replication, morphogenesis, and enterotoxin-like activity causing secretory diarrhea. A total of 11 partial nucleotide sequences of NSP4 coding gene were defined from group A rotavirus circulating in Brazilian swine herds. On comparing the viral sequences of diarrheagenic peptide area (amino acid 114-135), there was a single point mutation at amino acid 135 presented by two strains with amino acid alanine, and valine in the others. The NSP4 gene phylogeny showed that all strains clustered into E1 genotype, and the nucleotide identity between Brazilian strains ranged from 92.4% and 100%, while the putative amino acid identity, between 95.8% and 100%. Only one site (138aa) was positively selected and at least 119 were negatively selected. As a conclusion, these data demonstrate the occurrence of a common NSP4 genotype described elsewhere in pigs and low diversity between the samples from the surveyed areas.

DETECÇÃO DE ROTAVÍRUS EM EFLUENTES DE CRIAÇÕES DE SUÍNOS NO ESTADO DE SÃO PAULO, BRASIL.

Os rotavírus são um dos principais agentes virais envolvidos na ocorrência de gastroenterites em crianças e em animais de diferentes espécies. Sua elevada resistência ambiental aliada à via de transmissão fecal-oral, torna-o um agente propício de se propagar pela água, principalmente nos efluentes. O objetivo deste estudo foi o de se detectar a circulação e eliminação de rotavírus em criações de suínos de baixa tecnificação do Estado de São Paulo, Brasil. Um total de 25 amostras, incluindo fezes de leitões com diarréia e efluentes não tratados, de 7 diferentes propriedades, foram testadas em paralelo para detecção do rotavírus através da eletroforese em gel de poliacrilamida (PAGE) e ELISA, sendo as positivas confirmadas por RT-PCR (transcrição reversa - reação em cadeia pela polimerase). A PAGE evidenciou apenas uma amostra positiva (1/25 ou 4%) proveniente de material fecal, enquanto que pela ELISA, 6 (6/25 ou 24%) amostras positivas, das quais 4 de material fecal e 2 de efluentes. A RT-PCR confirmou todos os resultados positivos de PAGE e ELISA. Portanto, os rotavírus foram encontrados em 3 de 7 (42,86%) das criações pesquisadas, das quais em duas destas, o vírus foi detectado tanto no efluente quanto nos animais. Face a estes resultados, houve a detecção de rotavírus nos efluentes não tratados de criações de suínos, constituindo um risco para a disseminação do agente para h

DETECÇÃO DE ROTAVÍRUS EM EFLUENTES DE CRIAÇÕES DE SUÍNOS NO ESTADO DE SÃO PAULO, BRASIL.

Os rotavírus são um dos principais agentes virais envolvidos na ocorrência de gastroenterites em crianças e em animais de diferentes espécies. Sua elevada resistência ambiental aliada à via de transmissão fecal-oral, torna-o um agente propício de se propagar pela água, principalmente nos efluentes. O objetivo deste estudo foi o de se detectar a circulação e eliminação de rotavírus em criações de suínos de baixa tecnificação do Estado de São Paulo, Brasil. Um total de 25 amostras, incluindo fezes de leitões com diarréia e efluentes não tratados, de 7 diferentes propriedades, foram testadas em paralelo para detecção do rotavírus através da eletroforese em gel de poliacrilamida (PAGE) e ELISA, sendo as positivas confirmadas por RT-PCR (transcrição reversa - reação em cadeia pela polimerase). A PAGE evidenciou apenas uma amostra positiva (1/25 ou 4%) proveniente de material fecal, enquanto que pela ELISA, 6 (6/25 ou 24%) amostras positivas, das quais 4 de material fecal e 2 de efluentes. A RT-PCR confirmou todos os resultados positivos de PAGE e ELISA. Portanto, os rotavírus foram encontrados em 3 de 7 (42,86%) das criações pesquisadas, das quais em duas destas, o vírus foi detectado tanto no efluente quanto nos animais. Face a estes resultados, houve a detecção de rotavírus nos efluentes não tratados de criações de suínos, constituindo um risco para a disseminação do agente para h

Detection of rotavirus from pig livestock wastewater of São Paulo State, Brazil / Detecção de rotavírus em efluentes de criações de suínos no estado de São Paulo, Brasil

Rotavirus is one of the most important viral agents of gastroenteritis among child and animals from different species. It's high environmental resistance and the fecal-oral way of transmission makes this virus likely to be transmitted by wastewater. This study seeks to detect the wastewater elimination and circulation of group A rotavirus in low technified pig farms from São Paulo State, Brazil. A total of 25 samples, including piglet feces with diarrhea and untreated wastewater samples, from 7 different farms, were submitted in a parallel screening scheme of rotavirus infection through polyacrilamide gel electrophoresis (PAGE) and ELISA, which the positive samples were further confirmed by RT-PCR (reverse-transcription polimerase chain reaction). The PAGE revealed only one positive sample (1/25 or 4%) from feces, while by ELISA, 6 (6/25 or 24%) samples were positive, which 4 were from feces and 2 from wastewater. The RT-PCR confirmed all positive PAGE and ELISA results. Therefore, the rotavirus was found in 3 of 7 (42.86%) researched farms, which in 2 of these were detected both in animals and wastewater and one were found virus only in fecal samples. In view of these results, there was rotavirus detection from untreated pig farm wastewater, posing as a risk of spreading for humans and animals, implying the need of assuring microbiological and environmental safety measures with this material.

Os rotavírus são um dos principais agentes virais envolvidos na ocorrência de gastroenterites em crianças e em animais de diferentes espécies. Sua elevada resistência ambiental aliada à via de transmissão fecal-oral torna-o um agente propício de se propagar pela água, principalmente nos efluentes. O objetivo deste estudo foi o de se detectar a circulação e eliminação de rotavírus em criações de suínos de baixa tecnificação do Estado de São Paulo, Brasil. Um total de 25 amostras, incluindo fezes de leitões com diarréia e efluentes não tratados, de 7 diferentes propriedades, foram testadas em paralelo para detecção do rotavírus através da eletroforese em gel de poliacrilamida (PAGE) e ELISA, sendo as positivas confirmadas por RT-PCR (transcrição reversa - reação em cadeia pela polimerase). A PAGE evidenciou apenas uma amostra positiva (1/25 ou 4%) proveniente de material fecal, enquanto que pela ELISA, 6 (6/25 ou 24%) amostras positivas, das quais 4 de material fecal e 2 de efluentes. A RT-PCR confirmou todos os resultados positivos de PAGE e ELISA. Portanto, os rotavírus foram encontrados em 3 de 7 (42.86%) das criações pesquisadas, das quais em duas destas, o vírus foi detectado tanto no efluente quanto nos animais. Em face destes resultados, houve a detecção de rotavírus nos efluentes não tratados de criações de suínos, constituindo um risco para a disseminação do agente para humanos e animais, implicando na necessidade de assegurarem-se medidas de segurança ambiental e microbiológica deste material.

Enhancement of apoptosis with loss of cellular adherence in the villus epithelium of the small intestine after infection with the nematode Nippostrongylus brasiliensis in rats.

It has been reported that infection with Nippostrongylus brasiliensis induces villus atrophy with various histological alterations. In N. brasiliensis-infected rats, villus length in the jejunum was reduced significantly at day 10 p.i., when serum levels of rat mast cell protease (RMCP) II had increased significantly. To determine whether the villus atrophy is associated with enhancement of apoptosis, apoptotic nuclei were labelled using the nick end-labelling method. Numbers of labelled cells were markedly increased in the villus epithelium at 7-10 days p.i., while the numbers returned to normal 14 days p.i. when worms were rejected from the intestine and villus length became normal. Examination of the expression of the adhesion molecule E-cadherin showed granular immunoreactivity in the cytoplasm of atrophic villus epithelium with loss of normal localization to epithelial cell borders. In mast cell-deficient Ws/Ws rats, villus length was reduced as significantly as in +/+ counterparts at day 10 p.i. with marked increases in the numbers of apoptotic cells. These results suggested that villus atrophy was closely associated with enhanced apoptosis and loss of adhesion in epithelial cells. Mast cell activation appears not to be involved in these alterations.

Lack of active lung anaphylaxis in congenitally mast cell-deficient Ws/Ws rats sensitized with the nematode Nippostrongylus brasiliensis.

Ws/Ws rats are deficient in both mucosal- and connective tissue-type mast cells. To study the role of mast cells in active anaphylaxis, changes in vascular permeability in the trachea upon intravenous antigen challenge with Evans blue dye were examined in Ws/Ws, heterogenic Ws/+, and normal +/ + rats sensitized with the nematode Nippostrongylus brasiliensis. Antigen challenge resulted in fatal anaphylactic shock in some +/+ and Ws/+ rats, but not in Ws/Ws rats. Marked dye leakage developed within 30 min in the trachea of +/+ and Ws/+ rats, while Ws/Ws rats showed no substantial increases in the levels of vascular permeability. Ex vivo stimulation of sensitized lung fragments from +/+ animals with specific antigen induced significant releases of histamine and leukotriene (LT) C4, while sensitized Ws/Ws rat-lung fragments did not. In Ws/Ws rats, levels of nematode-specific IgE, IgG1 and IgG2a antibodies as well as levels of lung eosinophilia were not significantly different from those in +/+ rats. These results show that mast cell-deficient Ws/Ws rats fail to develop active anaphylaxis, and this is mediated probably by the lack of mast cell-derived mediators required for initiation of the reaction.

Lung granulomatous response induced by infection with the intestinal nematode Nippostrongylus brasiliensis is suppressed in mast cell-deficient Ws/Ws rats.

Certain nematode infections induce eosinophil infiltration and granulomatous responses in the lungs. To examine the role of mast cells in the development of lung lesions, normal +/+ and genetically mast cell-deficient Ws/Ws rats were infected with the nematode Nippostrongylus brasiliensis. In +/+ rats, numbers of eosinophils in bronchoalveolar lavage fluid (BALF) increased significantly 3-7 days after infection, and granulomatous responses composed of histiocytes/ macrophages and multinucleate giant cells were triggered in the lungs 3-14 days after infection. Challenge infection, which was carried out on day 28 after primary infection, induced much higher levels of granulomatous response than after primary infection, suggesting that the response is mediated at least in part by an immunological mechanism. In Ws/Ws rats, both the eosinophil percentage in BALF and the size of the granulomas in the lungs were significantly smaller than in +/+ rats after primary as well as after challenge infection. The amount of rat mast cell protease (RMCP) II in +/+ rat BALF was increased 1 day after primary infection and more significantly after challenge infection, suggesting that lung mucosal mast cells were activated more markedly after the challenge infection. In Ws/Ws rats, RMCP II was undetectable throughout the observation period. The time course of nematode migration in the lungs did not differ in +/+ and Ws/Ws rats. These results suggest that mast cell activation might be relevant to eosinophil infiltration and granulomatous response in the lungs, although the responses do not affect lung migration of the nematode.

Dissociation of early and late protective immunity to the nematode Nippostrongylus brasiliensis in Brown Norway and Fischer-344 rats.

Worm expulsion of, and IgE and interferon (IFN)-gamma responses to, Nippostrongylus brasiliensis were studied in 2 rat strains, Brown Norway (BN) and Fischer (F)-344. BN rats expelled the majority of worms by day 14 post-infection (p.i.) with approximately 6% of worms surviving for at least 3 weeks. In F-344 rats, worm expulsion was delayed by 2 days relative to that in BN, while the numbers of residual worms were significantly fewer than in BN, suggesting that different immune mechanisms are involved in early and late phases of immunity. Total serum IgE, as well as in vitro IgE production by mesenteric lymph node (MLN) cells, was increased 2 weeks p.i., the levels being markedly higher in BN than in F-344 rats. Serum rat mast cell protease II was also increased more significantly in BN than in F-344 rats. In contrast, production of IgG2a and IFN-gamma by MLN and spleen cells was found to be higher in F-344 than in BN rats. These results indicate that the early worm expulsion is correlated with the host IgE and mast cell responsiveness, whereas the persistence of infection in the late period may be controlled by different immune mechanisms.