Sibele Pinheiro de Souza

Rabies virus in insectivorous bats: implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology.

Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Abstract Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa=0.833) and substantial for rotavirus detection (kappa=0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.

Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene.

Abstract Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.

First detection and molecular diversity of Brazilian bovine torovirus (BToV) strains from young and adult cattle

Abstract Bovine torovirus (BToV) is an established enteric pathogen of cattle, but its occurrence in Brazilian cattle had not been reported until now. This article describes a survey on BToV in Brazil carried out on 80 fecal samples from diarrheic young and adult cattle, using a nested-RT-PCR targeting the nucleocapsid (N) gene. BToV was detected in 6.25% (5/80) of stool samples from three different geographic regions. Sequences analysis showed that Brazilian BToVs have a high degree of identity with European and Japanese BToVs and a lower degree of identity with North American Breda 1 strain. These results show that, albeit its low frequency and the scarce number of research on the field, BToV is still present amongst cattle populations.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

O Coronavirus bovino (BCoV) pertence ao grupo 2 do genero Coronavirus (Nidovirales: Coronaviridae) e e agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doenca respiratoria em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a deteccao do BCoV com base em sequencias representativas e recentes do gene do nucleocapsideo, regiao conservada do genoma dos coronavirus. Tres primers foram desenhados, a primeira amplificacao com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analitica foi determinada pela diluicao do BCoV cepa Kakegawa (titulo HA: 256) na base de 10 em agua ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensao fecal negativa para o BCoV, onde foram encontrados resultados positivos ate a diluicao de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitacao dos pellets pelo metodo de extracao utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analitica e ausencia de possiveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referencia, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnostico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo metodo proposto e o fato de que os primers foram desenhados baseados em sequencias atuais do BCoV, oferecem bases para o diagnostico mais acurado de infeccoes causadas pelo BCoV, assim como para novas perspectivas em protocolos de deteccao de outros Coronavirus de importância tanto em saninade animal quanto em saude publica.

A Multigene Approach for Comparing Genealogy of Betacoronavirus from Cattle and Horses

Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV) being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S), hemagglutinin-esterase protein (HE), and nucleoprotein (N) genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealed Betacoronavirus sequences indistinguishable from those found in cattle, a fact previously unknown.

Phylogeny of canine coronavirus (CCoV) from Brazilian dogs based on membrane protein partial sequences

Este artigo descreve a anteriormente desconhecida diversidade molecular de amostras brasileiras de Coronavirus canino (CCoV). Vinte e duas amostras foram submetidas a analise da sequencia parcial do gene codificador da proteina de membrana, sendo 12 classificadas como CCoV Tipo II e 10 como CCoV Tipo I e uma possivel sublinhagem tipicamente brasileira foi encontrada para o CCoV Tipo II.

Differentiation of Bovine coronavirus (BCoV) genotypes by a restriction enzyme assay

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Achados clínico patológicos durante um surto de disenteria de inverno em bovinos no Rio Grande do Sul

The report describes a winter dysentery outbreak that affected 10 dairy cows from a ranch located in the county of Viamao, Rio Grande do Sul. The most significant clinical sign was profuse and watery diarrhea, which ranged from a greenish to a brownish coloration and from occasional blood streaks with mucus to a bloody diarrhea. Most cases persisted for 5 days and also included depression, drastic decrease in milk production and in food consumption. Only one of the affected animals died. The necropsy revealed pale mucosa and sanguineous content with high quantity of blood clots, particularly within the spiral colon, and petechiae on the colonic mucosa. Histopathological lesions were predominant in the spiral colon, and consisted of a high number of dilated crypts without epithelium or with the replacement of a pavement epithelium with occasional immature cuboidal cells, which sometimes showed enlarged nucleus and proeminent nucleolus. Some crypts were filled with epithelial desquamation and polymorphnuclear cells. Bovine coronavirus (8F2) monoclonal antibody was used for immunohistochemistry on sections of the spiral colon that showed positive reactions in the cytoplasm of the infected crypt epithelium, sloughed necrotic cells, and within macrophages in the lamina propria of both.