Picobirnavirus is an unclassified dsRNA virus, which is associated with viral gastroenteritis in humans and animals. Picobirnavirus dsRNA has been detected in many cases when diagnostic PAGE screening for rotavirus dsRNA is performed. During this routine diagnosis, picobirnavirus dsRNA has been detected in the faeces of patients with and without viral gastroenteritis. Despite the common occurrence of picobirnavirus infection in humans and animals, its direct involvement in causing gastroenteritis has not been established. No molecular studies have been done on picobirnavirus except sequencing and epidemiology studies. Like all RNA viruses, picobirnavirus encodes a RNA-dependent RNA polymerase. The picobirnavirus RNA-dependent RNA polymerase has only been identified on the basis of its amino acid sequence. The catalytic activity of the polymerase has not been studied to date. In this study, a porcine picobirnavirus was studied at a molecular level to establish the activity of the protein encoded by segment 2 of its genome. To determine the identity of this putative picobirnavirus RNA-dependent RNA polymerase, its open reading frame (ORF) was successfully amplified by PCR, cloned and sequenced. Subsequently the ORF was successfully sub-cloned into baculovirus and bacterial expression vectors. The protein encoded by picobirnavirus segment 2 was successfully expressed as a recombinant protein in a soluble form in both baculovirus and bacterial expression systems. In the baculovirus system, two recombinant baculoviruses were constructed. One recombinant baculovirus expressing a histidine tagged protein and another one expressing an untagged protein. In bacterial expression systems, a recombinant protein fused to a Glutathione-S-Transferase (GST) tag at the N terminal end was expressed. The GST tag allowed easy purification of the expressed GST fusion protein by affinity chromatography on immobilized glutathione. Subsequently the GST tag could be removed from the purified recombinant protein by proteolysis with thrombin. Both tagged and untagged putative picobirnavirus RNA-dependent RNA polymerase from the bacterial expression system were shown to have an affinity for heparin. This implies that the protein might have an affinity for nucleic acids. Picobirnavirus genome segment 2 ssRNA was generated from full length picobirnavirus genome segment 2 cDNA by in vitro transcription. The recombinant E.coli expressed proteins (tagged and untagged) was tested for ssRNA binding and RNA replicase activity. No RNA binding or replicase activity was observed with either tagged or untagged recombinant protein. This study reports the first evidence other than conserved polymerase motifs, that the protein encoded by segment 2 of picobirnavirus is most likely a RNA-dependent RNA polymerase. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Picobirnavirus (PBV) pertencem à família Picobirnaviridae, divididos em duas espécies Human Picobirnavirus e Rabbit Picobirnavirus. São pequenos vírus constituídos de genoma bissegmentado de cadeia dupla de RNA (dsRNA), não envelopados, com capsídeo de simetria icosaédrica, sendo divididos em dois genogrupos, GI e GII. Já foram detectados em fezes humanas e de uma ampla gama de espécies animais, com ou sem sinais diarreicos, sendo considerados agentes emergentes e oportunistas, e seu potencial zoonótico foi sugerido. Entretanto, os estudos epidemiológicos e moleculares de PBV em bovinos são raros na literatura nacional e internacional. Devido à carência de dados a respeito de PBV em bovinos, o presente estudo foi realizado objetivando-se a detecção e caracterização moleculares de cepas de PVB bovinos dos genogrupos GI e GII em amostras fecais de bovinos com ou sem sintomatologia diarreica de diferentes idades e regiões do Brasil. O estudo foi conduzido a partir de 77 animais, obtendo-se 18 (23,3%) amostras positivas para GI, compreendendo animais provenientes dos estados de São Paulo, Minas Gerais e Goiás. Não foram detectadas amostras positivas para GII. A identidade nucleotídica das amostras obtidas apresentou média de 67,4% quando comparadas uma com as outras e de até 83,77% quando comparadas com amostras de PBV de referência. Na reconstrução filogenética, três amostras agruparam-se em clado de PVB humano e somente uma agrupou-se em clado de PVB bovino. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de PVB bovino pertencente ao genogrupo GI em diferentes estados brasileiros, com perfis filogenéticos heterogêneos. / Picobirnavirus (PBV) belong to the Picobirnaviridae family, divides into two species Human Picobirnavirus and Rabbit Picobirnavirus. They are small, non-enveloped, bisegmented double-stranded RNA (dsRNA) virus with an icosahedral capsid, being divided into two genogroups, GI and GII. They have been detected in feces of humans and many animal species, with or without diarrheal signs and are considered emerging and opportunistic agents, and its zoonotic potential has been suggested. However, epidemiological and molecular studies of bovine PBV are rare in the national and international literature. Due to lack of data on PBV in cattle, this study was conducted aiming to detect and molecularly characterize bovine PBV strains of GI and GII genogroups in feces from animals with or without diarrheal signs of different ages and regions of Brazil. Seventy-seven animals were sampled, resulting in 18 (23.3%) positive samples for GI, including animals from the states of São Paulo, Minas Gerais and Goiás. There were no positive samples for GII. The nucleotide identity of the samples obtained showed a mean of 67.4% compared to each other and up to 83.77% compared to PBV reference samples. In phylogenetic reconstruction, three samples were grouped in the human PBV clade and only one sample was clustered in the bovine PVB clade. In summary, the results indicate in an unprecedented way the circulation of the bovine PBV belong to GI genogroup in different Brazilian states, with heterogeneous phylogenetic profiles.

Picobirnavirus (PBV) pertencem à família Picobirnaviridae, divididos em duas espécies Human Picobirnavirus e Rabbit Picobirnavirus. São pequenos vírus constituídos de genoma bissegmentado de cadeia dupla de RNA (dsRNA), não envelopados, com capsídeo de simetria icosaédrica, sendo divididos em dois genogrupos, GI e GII. Já foram detectados em fezes humanas e de uma ampla gama de espécies animais, com ou sem sinais diarreicos, sendo considerados agentes emergentes e oportunistas, e seu potencial zoonótico foi sugerido. Entretanto, os estudos epidemiológicos e moleculares de PBV em bovinos são raros na literatura nacional e internacional. Devido à carência de dados a respeito de PBV em bovinos, o presente estudo foi realizado objetivando-se a detecção e caracterização moleculares de cepas de PVB bovinos dos genogrupos GI e GII em amostras fecais de bovinos com ou sem sintomatologia diarreica de diferentes idades e regiões do Brasil. O estudo foi conduzido a partir de 77 animais, obtendo-se 18 (23,3%) amostras positivas para GI, compreendendo animais provenientes dos estados de São Paulo, Minas Gerais e Goiás. Não foram detectadas amostras positivas para GII. A identidade nucleotídica das amostras obtidas apresentou média de 67,4% quando comparadas uma com as outras e de até 83,77% quando comparadas com amostras de PBV de referência. Na reconstrução filogenética, três amostras agruparam-se em clado de PVB humano e somente uma agrupou-se em clado de PVB bovino. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de PVB bovino pertencente ao genogrupo GI em diferentes estados brasileiros, com perfis filogenéticos heterogêneos. / Picobirnavirus (PBV) belong to the Picobirnaviridae family, divides into two species Human Picobirnavirus and Rabbit Picobirnavirus. They are small, non-enveloped, bisegmented double-stranded RNA (dsRNA) virus with an icosahedral capsid, being divided into two genogroups, GI and GII. They have been detected in feces of humans and many animal species, with or without diarrheal signs and are considered emerging and opportunistic agents, and its zoonotic potential has been suggested. However, epidemiological and molecular studies of bovine PBV are rare in the national and international literature. Due to lack of data on PBV in cattle, this study was conducted aiming to detect and molecularly characterize bovine PBV strains of GI and GII genogroups in feces from animals with or without diarrheal signs of different ages and regions of Brazil. Seventy-seven animals were sampled, resulting in 18 (23.3%) positive samples for GI, including animals from the states of São Paulo, Minas Gerais and Goiás. There were no positive samples for GII. The nucleotide identity of the samples obtained showed a mean of 67.4% compared to each other and up to 83.77% compared to PBV reference samples. In phylogenetic reconstruction, three samples were grouped in the human PBV clade and only one sample was clustered in the bovine PVB clade. In summary, the results indicate in an unprecedented way the circulation of the bovine PBV belong to GI genogroup in different Brazilian states, with heterogeneous phylogenetic profiles.