No description available.

No description available.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Pathology. Includes bibliographical references.

No description available.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 150 pages. Includes Vita. Includes bibliographical references.

Evidence is presented to show that variola and monkeypox viruses evolved independently from a common ancestor. An open reading frame (ORF), potentially coding for a protein of 341 amino acid residues, was found to be conserved in two strains of variola virus (Harvey and Somalia), but degenerate in the Denmark strain of monkeypox virus. Monkeypox virus had a deletion of 391 bp, two 24bp deletions and a single base pair deletion within the coding region of this single copy ORF. The ORF corresponds to the E5R ORF in the published sequence of the Copenhagen strain of vaccinia virus, and the DNA sequence was determined for an additional strain of vaccinia virus, Dairen. A number of other Orthopoxviruses were found to contain this ORF, strengthening confidence in its presence in an ancestral Orthopoxvirus. The equivalent DNA sequence was determined for a number of monkeypox virus strains from West and Central Africa. The Denmark strain was identical to one from Liberia, indicating that this virus probably originated from West Africa. A third virus from West Africa, Benin, was found to have >99% base similarity and the same pattern of deletions as the other two monkeypox viruses. The Zaire strains were identical to one another and different from the West African strains. Like the West African strains, they contained the two 24bp deletions and single base pair deletion. In place of the large deletion they had three smaller deletions of 5-, 9- and 127-bp as well as a single base pair insertion. They also had additional deletions of land 2-bp and an insertion of 3bp. The West African strains have the potential to code for a truncated gene product of 107 amino acid residues, whereas the Zaire strains have no significant ORF. This clearly shows that monkeypox virus has diverged into two geographically isolated groups (Zaire and West Africa). There was >99% base similarity between the two groups, suggesting that the divergence occurred recently. Phylogenetic analysis, by the neighbour-joining method, was undertaken on the corresponding DNA sequences from variola (2 strains), monkeypox (6 strains), vaccinia (1 strain + 2 published sequences), cowpox (2 strains), taterapox, camel pox and ectromelia viruses. For every species gerbilpox virus was the nearest neighbour, suggesting that taterapox virus may be the species most closely related to the common ancestral Orthopoxvirus. Within the variola and cowpox virus species there was >99% DNA sequence conservation. Between species, camelpox virus was the most closely related species to gerbilpox virus, with variola virus, and, more distantly, vaccinia virus, falling into the same group. Cowpox virus was the most diverged species examined. Ectromelia virus shared a branch with cowpox virus. A comparison was made of the intergenic DNA sequence between this ORF and the adjacent downstream ORF. Variation was found, both within and between species, in the form of insertions and deletions. The interrelationships between the different Orthopoxvirus species more or less parallels that of the E5R-equivalent comparison. Some of the viruses had clusters of direct repeats. A pentameric repeated unit was found in 2, 10 and 17 copies in camelpox, gerbilpox and ectromelia viruses respectively. Raccoon poxvirus had a 7bp unit in 13 adjacent copies. The two cowpox viruses had a more complex arrangement of repeated sequences. It was thought that the ESR ORF may prove to be nonessential for virus replication. This was tested by interruption of the E5R gene in vaccinia virus; this did not affect the ability of the virus to form plaques in cell culture, but appeared to reduce the pathogenicity of the 'virus for rabbits. The deduced amino acid sequences were analysed for conserved and variable regions within the gene, to which no specific function has yet been assigned.

No Brasil, casos de doença exantemática em bovinos e humanos têm sido relatados em diversas regiões, cujo agente causal é o virus vaccinia pertencente ao gênero Orthopoxirus da família Poxviridae. Classicamente, a varíola bovina é causada pelo Cowpoxvirus, entretanto, outros Poxvirus, como o vírus vaccinia podem desenvolver sintomatologia clínica semelhante. Além de comprometer a cadeia produtiva de leite, uma vez que dificulta a ordenha, predispõe a mastite e descarte do produto, é uma zoonose e atualmente está incluída no diagnóstico de doença vesicular. A origem desses casos bem como a epidemiologia da doença ainda é pouco conhecida. Assim, objetivou-se no presente estudo 1) Avaliar a soroprevalência de Orthopoxirus em rebanhos bovinos do circuito sete do Estado de São Paulo que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes e associar os possíveis fatores de risco envolvidos na transmissão da doença; 2) Detectar e caracterizar a presença de Orthopoxirus em amostras suspeitas de doença vesicular, no período de 2007 a 2009, provenientes de diversas regiões do Brasil utilizando técnicas convencionais e moleculares: microscopia eletrônica, isolamento viral, PCR e RFLP; 3) Sequenciamento e análise filogenética das amostras positivas para o vírus vaccinia. Para o inquérito soroepidemiológico foram analisadas 76 propriedades pertencentes ao circuito 7 que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes, estado de São Paulo, selecionadas aleatoriamente, totalizando 619 animais, estratificados em fêmeas acima de dois anos. A soroprevalência de Orthopoxirus no Vale do Paraíba foi de 32,3% (200/619) pela virusneutralização. Associação positiva foi encontrada para presença de animais silvestres. No diagnóstico virológico foram analisadas 227 amostras de epitélio, negativas para outras doenças vesiculares. Encontrou-se frequencia de 63,4% (144/227) positivas para o Orthopoxirus em praticamente todas as regiões do país. A análise por RFLP revelou perfil de padrão para o vírus vaccinia. O sequenciamento dos isolados confirmou que o vírus vaccinia é a estirpe circulante e está agrupado no grupo I de isolados de vaccinia brasileiros. / In Brazil, cases of rash illness in cattle and humans have been reported in several regions whose causal agent is the vaccinia virus belonging to the genus Orthopoxirus (OPV) family Poxviridae. Classically, cowpox is caused by Cowpoxvirus, however, other poxviruses such as vaccinia virus may develop same clinical signs. In addition to compromising the production chain of milk, as it hampers milking, predisposes to mastitis and discard the product, also it is a zoonosis and is currently included in the differential diagnosis of vesicular disease. The origin of these cases as well as the epidemiology of the disease is still unknown. Thus, the aim of the present study 1) assess the serum prevalence of OPV in cattle herds circuit seven Vale do Paraíba and associate the possible risk factors involved in disease transmission, 2) identify and characterize the presence of OPV in samples suspected vesicular disease in the period 2007-2009, from various regions of Brazil using conventional techniques and molecular electron microscopy, virus isolation, PCR and RFLP; 3) Sequencing and phylogenetic analysis of the samples positive for OPV. For epidemiological survey were analyzed 76 properties in the region of Vale do Paraíba, São Paulo, randomly selected, totaling 619 animals, stratified in females more than two years. The serum prevalence of OPV in the Paraíba Valley was 32.3% (200/619) by virus neutralization. Positive association was found for presence of wild animals. The virological diagnosis were analyzed 227 samples of epithelium, negative for other vesicular diseases. Met frequency of 63.4% (144/227) positive for OPV in practically all regions of the country. RFLP analysis revealed default profile for the vaccinia virus. The sequencing of the isolates confirmed that vaccinia virus strain is circulating and is clustered in group I isolates of Brazilians vaccinia.

No Brasil, casos de doença exantemática em bovinos e humanos têm sido relatados em diversas regiões, cujo agente causal é o virus vaccinia pertencente ao gênero Orthopoxirus da família Poxviridae. Classicamente, a varíola bovina é causada pelo Cowpoxvirus, entretanto, outros Poxvirus, como o vírus vaccinia podem desenvolver sintomatologia clínica semelhante. Além de comprometer a cadeia produtiva de leite, uma vez que dificulta a ordenha, predispõe a mastite e descarte do produto, é uma zoonose e atualmente está incluída no diagnóstico de doença vesicular. A origem desses casos bem como a epidemiologia da doença ainda é pouco conhecida. Assim, objetivou-se no presente estudo 1) Avaliar a soroprevalência de Orthopoxirus em rebanhos bovinos do circuito sete do Estado de São Paulo que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes e associar os possíveis fatores de risco envolvidos na transmissão da doença; 2) Detectar e caracterizar a presença de Orthopoxirus em amostras suspeitas de doença vesicular, no período de 2007 a 2009, provenientes de diversas regiões do Brasil utilizando técnicas convencionais e moleculares: microscopia eletrônica, isolamento viral, PCR e RFLP; 3) Sequenciamento e análise filogenética das amostras positivas para o vírus vaccinia. Para o inquérito soroepidemiológico foram analisadas 76 propriedades pertencentes ao circuito 7 que compreendem as regiões do Vale do Paraíba e Mogi das Cruzes, estado de São Paulo, selecionadas aleatoriamente, totalizando 619 animais, estratificados em fêmeas acima de dois anos. A soroprevalência de Orthopoxirus no Vale do Paraíba foi de 32,3% (200/619) pela virusneutralização. Associação positiva foi encontrada para presença de animais silvestres. No diagnóstico virológico foram analisadas 227 amostras de epitélio, negativas para outras doenças vesiculares. Encontrou-se frequencia de 63,4% (144/227) positivas para o Orthopoxirus em praticamente todas as regiões do país. A análise por RFLP revelou perfil de padrão para o vírus vaccinia. O sequenciamento dos isolados confirmou que o vírus vaccinia é a estirpe circulante e está agrupado no grupo I de isolados de vaccinia brasileiros. / In Brazil, cases of rash illness in cattle and humans have been reported in several regions whose causal agent is the vaccinia virus belonging to the genus Orthopoxirus (OPV) family Poxviridae. Classically, cowpox is caused by Cowpoxvirus, however, other poxviruses such as vaccinia virus may develop same clinical signs. In addition to compromising the production chain of milk, as it hampers milking, predisposes to mastitis and discard the product, also it is a zoonosis and is currently included in the differential diagnosis of vesicular disease. The origin of these cases as well as the epidemiology of the disease is still unknown. Thus, the aim of the present study 1) assess the serum prevalence of OPV in cattle herds circuit seven Vale do Paraíba and associate the possible risk factors involved in disease transmission, 2) identify and characterize the presence of OPV in samples suspected vesicular disease in the period 2007-2009, from various regions of Brazil using conventional techniques and molecular electron microscopy, virus isolation, PCR and RFLP; 3) Sequencing and phylogenetic analysis of the samples positive for OPV. For epidemiological survey were analyzed 76 properties in the region of Vale do Paraíba, São Paulo, randomly selected, totaling 619 animals, stratified in females more than two years. The serum prevalence of OPV in the Paraíba Valley was 32.3% (200/619) by virus neutralization. Positive association was found for presence of wild animals. The virological diagnosis were analyzed 227 samples of epithelium, negative for other vesicular diseases. Met frequency of 63.4% (144/227) positive for OPV in practically all regions of the country. RFLP analysis revealed default profile for the vaccinia virus. The sequencing of the isolates confirmed that vaccinia virus strain is circulating and is clustered in group I isolates of Brazilians vaccinia.

La variole est l'un des pires fléaux qu'ait connu l'humanité jusqu'à son éradication en 1980. Il existe aujourd'hui une menace terroriste de réémergence du virus de la variole comme arme biologique. Le manque de moyens thérapeutiques, la faible immunité de la population mondiale depuis l'arrêt de la vaccination antivariolique et les complications post-vaccinales liées à l'utilisation du vaccin réplicatif historique ont conduit ces dernières années à une intensification des recherches pour lutter contre la variole et les autres virus du genre orthopoxvirus. En complément d'assurer la production d'un nouveau vaccin antivariolique répondant aux normes sanitaires actuelles, il est indispensable de développer des molécules antivirales efficaces aux modes d'actions différents, utilisables immédiatement en cas d'attaque terroriste et pour pallier les complications post-vaccinales. L'objectif du travail de thèse est d'explorer de nouvelles stratégies thérapeutiques en ciblant plus particulièrement la réplication du virus de la vaccine, utilisé comme modèle substitutif au virus de la variole. La première stratégie est l'utilisation d'aptamères peptidiques ciblant A20, une protéine centrale du complexe de réplication formant avec l'uracile ADN glycosylase D4 le facteur de processivité pour l'ADN polymérase virale. Ces peptides sont sélectionnés in vivo en double-hybride en levure pour interagir avec une cible protéique et potentiellement l'inhiber. Ainsi nous avons sélectionné un aptamère interagissant avec une région critique de la protéine cible A20 et capable d'inhiber significativement la réplication du virus de la vaccine en culture cellulaire. La seconde stratégie est l'utilisation d'un criblage haut débit de molécules chimiques pour leur capacité à rompre les interactions entre notre cible de choix A20 et deux de ses interacteurs connus, la protéine D4 et la primase/hélicase D5, par une approche basée sur l'utilisation de deux rapporteurs luciférase en levure. Nous avons démontré que deux molécules issues du criblage permettaient l'inhibition significative et spécifique de la réplication de plusieurs orthopoxvirus, in vitro. Ces travaux viennent compléter le faible arsenal thérapeutique disponible destiné à lutter contre les infections à orthopoxvirus.

Nicht nur die mögliche Verwendung von Variolavirus als bioterroristischer Kampfstoff sondern auch das zoonotische Potential pathogener Pockenviren wird als eine erhebliche Gefahr für das öffentliche Gesundheitswesen diskutiert. Zurzeit liegt in der Bevölkerung kein ausreichender Impfschutz vor. Mit Ausnahme der Expositionsprophylaxe steht die Vakzination als einzige Prävention zur Verfügung, von der bekannt ist, dass sie mit erheblichen Impfkomplikationen assoziiert ist. Therapeutische Ansätze sind deshalb von großem Interesse. In dieser Arbeit wurden HEp-2-Zellen mit den Orthopockenviren (OPV) VACV-LE, VACV-MVA-BN, CMLV-CP-19 und Calpoxvirus sowie die Zelllinien 293, A-549, Huh-7 und HSB-2 mit Calpoxvirus infiziert und der Replikationszyklus analysiert. Die Genexpression von Faktoren der Transkription (D7R), der DNA-Replikation (K4L) oder für Strukturproteine (D8L, A56R, A27L) wurde unabhängig vom untersuchten Virus und der Zelllinie gleich reguliert, was auf eine wichtige Rolle während der Virusreplikation hinweist. Ferner wurde ein siRNA-System zur Hemmung der OPV-Gene D7R, K4L, D8L, A56R und A27L etabliert. Die Transkription von D7R, K4L, D8L und A27L wurde in infizierten Zellen effektiv durch spezifisch eingesetzte shRNAs inhibiert und dadurch die Virusreplikation von Calpoxvirus im Vergleich zu den Kontrollzellen 24 und 48 Stunden nach der Infektion gehemmt. Die Inhibition der A27L-Genexpression wurde zusätzlich im Western Blot für Calpoxvirus- und VACV-LE-infizierte 293-Zellen nachgewiesen. A56R wurde weder für die Replikation von Calpoxvirus noch von VACV-LE in der Zellkultur benötigt. Da die Gene D7R, K4L, D8L und A27L essentiell für die Replikation von OPV in der Zellkultur waren, könnten sie potentielle Zielstrukturen für die Entwicklung von antiviralen Substanzen für die Behandlung einer OPV-Infektion darstellen. / Not only the potential use of smallpox in a bioterrorist attack but also the zoonotic potential of other poxviruses infecting animals are discussed to be a public health threat. At present, there is no sufficient protection in the human population provided by vaccination. Prophylactic vaccination was effective but has been associated with serious adverse effects. No proven drug treatment is available. Therefore, therapies and new treatment strategies are greatly needed. In this study HEp-2 cells were infected with the orthopoxviruses (OPV) VACV-LE, VACV-MVA-BN, CMLV-CP-19 and Calpoxvirus. In addition, the cell lines 293, A-549, Huh-7 and HSB-2 were infected with Calpoxvirus alone to determine the OPV replication cycle. Irrespective of the analyzed virus and cell line gene expression was regulated similarly for factors involved in transcription (D7R), DNA replication (K4L) or for structural proteins (D8R, A56R, A27L), indicating an important role in virus replication. Furthermore, a siRNA-system for the inhibition of the OPV genes D7R, K4L, D8L, A56R and A27L was established. It could be shown, that specific shRNAs effectively inhibited transcription of D7R, K4L, D8L and A27L in infected cells and repressed Calpoxvirus replication 24 and 48 hours post infection in comparison to control cells. In addition, the inhibition of A27L gene expression was shown in Western Blot analyses for Calpoxvirus- and VACV-LE-infected 293 cells. A56R is not required for virus replication of Calpoxvirus and VACV-LE in cell culture. Since D7R, K4L, D8L and A27L seem to be essential for OPV replication in cell culture, these OPV genes might serve as potential targets for the development of antiviral substances for the treatment of OPV infections.