Memoria para optar al Título Profesional de Médico Veterinario / En el presente trabajo se realizó una caracterización molecular a aislados virales de Reovirus aviar. Las muestras se obtuvieron a partir de aves que presentaron signología clínica semejante a la descrita para la artritis viral o tendosinovitis. Se obtuvo líquido sinovial de las articulaciones afectadas, el que fue inoculado en la membrana corioalantoídea de huevos embrionados de pollo SPF de 10 días de incubación y se incubaron por 5 días más. A partir de las membranas que presentaron lesiones significativas se extrajo el RNA de los virus que pudieran haber mediante el kit PureLink® Viral RNA/DNA Mini Kit. Se realizó la prueba de RT-PCR usando las enzimas SuperScript III ® y Platinum Taq Polimerase ®. Se usaron 4 sets de partidores, cada set teniendo como blanco parte de cada uno de los segmentos cortos (S1, S2, S3 y S4) del genoma del virus. Los productos del RT-PCR se sometieron a electroforesis en agar noble al 1% mezclado con Red Gel® y fueron observados en un transiluminador UV. Desde el gel se extrajo el ADN amplificado de las muestras positivas usando el kit Nucleospin® Extract II, sólo para la parte del fragmento S1 del genoma que codifica la proteína σC. Este purificado fue secuenciado usando el método de Sanger y las secuencias fueron analizadas usando el programa computacional Geneious® versión 4.8.5. El análisis consistió en comparar secuencias genéticas codificantes de la proteína sigma C de cepas vacunales conocidas con respecto a las cepas aisladas, asimismo para las secuencias aminoacídicas. Los resultados mostraron que la secuencia genética que codifica a la proteína sigma C de cepas de campo difiere de cepas vacunales hasta en un 43% y que las secuencias aminoacídicas se diferencian hasta en un 52,2% entre estos aislados. Esta proteína tiene especial importancia en el desarrollo de una respuesta inmune protectiva contra la enfermedad. / In the present work, a molecular characterization of avian reovirus isolates is performed. Samples were obtained from birds that showed signology similar to that described for clinical viral arthritis or tenosynovitis. The samples consisted of synovial fluid from affected joints, which was inoculated on the chorioallantoic membrane of SPF embryonated chicken eggs. From the membranes that showed significant injuries, RNA was extracted using the PureLink® kit Viral RNA / DNA Mini Kit. RT-PCR was performed using the SuperScript III® and Platinum® Taq Polymerase enzymes. Four sets of primers were used, each set having as target part of each of the short segments (S1, S2, S3 and S4) of the virus genome. The RT-PCR products were electrophoresed on 1% agar Noble gel mixed with Red Gel® and observed on a UV transilluminator. From the gel, the DNA amplified from positive samples was extracted using the NucleoSpin Extract II kit, only for part of the S1 genome fragment encoding the protein σC. This purified DNA was sequenced using the Sanger method and the sequences were analyzed using the computer program Geneious® version 4.8.5. The analysis involved comparing genetic sequences coding for the protein sigma C from known vaccine strains against the isolates obtained from field samples. Also the amino acid sequence was compared. The results showed that the genetic sequence encoding the protein sigma C of field strains differ by up to 43% and amino acid sequences which differ by up to 52.2% when compared with vaccines strains. This protein is particularly important in developing a protective immune response against disease.

Orientador: Clarice Weis Arns / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T02:57:43Z (GMT). No. of bitstreams: 1 Barros_AlyneVieira_M.pdf: 608733 bytes, checksum: 93e4d814c0f99c65741a343e76d35fc4 (MD5) Previous issue date: 2011 / Resumo: Estudos de plantas medicinais com conhecimento tradicional têm sido uma fonte potencial de substâncias com atividades farmacológicas e biológicas significantes. Guettarda angelica Mart. ex Müll. Arg. (Rubiaceae) é uma planta medicinal no qual suas raízes são popularmente utilizadas para diversos fins terapêuticos, incluindo veterinário. Estudos antimicrobianos com raízes desta planta também relataram uma atividade in vitro contra bactérias. Como as infecções virais ainda continuam sendo um sério problema mundial, a etnofarmacologia fornece uma abordagem alternativa para descoberta de novos agentes antivirais. O objetivo do presente trabalho foi o estudo antiviral de extratos da casca das raízes, folhas e sementes de G. angelica frente aos herpesvírus bovino (BoHV-1), suíno (SuHV-1) e equino (EHV-1), reovírus (ARV) e metapneumovírus aviário (aMPV). A atividade antiviral foi testada in vitro em células Vero e MDBK utilizando os ensaios de redução do título viral e o ensaio quantitativo colorimétrico através do MTT. Inicialmente, a concentração máxima não-citotóxica (MNCC) dos extratos foi determinada nas células através da observação de suas alterações morfológicas. Estudos realizados através da redução dos títulos virais mostrou que apenas o extrato aquoso de sementes (AEs) apresentou uma atividade antiviral contra o BoHV-1, SuHV-1, EHV-1 e ARV. Assim, esse extrato foi posteriormente avaliado pelo método MTT para determinação do CC50 (concentração citotóxica a 50%), IC50 (concentração inibitória a 50%) e o SI (índice de seletividade). Os valores de CC50 do extrato AEs foram 400,60 e 920,50 para células Vero e MDBK, respectivamente. E os valores de IC50 e SI foram 22, 79 e 40,39 para BoHV-1; 91,30 e 10,08 para SuHV-1; 19,95 e 20,08 para EHV-1; e 23,59 e 17,00 para ARV. Esses resultados indicam que a semente de G. angelica contém compostos com atividade antiviral promissora e baixa toxicidade / Abstract: Study of medicinal plants with traditional knowledge has been a potential source of substances with significant pharmacological and biological activities. Guettarda angelica M. (Rubiaceae) is a medicinal plant where its roots are popularly used for various therapeutic purposes including veterinary. In vitro antimicrobial studies also related antibacterial activity of these roots against bacteria. Like viral infections still remain a serious worldwide problem, the ethnopharmacology provides an alternative approach for discovery of new antiviral agents. The aim of the present work was the antiviral study of extracts from roots bark, leaves and seeds of G. angelica against bovine (BoHV-1), swine (SuHV-1) and equine (EHV-1) herpesviruses, avian reovirus (ARV) and metapneumovirus (aMPV) The antiviral activity was tested in vitro on Vero and MDBK cells using the viral titer assay and the quantitative colorimetric assay through MTT. Initially, the maximum non-citotoxic concentration (MNCC) of extracts was determined in cells by observation of their morphological alterations. Studies through the reduction of viral titers showed that only the aqueous extract from seeds (AEs) presented an antiviral activity against BoHV-1, SuHV-1, EHV-1 and ARV. Then, this extract was further evaluated by MTT method to determine the CC50 (50% cytotoxic concentration), IC50 (50% inhibitory concentration) and SI (selectivity index). The values of CC50 of AEs extract were 400.60 and 920.60 to Vero and MDBK cells, respectively. And the values of IC50 and SI were 22.79 and 40.39 for BoHV-1; 91.30 e 10.08 for SuHV-1; 19.95 and 20.08 for EHV-1; 23.59 and 17.00 for ARV. These results indicate that the seeds from G. angelica contain composts with promising antiviral activity and low toxicity / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Made available in DSpace on 2016-12-08T16:24:09Z (GMT). No. of bitstreams: 1 PGCA11MA073.pdf: 3247783 bytes, checksum: 9d9513e50310db5c49782c22e75cc129 (MD5) Previous issue date: 2011-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infectious arthritis in broiler and breeders represents an economic and health problem resulting in severe losses due to retarded growth and down grading at slaughterhouse . The most commom agents associated with cases of infectious arthritis in avian are Mycoplasma synoviae (MS) and Orthoreovirus avian (ARV). The objective of this study was to evaluate the occurence of MS and ARV in infectious arthritis through molecular techniques, such as PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). In addition, we carried out histopathological studies of joints with arthritis and development of a multiplex PCR. For the study 150 samples were collected from lesions of broilers and 180 samples of breeders. MS were detected in 82.6% and ARV 20% of the simple analysis in breeders. The analysis of samples from broilers showed positivity to MS (58.9%) and ARV (33.9%). The histopathology analysis of joints demonstrated the presence of infiltrate of heterophils in the synovial capsule and the digital flexor tendon. The inflammatory process has also been found in samples from joints that were negative by PCR for both agents.The developed multiplex, with satisfactory results, amplifying genes from MS and ARV simultaneamente. In conclusions that molecular techniques, PCR and PCR-M, were effective for detecting MS and ARV in lesions of arthritis from breeders and broiler / A artrite infecciosa em frangos e matrizes de corte representa um problema sanitário e econômico de grande impacto, provocando elevadas perdas de produtividade e nos processos de produção e industrialização. O impacto econômico relacionado às perdas por problemas no aparelho locomotor normalmente são subestimados, pois geralmente são consideradas apenas as perdas no abatedouro, não contabilizando os descartes que ocorrem durante o período de alojamento.Os principais agentes etiológicos associados aos casos de artrites infecciosas em aves são o Mycoplasma synoviae (MS) e o Orthoreovirus aviario (ARV). O presente trabalho teve por objetivo detectar a presença de MS e ARV através de técnicas moleculares, PCR (Polymerase Chain Reaction) e RT-PCR (Reverse Transcription - Polymerase Chain Reaction), respectivamente, em casos de artrites em frangos e matrizes de corte. Além disso, realizou-se estudos histopatológicos das articulações com artrites e desenvolvimento de um multiplex PCR. Para o estudo foram coletadas 150 amostras de lesões artritícas de matrizes de corte e 180 amostras de frango de corte, durante o período de 2009 e 2010. Do total das 150 amostras de matriz de corte submetidas a PCR , sendo que 82,6% (124/150) foram positivas para MS e 20%(30/150) foram positivas para ARV. Em 83,3% (25/30) foi possível detectar a presença de ARV e MS na mesma articulação . A análise das amostras de frango de corte demonstrou positividade para o MS em 58,9% (106/180) das amostras e 33,9% (61/180) foram positivas para o ARV. A análise histopatológica das lesões demonstrou a presença de processo inflamatório crônico na cápsula sinovial e no tendão flexor digital. O processo inflamatório foi encontrado também nas amostras de articulações que foram negativas na PCR para ambos os agentes. A multiplex desenvolvida, apresentou resultados satisfatórios, amplificando genes do MS e ARV simultaneamente. Conclui-se que as técnicas moleculares, PCR, RT-PCR e M-PCR, mostraram-se eficientes para detecção de MS e ARV em lesões de artrite de matrizes e frango de corte, podendo ser incorporadas na rotina de diagnóstico para os respectivos agentes a partir de material articular

Public health professionals strive to understand how viruses are distributed in the environment, the factors that facilitate viral transmission, and the diversity of viral agents capable of infecting humans to characterize disease burdens and design effective disease intervention strategies. The public health discipline of conservation medicine supports this endeavor by encouraging researchers to identify previously unknown etiologic agents in wildlife and analyze the ecologic of basis of disease. Within this framework, this research reports the first examination of the prevalence in Southeast Asia of the orthoreovirus Nelson Bay virus in humans and in the Pteropus bat reservoir of the virus. Contact with Pteropus species bats places humans at risk for Nipah virus transmission, an important emerging infectious disease. This research furthermore explores the environmental determinants of Nelson Bay and Nipah viral prevalence in Pteropus bats and reports the characterization of two novel orthoreoviruses isolated from bat tissues collected in Bangladesh.

This thesis describes the molecular characterisation of Broome virus (BroV), a new fusogenic orthoreovirus species that was isolated from a little red flying-fox (Pteropus scapulatus) in Broome, Western Australia in 2002. The BroV genome consists of ten segments of dsRNA, each containing a plus-strand with a 3’ terminal pentanucleotide sequence that is conserved amongst all viruses in the genus Orthoreovirus, family Reoviridae, and a 5’ terminal pentanucleotide sequence that is unique to BroV. With the exception of S4, all genome segments are predicted to encode a single translation product producing a total of seven structural and four nonstructural proteins. All BroV proteins were identified as homologues of known orthoreovirus proteins and shown to have similar secondary structure and possess key conserved amino acid sequence motifs and structural features implicated in biological function. Notably, no cell-attachment protein gene homologue was identified in the BroV genome suggesting the use of an alternate cell entry mechanism to that employed by most orthoreoviruses. The amino acid sequence identity between cognate BroV proteins and those of other orthoreoviruses ranges from 13-50%, which is too low for BroV to be considered a new isolate of any established orthoreovirus species group. Phylogenetic analyses based on both structural and nonstructural proteins provide additional evidence to support this claim. It is proposed that BroV is the prototype member of a new sixth species group Broome virus, in the genus Orthoreovirus. The complete genome characterisation of BroV provided an opportunity to produce recombinant proteins in Escherichia coli and to generate polyclonal antibodies in rabbits for use in research and surveillance. Such reagents proved valuable in the experimental identification of the fusion-associated small transmembrane (FAST) protein p13 that is responsible for the syncytia observed in BroV-infected cells. Despite the low amino acid sequence identity between the FAST proteins of different orthoreovirus species they possess conserved structural features that have been implicated in biological function. Of these conserved features, the BroV p13 protein is predicted to possess one transmembrane domain, a C-terminal polybasic region, a C-terminal hydrophobic patch and an N-terminal myristoylation consensus sequence. The unique repertoire and arrangement of sequence-predicted structural features identified in p13 indicate that it is a novel fifth member of the FAST protein family. The BroV-specific immunological reagents were also used to develop an enzyme-linked immunosorbent assay (ELISA) suitable for serological screening. A survey of flying-foxes from Papua New Guinea (PNG) revealed that BroV or BroV-like viruses are currently circulating in these animals. This demonstrates that BroV is not limited to the Australian continent.

Les maladies émergentes représentent actuellement 65% de toutes les pathologies infectieuses récentes. Récemment, un nombre croissant de nouveaux virus a été associé à de petits mammifères terrestres, plus particulièrement à des rongeurs, désignant ce groupe comme étant l'une des possibles sources de dangereuses pathologies émergentes et ré-émergentes. Actuellement, la réaction en chaîne par polymérase (PCR) est l'outil principal utilisé pour la détection d'agents pathogènes dans la diagnostique de routine et dans la recherche. Or, plusieurs recherches ont montré que certaines substances inhibent la PCR, causant de faux résultats. Aussi, nous avons lancé un programme de capture de rongeurs pour le dépistage de virus connus et non identifiés. Au total 1441 rongeurs ont été capturés pendant des campagnes organisées en Europe et Afrique entre 2002 et 2011. Tout d'abord, nous avons examiné l'inhibition de la PCR et étudié les différentes techniques de traitement d'échantillons qui favorisent la réduction de la quantité d'inhibiteurs dans les échantillons de rongeurs. Parmi les techniques d'extraction évaluées, l'EZ1 virus mini kit et le réactif d'extraction RNAnow se sont avéré plus efficaces que le NucleoSpin virus kit ou le réactif d'extraction TRIzol. De même, l'utilisation des poumons et de reins était préférable à l'utilisation du foie et de la rate. Aucune différence significative n'a été observée entre le stockage à -80°C et le stockage dans le réactif RNAlater. Nous avons conduit le dépistage des virus, en utilisant les tests moléculaires et la culture cellulaire. Deux nouvelles souches de virus ont été isolées, séquencées et caractérisées. / Emerging diseases currently represent 65% of recent major disease outbreaks. Of them, 75% are associated with wildlife. Recently, an increasing number of newly discovered viruses have been associated with small terrestrial mammals, particularly with rodents, pointing at this group as one of the most dangerous potential sources of emerging or re-emerging diseases. To meet these challenges for public health, a proper surveillance becomes necessary, which passes by detection of pathogens in human and risky groups of animals, including field investigations. Yet this can be achieved only by using proper techniques of samples treatment and pathogen detection. Currently, polymerase chain reaction (PCR) is the main tool used for the detection of pathogens in routine diagnostic and research. Yet, several researches showed that some substances can inhibit PCR, causing false-negative results. Therefore, we initiated a screening program targeting rodents for the presence of known and unidentified viruses. A total of 1441 rodents were trapped during field campaigns organized in Europe and Africa, between 2002 and 2011. At first we investigated on PCR inhibitors and discussed techniques of treatment of samples allowing reducing the influence of inhibitors in rodent samples. Among the extraction techniques tested, EZ1 virus mini kit and RNAnow extraction reagent were more effective than NucleoSpin virus kit or TRIzol extraction reagent. Also, the use of lungs and kidneys was preferable to the use of liver and spleen, the quantity of inhibitors being higher in the last two organs. No significant difference was observed between storage at -80°C, or in RNAlater RNA stabilization reagent.