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Made available in DSpace on 2016-12-08T16:24:07Z (GMT). No. of bitstreams: 1 PGCA09MA052.pdf: 726948 bytes, checksum: 2a141d4d92b1c5ac552655f7c9ad3c1a (MD5) Previous issue date: 2009-11-06 / Many infectious agents have been associated with reproductive failure in swine, representing significantly economic losses for production. Recently, Porcine Circovirus Type 2 (PCV2), etiologic agent of PCVAD or PCV2 associated diseases, was associated with reproductive failure in swine around the world. To confirm the pathogenic potential of PCV2 inducing reproductive failure in sows, it s necessary the viral isolation and antigen and nucleic acid demonstration in fetuses. Other viral agent, Torque Teno Vírus (TTV), also have been recently associated with infections caused by PCV2. TTV alone has not showed pathogenic signals in swine, but, its role in co-infections with other pathogens has been investigated. The present study aimed the diagnostic of PCV2 in natural infections where there was reproductive failure, as well as to establish and apply the Polimerase Chain Reaction (PCR) technique for TTV from organs. Samples from field cases, as aborted fetuses, mummified, stillborn, fragile piglets and material from abattoir sows were collected and processed to diagnostic infection in order to detect PCV2 by PCR and immunohistochemistry (IHC). Samples were collected from 21 farms; and a total of 169 fetuses were necropsied. Moreover, reproductive samples from 83 abattoir sows were collected in 4 slaughterhouses of Santa Catarina State. In the present study was possible detect viral DNA by PCR in 29 (17,1%) of 169 analyzed fetuses, where heart and lymphoid tissues showed virus DNA more frequently, 41,4% and 37,8%, respectively. Viral presence was confirmed by IHC in tissues, which detected viral antigens in 17 PCV2 positives fetuses by PCR. Samples of reproductive tissues from sows also were tested by PCR and PCV2 was identified in 4 sows (4,8%). PCR technique aimed to detect TTV was established for viral DNA from organs. Samples of reproductive tissues from sows were tested, and were found both genogroups of TTV (TTV1 and TTV2), in 25 (30,1%) and 41 (49,3%) sows, respectively. Fetuses samples that resulted positive to PCV2 by PCR were also tested to TTV, and it was observed the occurrence of co-infection between these agents. The results obtained here suggest the involvement of PCV2 in reproductive failure in sows, besides show that TTV was present in analyzed samples, corroboring the association with PCV2 / Muitos agentes infecciosos têm sido associados às falhas reprodutivas na produção de suínos, representando significativas perdas econômicas para os suinocultores. Recentemente o Circovirus Suíno tipo 2 (PCV2), agente etiológico da circovirose suína, foi associado a falhas reprodutivas em suínos em diversas partes do mundo. Para confirmar o potencial patogênico do PCV2 causando falhas reprodutivas em porcas, é necessário o isolamento do vírus e demonstração de antígeno e ácido nucléico viral em fetos. Outro agente viral, o Torque Teno Vírus (TTV), também foi recentemente associado às infecções causadas pelo PCV2. O TTV sozinho ainda não tem se mostrado patogênico em suínos, porém, seu papel em co-infecções com outros patógenos vem sendo investigado. O presente trabalho teve por objetivos diagnosticar o PCV2 em infecções naturais onde existiam falhas reprodutivas, assim como padronizar e aplicar a técnica de Reação em Cadeia da Polimerase (PCR) para TTV a partir de órgãos. Amostras provenientes de casos clínicos de campo, como fetos abortados, mumificados, natimortos, leitões inviáveis e material de fêmeas descartadas foram coletadas e processadas para diagnóstico da infecção pelo PCV2 através de PCR e imunoistoquímica (IHQ). Foram colhidas amostras de 21 granjas produtoras de suínos, totalizando 169 fetos, que foram necropsiados para coleta de órgãos. Além disso, amostras de órgãos reprodutivos de 83 fêmeas descartadas foram colhidas em 4 abatedouros da região oeste catarinense. No presente estudo foi possível detectar DNA viral por PCR em 29 (17,1%) dos 169 fetos analisados, sendo coração e tecidos linfóides os órgãos onde o vírus foi identificado com maior freqüência, 41,4% e 37,8%, respectivamente. A presença do vírus foi confirmada por teste de IHQ dos tecidos, sendo encontrado antígeno viral em 17 fetos positivos para PCV2 por PCR. As amostras de tecido reprodutivo das fêmeas também foram testadas por PCR e o PCV2 foi identificado em 4 porcas (4,8%). Visando a detecção de TTV foram testadas por PCR amostras de órgãos reprodutivos de fêmeas suínas, sendo diagnosticados os dois genogrupos de TTV, TTV1 e TTV2 em 25 (30,1%) e 41 (49,3%) fêmeas, respectivamente. As amostras de fetos que resultaram positivas para PCV2 pela técnica de PCR também foram testadas para TTV, observando-se a ocorrência de coinfecção entre estes agentes. Os resultados obtidos evidenciam o provável envolvimento do PCV2 em falhas reprodutivas em fêmeas suínas, bem como mostram que o TTV está presente nas amostras analisadas, confirmando a associação com o PCV2

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease (PCVAD). Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV). Both PCV2 and PRRSV have caused devastating diseases in the swine industry worldwide, resulting in immense economic losses. One of the most common co-infections in the swine industry is PCV2 and PRRSV. The aim of this dissertation research is to explore different experimental approaches to develop novel vaccines against the two major pathogens affecting swine production and study the basic mechanisms that may be involved in viral pathogenesis. Two types of porcine circovirus (PCV), PCV1 and PCV2, have been identified thus far. PCV1, first identified as a contaminant of the PK-15 cell line, is non-pathogenic and has a low prevalence in swine herds. PCV2 is highly prevalent in most swine-producing countries and is associated with clinical PCVAD. The non-pathogenic PCV1 shares similar genomic organization with PCV2. Previously, it has been demonstrated that a genetically modified infectious chimeric PCV1-2a virus can tolerate up to a 27 aa insertion in the C-terminus of the ORF2 without affecting infectivity and produce a dual immune response against PCV2cap and the inserted epitope tag. Therefore, we evaluated the use of the non-pathogenic PCV1 wild-type (wt) virus and chimeric PCV1-2a vaccine virus (vs) to express four known B-cell epitopes of PRRSV. Peptide epitopes of PRRSV-VR2385, including GP2II (aa 40–51, ASPSHVGWWSFA), GP3I (aa 61–72, QAAAEAYEPGRS), GP5I (aa 35–46, SSSNLQLIYNLT), and GP5IV (aa 187–200, TPVTRVSAEQWGRP) were inserted in frame into the C-terminus of the ORF2 of PCV1wt as well as the PCV1-2avs. Four PCV1-PRRSVEPI chimeric viruses and four PCV1-2a-PRRSVEPI chimeric viruses were successfully rescued and shown to be infectious in vitro and co-expressed PCV1cap or PCV2cap with each specific PRRSV epitope. Two independent animal studies were conducted to evaluate whether the non-pathogenic PCV1 can serve as a vaccine delivery vector and whether the PCV1-2a vaccine virus can be used to develop a bivalent vaccine against both PCV2 and PRRSV. We demonstrated that three PCV1-PRRSVEPI chimeric viruses and two PCV1-2a-PRRSVEPI chimeric viruses were infectious in pigs. Importantly, we demonstrated that the PCV1-PRRSVEPI and PCV1-2a-PRRSVEPI chimeric viruses not only induced specific PCV1 or PCV2 IgG antibody but also specific anti-PRRSV epitope antibody responses as well. Regardless of the PCV backbone used, we showed that the PCV-PRRSV chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. These results provided a proof of concept for the potential use of the non-pathogenic PCV1 as a vaccine delivery system for PRRSV or other swine pathogens and the use of PCV1-2a vaccine virus to generate a bivalent vaccine against both PCV2 and PRRSV. PRRSV causes a persistent infection and immunosuppression. Immunomodulation of the host immune system is caused by modulation of numerous interleukins, such as type I interferons, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-12 (IL-12) in infected pigs. Antigen-presenting cells (APCs) are the first line of defense, and their infection plays an important role in innate-mediated immune regulation during early immune responses. Among the APCs, pulmonary alveolar macrophages (PAMs), pulmonary interstitial macrophages (PIMs), and dendritic cells (DCs) are the main targets for PRRSV replication. The role of PRRSV-DCs interaction is not fully understood, and current research focuses on the production and regulation of interferons through DC-SIGN receptors. In this study, we evaluated the immunomodulation of MoDCs by PRRSV through interactions with the pDC-SIGN receptor, by blocking pDC-SIGN with recombinant hICAM-3-Fc or anti-pDC-SIGN mAb. Our results indicate that recombinant hICAM-3-Fc enhances mRNA expression of proinflammatory cytokines and that anti-pDC-SIGN mAb inhibits mRNA expression of TNF-α and IL-1α and enhances the expression of IL-12 induced by PRRSV in MoDCs. The results will help understand the molecular mechanisms of PRRSV pathogenesis. / Ph. D.