The diagnosis of porcine circovirus-associated disease (PCVAD) in pigs requires the detection of characteristic clinical signs and pathological changes, and the detection of virus in tissues of affected pigs. To increase Australia’s capacity to independently diagnose PCVAD in Australia, techniques for the detection of Porcine circovirus type 2 (PCV2) infection in pigs were developed and are reported in this thesis. These techniques were applied to samples obtained from normal pigs and pigs with disease and confirmed the presence of PCV2 and PCVAD in the Australian pig herd. Viral DNA was detected in tissues of infected pigs by both standard PCR and real-time PCR techniques. The real-time PCR was more sensitive. While the conventional PCR was able to detect approximately 100 copies of the viral genome, the real-time PCR was able to detect 20 copies of the genome. An immunohistochemical (IHC) technique which was also developed enabled the visualisation of PCV2 antigen in fixed tissues of pigs with PCVAD. The techniques that were developed were applied to an examination of tissues from pigs affected by illthrift and increased weaner mortality in herds in South Australia, New South Wales and Western Australia. Lesions suggestive of the PCVAD postweaning multisystemic wasting syndrome (PMWS) were detected and virus antigen was detected in association with lesions. The nature of the clinical signs and histopathological lesions detected, coupled with the presence of PCV2 antigen, suggested that PCVAD was present in some Australian pig herds. Phylogenetic analysis of the strains of PCV2-associated with these disease outbreaks demonstrated they were of a type not previously detected in Australia and similar to strains associated with PMWS in North America. To further assist in investigation of PCV2 infections in the Australian pig herd, an enzyme-linked immunosorbent assay (ELISA) was developed that specifically detected antibody to PCV2 and not the related and non-pathogenic Porcine circovirus type 1. The development of this assay required the production of a virus capsid protein antigen using a prokaryotic protein production system. The ELISA was used to test serum samples form the Australian national pig serum bank. A high prevalence of PCV2 infection was detected in most pig herds examined in all Australian states. International trade in pig meat has resulted in many countries placing restrictions on the importation of pig meat, requiring imported pig meats to be cooked to destroy viral agents. This study investigated the in vitro resistance of an Australian strain of PCV2 to heat treatment at temperatures between 56°C and 85°C. The viability of the virus was determined by a combination of reverse transcriptase polymerase chain reaction (RT-PCR), and IHC to visualise viral capsid antigen within infected cells. This study indicated that PCV2 retained its infectivity following heating up to and including 75°C for 15 mins, but was inactivated following heating to 80°C and above. The investigations reported make a significant contribution to PCV2 research in Australia and ensure Australia’s capacity to independently investigate PCVAD in the Australian pig herd.

This thesis reports for the first time the detection of porcine circovirus virus (PCV) in the Australian pig herd. PCV DNA was detected in the tissues of pigs from several Australian states using a multiplex polymerase chain reaction (PCR) assay, the primers for which were based on the sequence of PCV1 and PCV2 strains detected in North America and Europe. PCV type 1 or 2 was detected in 80 of 367 (21.7%) pigs tested. In the 80 positives, both PCV1 and PCV2 were detected in 14 samples. Virus was detected in pigs from all states from which samples were obtained: Western Australia, South Australia, New South Wales and Queensland. The complete genomes of 13 strains of Australian PCV were sequenced. Analysis of the data indicated there was extremely high homology between the Australian strains of PCV1 and PCV2 and previously published sequences of PCV1 and PCV2 strains from North America and Europe.There were no consistent differences between the genome of the Australian strains and strains in North America and Europe. The widespread occurrence of PCV in the tissues of pigs was confirmed by a small scale serological study of the Western Australian pig herd using an immunofluorescence assay, which did not discriminate antibody to PCV1 and PCV2. This assay detected PCV antibody in 11 of 14 pig herds in Western Australia, with a prevalence rate in positive herds varying from 25 to 47%, but it was unable to differentiate antibody to PCV1 and PCV2. A PCV2-specific recombinant viral capsid protein was produced in insect cells with a baculovirus expression system and this was used to develop a PCV2-specific ELISA and a Western immunoblotting assay. These assays were applied to samples from a national pig serum bank and detected PCV2 antibody in 33% of 3933 serum samples. The highest seroprevalence to the recombinant PCV2 capsid antigen was detected in the samples from Victoria where there was a 51.3% seroprevalence rate, and the lowest in Western Australia where there was an 11.4% seroprevalence rate. An in situ hybridisation (ISH) technique was developed for the detection of PCV in tissues of infected pigs and infected cell cultures. A monoclonal antibody specific for the capsid protein of PCV2 was also produced and has application for the development of immunocytochemical procedures for the detection of PCV2 in tissues and cell cultures. The high prevalence of PCV in the Australian pig herd and the absence of reports of PMWS suggested that the Australian strains of PMWS detected may have been of low virulence. To examine the pathogenicity of Australian strains, two animal experiments were conducted where the type species of PCV1 present in persistently-infected PK15 pig kidney cells and an Australian PCV2 strain were cultured in vitro in cell cultures and inoculated into weaner pigs. As expected, the PCV1 replicated well in pigs but did not result in the induction of clinical signs or lesions in the inoculated pigs. The inoculation into weaner pigs of cell culture replicated PCV2 with an apparent virus titre of 103 virus particles/mL resulted in infection of only some of the inoculated pigs and it was concluded that the PCV2 inoculum contained insufficient virus to infect all pigs into which it was inoculated. The PCV2 did not induce any disease syndrome and could not be visualised in tissue sections of infected pigs using immunohistochemical techniques. In conclusion, techniques were developed for the detection of PCV in the Australian pig herd. PCV of both genetic types were detected at prevalence rates similar to those reported in other countries where PMWS has occurred, and the widespread occurrence of PCV was confirmed by serological assays. The PCV strains present were genetically indistinguishable from those present in North America and Europe. The reason for the absence of PMWS in Australia is most likely not due to differences in the characteristics of the PCV strains present.

No description available.

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-10-05T14:03:09Z No. of bitstreams: 1 texto completo.pdf: 298725 bytes, checksum: 7bee1975846b19c9ac200de64bd75213 (MD5) / Made available in DSpace on 2016-10-05T14:03:09Z (GMT). No. of bitstreams: 1 texto completo.pdf: 298725 bytes, checksum: 7bee1975846b19c9ac200de64bd75213 (MD5) Previous issue date: 2003-04-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O circovírus suíno (PCV) é um agente amplamente distribuído na Europa, América do Norte e sul da Ásia. O PCV é um pequeno vírus de cadeia simples de DNA (17 nm) que foi reconhecido, a partir da década de 90, como um patógeno de suíno. Dois tipos de PCV tem sido caracterizados e designados PCV tipo 1 (PCV1) e PCV tipo 2 (PCV2). O PCV1 foi primeiramente isolado em 1974 como um contaminante persistente da linhagem de células PK-15 de rim de suíno (ATCC CCL 31) e a cepa de PCV isolada de células PK-15 tem sido bem caracterizada. O PCV1 é considerado como um vírus não patogênico, enquanto que a infecção de um suíno pelo PCV2 é normalmente associada ao desenvolvimento de Síndrome Multissistêmica Pós-Desmame (PMWS), em animais de 5 a 12 semanas de idade, e ao tremor congênito (CT), que acomete animais no período neonatal. A PMWS é uma nova doença emergente de suínos, caracterizada clinicamente por dispnéia progressiva, aumento dos nódulos linfáticos e patologicamente caracterizada por uma ampla extensão de lesões inflamatórias. Recentemente, pesquisadores da EMBRAPA iniciaram um estudo da PMWS em leitões, mas no Brasil a presença do PCV ainda não é reconhecida oficialmente. O objetivo desse trabalho foi (1) padronizar técnicas de diagnóstico para o genoma e antígeno do PCV, assim como anticorpos contra o agente; (2) avaliar a susceptibilidade de diferentes linhagens celulares ao PCV; (3) diagnosticar a infecção do PCV em suínos da Zona da Mata de Minas Gerais; (4) isolar o PCV de amostras positivas. O PCV, proveniente de tecidos de animais normais e com diagnóstico de CT, foi isolado em células SK6 e analisadas por PCR. O padrão de bandas foi o mesmo encontrado em células PK15 contaminadas com PCV2, gentilmente cedidas pela EMBRAPA. Os oligos usados diferenciavam o PCV1 do PCV2. Todos os leitões de maternidade testados por PCR foram positivos para o PCV2. Porém, em 59 animais de abate testados por PCR não foi observada a presença do PCV. No teste de susceptibilidade as células PK15, SK6, VERO e MDCK foram susceptíveis ao PCV, mas somente as PK-15 estavam persistentemente infectadas. No ensaio de imunofluorescência indireta, foi utilizado um conjugado anti-IgG suína previamente padronizado e anticorpos contra PCV foram identificados em soros de 24 em 44 animais de abate testados e nenhum anticorpo foi encontrado nos animais com diagnóstico de CT positivos para PCV2 por PCR. Com esses resultados podemos concluir que os 24 suínos de abate soropositivos entraram em contato com o agente e desenvolveram a infecção em alguma fase durante o estagio de produção. A ausência de soropositivos entre os leitões recém nascidos, aliada a presença de infecção, pode ser explicada pela incapacidade de produção de anticorpos por esses animais neste estágio de desenvolvimento. Estudos adicionais da epidemiologia e da imunologia de infecções pelo PCV são necessários para o melhor entendimento e efetivo controle das doenças associadas a esse vírus. / Porcine circovirus (PCV) is thoroughly an agent distributed in Europe, North America and south of Asia. PCV is a small virus of simple chain of DNA (17 nm) that was recognized, starting from the decade of 90, as a swine pathogen. Two types of PCV have been characterized and designated PCV type 1 (PCV1) and PCV type 2 (PCV2). PCV1 was isolated firstly in 1974 as a persistent contaminant of the lineage of cells PK-15 of swine kidney (ATCC CCL 31) and the stump of isolated PCV of cells PK-15 has been well characterized. PCV1 is considered as a non- pathogenic virus, while the infection of a swine for PCV2 is usually associated to Post weaning Multisistemic Wasting Syndrome (PMWS), in animals from 5 to 12 weeks of age, and to the congenital tremor (CT), that attack animals in the neonatal period. PMWS is a new emergent disease of swine, clinically characterized by progressive dispnea, increase of the lymphatic nodules and pathologically characterized by a wide extension of inflammatory lesions. Recently, researchers of EMBRAPA began a study of PMWS in pigs, but in Brazil the presence of PCV is not still recognized officially. The objective of that work was (1) to standardize diagnosis techniques for the genome and antigen of PCV, as well as antibodies against the agent; (2) to evaluate the susceptibility of different cellular lineages to PCV; (3) to diagnose the infection of PCV in swine of the Zona da Mata of Minas Gerais; (4) to isolate PCV of positive samples. PCV, originating from tissues of normal animals and with diagnosis of CT, it was isolated in SK6 cells and analyzed by PCR. The pattern of bands was the same found in contaminated cells PK15 with PCV2, kindly by EMBRAPA. The used oligos differentiated PCV1 of PCV2. All the pigs of maternity tested by PCR were positive for PCV2. However, in 59 slaughtering animals tested by PCR, PCV was not found. In susceptibility test, PK15, SK6, VERO and MDCK cells were susceptible for both PCV but only PK15 cells were persistently infected. Anti-PCV antibodies were found to be positive in 54,5% of slaughtering animals serum and any anti-PCV antibody was found in animals with clinical CT. Rapid and accurate diagnosis and removal of disease animals from farms, combined with good husbandry practices, would appear to be the only current method of controlling losses attributable to PCV2 infections. However, additional studies into the epidemiology and immunology of PCV infections are now required if better understanding and eventual control of the disease syndromes associated with these viruses are to be achieved. / Dissertação importada do Alexandria.

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

O Circovirus suíno tipo 2 (PCV2) é um vírus ubíquo que tem sido associado a um número de síndromes em suínos. Entre elas, a Síndrome Multissistêmica do Definhamento dos Suínos (SMDS) tornou-se uma das principais causas de perdas econômicas na suinocultura nacional. No entanto, existe incerteza se o PCV2 é, de fato, o único agente responsável por esse quadro, essencialmente porque a administração isolada do vírus a animais suscetíveis não tem sido capaz de reproduzir experimentalmente a síndrome. Em vista disso, um número de outros agentes infecciosos (e não infecciosos) tem sido examinados e sua potencial participação no desenvolvimento da SMDS tem sido pesquisada. No presente estudo foram realizados experimentos visando determinar se outro(s) agente(s) com genoma de DNA circular poderia(m) desempenhar algum papel no desenvolvimento da SMDS. Para tanto, a técnica denominada “amplificação por círculo rolante com múltiplos primers” (ACRMP) foi empregada. A ACRMP é baseada na atividade da DNA polimerase do fago phi29, uma enzima capaz de sintetizar novas moléculas de DNA a partir de um molde de DNA circular. Numa segunda etapa, o DNA amplificado é clivado com enzimas de restrição, ocasionando a linearização de grande quantidade de cópias do DNA alvo original. Como a ACRMP é realizada com primers aleatórios, nenhum conhecimento prévio da seqüência de nucleotídeos alvo é necessário. Portanto, pode-se teoricamente amplificar DNA circular de qualquer microorganismo, o que a torna ideal para o propósito do presente estudo. O DNA extraído de soros de 67 suínos com sinais clínicos de SMDS, assim como de 63 suínos saudáveis, foram submetidos à ACRMP. O principal achado deste estudo foi que o genoma de um (ou mais) anelovírus foi(ram) detectado(s) em 88,9% (56/63) dos suínos saudáveis, ao passo que o(s) mesmo(s) agente(s) somente foi(ram) detectado(s) em 16,4% (11/67) dos soros de suínos com sinais clínicos da SMDS. Alguns fragmentos de DNA potencialmente correspondentes a fragmentos de genomas virais foram seqüenciados, revelando que pelo menos um deles corresponde a uma seqüência de anelovírus suíno ainda não descrita. No entanto, outro genoma correspondente a um anelovírus foi encontrado na mesma amostra, sugerindo que mais de um vírus pode estar presente em amostras de soro. Estes resultados demonstraram que os anelovírus, de grande variabilidade genética, são significativamente mais prevalentes em suínos clinicamente saudáveis do que em suínos com SMDS. / Porcine circovirus type 2 (PCV2) is an ubiquitous virus that has been associated to a number of syndromes in swine. Among these, Postweaning Multisystemic Wasting Syndrome (PMWS) has become a major cause of economic losses in swine worldwide. However, there is uncertainty as to whether PCV2 is in fact the sole agent responsible for the disease, essentially because the disease has not been experimentally reproduced when PCV2 is inoculated onto susceptible animals. In view of that, a number of other infectious (and non infectious) agents have been examined and their potential role in PMWS searched for. This study was carried out to determine whether any other agent(s) with circular DNA genome might be playing some role in PMWS. In order to achieve that, a technique called “randomly primed rolling circle amplification” (RPRCA) was employed. RPRCA is based on the activity of bacteriophage phi29 DNA polymerase, an enzyme that synthesizes new DNA molecules starting from a circularized DNA template. In a second phase, the amplified DNA is cleaved with restriction enzymes, so giving rise to large amounts of linearized copies of the original target DNA. As RPRCA is performed with random priming, no previous knowledge of the target nucleotide sequence is necessary. Therefore, it is theoretically possible to amplify circular DNA of any microorganism, thus making it ideal for the purpose of the present study. DNA extracted from sera of 67 pigs with clinical signs of PMWS as well as from 63 healthy pigs was submitted to RPRCA. The major finding of this study was that the genome of one (or more) anelloviruses was detected in 88,9% (56/63) of the healthy pigs, whereas the same agent was only detected in 16,4% (11/67) of pigs with clinical signs of PMWS. Some of the DNA fragments corresponding to the putative virus genomes were sequenced and revealed at least one non-previously described anellovirus sequence. However, other anellovirus could be found on the same sample, suggesting that more than one genome are present in samples of serum. These results demonstrate that anelovírus, of great genetic variability, were significantly more prevalent in healthy pigs than in pigs with PMWS.

Le circovirus porcin de type 2 est responsable de la maladie d’amaigrissement du porcelet. Il est différent du circovirus porcin de type 1 qui est non pathogène. Nous disposons de peu de données pouvant expliquer pourquoi le virus PCV2 est pathogène et le virus PCV1 ne l’est pas. De plus les bases moléculaires soutenant la pathogénicité du PCV2 et l’immunodépression induite par le PCV2 sont mal comprises. Dans cette étude nous avons montré que la protéine gC1qR était capable d’interagir de façon différentielle avec les protéines de capside (Cap) de circovirus pathogène PCV2 et non pathogène PCV1. La protéine de capside de PCV1 isolée d’un porcelet issu d’un élevage était incapable d’interagir avec la protéine gC1qR. Il a été également montré que la région de la protéine Cap PCV2 impliquée dans l’interaction avec gC1qR était comprise dans les 59 acides aminés N-terminaux, région riche en arginine. Il a été également mis en évidence que les transcrits de gC1qR étaient sous-régulés in vitro et in vivo après une infection par le virus PCV2 au temps court de l’infection. Une sous-régulation de gC1qR induite par ARN interférence en cellules permissives rénales de porc PK15 n’induisait cependant ni une diminution de la réplication du virus PCV2, ni une diminution de formation de ses particules infectieuses. Ce travail apporte de nouveaux éléments pour comprendre l’adaptation des souches de circovirus porcins à leur hôte ainsi que son interaction avec les protéines de son hôte. / The porcine circovirus type 2 is the causal agent of the post-weaning multisystemic wasting syndrome. It is different from porcine circovirus type 1 which is non-pathogenic. We have little data that could explain why the PCV2 is pathogenic and PCV1 is not. The molecular basis supporting the pathogenicity of PCV2 and the induced immune-depression is misunderstood. It has been shown that the capsid protein (Cap) of the porcine circovirus was able to interact differentially with the capsid protein of the pathogenic circovirus PCV2 and nonpathogenic PCV1. Cap proteins from PCV1 virus isolated from a piglet was unable to interact with gC1qR. It has also been shown that the Cap PCV2 region involved in the interaction with gC1qR was included among the 59 N-terminal amino acids, an arginine-rich region. It was also shown that gC1qR transcripts were down-regulated in vitro and in vivo after infection with PCV2 virus at the beginning of infection. A siRNA-mediated downregulation of gC1qR in the PK15 permissive cells did not induce a modification of the replication of PCV2 virus and neither the production of infectious viral particles. This work provides new evidence for understanding the adaptation of porcine circovirus strains to their host as well as its interaction with its host proteins.

O circovírus suíno tipo 2 (PCV2) é o principal agente da síndrome multissistêmica do definhamento do suíno (SMDS), uma doença mundialmente disseminada e que provoca perdas econômicas significativas para a suinocultura. Visando contribuir no diagnóstico da síndrome, o presente trabalho padronizou e comparou testes para a detecção do PCV2. Para isso, foram utilizadas as técnicas de amplificação por círculo rolante (ACR) e variações da PCR (convencional, tempo-real e competitiva). Utilizando a ACR foi possível obter a amplificação total de genomas do PCV2, os quais foram clonados, sequenciados e agrupados no genótipo PCV2b. Os genomas clonados foram isolados, recircularizados e transfectados em células PK-15. Este procedimento possibilitou a recuperação do vírus infeccioso em títulos de até 105,55 DICC50/mL. Portanto, a ACR foi uma ferramenta útil em estratégias de isolamento e sequenciamento do vírus. No entanto, a ACR foi menos sensível que a PCR para fins de detecção do PCV2. No segundo estudo, buscando métodos auxiliares no diagnóstico da SMDS, dois ensaios para a quantificação do PCV2 foram desenvolvidos. Estes ensaios foram baseados nas técnicas de PCR competitivo (cPCR) e de PCR em tempo real. Visando determinar qual seria o mais adequado para estimar a carga viral do PCV2, os dois métodos foram comparados. Ambos os ensaios foram capazes de detectar diferenças significativas entre o número de cópias de DNA de PCV2 encontradas em tecidos de animais saudáveis e acometidos pela SMDS (≥ 2,5 log10). No entanto, uma diferença média de 1,8 log10 na carga viral foi encontrada entre ensaios, onde as maiores cargas virais foram detectadas pela PCR em tempo real. Outro objetivo deste trabalho foi gerar vacinas baseadas na proteína do capsídeo (Cap) do PCV2. Assim, no terceiro estudo, três baculovírus recombinantes foram construídos de modo a expressar a proteína Cap. Em dois recombinantes, a seqüência de nucleotídeos do peptídeo sinal (PS) da glicoproteína I do herpesvírus bovino (BoHV-gI) foi inserida na extremidade 5’ do gene cap (ORF2). Além disso, um recombinante contendo a seqüência de nucleotídeos do PS foi construído sem o sinal de localização nuclear (NLS) de proteína Cap. Através do ensaio de imunoperoxidase em monocamada (IPMA), antígenos de PCV2 foram detectados em células Sf21 infectadas pelos três vírus recombinantes. Este resultado sugere que os recombinantes construídos são potenciais candidatos vacinais, uma vez que eles foram capazes de produzir antígenos de PCV2. / Porcine circovirus type 2 (PCV2) is the major agent of postweaning multisystemic wasting syndrome (PMWS), a worldwide spread disease that causes significant economic losses to the swine productive chain. Aiming to contribute in the diagnosis of the syndrome, this thesis compared and developed tests for PCV2 detection. For this, multiply-primed rolling-circle amplification (MPRCA) and PCR-based assays (conventional, real-time and competitive) were tested. The MPRCA allowed amplifying the full-length PCV2 genomes, which were cloned, sequenced and grouped on PCV2b genotype. The cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 105.55 TCID50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes in sight of sequencing and virus isolation strategies. However, it was less sensitive than PCR for diagnostic purposes. In the second study, searching for methods in support to PMWS diagnosis, two PCR assays were developed: a competitive PCR (cPCR) and a SYBR green real-time PCR. The quantitative PCR methods were compared to determine which would be more suitable to estimate the PCV2 DNA load. Both assays were able to detect significant differences between the numbers of PCV2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥2.5 log10). However, a mean difference of 1.8 log10 on the viral load was found between assays, where the highest viral loads were detected by SYBR green real-time PCR. In the work outlined herein, another purpose was to generate vaccine candidates based on PCV2 capsid protein (Cap). Therefore, in the third study, three types of recombinant baculoviruses were constructed to express the Cap protein. In two recombinants, the nucleotide sequence from the signal peptide (SP) of bovine herpesvirus glycoprotein I (BoHV-gI) was inserted at the 5’ end of the cap gene (ORF2). Additionally, one recombinant containing the SP nucleotide sequence was constructed lacking the nuclear localization signal (NLS) of Cap protein. Through immunoperoxidase monolayer assay (IPMA), the PCV2 antigen was detected in Sf21 cells infected by the three recombinant viruses. This result suggests that the recombinants here constructed are potential vaccine candidates, once they were able to produce PCV2 antigens.

O circovírus suíno tipo 2 (PCV2) é o principal agente da síndrome multissistêmica do definhamento do suíno (SMDS), uma doença mundialmente disseminada e que provoca perdas econômicas significativas para a suinocultura. Visando contribuir no diagnóstico da síndrome, o presente trabalho padronizou e comparou testes para a detecção do PCV2. Para isso, foram utilizadas as técnicas de amplificação por círculo rolante (ACR) e variações da PCR (convencional, tempo-real e competitiva). Utilizando a ACR foi possível obter a amplificação total de genomas do PCV2, os quais foram clonados, sequenciados e agrupados no genótipo PCV2b. Os genomas clonados foram isolados, recircularizados e transfectados em células PK-15. Este procedimento possibilitou a recuperação do vírus infeccioso em títulos de até 105,55 DICC50/mL. Portanto, a ACR foi uma ferramenta útil em estratégias de isolamento e sequenciamento do vírus. No entanto, a ACR foi menos sensível que a PCR para fins de detecção do PCV2. No segundo estudo, buscando métodos auxiliares no diagnóstico da SMDS, dois ensaios para a quantificação do PCV2 foram desenvolvidos. Estes ensaios foram baseados nas técnicas de PCR competitivo (cPCR) e de PCR em tempo real. Visando determinar qual seria o mais adequado para estimar a carga viral do PCV2, os dois métodos foram comparados. Ambos os ensaios foram capazes de detectar diferenças significativas entre o número de cópias de DNA de PCV2 encontradas em tecidos de animais saudáveis e acometidos pela SMDS (≥ 2,5 log10). No entanto, uma diferença média de 1,8 log10 na carga viral foi encontrada entre ensaios, onde as maiores cargas virais foram detectadas pela PCR em tempo real. Outro objetivo deste trabalho foi gerar vacinas baseadas na proteína do capsídeo (Cap) do PCV2. Assim, no terceiro estudo, três baculovírus recombinantes foram construídos de modo a expressar a proteína Cap. Em dois recombinantes, a seqüência de nucleotídeos do peptídeo sinal (PS) da glicoproteína I do herpesvírus bovino (BoHV-gI) foi inserida na extremidade 5’ do gene cap (ORF2). Além disso, um recombinante contendo a seqüência de nucleotídeos do PS foi construído sem o sinal de localização nuclear (NLS) de proteína Cap. Através do ensaio de imunoperoxidase em monocamada (IPMA), antígenos de PCV2 foram detectados em células Sf21 infectadas pelos três vírus recombinantes. Este resultado sugere que os recombinantes construídos são potenciais candidatos vacinais, uma vez que eles foram capazes de produzir antígenos de PCV2. / Porcine circovirus type 2 (PCV2) is the major agent of postweaning multisystemic wasting syndrome (PMWS), a worldwide spread disease that causes significant economic losses to the swine productive chain. Aiming to contribute in the diagnosis of the syndrome, this thesis compared and developed tests for PCV2 detection. For this, multiply-primed rolling-circle amplification (MPRCA) and PCR-based assays (conventional, real-time and competitive) were tested. The MPRCA allowed amplifying the full-length PCV2 genomes, which were cloned, sequenced and grouped on PCV2b genotype. The cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 105.55 TCID50/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes in sight of sequencing and virus isolation strategies. However, it was less sensitive than PCR for diagnostic purposes. In the second study, searching for methods in support to PMWS diagnosis, two PCR assays were developed: a competitive PCR (cPCR) and a SYBR green real-time PCR. The quantitative PCR methods were compared to determine which would be more suitable to estimate the PCV2 DNA load. Both assays were able to detect significant differences between the numbers of PCV2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥2.5 log10). However, a mean difference of 1.8 log10 on the viral load was found between assays, where the highest viral loads were detected by SYBR green real-time PCR. In the work outlined herein, another purpose was to generate vaccine candidates based on PCV2 capsid protein (Cap). Therefore, in the third study, three types of recombinant baculoviruses were constructed to express the Cap protein. In two recombinants, the nucleotide sequence from the signal peptide (SP) of bovine herpesvirus glycoprotein I (BoHV-gI) was inserted at the 5’ end of the cap gene (ORF2). Additionally, one recombinant containing the SP nucleotide sequence was constructed lacking the nuclear localization signal (NLS) of Cap protein. Through immunoperoxidase monolayer assay (IPMA), the PCV2 antigen was detected in Sf21 cells infected by the three recombinant viruses. This result suggests that the recombinants here constructed are potential vaccine candidates, once they were able to produce PCV2 antigens.