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.

Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated disease (PCVAD), an economically important swine disease that causes wasting in pigs 5-18 weeks of age. There exist two different types of porcine circoviruses: porcine circovirus type 1 (PCV1) was discovered as a contaminant of porcine kidney (PK-15) cells and was determined to be nonpathogenic in swine; whereas porcine circovirus type 2 (PCV2) is pathogenic. A recently released vaccine for PCVAD was generated by inserting the gene encoding the immunogenic capsid protein of PCV2 into the genetic backbone of the non-pathogenic PCV1. This chimeric PCV vaccine, called PCV1-2, was shown to induce protective immunity against PCV2 infection in pigs. The vaccine is currently on the market in a killed form. In order to develop a live version of the vaccine, the genetic stability of the chimeric PCV1-2 vaccine virus was investigated by in vitro and in vivo passaging of the vaccine virus. In vitro passaging of the PCV1-2 vaccine virus was done in a porcine kidney PK-15 cell line. Cells were infected with the PCV1-2 vaccine virus and then serially passaged 11 times. The passaged vaccine viruses recovered from passages 5 and 11 were sequenced, and the sequences were compared to that of the original PCV1-2 vaccine virus. The in vitro serial passage result showed that no mutation occurred during the 11 in vitro passages. The in vivo passaging was done using specific-pathogen-free (SPF) pigs. In in vivo "passage 1", nine piglets were divided into 3 groups of 3 each: group 1 each inoculated with 200ug of PCV1-2 plasmid, group 2 each with 1Ã 103 TCID50 live PCV1-2 vaccine virus, and group 3 each with 3ml phosphate buffered saline (PBS) buffer as a control. One pig from each group was necropsied at 14, 21, and 28 days post-inoculation (DPI), respectively. A panel of tissue samples including lymph nodes and thymus were collected from each pig. Tissue homogenates from DPI 28 that were positive by PCR for PCV1-2 DNA were used to inoculate new piglets in the in vivo passage 2 experiment. Viruses recovered from passage 2 pigs were subsequently used for inoculation in the in vivo passage 3 experiment. The PCV1-2 vaccine virus DNA from pigs in each passage was amplified and sequenced. The results of the in vivo serial passage experiment showed that, after 3 passages of the PCV1-2 vaccine virus in pigs, there were no new mutations in the viruses recovered from pigs. The PCV1-2 vaccine contained an introduced marker mutation at amino acid position number 79, which is in the capsid region. During the in vivo passaging of the vaccine virus in pigs, this marker mutation quickly reverted back to its original nucleotide. This marker back mutation occurred between DPI 21 and DPI 28 of passage 1 in the PCV1-2 live vaccine virus group, and between DPI 28 of passage 1 and DPI 14 of passage 2 in the PCV1-2 vaccine plasmid group, and remained stable throughout the reminder of the in vivo study. Based upon the results from this study, we conclude that the PCV1-2 chimeric vaccine virus is genetically stable in vitro and in pigs, and thus should serve as a good candidate for a live vaccine against PCV2. / Master of Science

Made available in DSpace on 2014-07-29T15:07:34Z (GMT). No. of bitstreams: 1 Dissertacao Tatyane P Sales.pdf: 1783169 bytes, checksum: 234cc6cbf4d24d8487721c1017369423 (MD5) Previous issue date: 2011-03-25 / Circovirosis, a disease associated to porcine circovirus 2 (PCV2), has been clinically reported as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS) and other diseases presenting reproductive, respiratory and digestive clinical manifestations. Such diseases are considered emergent and economically important for the pig industry. This study aimed to identify the agent and characterize the clinical, gross and microscopic changes of circovirosis in pig intensive production farms in the state of Goiás, Brazil. For that purpose, 20 pigs with clinical signs of circovirosis from six farms were used. After clinical examination, the pigs were slaughtered, submitted to gross inspection and samples from the spleen, kidneys, lungs, liver and lymph nodes were collected for microscopic analysis and PCV2 identification by PCR. Microscopic changes typical of PCV2 infection were found. PCV2 DNA was identified in all tested animals; however, when compared to clinical signs and gross findings data, only 16 of 20 pigs showed signs of circovirosis. Taken together, the data may support a routine protocol of circovirosis identification and prevention in the state of Goias. / A circovirose, causada por circovírus suíno tipo 2 (PCV2), tem se manifestado clinicamente como Síndrome Multissistemica do Definhamento dos Suínos (SMDS), Síndrome Dermatite Nefropatia dos Suínos (SDNS) e outras doenças que cursam com sintomas reprodutivos, pneumônicos e entéricos. Essas doenças são consideradas emergentes e de significativo impacto econômico na suinocultura. O objetivo deste estudo foi identificar o PCV2, caracterizar alterações macroscópicas e microscópicas em animais com suspeita clinica de circovirose em rebanhos suínos criados de forma intensiva no estado de Goiás. Para isso, foram identificadas seis propriedades e 20 animais com suspeita clínica. Esses animais foram submetidos à eutanásia e necropsia na qual foram identificadas alterações macroscópicas e colhidos baço, rim, pulmão, fígado e linfonodos para exames histopatológico e identificação do agente por PCR. Foram observados achados microscópicos compatíveis com os já descritos para a circovirose. Foi identificado o DNA do PCV2 em todos os animais testados advindos de granjas de sistema intensivo de produção do estado de Goiás. Entretanto, quando comparado com as lesões macroscópicas, em apenas 16 dos 20 suínos foi identificada a doença clínica.

O Torque teno vírus suíno é classificado como pertencente à família Anelloviridae gênero Iotatorquevirus que compreende as espécies: Torque teno sus virus 1a (TTSuV1a) e Torque teno sus virus 1b (TTSuV1b); e o gênero Kapatorquevirus que compreende apenas a espécie Torque teno sus virus k2 (TTSuVk2). O TTSuV foi identificado como potencial agente causador de falhas reprodutivas em porcas, como agente desencadeante nos quadros de doenças associadas ao circovírus suíno tipo 2 (PCVAD) e em associação com outros agentes como o vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV), vírus da influenza suína (SIV) e Mycoplasma hyopneumoniae como co-agentes na manifestação clínica do complexo respiratório suíno (PRDC). No presente estudo foi utilizada uma PCR direcionada a região não traduzida do genoma viral (UTR), e foi investigado um total de 391 amostras divididas entre fetos abortados e mumificados, leitões natimortos, soro, fezes e pulmão de suínos de dez propriedades localizadas no Estado de São Paulo. Diferenças entre as frequências do TTSuV1 e do TTSuVk2 foram comparadas entre amostras de porcas com problemas reprodutivos (fetos abortados e mumificados e leitões natimortos), diferentes fases da criação de suínos (porcas, leitões da creche e crescimento), leitões apresentando ou não diarréia, entre animais da terminação vacinados ou não contra o PCV2 e ainda entre amostras positivas e negativas para o PCV2. Amostras positivas de pulmão de suíno e um pool de soro de Porco Monteiro do Pantanal foram submetidos a uma PCR para amplificação da ORF1 de ambos os gêneros do TTSuV, seguida pela clonagem e posterior sequenciamento para reconstrução da filogenia. Foi investigada a ocorrência de eventos de recombinação entre as amostras, pressão de seleção e propriedades da proteína relacionadas à ligação com anticorpos. O TTSuVk2 foi mais presente infectando tanto fetos abortados quanto leitões natimortos. Porcas e leitões da creche foram mais frequentemente infectados pelo TTSuV1 enquanto que leitões do crescimento pelo TTSuVk2. A coinfecção (TTSuV1 + TTSuVk2 + PCV2) foi mais frequente em amostras fecais diarreicas que nas não diarreicas. Animais da terminação apresentaram alta frequência de coinfecção (TTSuV1 + TTSuVk2), sendo ainda maior na associação com o PCV2. A filogenia construída pelo método neighborjoing baseada na sequência da ORF1 revelou existir quatro tipos do TTSuV1 (1a, 1b, 1c e 1d) e sete subtipos do TTSuVk2 (2a, 2b, 2c, 2d, 2e, 2f, 2g) circulantes nas populações de suínos domésticos e ferais do Brasil. Entre as estirpes virais circulantes foram detectados eventos de recombinação intra-hospedeiro, inter-hospedeiro, intra-genótipo e inter-genótipo. A região carboxi-terminal da proteína demonstrou agrupar características relacionadas à ligação com anticorpos. Este estudo é o primeiro a caracterizar geneticamente amostras de TTSuV circulantes em suínos domésticos e ferais do Brasil e contribui para um melhor entendimento sobre a epidemiologia do vírus. / The porcine torque teno virus is classified within the Anelloviridae family, genus Iotatorquevirus comprising the species: Torque teno sus virus 1a (TTSuV1a) and Torque teno sus virus 1b (TTSuV1b). The genus Kapatorquevirus comprises only one species, the Torque teno sus virus k2 (TTSuVk2). TTSuV was identified as a potential agent of reproductive failure causes in sows, as a trigger agent associated with porcine circovirus associated diseases (PCVAD) and in combination with other agents such as porcine reproductive and respiratory syndrome (PRRSV), swine influenza virus (SIV) and Mycoplasm hyopneumoniae as a co-agent in the clinical manifestation of porcine respiratory disease complex (PRDC). In this study, PCR targeting the untranslated region (UTR) of the virus genome was used to investigate a total of 391 samples within aborted and mummified fetuses, stillborn piglets, serum, feces and lungs of pigs from ten different properties located at São Paulo State. Differences between frequencies of TTSuV1 and TTSuVk2 were compared among samples of sows with reproductive failure (aborted and mummified fetuses and stillborn piglets), different phases of pig breeding (sows, nursery and growing piglets), samples from piglets with diarrhea or not, finishing pigs vaccinated or not against PCV2 and between positive and negative samples for PCV2. Positive lung samples from pigs and a pool of sera from feral pigs were used to PCR amplification for the ORF1 of both genera of TTSuV, followed by cloning and subsequent sequencing to reconstruct the phylogeny. The occurrence of recombination events among the samples, selection pressure and protein-related antibodies binding properties was investigated. The TTSuVk2 was more frequent infecting both aborted fetuses as stillborn piglets. Sows and nursery piglets were frequently more infected by TTSuV1 while growing piglets by TTSuVk2. The coinfection (TTSuV1 + TTSuVk2 + PCV2) was more frequent in diarrheic stool samples than in the non-diarrheic. Finishing pigs showed high frequency of coinfection (TTSuV1 + TTSuVk2) and and increase of the coinfection in association with PCV2. Phylogeny constructed by neighborjoing method based on the ORF1 sequence revealed the existence of four types TTSuV1 (1a, 1b, 1c and 1d) and seven subtypes of TTSuVk2 (2a, 2b, 2c, 2d, 2e, 2f, 2g) circulating within populations of domestic and feral pigs in Brazil. Among circulating viral strains, intra-host, inter-host, intra-and inter-genotype recombination events were detected. The carboxy-terminal region of the protein showed the have characteristics related with antibodies binding activity. This study is the first to genetically characterize samples of TTSuV circulating in domestic and feral pigs from Brazil and contributes to a better understanding of the epidemiology of the virus.

O Torque teno vírus suíno é classificado como pertencente à família Anelloviridae gênero Iotatorquevirus que compreende as espécies: Torque teno sus virus 1a (TTSuV1a) e Torque teno sus virus 1b (TTSuV1b); e o gênero Kapatorquevirus que compreende apenas a espécie Torque teno sus virus k2 (TTSuVk2). O TTSuV foi identificado como potencial agente causador de falhas reprodutivas em porcas, como agente desencadeante nos quadros de doenças associadas ao circovírus suíno tipo 2 (PCVAD) e em associação com outros agentes como o vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV), vírus da influenza suína (SIV) e Mycoplasma hyopneumoniae como co-agentes na manifestação clínica do complexo respiratório suíno (PRDC). No presente estudo foi utilizada uma PCR direcionada a região não traduzida do genoma viral (UTR), e foi investigado um total de 391 amostras divididas entre fetos abortados e mumificados, leitões natimortos, soro, fezes e pulmão de suínos de dez propriedades localizadas no Estado de São Paulo. Diferenças entre as frequências do TTSuV1 e do TTSuVk2 foram comparadas entre amostras de porcas com problemas reprodutivos (fetos abortados e mumificados e leitões natimortos), diferentes fases da criação de suínos (porcas, leitões da creche e crescimento), leitões apresentando ou não diarréia, entre animais da terminação vacinados ou não contra o PCV2 e ainda entre amostras positivas e negativas para o PCV2. Amostras positivas de pulmão de suíno e um pool de soro de Porco Monteiro do Pantanal foram submetidos a uma PCR para amplificação da ORF1 de ambos os gêneros do TTSuV, seguida pela clonagem e posterior sequenciamento para reconstrução da filogenia. Foi investigada a ocorrência de eventos de recombinação entre as amostras, pressão de seleção e propriedades da proteína relacionadas à ligação com anticorpos. O TTSuVk2 foi mais presente infectando tanto fetos abortados quanto leitões natimortos. Porcas e leitões da creche foram mais frequentemente infectados pelo TTSuV1 enquanto que leitões do crescimento pelo TTSuVk2. A coinfecção (TTSuV1 + TTSuVk2 + PCV2) foi mais frequente em amostras fecais diarreicas que nas não diarreicas. Animais da terminação apresentaram alta frequência de coinfecção (TTSuV1 + TTSuVk2), sendo ainda maior na associação com o PCV2. A filogenia construída pelo método neighborjoing baseada na sequência da ORF1 revelou existir quatro tipos do TTSuV1 (1a, 1b, 1c e 1d) e sete subtipos do TTSuVk2 (2a, 2b, 2c, 2d, 2e, 2f, 2g) circulantes nas populações de suínos domésticos e ferais do Brasil. Entre as estirpes virais circulantes foram detectados eventos de recombinação intra-hospedeiro, inter-hospedeiro, intra-genótipo e inter-genótipo. A região carboxi-terminal da proteína demonstrou agrupar características relacionadas à ligação com anticorpos. Este estudo é o primeiro a caracterizar geneticamente amostras de TTSuV circulantes em suínos domésticos e ferais do Brasil e contribui para um melhor entendimento sobre a epidemiologia do vírus. / The porcine torque teno virus is classified within the Anelloviridae family, genus Iotatorquevirus comprising the species: Torque teno sus virus 1a (TTSuV1a) and Torque teno sus virus 1b (TTSuV1b). The genus Kapatorquevirus comprises only one species, the Torque teno sus virus k2 (TTSuVk2). TTSuV was identified as a potential agent of reproductive failure causes in sows, as a trigger agent associated with porcine circovirus associated diseases (PCVAD) and in combination with other agents such as porcine reproductive and respiratory syndrome (PRRSV), swine influenza virus (SIV) and Mycoplasm hyopneumoniae as a co-agent in the clinical manifestation of porcine respiratory disease complex (PRDC). In this study, PCR targeting the untranslated region (UTR) of the virus genome was used to investigate a total of 391 samples within aborted and mummified fetuses, stillborn piglets, serum, feces and lungs of pigs from ten different properties located at São Paulo State. Differences between frequencies of TTSuV1 and TTSuVk2 were compared among samples of sows with reproductive failure (aborted and mummified fetuses and stillborn piglets), different phases of pig breeding (sows, nursery and growing piglets), samples from piglets with diarrhea or not, finishing pigs vaccinated or not against PCV2 and between positive and negative samples for PCV2. Positive lung samples from pigs and a pool of sera from feral pigs were used to PCR amplification for the ORF1 of both genera of TTSuV, followed by cloning and subsequent sequencing to reconstruct the phylogeny. The occurrence of recombination events among the samples, selection pressure and protein-related antibodies binding properties was investigated. The TTSuVk2 was more frequent infecting both aborted fetuses as stillborn piglets. Sows and nursery piglets were frequently more infected by TTSuV1 while growing piglets by TTSuVk2. The coinfection (TTSuV1 + TTSuVk2 + PCV2) was more frequent in diarrheic stool samples than in the non-diarrheic. Finishing pigs showed high frequency of coinfection (TTSuV1 + TTSuVk2) and and increase of the coinfection in association with PCV2. Phylogeny constructed by neighborjoing method based on the ORF1 sequence revealed the existence of four types TTSuV1 (1a, 1b, 1c and 1d) and seven subtypes of TTSuVk2 (2a, 2b, 2c, 2d, 2e, 2f, 2g) circulating within populations of domestic and feral pigs in Brazil. Among circulating viral strains, intra-host, inter-host, intra-and inter-genotype recombination events were detected. The carboxy-terminal region of the protein showed the have characteristics related with antibodies binding activity. This study is the first to genetically characterize samples of TTSuV circulating in domestic and feral pigs from Brazil and contributes to a better understanding of the epidemiology of the virus.

O resultado deste estudo foi dois artigos relacionados às lesões intestinais encontradas e presença de agentes associados a infecção por PCV2 e um terceiro artigo sobre a associação entre a presença de lesões histopatológicas em diferentes porções do trato intestinal e a carga viral presente nestas regiões e nas fezes. O primeiro artigo apresenta as lesões macroscópicas e microscópicas encontradas em 79 animais da fase de crescimento. As principais lesões macroscópicas foram: aumento de tamanho dos linfonodos mesentéricos (56), enterite necrótica (24), edema mesentérico (20), linfangectasia (15) e espessamento da parede do íleo (5). Marcação IHQ anti-PCV2 foi associada com depleção de células caliciformes (24), atrofia e fusão de vilosidades do íleo (18) e dilatação de vasos linfáticos (11). Foi observada a presença de co-infecções por Escherichia coli (5) Lawsonia intracellularis (1), Brachyspira spp.(4), Salmonella spp. (17), rotavírus (6) e calicivírus entérico (6). O segundo artigo avaliou 63 casos de enterite necrótica, nos quais o cólon foi o principal local onde foi observada necrose, aparecendo em 55 casos. Em 24 casos observou-se co-infecção de PCV2 e Salmonella spp. As alterações descritas: depleção de células caliciformes, índice apoptótico elevado em enterócitos do cólon, e marcação de IHQ positiva no citoplasma de enterócitos sugerem o envolvimento de PCV2 nos casos de enterocolite necrótica. A carga viral de PCV2 no trato intestinal, fezes e sangue de suínos, avaliada pela técnica de PCR em tempo real, associada com lesões histológicas e marcação de IHQ, bem como a interpretação destes resultados foi detalhadamente descrita no terceiro artigo. / The result of this study are presented in two articles describing intestinal lesions and the presence of associated agents with PCV2 and a third article that describes the association between the presence of histopathological lesions in different areas of the intestinal tract and viral load present in those areas and in the feces. The first report showed macroscopical and microscopical lesions in samples collected from 79 growing pigs. The main macroscopical lesions were: enlargement of mesenteric lymph-nodes (56), necrotic enteritis (24), mesenteric edema (20), limphangectasy (15) and thickening of the ileal wall (5). Immunohistochemistry (IHC) staining anti-PCV2 was associated with depletion of goblet cells (24), villous fusion and atrophy of the ileum (18) and dilatation of lymphatic vessels (11). It was observed the presence of co-infections with Escherichia coli (5) Lawsonia intracellularis (1), Brachyspira spp. (4), Salmonella spp. (17), rotavirus (6) and enteric caliciviruses (6). The second article evaluated 63 cases of necrotic enteritis, in which the colon was the main target of necrotic lesions, observed in 55 cases. In 24 cases co-infection with PCV2 and Salmonella spp. was observed. The main findings described: depletion of goblet cells, high apoptotic rate in colon enterocites and positive anti-PCV2 IHC staining in cytoplasm of enterocites suggests the involvement of PCV2 in cases of necrotic enterocolitis. The viral load of PCV2 in intestinal tract, feces and blood of swine, associated with histological lesions and IHC staining, evaluated by Real time PCR technique, as well as the interpretation of these results were described in details in the third article.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.