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.

The non-pathogenic porcine circovirus type 1 (PCV1) was originally isolated as a persistent contaminant of the porcine kidney cell line PK-15. Whereas, porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) in pigs, which is devastating to the swine industry. My objectives were to determine the effect of maternally derived antibodies on PCV2 infection, assess the role of 2 amino acid substitutions in the PCV2 capsid protein in PCV2 attenuation, evaluate the effect of Rep gene exchange between PCV1 and PCV2 on growth characteristics of a chimeric PCV2, and evaluate the role of open reading frame (ORF) 3 of PCV2 in virus replication and pathogenesis in pigs. Under field conditions, PCV2 infection is widespread and most breeding pigs are seropositive. Assessment of the role of PCV2 maternal antibodies in preventing PCV2 infection in piglets provided evidence that higher levels of maternal antibody provide more protection to piglets. Two amino acid substitutions in the PCV2 capsid protein that enhanced virus replication in vitro and attenuated the virus in vivo were evaluated for their pathogenicity in pigs. The results indicated that P110A and R191S are collectively responsible for virus attenuation. PCV1 replicates better in PK-15 cells and grows at least 1-log titer higher than PCV2. A chimeric PCV with the rep gene of PCV1 replacing that of PCV2 in the genomic backbone of PCV2 replicated more rapidly than PCV1 and PCV2, and more efficiently than PCV2, although to a titer similar to PCV1. The ORF3 of PCV2 is believed to encode a protein involved in apoptosis. The ORF3 start codon was mutated from ATG to GTG and the resulting mutant muPCV2 was infectious in vitro and in pigs; therefore ORF3 is dispensable for virus replication. The pathogenicity of muPCV2 was compared with PCV2 in vivo. Delayed viremia and seroconversion, decreased viral loads, lower level of IgG antibodies, and lower amounts of PCV2 antigen in mesenteric lymph nodes suggested attenuation of muPCV2. However, there was no significant difference in histological or gross lesions in tissues between PCV2- and muPCV2-inoculated groups. The role of ORF3 in attenuation needs to be further elucidated. / Ph. D.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen. / Ph. D.