Dissertations / Theses on the topic 'Porcine circovirus'
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1
Kolyvushko, Oleksandr Hryhorovych. "A Porcine Circovirus Vaccine with Enhanced Capabilities." Thesis, North Dakota State University, 2016. https://hdl.handle.net/10365/28059.
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Porcine circovirus type 2 (PCV2) is a pathogen of swine. Vaccines against PCV2 are available, although none are capable of differentiating infected from vaccinated animals (DIVA). Positive and negative DIVA markers were introduced in the vaccine constructs. Decoy epitopes were modified by site directed mutagenesis to avoid possible subversion of host immunity and achieve a rationalized vaccine design. Immunization of pigs with the modified vaccines, followed by challenge with a virulent field strain showed that the efficacy of the vaccine was comparable to a commercial vaccine. The average weight gain was significantly higher group vaccinated with experimental construct if compared to the group that received commercial vaccine. An appropriate response to the positive and negative DIVA tags was detected. Therefore, the strategy used in this study is the first to enable a DIVA capable vaccine and accompanying immuno-assay, while using an epitope based approach to target improved immunogenicity.
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Halami, Mohammad Yahya. "Circovirus Infection in Cattle." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-155666.
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Circoviren sind kleine, unbehüllte Viren mit einem einzelsträngigen zirkulären DNA Genom mit eine Größe von 1,7 bis 2,4 kb. Das Porcine Circovirus Typ 2 (PCV2), welches zum Genus Circovirus gehört, ist mit einer Anzahl von Krankheitsmanifestationen verbunden worden, die heute als Porcine Circovirus Assoziierte Krankheiten (PCVAD) zusammengefasst sind. Die PCV2-Infektion bei Rindern ist bis zum jetzigen Zeitpunkt marginal erforscht worden. Serologische Untersuchungen auf Circovirus spezifische Antikörperführten zu widersprüchlichen Ergebnissen. Im Jahr 2007 wurde von der Bovinen Neonatalen Panzytopenie (BNP) in Europa mit unklarer Genese berichtet. Das klinisch - pathologische Bild der Hämorrhagien ähnelte dem Krankheitsbild der Infektiösen Anämie, welche durch ein Circovirus bei Hühnern verursacht wird. Deshalb wurde in dieser Studie eine Breitspektrum PCR zum Nachweis von Cirocvirus-Genomen durchgeführt. In 5 von 25 BNP betroffenen Kälbern konnte circovirale DNA nachgewiesen werden. Das komplette Genom wurde nachfolgend amplifiziert, kloniert und sequenziert. Das nachgewiesene Genom (PCV2-Ha08) hat eine Länge von 1768 Nukleotiden und zeigte eine hohe Homologie (bis zu 99%) mit PCV2-Genotyp b (siehe Publikation 1). Als Ursache der BNP ist vor kurzen die Übertragung von Alloantikörpern über das Kolostrum beschrieben wurden, welche die Zerstörungen von Leukozyten und Thrombozyten sowie deren Vorläuferzellen bewirken. Ungeachtet dessen war es wichtig, die Empfänglichkeit und Immunantwort von Kälbern nach experimenteller Infektion mit PCV2 zu studieren. Für diesen Zweck wurden weitere 181 Proben von BNP-Kälbern aus Deutschland mit Hilfe einer Breitspektrum-PCR getestet. In zwei von 181 Proben wurde PCV2 DNA nachgewiesen. Die vollständigen Sequenzen konnten amplifiziert werden. Während das erste Genom aus einer Blutprobe eines Kalbs in Bayern stammte (PCV2-Ha09), stammte das zweite nachgewiesene Genom aus Lunge und Gehirn von einem Kalb in Sachsen (PCV2-Ha10). Das Genom (PCV2-Ha09) besteht aus 1768 nt, währenddessen das Genom (PCV2-Ha10) aus 1767 nt aufgebaut ist (siehe Publikation 2). Weiterhin wurden die PCV2 Empfänglichkeit und die Immunantwort von Kälbern durch experimentellen PCV2 Inokulation sowie die Möglichkeit, eine Serokonversion nach Impfung mit einer kommerziellen PCV2 Vakzin zu entwickeln, untersucht. PCV2-spezifische Antikörper wurden in den PCV2-infizierten Tieren und in den PCV2-immunisierten Tieren im Tag 11 und 7 nach Inokulation (p.i.) nachgewiesen. PCV2-Genome wurden durch quantitative Realtime-PCR zwischen Tag 4 und Tag 46 p.i. nur in den Blutproben sowie in verschiedenen Geweben (z.B. Milz, Lymphknoten, Thymus) der PCV2-infizierten Tiere nachgewiesen. Das Genom, welches von den Lymphknoten der PCV2-infizierten Kälber erneut isoliert wurde, zeigt eine Identität von 99,9% gegenüber dem Inokulum. Dies weist möglicherweise auf adaptierte Mutationen im PCV2 Genom hin. Die Mutationen C1708T und G365C sind während der Infektionen aufgetreten. Die Sequenzanalyse zeigt eine mögliche adaptierte Mutation an der Aminosäure Nr. 105 in Replikationsgen (Met zu Ile) (siehe Publikation 3). Zusammenfassend kann geschlussfolgert werden, dass der Nachweis der PCV2 Genomen und eine experimentell induzierte Serokonversion möglich war. Es konnte gezeigt werden, dass die Empfänglichkeit von PCV2 nicht allein auf Schweine begrenzt ist und eine Übertragung von PCV2 auf Rinder möglich ist.
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Richmond, Owen Benjamin. "Immune modulation mechanisms of porcine circovirus type 2." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73766.
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Porcine circovirus associated disease (PCVAD) is an umbrella term for a multitude of diseases and syndromes that have a negative impact on the health and economics of pig production operations throughout the world. Porcine circovirus type 2 is the causative agent of PCVAD; however the presence of PCV2 alone is rarely enough to cause clinical disease. In order for the full development of PCVAD the presence of a co-infecting pathogen is required. The mechanisms by which co-infection leads to disease remain ongoing areas of research, but it is thought that host immune modulations by PCV2 or a co-infecting pathogen are critical in the pathogenesis of PCVAD. In the first study of this dissertation the ability of PCV2 to induce regulatory T-cells (Tregs) and alter cytokine production was evaluated in vivo. The addition of PCV2 to a multiple viral challenge resulted in a significant increase in Tregs. Levels of IL-10 and IFN-γ were also found to be altered when PCV2 was added to a multiple viral challenge. In further experiments, monocyte derived dendritic cells (MoDC) were infected with different combinations and strains of PCV2 and PRRSV in vitro and evaluated for expression levels of programmed death ligand-1 (PD-L1), IL-10, CD86, swine leukocyte antigen-1 (SLA-1), and swine leukocyte antigen-2 (SLA-2). Expression levels of PD-L1 were significantly increased in PCV2 and PRRSV co-infected MoDCs. SLA-1, SLA-2, and CD86 expression levels were significantly decreased in the MoDC treatment groups containing both PCV2 and virulent stains of PRRSV. MoDC IL-10 expression was significantly increased by PCV2 and virulent strains of PRRSV co-infection. Finally, we investigated the role of the PD-L1/programmed death ligand-1 (PD-1) axis in porcine lymphocyte anergy, apoptosis, and the induction of Tregs. Lymphocyte populations with normal PD-1 expression had significantly higher percentages of anergic and apoptotic lymphocytes, and CD4+CD25HighFoxP3+ Tregs when compared to a PD-1 deficient lymphocyte population. The findings from these studies indicate host immune modulation by PCV2 in vivo and the development of a regulatory phenotype of dendritic cell following PCV2/PRRSV co-infections in vitro that may contribute to a dysfunctional adaptive immune response and the overall pathogenesis of PCVAD.Ph. D.
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López, Soria Sergio. "Puzzling over the epidemiology of porcine circovirus type 2." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285056.
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El objetivo de la tesis aquí presentada fue el de proporcionar información sobre la epidemiología del circovirus porcino tipo 2 (PCV2). Los cuatro estudios incluidos en esta Tesis Doctoral se resumen a continuación:
El primer estudio fue dirigido a averiguar la prevalencia de PCV2 y otros virus porcinos, en concreto el virus reproductivo y respiratorio porcino (PRRSV), el virus de la influenza porcina (SIV), el virus de la enfermedad de Aujeszky (ADV) y parvovirus porcino (PPV) en granjas porcinas españolas. Se obtuvo que a principio-mitad de los 2000, PCV2 y PPV mostraron evidencias de una distribución ubicua en cerdos; PRRSV y SIV también estaban extendidos. La seroprevalencia del virus salvaje de ADV disminuyó con el tiempo. La seroprevalencia en verracos era inferior que en madres y engorde.
El Segundo trabajo consistió en un estudio exploratorio de casos-controles dirigido a encontrar factores de riesgo que, en asociación con la infección por PCV2, inducían la expresión de la enfermedad sistémica por PCV2 (ES-PCV2), una enfermedad multifactorial. Se concluyó que la infección temprana por PCV2, medida por la evidencia de seroconversión, es un factor predisponente para el desarrollo de ES-PCV2.
El tercer estudio se enfocó a los antecedentes genéticos, un factor de riesgo específico para la ES-PCV2. Se concluyó que los antecedentes genéticos son un factor de riesgo para el desarrollo de ES-PCV2. Los lechones procedentes de verracos Pietrain mostraron el mejor rendimiento clínico seguido de los de verracos Large White x Pietrain. Los lechones de verracos Large White x Duroc fueron los más afectados por ES-PCV2.
Finalmente, el último estudio fue dirigido a averiguar el efecto de la carga de PCV2 en suero en la ganancia media diaria de peso (ADWG) durante el periodo post-destete. Se concluyó que la variación de ADWG entre cerdos en granjas afectadas por ES-PCV2 está parcialmente explicada por la cantidad de PCV2 en suero desde el destete al sacrificio. Se identificaron 3 subpoblaciones de cerdos con diferentes cargas de PCV2 en este periodo. Estas subpoblaciones experimentaron diferentes ADWG, donde cuanto mayor era la carga de PCV2 menor era el ADWG.The present thesis aimed to provide information on porcine circovirus type 2 (PCV2) epidemiology. The four studies included in this PhD Thesis are summarised below: The first study aimed to assess the prevalence of PCV2 and other swine viruses, namely reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Aujeszky’s disease virus (ADV) and porcine parvovirus (PPV) in Spanish pig herds. It was obtained that in the early-mid 2000s, PCV2 and PPV showed evidence of ubiquitous distribution in pigs; PRRSV and SIV were also widespread. Seroprevalence against ADV wild virus decreased over time. Boar studs had lower seroprevalences than sow and fattening herds. The second work consisted in an exploratory case-control study aimed to assess risk factors that, in association with PCV2 infection, induced the expression of porcine circovirus type 2-systemic disease (PCV2-SD), a multifactorial disease. It was concluded that early infection by PCV2, measured by evidence of seroconversion, is a predisposing factor for PCV2-SD occurrence. The third study focused on the pig genetic background, a specific risk factor for PCV2-SD. It was concluded that the genetic background is a risk factor for PCV2-SD development. Piglets from pure Pietrain boars showed the best clinical performance followed by piglets from Large White x Pietrain boars. Piglets from Large White x Duroc boars were the most affected by PCV2-SD. Finally, the last study aimed to assess the effect of PCV2 loads in pig serum on average daily weight gain (ADWG) during the postweaning period. It was concluded that ADWG variation among pigs in PCV2-SD affected farms is partly explained by serum PCV2 load from weaning to slaughter age. Three subpopulations of pigs with different serum PCV2 loads from weaning to slaughter age were identified. These subpopulations experienced significantly different ADWG, in which the higher the PCV2 load the lower the ADWG.
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Allan, Gordon Moore. "Studies on the epidemiology and pathogenicity of porcine circovirus." Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241324.
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Fusaro, Laura <1981>. "Patologie da Porcine Circovirus tipo 2 (PCV2) nel suino." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3640/.
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The main work involved the PMWS (Post-weaning multisystemic Wasting Syndrome), caused by PCV-2 (Porcine Circovirus type 2) that involved post-weaned pigs. Merial Italy has funded a study activity in which groups of 3-5 animals were sampled for lungs, tracheo-bronchial and superficial inguinal lymph nodes, ileum and tonsils. The protocol applied can be identified as a more diagnostic potential on the individual than on the group.
PNP. Another investigation has been conducted to study proliferative and necrotizing pneumonia (PNP), a form of interstitial pneumonia in weaning and post-weaning pigs characterized by hypertrophy and hyperplasia of type II pneumocytes, coagulative necrosis and granular debris within alveolar spaces. Many studies suggest porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as the main causes of the disease, but Aujeszky disease virus (ADV) and swine influenza virus (SIV) are also considered. An immunohistochemical study was carried out to evaluate the role of these viruses in PNP lesions in Italy. PNP results primarily associated with PRRSV, even if co-infection is characterized by more severe histological features.
Reproductive pathology. A major risk factor for PCV2 infection is a viraemic episode taking place in pregnant sows with low antibody titer which is transmitted by specific PCV2 products of conception. PCV2 can infect the fetus even by vehicles through infected semen or ova, or as a result of infection of the genital tract. An investigation was carried out to identify the presence and localization of PCV2 in the genital tracts of sows experimentally infected with PCV2 and in their fetuses. The results obtained suggest that: conventional sows can be infected by intrauterine exposition; low antibody titres increase the probability of infection; PCV2 infection close to insemination time reduces the pregnancy rate; placental lesions may represent an additional cause of fetal suffering.
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Juhan, Nicole McKeown. "Molecular mechanisms of porcine circovirus 2 replication and pathogenesis." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27329.
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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.
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Gilpin, D. F. "Studies on the interaction between porcine circovirus type 2 and the porcine immune system." Thesis, Queen's University Belfast, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273035.
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au, warren raye@vcp monash edu, and Warren Raye. "An investigation into the status of porcine circovirus in Australia." Murdoch University, 2004. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050705.135219.
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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.
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Smith, Sara Marie. "Molecular Breeding of Porcine Circovirus Type 2 by Synthetic DNA Shuffling." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/76809.
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Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded DNA virus that causes disease in pigs and is an economically important pathogen affecting pig populations worldwide. PCV2 contains two major open reading frames (ORF): ORF1 encodes two replicase proteins and ORF2 encodes the immunogenic capsid protein. There are three genotypes of PCV2 (PCV2a, PCV2b, and PCV2c), but vaccines available for PCV2 infection are only targeted against PCV2a. The objective of this thesis was to create viable chimeric PCV2 viruses with an ORF2 displaying genetic diversity from all PCV2 genotypes by synthetic DNA shuffling. Variation was identified at 55 amino acid positions in the ORF2 gene among 853 PCV2 capsid gene sequences available in the GenBank database. Degenerate oligonucleotide primers spanning ORF2 were synthesized to contain this naturally observed sequence diversity. Sets of overlapping oligonucleotide primers were fused together using overlap extension PCR to create full-length shuffled ORF2 sequences. The shuffled library of the ORF2 genes was subsequently cloned into the genomic backbone of a wildtype PCV2a infectious DNA clone and transfected into porcine kidney cells (PK-15). After transfection and infection of PK-15 cells, viability of chimeric viruses was screened by immunofluorescence assay (IFA) using anti-PCV2 Rep antibodies. PCR was used to amplify the genomes of viable shuffled viruses from infected cells. PCV2 viruses containing an ORF2 displaying genetic diversity from PCV2a, PCV2b, and PCV2c were isolated in vitro. These shuffled PCV2 viruses may be used as potential candidates for a broadly-protective PCV2 vaccine, although additional studies are warranted to determine in vivo infectivity and pathogenicity.Master of Science
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Raye, Warren. "An investigation into the status of porcine circovirus in Australia." Raye, Warren (2004) An investigation into the status of porcine circovirus in Australia. PhD thesis, Murdoch University, 2004. http://researchrepository.murdoch.edu.au/273/.
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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.
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Raye, Warren Sean. "An investigation into the status of porcine circovirus in Australia /." Access via Murdoch University Digital Theses Project, 2004. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050705.135219.
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Madson, Darin Michael. "Vertical transmission of porcine circovirus type 2 in breeding herds." [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3369857.
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Teixeira, Thais Fumaco. "Detecção de possíveis agentes virais associados à circovirose suína." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2008. http://hdl.handle.net/10183/13371.
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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.
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Klaumann, Francini. "Molecular epidemiological studies of Porcine circovirus 3, a novel virus identified in domestic pig and wild boar." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/665495.
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El Circovirus porcino 3 (PCV-3) es un virus descubierto recientemente en cerdos domésticos y jabalíes. El virus fue hallado por primera vez en 2016 mediante estudios metagenómicos, en animales afectados por fallo reproductivo, e inflamación cardíaca y multisistémica. Desde entonces, el virus se ha descrito circulando de forma generalizada en animales con diferentes presentaciones clínico/patológicas como en cerdos sanos. Por lo tanto, el objetivo principal de esta Tesis fue generar nueva información sobre la epidemiología molecular del PCV-3 en muestras de cerdos domésticos y jabalíes en España.
En el primer estudio, la presencia de PCV-3 en la población porcina española se evaluó retrospectivamente de 1996 a 2017 en sueros de animales de diferentes fases de producción y condiciones clínico/patológicas. La detección del genoma de PCV-3 en estas muestras se realizó mediante PCR y secuenciación del genoma. Los datos obtenidos confirmaron que PCV-3 ha estado circulando en la población porcina española desde el año 1996. La frecuencia global de muestras PCR positivas para PCV-3 en el período de estudio fue 11.47% (75 de 654). El análisis filogenético de las secuencias obtenidas de PCV-3 mostró una alta identidad con las secuencias de PCV-3 ya conocidas. Aunque la información obtenida fue limitada, la presencia de PCV-3 no pareció estar relacionada con ninguna condición patológica específica ni asociada a ninguna fase de producción del cerdo.
En el segundo estudio se evaluó la dinámica de la infección por PCV-3. Para ello se analizaron mediante PCR los sueros de 152 cerdos de 4 granjas de alto estatus sanitario y sin problemas clínicos. Los animales fueron monitorizados longitudinalmente 5-6 veces desde las 2 a 4 semanas de edad hasta el final de la fase de engorde. El genoma del PCV-3 se detectó en cerdos de todas las edades y granjas evaluadas; algunos animales presentaron una aparente infección a largo plazo durante un período que varió de 4 a 23 semanas. El análisis filogenético mostró una gran similitud entre las secuencias obtenidas y los genomas de PCV-3 de diferentes países disponibles en las bases de datos. Los resultados confirman que PCV-3 circuló en las granjas estudiadas en España, lo que sugiere que la infección probablemente sea generalizada en el país. La mayoría de los cerdos se infectaron durante su vida productiva, aunque no se encontró asociación con una edad específica.
En el tercer estudio, se verificó la frecuencia retrospectiva de la infección por PCV-3 entre 2004 y 2018, así como en una población española de jabalíes capturados y recapturados. Los resultados obtenidos confirmaron la susceptibilidad del jabalí a la infección por el virus, mostrando alta frecuencia de detección de PCV-3 (221 de 518, 42.66%) y demostrando circulación al menos desde el año 2004. Los datos compilados sugieren que PCV-3 es aparentemente capaz de causar una infección persistente, ya que 5 de 10 jabalíes capturados/re-capturados positivos a PCV-3 mostraron positividad en muestreos separados por más de 5 meses. La frecuencia de detección del genoma de PCV-3 también fue investigada por primera vez en diferentes muestras de tejido y heces. Se detectó el genoma de PCV-3 en todos los tipos de tejido analizados. La cantidad de ADN en todas las muestras de PCR positivas para PCV-3 analizadas fue de moderada a baja. Todas las secuencias parciales y completas de PCV-3 obtenidas de jabalíes mostraron una elevada similitud nucleotídica (> 98%).
En conclusión, los resultados obtenidos en esta Tesis proporcionan datos relevantes sobre la epidemiología de este nuevo virus, PCV-3, tanto en cerdos domésticos como en jabalíes. Además, la información filogenética sugiere una baja variabilidad genética de PCV-3, en contraste con otros virus de ADN monocatenario.Porcine circovirus 3 (PCV-3) is a recently discovered circovirus species found in domestic pigs and wild boar. The virus was found in 2016, through metagenomic sequencing approach, in animals affected by reproductive failure, cardiac and multisystemic inflammation. Since then, the virus has been described in pigs with different clinical/pathological presentations as well as in healthy ones, with a widespread circulation. Therefore, the main objective of this Thesis was to gain insights into the molecular epidemiology of PCV-3 in samples from domestic pigs and wild boar from Spain. In the first study, the presence of PCV-3 in the Spanish pig population was retrospectively evaluated from 1996 to 2017 in sera from animals of different production phases and clinical/pathological conditions. The detection of PCV-3 genome in such samples was attempted by PCR and partial genome sequences were obtained from selected PCV-3 positive samples from different years. Compiled data confirmed that PCV-3 has been circulating in the Spanish pig population since 1996. The overall frequency of PCV-3 PCR positive samples in the study period was 11.47% (75 out of 654). Phylogenetic analysis of the PCV-3 obtained sequences showed high identity with the already known PCV-3 sequences, with low variations among years. Although the available information was limited, PCV-3 did not appear to be linked to any specific pathological condition or pig age-group. The second study aimed to assess the dynamics of PCV-3 infection by means of PCR in serum. A total of 152 pigs from 4 different healthy farms, which were sampled longitudinally five or six times from 2-4 weeks of age until the end of the fattening period, were analyzed. PCV-3 genome was found in pigs from all tested ages and farms; few animals had an apparent long-term infection during a period ranging from 4 to 23 weeks. Phylogenetic analysis showed high similarity among the obtained sequences and with available PCV-3 genomes from different countries. Results confirmed that PCV-3 circulated in all studied farms from Spain, suggesting that infection is probably widespread in the country. Most pigs got infection during their life, although PCV-3 did not appear to circulate mostly at any specific age. In the third study, the frequency of PCV-3 infection was retrospectively assessed in Spanish wild boar from 2004 to 2018, as well as in captured and re-captured animals. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV-3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data suggests that PCV-3 is apparently able to cause persistent infection, since 5 out of 10 PCV-3 PCR positive captured/re-captured boars showed positivity in samplings separated for more than 5 months. The frequency of PCV-3 genome was also investigated for the first time in different tissue samples and feces, where all tested tissue types’ harbored PCV-3 genome. The amount of DNA in all tested PCV-3 PCR positive samples was moderate to low. All partial and complete PCV-3 sequences obtained from wild boar displayed high nucleotide similarity (>98%). In conclusion, the obtained results of this Thesis provide relevant data on the epidemiology of this novel virus, in both domestic pig and wild boar, which appear to be widespread. Moreover, the phylogenetic information suggests low genetic variability of PCV-3, in contrast with other single stranded-DNA viruses.
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Pineyro, Pineiro Pablo Enrique. "Novel approaches towards vaccine developments against porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/77542.
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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.
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Li, Yick-yeung, and 李亦揚. "Molecular and phylogenetic analysis of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2(PCV2)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29297102.
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Ferrara, Domenico <1977>. "Ruolo del Porcine Circovirus tipo 2 (PCV2) nella patologia riproduttiva del suino." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4560/.
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La tesi è organizzata in 4 capitoli:
-nel primo vengono brevemente riferite le patologie associate all’infezione da PCV2 con particolare riferimento all’iter diagnostico ed al ruolo rivestito dall’esame istologico e dalla identificazione dell’agente eziologico in situ contestualmente alle lesioni istologiche;
-nel secondo viene presentato un iter diagnostico originale da applicare in condizioni di campo, qualora si voglia accertare la presenza del PCV2 nei tessuti dei prodotti di natimortalità/aborto del suino. In specifico si riferisce all’applicazione del protocollo in 2 aziende ed i risultati vengono analizzati per una revisione critica del protocollo impiegato;
-nel terzo vengono presentati i risultati di un protocollo di infezione con PCV2 per via genitale tramite seme infetto. Scrofe convenzionali sono state sincronizzate per l’estro e fecondate con un’unica dose di seme PCV2 negativo alla PCR (gruppo controlli) o sperimentalmente esposto al PCV2 (gruppo infette). I risultati vengono analizzati in funzione delle ripercussioni che l’infezione precoce in gravidanza può produrre sulla scrofa (mancata gravidanza, ritorno in calore), sui feti e sugli invogli fetali. Viene stabilito il ruolo protettivo degli anticorpi circolanti al momento dell’infezione, stante l’evenienza che un basso titolo anticorpale si associa a viremia prolungata e maggiore numero di feti positivi al virus;
-nel quarto viene presentato un esperimento sovrapponibile a quello riferito nel capitolo 3, però con la presenza anche di un gruppo di soggetti convenzionali vaccinati ed infettati con PCV2 durante la fecondazione artificiale usando seme sperimentalmente esposto al virus. Nella discussione dei risultati vengono enfatizzati 2 aspetti importanti nell’epidemiologia dell’infezione da PCV2: la eliminazione di virus è fortemente ridotta dalla vaccinazione, con conseguenze verosimilmente positive sulla circolazione del virus negli effettivi dell’allevamento; l’esposizione uterina è protetta dalla vaccinazione, stante la bassa percentuale di placente infette nel gruppo dei soggetti vaccinati rispetto a quelli non vaccinati e nei controlli.The thesis is organized into 4 chapters: -in the first chapter, it is briefly overviewed the association of PCV2 with several diseases with particular emphasis to the diagnostic protocols and to the in situ identification of the virus in histological lesions; -in the second chapter, it is presented an original diagnostic protocol to be applied in field conditions, to check for the presence of PCV2 in piglet tissues obtained from stillbirth/abortion. It refers to the application of the protocol in 2 herds and the results are analyzed for a critical review of the used protocol; -in the third chapter, it is presented an experimental trial aimed to infect gilts during artificial insemination by PCV2 infected semen. Conventional gilts were synchronized for oestrus and inseminated with a single dose of semen PCV2 PCR-negative (control group) or experimentally exposed to PCV2 (infected group). The results are analyzed to evaluate the impact that infection in early pregnancy may have on the sow (no pregnancy, return to oestrus), foetuses and foetal membranes. It emphasizes the protective role of circulating antibodies at the time of infection, given the possibility that a low antibody titre is associated with prolonged viremia and increased number of PCV2 positive foetuses; -in the fourth chapter, it is presented a protocol similar to that of Chapter 3, but with the presence of a third group of animals: gilts vaccinated and infected with PCV2 using semen experimentally exposed to the virus. In the discussion 2 important aspects are emphasized: the shedding of the virus is greatly reduced by vaccination, with positive effects on the reduction of the circulation of the virus in the herds; uterine exposure is protected by vaccination, given the low percentage of infected placentas in the vaccinated group compared with not vaccinated and control groups.
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Pinheiro, Albanno Leonard Braz Campos. "Investigação do potencial de roedores peridomésticos como reservatório do porcine circovirus 2 (PCV2)." Universidade Federal de Viçosa, 2011. http://locus.ufv.br/handle/123456789/5090.
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Previous issue date: 2011-11-22Porcine circovirus-2 (PCV2) has been related as the causative agent of the Postweaning Multissystemic Wasting Syndrome (PMWS) and other diseases called porcine circovirus associated diseases (PCVAD). They are responsible for economic losses in pork production worldwide. There is only a few scientific studies describing the infection in other species but swine and their hole at the epidemiologic dynamics of the diseases related to the PCV2. The aim of this study is to investigate the occurrence of infection by the PCV2 in wild mice (Mus musculus and Rattus rattus) captured in hog farms. The capture of the 40 sorted mice was made at 5 pig wean tofinish farms in Minas Gerais, an important state of pork production in Brazil. Samples of tissues (lymph nodes, spleen, kidney, heart and lungs) and blood were collected from the mice. The tissue fragments collected were submitted to immunohistochemistry and Nested PCR. Additionally, samples from spleen and lungs were analyzed by histology assays. Presence of antibodies anti-PCV2 was tested by ELISA assays. Immunohistochemical analysis showed positive prints in 12 animals, mostly on spleen (sub scapular area), lungs (alveolar macrophages) and kidney (inside the tubules). The 12 serum analyzed by ELISA hasn t detected antibodies anti-PCV2. Histopathological analyses revealed in some samples, a multifocal and lympho-neutrophilic interstitial bronchopneumonia, with some node formations. Moreover, spleen samples showed a mild to moderate lymphocyte depletion related to the PCVAD. The Nested PCR assays showed the presence of viral DNA at different tissues from 6 tested rodents. Thus, the results found in this work, indicate that mice from the species Mus musculus and Rattus rattus can be naturally infected by the PCV2 and they would play a hole in the epidemiology of PCVAD. However, more studies are necessary to confirm the transmission of the PCV2 from wild rodents to pigs.
O porcine circovirus-2 (PCV2) é atribuído como um dos agentes relacionados a doenças associadas ao circovírus (PCVAD), ocasionando perdas econômicas significativas na produção mundial de suínos. Poucos trabalhos são realizados a respeito da infecção em outras espécies pelo PCV2 e sua participação na epidemiologia das doenças associadas ao vírus. O propósito desse estudo foi investigar a ocorrência de infecção em roedores peridomésticos das espécies Mus musculus e Rattus rattus pelo PCV2 em granjas comerciais de suínos. Animais dessas espécies foram capturados em importantes centros de produção no estado de Minas Gerais. Amostras de órgãos (linfonodos, baço, rins, fígado, pulmão) e sangue foram coletadas. Os fragmentos de tecidos coletados foram submetidos ao teste de imunohistoquímica e Nested PCR. Adicionalmente, foram realizadas avaliações histológicas em amostras de baço, rim e pulmão. Presença de anticorpos anti-PCV2 foram avaliados pela técnica de ELISA. O teste de imunohistoquímica demonstrou marcações encontradas em 12 animais, principalmente no baço (região subcapsular), no pulmão (macrófagos alveolares) e nos rins (interior dos túbulos). A análise do soro pela técnica de ELISA não detectou anticorpos contra o PCV-2 nas 12 amostras avaliadas.. A histopatologia demonstrou em algumas amostras, uma pneumonia bronco-intersticial neutrofílica e linfocítica, multifocal e moderada, com formação de nódulos linfóides associados a vasos e bronquíolos. No ensaio de nested-PCR foi detectado DNA viral em diferentes tecidos avaliados de seis animais. Os resultados citados demonstram que os roedores domésticos das espécies estudadas podem exercer importante papel na epidemiologia das doenças relacionadas ao PCV2. No entanto, mais estudos são necessários para comprovar a transmissão do PCV2 dos roedores para os suínos.
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au, maodea@agric wa gov, and Mark O'Dea. "Pathogenesis and Detection of Porcine Circovirus Type 2 in the Australian Pig Herd." Murdoch University, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20081128.125816.
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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 Australias 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 Australias capacity to independently investigate PCVAD in the Australian pig herd.
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Gillespie, Jennifer Ann. "Genetic Stability of a Genetically-Engineered Chimeric Porcine Circovirus (PCV) Vaccine, PCV1-2." Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/31475.
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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 â passage 1â , 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
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O'Dea, Mark. "Pathogenesis and detection of porcine circovirus type 2 in the Australian pig herd." O'Dea, Mark (2008) Pathogenesis and detection of porcine circovirus type 2 in the Australian pig herd. PhD thesis, Murdoch University, 2008. http://researchrepository.murdoch.edu.au/739/.
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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.
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Palinski, Rachel. "The application of metagenomic sequencing to detect and characterize emerging porcine viruses." Diss., Kansas State University, 2016. http://hdl.handle.net/2097/34468.
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Doctor of PhilosophyDepartment of Diagnostic Medicine/Pathobiology
Raymond R. R. Rowland
Emerging viral diseases threaten the health of the US swineherd and have the potential to impact the industry. Parvoviruses are capable of infecting birds, livestock and humans, however, in swine, parvoviruses cause reproductive failure and contribute to a devastating set of diseases termed porcine circovirus associated disease (PCVAD). Here, a divergent porcine parvovirus, porcine parvovirus 7 (PPV7), distantly related to known parvovirus sequences, was identified in market pigs in the US. The PPV7 non-structural protein displayed 42.4% similarity to Eidolon helvum parvovirus 2 and 37.9% similarity to turkey parvovirus. Conserved parvovirus replicase motifs including three rolling circle replication (RCR), two NTP-binding motifs and a helicase- binding domain, were present in PPV7. Analysis by qPCR of 182 porcine samples found 16 (8.6%) positive, suggesting moderate nucleic acid prevalence in US swine. Paramyxoviruses are capable of infecting various species including cattle, pigs and humans, causing respiratory disease and importantly, can overcome species barriers causing disease. In 2013, a novel paramyxovirus sequence was described in Hong Kong, China in slaughterhouse pigs, and subsequently named porcine parainfluenza virus 1 (PPIV1). The second study identifies two complete PPIV1 genomes in US pigs originating in Oklahoma and Nebraska that display 90.0-95.3% identity to the Chinese strains. Molecular analysis by qPCR resulted in 6.1% prevalence in 279 porcine respiratory samples. Further serological analysis revealed 66.1% of 59 porcine sera samples were positive by PPIV1 F ELISA. Eleven 3-week old nursery pigs from a PPIV1 naturally infected herd were monitored for signs of infection. No clinical signs were seen in the animals, however, six pigs and the lungs of one animal tested qPCR positive by the conclusion of the study. Taken together, PPIV1 is moderately prevalent in US swine-herds. Previously known to infect avian species, canines and swine, recent reports have identified circoviruses in bats, mink, and human feces. In pigs, porcine circovirus 2 (PCV2) is essential to PCVAD, a group of diseases including reproductive failure, respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (PDNS) and postweaning multisystemic wasting syndrome (PMWS). Additionally, PCV2 nucleic acid has been detected in mammalian species other than swine such as cattle and mink. The final study focuses on the identification and characterization of a divergent circovirus, porcine circovirus 3, identified in aborted mummies taken from sows displaying clinical and histological signs of PDNS. Putative capsid and replicase open reading frames display 37% and 55% identity to PCV2, respectively. A retrospective study of 48 PDNS cases, PCV2 negative by immunohistochemistry (IHC), identified 45 positive and 60% of a subset, positive for PCV3 by IHC. Molecular and serological prevalence studies revealed 12.5% nucleic acid and 55% antibody prevalence in US swine samples. Collectively, these studies identify emerging porcine viruses with the potential to cause disease using metagenomic sequencing. The results of these studies will help to mitigate the risk attributed to emerging swine viruses causing disease outbreaks.
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Cecere, Thomas E. "Porcine circovirus associated disease: Modulation of the host immune response to PCV2 and PRRSV by regulatory T cells." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77098.
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Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases facing the global swine industry. Porcine circovirus type 2 (PCV2) is the primary and essential causative agent of PCVAD, but development of clinical disease typically requires co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). The specific mechanisms of co-infection that lead to clinical disease are not fully understood, but immune modulation by the co-infecting viruses is thought to play a critical role. The ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) was evaluated in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). This Treg induction was found to be dependent on TGF-β and not IL-10. Further investigation of the in vivo swine immune response to acute co-infection with PCV2 and PRRSV failed to detect activation of Tregs in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage samples. The Treg response to in vitro and in vivo PRRSV challenge in pigs persistently infected with PCV2 or vaccinated against PCV2 was evaluated. There was no significant difference in Tregs in PBMCs among chronically PCV2-infected, vaccinated PCV2 challenged or negative control pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV, or both viruses, co-cultured lymphocytes from chronically infected and PCV2 vaccinated pigs had significantly (p<0.05) decreased Treg expression in the virus infected groups compared to the negative controls. In separate experiments, pigs vaccinated against PCV2 and subsequently challenged with an attenuated PRRSV strain and its pathogenic parental strain developed increased CD4+CD25+FoxP3+ Tregs (p<0.05) in PBMC samples compared to uninfected controls, and this correlated with increased suppressor activity and IL-10 expression. The findings from these studies indicate that the interaction of PCV2 and PRRSV in swine modulates the host immune response mediated in part through the activity of Tregs. However, the extent to which Tregs orchestrate a dysregulated immune response in the pathogenesis of PCVAD in vivo remains to be determined.Ph. D.
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Ma, Ching-man. "Molecular epidemiology and characterization of the receptor binding of porcine circovirus type 2 (PCV2)." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B38227204.
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Kirwan, Jennifer Anne. "A GC-TOF-MS metabolomics strategy to investigate porcine circovirus 2 infection in pigs." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533998.
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Gauger, Phillip C. "Characterization of porcine circovirus type 2a and 2b infection and lesions in gnotobiotic pigs." [Ames, Iowa : Iowa State University], 2008.
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Fenaux, Martijn. "Molecular Pathogenesis and Development of a Genetically Engineered Vaccine for Type-2 Porcine Circovirus." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/27171.
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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.
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Alberti, Kyle Anthony. "Effect of vaccination against porcine circovirus type 2 (PCV2) on ejaculate characteristics and the shedding of virus in boar semen." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/32965.
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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
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Teixeira, Fernandes Lana. "Microarray-based gene expression analysis in natural and experimental cases of porcine circovirus type 2 infection." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/299793.
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La presente Tesis se orientó a la caracterización molecular de la infección causada por PCV2 utilizando la tecnología de los microarrays. Este virus es el agente infeccioso esencial de la enfermedad sistémica (ES) por PCV2, una enfermedad de carácter multifactorial que afecta principalmente a cerdos en las fases de transición y engorde, y es una de las enfermedades porcinas más importantes económicamente a nivel mundial. La tecnología de los microarrays permite la determinación simultánea de los niveles de ARNm de miles de genes y ha sido utilizada en los últimos años para investigar los perfiles de expresión génica de tejidos y líneas celulares, ayudando a descifrar las interacciones huésped-patógeno que son relevantes para la patogénesis de varias enfermedades. Es por ello que esta Tesis se orientó a identificar genes y procesos biológicos implicados en la respuesta inmune de cerdos subclinicamente infectados por el PCV2 y también de animales naturalmente afectados por la ES-PCV2.
La primera parte de esta Tesis consistió en un trabajo exploratorio dirigido a evaluar la utilidad de la plataforma Affymetrix Porcine GeneChip® para estudiar el perfil global del transcriptoma de lechones de la raza Duroc, derivados por cesárea y privados de calostro (DCPC), experimentalmente infectados por PCV2. A partir del análisis de los datos de los microarrays, se seleccionaron 25 y 33 genes que resultaron diferencialmente expresados (DE) entre los grupos control e inoculados con PCV2 en linfonodos mesentéricos y pulmones, respectivamente. La gran mayoría de los genes sobre-expresados en el grupo inoculado con PCV2 estaba relacionada a la respuesta inmune. Desde el punto de vista del transcriptoma, los animales inoculados con PCV2 fueron capaces de activar la respuesta mediada por células y desarrollar anticuerpos específicos para PCV2, hechos que se asocian a la generación de una infección subclínica. Los resultados de este trabajo indicaron que la técnica de los microarrays es una herramienta útil para el estudio de la patogénesis de la infección por el PCV2.
El segundo estudio fue dirigido a caracterizar los mecanismos moleculares de la respuesta inmune que tienen lugar en las fases temprana y tardía de la infección subclínica por PCV2. Los genes sub-expresados en las muestras de linfonodo mediastínico (LM) se relacionaron principalmente a la adhesión celular y migración, lo que sugiere la participación de esos genes en los procesos inflamatorios observados en la infección por PCV2. La inmunidad innata se desarrolló en la primera semana p.i. y se demostró por la sobre-expresión de varios genes estimulados por interferón (ISGs) entre las muestras de LM y de sangre total (ST) de los animales inoculados con PCV2. También se detectó un aumento en la expresión de genes relacionados con la activación linfocitaria en la primera semana p.i. en las muestras de LM de los cerdos infectados, lo que indica la activación temprana de la respuesta adaptativa. Se obtuvieron resultados similares en las fases tardías de la infección, dada la sobre-expresión de los genes que codifican para el interferón (IFN)-γ y para la inmunoglobulina (Ig)-G en el día 29 p.i en las muestras de LM.
El tercer estudio consistió en investigar los cambios globales en el transcriptoma de animales naturalmente afectados por ES-PCV2 y congéneres sanos. Tras los análisis de datos, se encontraron 366 tránscritos con significativa abundancia diferencial en el grupo de animales afectados por ES-PCV2 en relación al grupo control. Los resultados de este estudio identificaron mecanismos (daño mediado por el complemento e inmunosupresión) potencialmente involucrados en la inflamación y depleción linfocitaria en tejidos linfoides, características histopatológicas claves de la ES-PCV2.This PhD Thesis aimed to characterize the molecular mechanisms underlying the porcine circovirus type 2 (PCV2) infection using the microarray technology. This virus is the essential infectious agent of PCV2- systemic disease (SD), a multifactorial condition that mainly affects nursery and growing pigs, and is considered one of the most economically important pig diseases worldwide. Microarray technology allows simultaneous measurement of the mRNA levels of thousands of genes and have been used during recent years to examine gene expression profiles of tissues or cell lines subjected to infection, helping to unravel host–pathogen interactions relevant to pathogenesis of a variety of diseases. Therefore, this Thesis aimed to identify genes and biological processes implicated in the immune response of pigs subclinically infected by PCV2 and also of animals naturally affected by PCV2-SD. In the study of this Thesis, an exploratory work was conducted to evaluate the technical feasibility of utilizing the Affymetrix Porcine GeneChip® platform to study the global transcriptional profile of caesarean-derived, colostrum-deprived (CDCD) Duroc piglets experimentally infected with PCV2. The microarray analysis detected 25 and 33 significantly differentially expressed (DE) between control and PCV2 groups for mesenteric lymph node and lung, respectively. Most up-regulated genes in PCV2 group were closely related to the immune response. From a transcriptional point of view, PCV2-inoculated pigs were able to activate a cell-mediated response and develop PCV2-specific antibodies, which probably led to a subclinical infection. The results from this study also indicate that a microarray-based approach is a helpful tool to better understand the pathogenesis of PCV2 infection. The second study was aimed to characterize the early and late molecular events underlying the immune response taking place during a subclinical PCV2 infection. Down-regulated genes from mediatinal lymph nodes (MLN) samples were mainly related to cell adhesion and migration, suggesting the participation of these genes in the inflammatory processes (granulomatous infiltration) observed in the PCV2 infection. Innate immunity developed within the first week post-infection (p.i.) and it was demonstrated by the up-regulation of several interferon-stimulated genes (ISGs) both in MLN and whole blood (LWB) samples from PCV2-infected pigs. An increased expression of genes related to lymphocyte activation was also detected during the first week p.i. in LWB samples of infected animals, indicating an early activation of adaptive responses. Similar results were obtained at late stages of infection by the up-regulation of genes coding for interferon (IFN)-γ and the immunoglobulin (Ig)-G at 29 days p.i. in MLN samples. The aim of the third study was to investigate the global transcriptional profile of MLNs from pigs naturally affected by PCV2-SD, as well as healthy counterparts. The microarray data analysis detected 366 transcripts with significant differential abundance in the PCV2-SD group of pigs relative to healthy animals. Results from this study identified potential mechanisms (complement mediated damage and immunosuppression) underlying the inflammation and lymphocyte depletion in lymphoid tissues, which are key features of PCV2-SD.
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Fort, de Puig Maria. "Characterization of immune responses to porcine circovirus type 2 (PCV2) infection and vaccination in pigs." Doctoral thesis, Universitat Autònoma de Barcelona, 2009. http://hdl.handle.net/10803/5619.
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El Circovirus porcí tipus 2 (PCV2) és l'agent causal de la circovirosi porcina (CP), una malaltia multifactorial que afecta porcs en fases de transició i engreix i que causa importants pèrdues econòmiques a la indústria porcina d'arreu del món. Les característiques histopatològiques de la CP i el fet que els animals malalts pateixin sovint infeccions secundàries i oportunístiques indica que aquesta malaltia implica una greu alteració del sistema immunitari del porc. Durant molts anys, el control de la CP es centrava principalment en la millora d'estratègies de maneig i en el control dels factors de risc que influeixen la presentació clínica de la malaltia. A dia d'avui, s'han introduït al mercat internacional quatre vacunes enfront PCV2 i la seva implementació al camp ha disminuït dràsticament la incidència de la CP. La present Tesi tenia com objectiu la caracterització de les respostes immunitàries desenvolupades pel porc en el curs de la infecció i vacunació per PCV2. En la primera part (Estudis I i II), es va caracteritzar el perfil immunològic de porcs sub-clínicament infectats amb PCV2, amb especial èmfasi en les respostes mediades per cèl·lules i el paper de les proteïnes capside (Cap) i replicasa (Rep) de PCV2 en el seu desenvolupament. Es va demostrar que el virus sencer, però no Cap i Rep indueix l'alliberació d'interleuquina (IL)-10 en cèl·lules mononuclears de sang perifèrica (CMSP), fins i tot en els cultius procedents d'animals verges, indicant l'origen innat d'aquesta resposta. Addicionalment, es va observar que, en fases inicials de la infecció per PCV2, es pot detectar interferó (IFN)-α en sèrum (dia 5 post-inoculació (PI)). Per altra banda, nivells detectables d'IL-10 només s'observaren de forma esporàdica, suggerint que els animals infectats sub-clínicament per PCV2 no es caracteritzen per tenir nivells elevats d'aquesta citoquina en sèrum. En relació a la immunitat adaptativa enfront a PCV2, es va veure que la resposta humoral es desenvolupa entra la segona i tercera setmana PI i que es caracteritza per la producció d'anticossos totals i neutralitzants, essent els neutralitzants d'aparició més tardana. La immunitat mediada per cèl·lules apareix entre la primera i segona setmana PI, i tant Cap com Rep estan implicades en el seu desenvolupament. A la segona part d'aquesta Tesi (Estudis III i IV), es va avaluar la immunogenicitat i eficàcia d'una vacuna comercial (una i dues dosis) basada en la Cap d'una soca de genotipus PCV2a en porcs convencionals. Els resultats d'aquests estudis van demostrar que la vacunació indueix el desenvolupament d'immunitat humoral i cel·lular i redueix la viremia, l'excreció i la càrrega vírica en teixits després de la infecció amb PCV2, tant amb soques de genotipus PCV2a com PCV2b. També es va observar que els anticossos maternals (AM) protegeixen enfront la infecció per PCV2 i influeixen en el desenvolupament de la resposta humoral després de la vacunació. Els resultats de l'estudi IV suggereixen que porcs amb títols d'IPMA per sota 5 log2 són potencialment més susceptibles a infectar-se amb PCV2. D'altra banda, es va veure que títols d'IPMA per sobre 10 log2 poden interferir amb el desenvolupament d'anticossos en resposta a la vacunació. En base a aquestes observacions, es proposa una "finestra de vacunació", definida com el rang de títols d'anticossos en el que els porcs s'haurien de vacunar per minimitzar la interferència amb els AM i, al mateix temps, assegurar el desenvolupament d'immunitat protectiva abans que els animals s'exposin a PCV2.Porcine circovirus type 2 (PCV2) is the causative agent of Postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease of nursery and fattening pigs that causes considerable economic losses to the swine industry worldwide. The histopathological features of PMWS and the fact that secondary and opportunistic infections are common in PMWS-affected pigs indicate that this disease involves a deep alteration of the immune system. For years, control of PMWS was limited mainly to the improvement of management strategies and to the control of risk factors thought to influence the infection outcome. At present, four vaccines against PCV2 are being sold on the international market and their application in the field drastically reduced the incidence of PMWS. This Thesis aimed to characterize the immune responses developed by pigs upon PCV2 infection and vaccination. In the first part (Studies I and II), the immune features of PCV2 experimental sub-clinical infections were characterized, with particular emphasis on cell-mediated responses, and on the role of PCV2 capsid (Cap) and replicase (Rep) proteins on it. Results from these studies showed that whole PCV2, but not Cap or Rep, induces the release of interleukin (IL)-10 in peripheral blood mononuclear cells (PBMC), even in those cultures obtained from PCV2-naïve pigs, a fact that indicates the innate origin of this response. In addition, it was found that interferon (IFN)-α can be detected in serum at early stages of the sub-clinical infection (5 days post inoculation (PI)). In contrast, IL-10 in serum could only be sporadically detected, suggesting that increased levels of this cytokine are not characteristic of PCV2 sub-clinically infected pigs. With regards to the acquired immunity to PCV2, humoral responses were developed mostly between the second and third week PI, characterized by the production of PCV2 total and neutralizing antibodies (NA), appearing NA later than total antibodies. Cell-mediated immunity developed within the first two weeks PI and it was demonstrated that both Cap and Rep proteins of PCV2 are involved in its development. In the second part of this thesis (Studies III and IV), the immunogenicity and efficacy of a commercial PCV2a-based sub-unit vaccine used in one- and two-dose schedules was evaluated in conventional piglets. Results from these studies demonstrated that vaccination induced the development of humoral and cell-mediated responses and significantly reduced viremia, shedding, and viral load in tissues upon challenge with either PCV2a or PCV2b isolates. It was also found that maternally derived PCV2 antibodies (MDA) protect against PCV2 infection and influence the humoral response developed after vaccination. Results from study IV suggest that pigs with IPMA titres below 5 log2 are potentially more susceptible to PCV2 infection. Besides, IPMA titres beyond 10 log2 were seen to interfere with the development of antibodies following PCV2 vaccination. Based on these observations a "vaccination window" was proposed, defined as the range of antibody titres at which piglets should be vaccinated to minimize interference with MDA and, at the same time, ensure the development of protective immunity before PCV2 exposure.
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Oliver, Ferrando Salvador. "Effect of Porcine circovirus 2 (PCV2) sow or piglet vaccination in different PCV2 subclinical infection scenarios." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/461187.
Full textAbstract:
La present Tesi doctoral esta formada per tres estudis. El primer estudi va avaluar l'efecte de la vacunació de truges enfront a PCV2 sobre els paràmetres reproductius durant dos cicles gestacionals consecutius. Es van immunitzar 94 truges gestants amb una vacuna comercial enfront a PCV2, i 97 van ser injectades amb una solució salina tamponada amb fosfat (PBS). En el primer cicle reproductiu el tractament va ser aplicat a les 6 i 3 setmanes abans del part, i en el segon cicle es va aplicar una dosi de record o PBS a les 2 setmanes abans del part. Les truges vacunades van mostrar nivells d'anticossos significativament més elevats en comparació amb les no vacunades. L'ADN de PCV2 només es va detectar al part en 2 (4,2%) truges no vacunades. Les truges vacunades van tenir 1,3 garrins nascuts vius més per camada en el segon cicle que les no vacunades. El present estudi va demostrar per primera vegada que la vacunació de truges enfront a PCV2 pot tenir una influència positiva sobre la prolificitat i la vitalitat de la seva descendència en una explotació de reproductores subclínicament infectades per PCV2. En el segon estudi, es va avaluar l'efecte de la vacunació de truges enfront a PCV2 sobre la resposta immune humoral i cel·lular en truges i la seva progènie. A les 7 setmanes abans del part, es van seleccionar 15 truges negatives a PCV2 per PCR i amb valors d'anticossos mitjans-baixos. Aquestes truges es van distribuir aleatòriament en dos grups de tractament segons els seus nivells d'anticossos. A les 6 i 3 setmanes abans del part, 7 truges van ser vacunades amb una vacuna comercial enfront a PCV2 i 8 van ser injectades amb PBS. Els garrins nascuts de truges vacunades tenien nivells significativament més alts de citoquines vinculades a les cèl·lules Th1 de memòria (IFN-γ i TNF-α) en comparació amb els provinents de les femelles no vacunades. En conclusió, la vacunació de truges enfront a PCV2, a més de desencadenar una resposta immune humoral en truges i la seva progènie, podria estar associada a una major transferència d'immunitat cel·lular de la mare al garrí. L'objectiu del tercer estudi va ser determinar la dinàmica serològica i virològica de la infecció per PCV2 en garrins vacunats a diferents edats en un escenari de PCV2-SI. Es van seleccionar 644 garrins sans de 2 setmanes d'edat que foren distribuïts aleatòriament en quatre grups de tractament: vacunació enfront a PCV2 a les 3, 6 o 10 setmanes d'edat (grups 3W-VAC, 6W-VAC i 10W-VAC, respectivament) i porcs no vacunats (grup NON-VAC). Específicament, amb la vacunació enfront a PCV2 a les 3 o 6 setmanes d'edat es van obtenir resultats similars, ja que les dues pautes van desencadenar una seroconversió eficient i van reduir, en diferents punts de mostreig, la proporció d'animals virèmics en comparació amb el grup no vacunat. Per altra banda, la vacunació enfront a PCV2 a les 10 setmanes d'edat només va aconseguir aquesta reducció a les 25 setmanes d'edat; en aquest cas, la vacunació va coincidir amb l'augment del percentatge de porcs virèmics. En conclusió, sota les condicions del present estudi, el temps òptim de vacunació dels garrins per controlar la infecció per PCV2 va ser a les 3 o 6 setmanes d'edat. A més, els OF van demostrar ser una matriu útil per a la avaluació de la dinàmica de seroconversió, en canvi, la detecció del ADN de PCV2 en OF no va mostrar ser un mètode efectiu per a la avaluació del control de la infecció durant els programes vacunals estudiats.The present PhD thesis consists of three studies. The first study sought to evaluate the effect of sow vaccination against PCV2 on reproductive parameters during two consecutive reproductive cycles. Ninety-four pregnant sows were primo-immunized with a commercial PCV2 vaccine and ninety-seven were injected with phosphate-buffered saline at 6 and 3 weeks before farrowing, and then boosted at 2 weeks before the second one. Vaccinated sows showed significantly higher antibody levels compared to the non-vaccinated counterparts. PCV2 DNA was only detected at farrowing in 2 (4.2%) non-vaccinated sows. Vaccinated sows had 1.3 more live-born piglets per litter at the second cycle than non-vaccinated counterparts. The present study represents the first attempt to demonstrate that PCV2 sow vaccination may have a positive influence on prolificacy and vitality of the offspring in a subclinically infected breeding herd. In the second study, the effect of PCV2 sow vaccination on humoral and cell-mediated immune responses in sows and their progeny was assessed. At 7 weeks before farrowing, fifteen PCV2 PCR negative pregnant sows with medium-low antibody values were selected and randomly distributed in two groups according to the antibody levels. Seven sows were vaccinated with a commercial PCV2 vaccine and eight were injected with phosphate-buffered saline at 6 and 3 weeks before farrowing. Piglets from vaccinated sows had significantly higher levels of cytokines linked to Th1 memory cells (IFN-γ and TNF-α) in comparison to the ones from non-vaccinated dams. In conclusion, PCV2 sow vaccination, apart from triggering a humoral immune response in sows and their progeny, might be associated to an increased transfer of cell-mediated immunity from the dam to the piglet. The purpose of the third study was to determine the PCV2 serological and virological infection dynamics in piglets vaccinated at different ages in a PCV2-SI scenario. Six hundred and forty-four 2 week-old healthy piglets were selected and distributed into four treatment groups: vaccination at 3, 6 or 10 weeks of age (3W-VAC, 6W-VAC and 10W-VAC groups, respectively) and unvaccinated pigs (NON-VAC group). Specifically, PCV2 vaccination at 3 or 6 weeks of age yielded similar results, since they produced an earlier seroconversion and reduced, at different sampling points, the proportion of viraemic animals in comparison to the unvaccinated group. In contrast, PCV2 vaccination at 10 weeks of age only achieved such reduction at 25 weeks of age; in this case, vaccination coincided with the increase of the percentage of viraemic pigs in the population. In conclusion, under the present study conditions, the optimal time for piglet vaccination to control PCV2 infection was at either 3 or 6 weeks of age. In addition, OF proved to be a useful matrix for the evaluation of seroconversion dynamics, however, PCV2 DNA detection in OF did not show to be an effective method for the infection control assessment during the studied vaccine programs.
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Feng, Hua. "New insights on PCV2 vaccination: thinking out of the box." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/330925.
Full textAbstract:
La present tesi doctoral tenia com a objectiu complementar el coneixement actual sobre l’eficàcia de la vacuna enfront a PCV2 en condicions d’infecció subclínica i explorar dos conceptes nous (en quan a estratègies de vacunació enfront a aquest patogen) que poguessin augmentar l’eficàcia d’aquest tractament.
El primer estudi pretenia avaluar la possible interferència de la presència de diferents nivells d’anticossos d’origen maten (AOM) en el moment de la vacunació, en l’evolució del guany mig diari de pes (GMDP). En aquest estudi, només es va detectar una possible interferència en l’eficàcia de la vacuna sobre el GMDP, quan es va considerar la subpoblació d’animals amb els valors S/P més alts. Per tant, l’impacte d’aquesta possible interferència en condicions de camp es probablement negligible en la majoria d’animals i de les granges.
En el segon estudi, es va avaluar la viabilitat d’eradicar la infecció de PCV2 en una granja convencional infectada subclínicament amb PCV2 mitjançant una estratègia de vacunació en massa. L’aplicació durant una any de la vacunació en massa enfront a PCV2 (sense implementar mesures específiques de maneig o de bioseguretat) no va ser capaç d’eliminar l’infecció per PCV2. De fet, un cop la vacunació es va aturar, el virus es va detectar de nou. De totes maneres, la disminució dels nivells d’anticossos i la no detecció del virus durant la segona meitat del període de vacunació en massa deixa entreveure que la eradicació de la infecció de PCV2 mitjançant un programa de vacunació més llarg i més extensiu podria ser possible.This thesis aimed to complement the current knowledge on PCV2 vaccination efficacy under subclinical infection conditions and give new creative concepts (“thinking out of the box”) for future related studies. The first study had the objective to assess the putative interference of different maternally derived antibody (MDA) levels at the time of vaccination on the average daily weight gain (ADWG) evolution. In this study, an apparent interference of vaccine efficacy on ADWG was noticed only when a small subpopulation of pigs with the highest ELISA S/P ratios was considered, Therefore, the impact of this possible interference under field conditions is probably negligible for most of the animals and farms. In the second study, the feasibility to eradicate PCV2 in a conventional PCV2 infected farm by using a mass vaccination strategy was assessed.. One year period of mass PCV2 vaccination (without implementing further farm management practices or biosafety measures) was not able to clear out PCV2 infection. Indeed the virus became detectable again when vaccination was stopped. However, the decreasing antibody levels and the lack of viral detection during the second half of the vaccination period shed a light on eradicating this virus by applying a longer term vaccination in a wider area would be feasible.
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Cottrell, Tiffany Sinclair. "Epidemiology of post-weaning multi-systemic wasting syndrome and pathogenic strains of porcine circovirus in southern Ontario." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ40404.pdf.
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Trible, Benjamin R. "Expression of recombinant porcine circovirus 2 (PCV2) capsid polypeptides for mapping antibody epitopes following vaccination, infection, and disease." Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/13350.
Full textAbstract:
Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Raymond R. R. Rowland
Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease.
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Bulos, Luiz Henrique Silva. "Perfil sorológico e virêmico de suínos da raça Piau e linhagem comercial naturalmente infectados com o Porcine circovirus 2 em diferentes fases de produção." Universidade Federal de Viçosa, 2013. http://locus.ufv.br/handle/123456789/5149.
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The diseases associated with PCV2 (PCVAD) require various factors to occur, however, the virus infection is critical to the development of any one of the syndromes. The present study aimed to determine differences in serologic and viremic profiles for PCV2 in the swine breed Piau and a commercial line (Landrace x Large White x Pietrain) at a subclinically infected farm by the virus studied. The experiment was conducted at the Genetic Improvement Pig Farm (GMS), Federal University of Viçosa (UFV), in which it isn´t carried out vaccination against PCV2. This study conducted a cross-sectional sample of sows (> 2 parity), pigs for 1-3 weeks, 3-8 weeks and 8-22 weeks of age. The serum samples were used to measure the level of total antibodies by ELISA and quantitation of viremia by real time PCR. The results showed that, at the age of 3-8 weeks, the Piau breed piglets seroconverted earlier than the commercial line piglets and the Piau breed sows showed lower levels of total antibodies in relation to the commercial line. There were no differences in viremia between the different stages of production within each genetic group or between groups. This work provides evidence that the breed Piau has a different humoral immune response than the commercial line studied when facing a natural PCV2 subclinical infection. The results of this study reinforce the importance of the conservation of native breeds that have not been used for development of high productivity commercial lines.
As doenças associadas ao PCV2 (PCVAD) necessitam de vários fatores para ocorrer, no entanto, a infecção pelo vírus é fundamental para o desenvolvimento de qualquer uma das síndromes. O presente estudo teve como objetivo verificar diferenças nos perfis sorológicos e virêmicos para o PCV2 entre suínos da raça Piau e de uma linhagem comercial (Landrace x Large White x Pietrain) em uma granja subclinicamente infectada pelo vírus estudado. O experimento foi realizado na Granja de Melhoramento Genético (GMS) da Universidade Federal de Viçosa (UFV), na qual não é realizada a vacinação contra o PCV2. O presente estudo realizou uma amostragem transversal em porcas (>2º parto), suínos de 1-3 semanas, 3-8 semanas e 8-22 semanas de idade. As amostras de soro obtidas foram utilizadas para mensuração dos níveis de anticorpos totais por ELISA indireto e quantificação da viremia por PCR em tempo real. Os resultados demonstraram que, na idade de 3-8 semanas, os leitões da raça Piau soroconverteram mais precocemente em relação aos leitões da linhagem comercial e as porcas da raça Piau apresentaram menores níveis de anticorpos totais em relação às da linhagem comercial. Não houve diferença na viremia entre as diferentes fases de produção dentro de cada grupo genético ou entre os grupos. Este trabalho fornece indícios de que a raça Piau apresenta uma resposta imune humoral diferente da desenvolvida pela linhagem comercial estudada diante de uma infecção subclínica natural pelo PCV2. Os resultados obtidos neste estudo reforçam a importância da conservação das raças nativas que não foram utilizadas para formação de linhagens de alta produtividade.
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Núñez, Hernández Fernando. "Identification and characterization of microRNAs in porcine circovirus type 2 and African swine fever virus infections in vivo." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/399553.
Full textAbstract:
Cuatro estudios diferentes fueron llevados a cabo con el fin de analizar la expresión
de microRNAs (miRNAs) porcinos y virales en dos infecciones in vivo diferentes con
circovirus porcino tipo 2 (PCV2) y el virus de la peste porcina africana (VPPA).
El objetivo del primer estudio fue identificar el patrón de expresión de miRNAs en
cerdos infectados y no infectados con PCV2. Para ello, se llevó a cabo una infección
experimental y se construyeron librerías de ARN de pequeño tamaño de tonsila y
linfonodo mediastínico de estos animales. La secuenciación masiva determinó
diferencias de expresión de miRNAs porcinos entre animales infectados y no
infectados. La predicción del análisis funcional mostró que estos miRNAs pueden
estar involucrados en rutas biológicas relacionadas con el sistema inmune y en
procesos relacionados con la patogénesis provocada por PCV2.
El objetivo del segundo estudio fue investigar si PCV2 puede codificar miRNAs
virales. A partir de las librerías ya construidas, los resultados de la secuenciación
masiva revelaron que PCV2 no expresa miRNAs en una infección subclínica in vivo.
En el tercer estudio se analizó la diferencia de expresión de miRNAs en bazo y
linfonodo submandibular en infecciones in vivo con el VPPA entre i) animales
infectados con la cepa virulenta E75 de VPPA a distintos tiempos (3 y 7 días postinfección)
y ii) entre animales infectados con la cepa virulenta E75 y animales
infectados con la cepa derivada de ésta y atenuada E75CV1 a un mismo tiempo
post- infección (3 días post- infección). Se crearon librerías de ARN de pequeño
tamaño y la secuenciación masiva de las mismas mostró diferencias de expresión
de miRNAs en ambos casos. Además, se observó la asociación de los miRNAs
diferencialmente expresados con genes porcinos y virales involucrados en la
respuesta inmune y las interacciones virus- hospedador.
En el cuarto estudio se analizó la posibilidad de que VPPA codificase miRNAs virales.
Para ello se emplearon las muestras previas de E75 y E75CV1 y se añadieron al
estudio muestras de animales infectados con la cepa atenuada E75CV1 y
sacrificados a día 31 post- infección aparte de animales infectados con esta misma
cepa y re-inoculados a día 31 post- infección con la cepa virulenta Ba71 y
eutanasiados a día 38 post- infección. La secuenciación masiva de todas las librerías
de ARN de pequeño tamaño mostró que VPPA no expresa miRNAs virales en
ninguna de las condiciones estudiadas.Four studies were carried out in order to explore the expression of porcine and viral microRNAs (miRNAs) in two different in vivo infections with porcine circovirus type 2 (PCV2) and African swine fever virus (ASFV). The objective of the first study was to analyze the porcine miRNAs expression pattern in subclinically infected and non- infected pigs with PCV2. For this end, an experimental infection was carried out and small RNA libraries were constructed from tonsil al mediastinal lymph node from infected and non- infected animals. Highthroughput sequencing determined differences in the expression of porcine miRNAs between infected and noninfected animals. The prediction analysis showed that these differentially expressed miRNAs could be involved in biological pathways related to immune system and processes related to PCV2 pathogenesis. The objective in the second study was to explore if PCV2 encodes viral miRNAs. From the previously constructed libraries, highthroughput sequencing revealed that PCV2 does not encode viral miRNAs in an in vivo subclinical infection. In the third study the expression pattern of porcine miRNAs in spleen and submandibular lymph node in ASFV in vivo infections was assessed between: i) animals infected with E75 ASFV virulent strain at different times (3 and 7 days post- infection) and ii) between animals infected with E75 virulent strain and E75CV1 attenuated strain at the same time point postinfection (3 days post- infection). Small RNA libraries were constructed and the highthroughput sequencing revealed miRNAs differentially expressed in both cases. In addition, it was observed that these miRNAs were associated to porcine and viral genes involved in immune response and host- pathogen interactions. In the fourth study, it was analyzed the capability of ASFV to encode viral miRNAs. For this end, previous samples from the infections with E75 and E75CV1 were used in addition to samples from animals infected with the attenuated strain and sacrificed at day 31 postinfection and also, animals infected with this attenuated strain, re- inoculated at day 31 with the virulent strain Ba71 and euthanized at day 38 post- infection. Highthroughput sequencing from all the small RNA libraries revealed that ASFV does not encode viral miRNAs in any of the studied conditions.
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Angobe, Aune Tuyoleni. "Development of a plant-made immunoassay for the detection of Porcine circovirus infections in South African swine herds." Master's thesis, Faculty of Science, 2021. http://hdl.handle.net/11427/33629.
Full textAbstract:
Porcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirusassociated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication-associated protein. Recombinantly expressed CP can selfassemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLPbased and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study was to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. Production of PCV-2 CP in Nicotiana benthamiana via transient Agrobacterium-mediated expression was optimised by comparing two expression vectors, pEAQ-HT and pCBP2, and VLPs were also expressed in Escherichia coli. VLPs produced in plants and in E. coli were used to set up a lateral flow device. In addition, various purification methods of VLPs such as ion exchange chromatography (IEC) and sucrose gradient ultracentrifugation were explored to obtain pure VLPs free of bacterial contamination. The VLPs were successfully expressed in N. benthamiana with both pEAQ-HT and pCBP2, and VLPs were subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs. To further purify the VLPs IEC was used, and fully assembled VLPs which were free of contamination were prepared. Purified VLPs expressed in plants and E. coli were successfully used as coating antigen in lateral flow devices, which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera. E. coli-made VLPs showed higher affinity to PCV-2 antibodies compared to plant-made VLPs. In conclusion, this study has successfully demonstrated the potential to use a plantbased transient expression system to produce affordable diagnostic reagent, especially for developing countries. This is the first study that expressed PCV-2 VLPs using a pCBP-2 expression vector and used PCV-2 VLPs as a coating reagent in the development of a lateral flow test as a proof of concept.
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Ober, Rebecca Ariel. "Pre and post-infection microbiome associations with weight gain in pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2)." Thesis, Kansas State University, 2017. http://hdl.handle.net/2097/38431.
Full textAbstract:
Master of ScienceDepartment of Diagnostic Medicine and Pathology
Megan Niederwerder
Evidence has shown that the gastrointestinal microbiome plays an important role in response to infectious disease. Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial microbiome work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70 days post-infection (dpi). However, it remained uncertain if microbiome characteristics could predispose response to viral challenge. The purpose of this study was to determine if microbiome characteristics present at the time of viral challenge were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces on 0 dpi from pigs identified as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced PRRSV replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of the high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. Our results indicate that both microbiome diversity and composition prior to virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection. We followed this study by investigating the microbiome characteristics that are present after co-infection and the role of the microbiome in subclinical infections. Microbiome analysis at 3 and 6 weeks post-infection showed no significant difference between high and low growth rate pigs. The results from both exploring the impact that the initial microbiome has on outcome as well as examining the trends in the microbiome during the post-infection period demonstrate that microbiome pre-infection composition may play a larger role in the outcome of subclinical disease in pigs than microbiome composition during viremia or after viral clearance.
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Rath, Katharina [Verfasser], and Mathias [Akademischer Betreuer] Ritzmann. "Porcine circovirus diseases in drei deutschen Mastbeständen : Auswertung vorangegangener Bestandsdiagnostik sowie diagnostische Querschnittsuntersuchung im Ferkelerzeugerbestand / Katharina Rath ; Betreuer: Mathias Ritzmann." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1211957659/34.
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Turner, Megan Jenny. "Epidemilogical Studies of the Emerging Pig Disease Postweaning Multisystemic Wasting Syndrome (PMWS): The role of Porcine Circovirus Type 2 (PCV2)." Thesis, University of Warwick, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488499.
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Jacela, Jay Yanoria. "Effects of porcine circovirus type 2 vaccination, biofuel co-products, and dietary enzymes on finishing pig performance under field conditions." Diss., Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/2216.
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Tsai, Yi-Chieh, and 蔡依潔. "Immunopathogenesis of Porcine Circovirus Type 2." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/69828844612880150663.
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博士國立臺灣大學
獸醫學研究所
101
Abstract Porcine circovirus type 2 (PCV2) is a small, non-enveloped virus possessing a covalently closed, circular and single-stranded DNA genome. PCV2 has been demonstrated as the major etiologic pathogen of porcine circovirus disease (PCVD) or porcine circovirus-associated disease (PCVAD). PCVD or PCVAD can be subclinical or include 1 or more clinical manifestations of the syndrome. Histopathologically, PCVAD-affected pigs display T- and B-lymphocyte depletion, monocyte (Mo)/macrophage-lineage cell infiltration in lymphoid organs, and altered patterns of cytokine responses. PCV2 infection can remain asymptomatic in pigs for a long period of time by eliciting immune evasion strategies in target cells, which may subsequently lead to the impairment of the host immunity. To elucidate the possible role of PCV2 in the development of PCVAD, first, the PCV2 antigen-containing rate, cell fusion rate, cell migration, chemokine mRNA expression, such as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1), of target cells (Chapter II) were determined to evaluate the formation of granulomatous inflammation by using blood Mos and Mos-derived macrophages (MDMs); second, the phagocytotic and microbial killing capabilities, cytokine profiles (IL-8, TNF-α, and IFN-α) , FasL transcripts (Chapter III), and innate immune response-modulating genes (Chapter IV and V) were investigated in vitro to elucidate how PCV2 alter and dis-regulate the functions of the target cells to facilitate the disease development by using alveolar macrophage (AMs) and blood Mos, respectively. It was found that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PCVAD-affected pigs (Chapter II). Swine AMs infected with PCV2 first then PRRSV later or infected with PCV2 and PRRSV simultaneously displayed marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level (Chapter III). Under the condition of no further treatment, Mos from pigs with subclinical PCV2 infection displayed significantly lower mRNA expression levels in TLR-9, IRF-3, IRF-6, IRF-7, IL-6, IL-12p35, IL-12p40, and interferon (IFN)-α than those from PCV2-free pigs (Chapter IVand V). A broader and/or more obvious spectrum of significant reduction in TLRs, IRFs, IL-12, and IFN-α were observed following PCV2 superinfection (Chapter V) and LPS stimulation (Chapter IV) in vitro. On the contrary, the mRNA expression level of NF-κB was consistently up-regulated with or without PCV2 superinfection (Chapter V) and LPS stimulation (Chapter IV). The evidences discovered in the present study suggest that PCV2 alone could induce formation of the granulomatous inflammation (Chapter II), the subclinically PCV2-infected pigs are actually in an immune dis-regulated status (Chapter IV and V), and many of the functions of PCV2-infected macrophages are altered (Chapter III). All of the above mentioned evidences further support the fact that PCV2-infection predisposes the affected pigs to the secondary bacterial and viral infection and leads to the development of PCVAD.
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Yu, Shan. "Effect of porcine circovirus type 2 on porcine cell populations /." 2006.
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Hung, Ling-Chu, and 洪鈴柱. "Epitope Determination of Porcine Circovirus Type 2." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/3n7s7e.
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博士國立臺灣大學
獸醫學研究所
106
Porcine circovirus 2 (PCV2) is a small, non-enveloped DNA virus causing swine lymphocyte depletion and severe impact on the swine industry. The aim of this study was to evaluate the antigenicity and immunogenicity of specific peptides, and to seek the potential candidate of PCV2 peptide-based vaccine. It was initiating from peptides reacting with PCV2-infected pig sera and peptide-immunized mouse sera. The synthetic peptides (C1, C2, C3, N1, N2, and N3) were analyzed for the binding with field sera collected from PCV2-infected herds. This study involved 22 newborn piglets of TLRI Black Pig No.1 (TBP), delivered from 11 sows during 4 seasons of one year. One male and one female piglet were selected from each litter, which body weights were close to the average of their littermate’s. All of pigs had not been immunized with PCV2 vaccine. Blood samples from each pig were collected 4 times during this experiment: on the 1st day (after colostrum uptake), 1st month, 3rd month, and 6th month of life, respectively. Serum samples from these pigs were used to detect anti-PCV2 specific antibodies by an indirect enzyme-linked immunosorbent assay. We also explored specific peptides could be candidates of immunogen involved during humoral immunity. To demonstrate these peptides can mimic the epitopes present on the native PCV2 CP, we utilized the conjugated peptides to inoculate mice and generate mAbs. We generated mAbs and defined their minimal binding region on PCV2 CP using epitope mapping and liquid phase blocking immunoassay. The data showed that the sera from PCV2-infected pigs could react with the N-terminal (C1), middle region (C2), and C-terminal peptide (C3) of PCV2 capsid protein (CP), ORF3 protein (N1), ORF6 protein (N2) and ORF9 protein (N3). This study demonstrated that anti-PCV2 mouse antisera could be generated by specific synthetic peptides (C3 and N2) and recognized PCV2 viral protein. We found that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the C1 and the C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. The results indicated that these pigs had the highest C3-specific IgA level on Day 1 in 6 months of life (p < 0.01, paired Student’s t-test). These data demonstrated that PCV2-infected pigs had higher OD405 value of anti-C3 IgG on Day 1, Month 3 and Month 6 than that in Month 1 (p < 0.05, paired Student’s t-test). This suggested that suckling newborn piglets absorbed maternal transferring antibodies (C3-specific IgA and IgG) from colostrum and milk in the first 24 h. These pigs had higher anti-C3 IgM level in Month 3 and Month 6 than that on Day 1 (p < 0.01, paired Student’s t-test). This suggested that piglets developed the adaptive immune response by increase synthesis of globulin (C3-specific IgA, IgG, and IgM) at aged 3 months or after weaning. The specific antibody against the C3 were confirmed as the serological marker in PCV2-infected pigs. Further, we utilized the peptide (C3) mimetic carboxyl-terminus (C-terminus) of PCV2b CP (PCV2b-1A/1B) to induce humoral immunity for hybridomas preparation. The positive reactivity of the mAbs to PCV2 CP was demonstrated by western blot assay. Those mAbs also showed positive signals on PCV2b infected swine lymphocytes by indirect immunofluorescence staining. The mAb 1H3 bound to three minimal linear epitopes (P62, DPPLNP; P67, DPPLNPK; P73, LKDPPLKP), which was located at C-terminus of the capsid protein of PCV2b-1A/1B, PCV2b-1C, and PCV2a-2A respectively. The mAbs 3B2 bound to only one minimal linear epitopes (P59, KDPPLNP). The mAbs 6B8 bound to two minimal linear epitopes (P59 and P67). This data demonstrate the core motif (P62) within the P59 could be recognized by mAbs (3B2 and 6B8) in the free status by liquid phase blocking immunoassay (LPBI) but not be recognized in the fixed form on the plate by indirect ELISA (iELISA). However, the P73 could be recognized by mAb 1H3 by iELISA but no inhibition of the interactive binding of C3 and mAb 1H3 by LPBI. This study also indicated that IgM mAbs and defective Ig mAb have broad binding, moderate specificity and low affinity. This study confirm that mAbs have pluripotency of binding. It might be a phenomenon of antibody response to C-terminus of PCV2b CP.
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劉旭展. "Molecular Diagnosis and Gene Analysis of Porcine Circovirus." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/89645731964085126883.
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碩士國立屏東科技大學
獸醫學系
91
Porcine circovirus (PCV) is a single strand DNA virus with circular structure and containing 1.76 Kb. Two types of PCV have been identified and characterized, PCV-1 and PCV-2. PCV-1 is non-pathogenic and PCV-2 is found in pigs with postweaning multisystemic wasting syndrome (PMWS) . Totally 512 pig tissue samples of tonsils, lymph nodes, and spleen were collected from different source of pigs located at centeral and southern Taiwan. Mutiplex PCR was used to detect PCV in porcine samples. The results indicated that the positive of PCV-1 was 11.8% (18/152) and 30.3% (46/152) of PCV-2. Both PCV-1 and PCV-2 positive was 4.0% (61/52) . In this study, the first cases in Taiwan was detected in 1990. Nde I, Kpn I and BstE II was used for restriction fragment length polymorphism (RFLP) assay, and this technicqe was capable of discriminating between PCV-1 and PCV-2. The complete nucleotide sequence of the five PCV-2 isolate in this study was determined and compared with the sequence of PCV strain in GenBank. Sequence comparison revealed significant difference between the PCV-2 strains, showing 95.2~ 99.7% identity. In contrast, sequence comparison between PCV-1 and PCV-2 showed only 76.8~ 77.6% identity. Open reading frame-1(ORF-1) is predicted to encoded a replication-associated protein, and ORF-2 is encoded the major structural protein. ORF-1 was more conserved between the two strains, with 83.0~ 84.0% nucleotide homology and 85.3~ 87.5% amino acid homology. ORF-2 was more variable, with nucleotide homology of 66.3~ 68.1% and amino acid homology of 64.8~ 69.1% . ORF-1 of PCV-2 gene was linked to pET32a vector expressed by bacterial system. The fusion protein showed 53.8 kDa, western blot and staining of 6X His protein can be sure the vallidness of expression.
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Wang, Chun, and 王羣. "Molecular Epidemiological Studies of Porcine Circovirus in Taiwan." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/04838209107440463013.
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博士國立臺灣大學
獸醫學研究所
102
Porcine circovirus type 2 (PCV2) is one of the major swine viral diseases and caused significant economic loss to pig producers worldwide, including Taiwan. PCV2 has been considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) as well as other clinical diseases. All these associated syndromes have been categorized as PCV2 associated diseases (PCVAD). PCV2 infection is commonly present in pig farms in Taiwan; however, the information regarding evolution and phylogenic analysis of PCV2 in this area is rare. In addition, application of loop-mediated isothermal amplification (LAMP) in PCV infection, including commonly PK cell line-contaminated PCV1, has rarely been reported. The purposes of this thesis were to perform molecular epidemiological studies of PCV2 in Taiwan and establish a novel diagnostic tool, LAMP, to detect PCV. In the first study, complete genomes of PCV2 were amplified by polymerase chain reaction (PCR) and sequenced directly from 571 Taiwanese PCV2 isolates collected during the period of 2001 and 2011. The phylogenetic tree analysis indicated that these isolates could be divided into 2 distinct genotypes, PCV2a and PCV2b. Among the 571 isolates, 131 isolates were clustered within genotype PCV2a (further classified as 2B, 2D, and 2E), and 440 isolates were clustered within genotype PCV2b (further classified as 1A, 1B, and 1C). PCV2a genotype predominated in 2001, and then PCV2b became the major prevalent genotype since 2003. In the second study, a LAMP method with a real-time monitoring system was developed based on open reading frame 2 (ORF2) in the viral genome for the simultaneous detection and differentiation of PCV2a and PCV2b in clinical samples. The LAMP reaction could be completed within 50 min, and the PCV2a and PCV2b could be detected and accurately distinguished without cross-reaction. The detection limit of the LAMP were 10 copies/μl of the PCV2a and PCV2b recombinant plasmids, demonstrating detection limit comparable to that obtained using nested polymerase chain reaction (nested PCR). On the basis of the results of 168 clinical samples using nested PCR as the gold standard, the relative sensitivity of LAMP was 97.7% and the relative specificity was 100%. Thus, LAMP can be a simple, rapid and valuable tool for the differential diagnosis of PCV2a and PCV2b. In the third study, we have developed a LAMP method with a real-time monitoring system for the detection of PCV type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1, no cross-reaction to other common swine viral pathogens was observed and the LAMP reaction could be completed within one hour. The detection limit of the LAMP for PCV1 DNA was 10 copies/μl of the positive recombinant plasmid, comparable to that obtained from nested PCR. In addition, 25 commercial swine vaccines were tested by both LAMP and nested PCR, and PCV1 DNA was detected in 3/25 commercial swine vaccines (11%) by either method. The LAMP method holds promise for using as a highly specific, sensitive, and rapid diagnostic assay for PCV1 DNA detection in commercial swine vaccines.
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Lin, Chun-Ming, and 林俊明. "The Susceptibility of Swine Lymphocytes to Porcine Circovirus Type Ⅱ." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/33139713521888108081.
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碩士國立臺灣大學
獸醫學研究所
94
The porcine circovirus type Ⅱ (PCV2)-associated newly emerged disease, postweaning multisystemic wasting syndrome (PMWS), is characterized by lymphoid depletion in lymphoid organs and replacement by macrophages. Until now, whether PCV2 replicates in immune system and the pathogenesis of PMWS are still not well understood. In this study, we established a model of PCV2 infection in mitogen-treated peripheral blood lymphocytes (PBL). In the first part of the study, it was demonstrated that there was only 0.1% of PCV2 antigen-containing rate in PBL isolated from PCV2-free pigs and treated simultaneously with mitogen and PCV2. The results of both MTT assay and blastogenesis indicated that PCV2 could reduce the responsiveness of PBL to mitogen stimulation, but the results of survival and apoptotic rates indicated that PCV2 had no effects on the viability of PBL. In the second part of study, it was found PCV2 nucleic acid and antigens could be more easily detected in mitogen-stimulated PBL from clinically healthy PCV2-carrier pigs by in situ hybridization and immunofluorescence analysis, respectively. After 4 days of mitogen treatment, the PCV2 antigen-containing rate in PBL could reach 20% with IgM-positive cells predominant. The mitogen-stimulated PBL had higher PCV2 titer. Additionally, PCV2 ORF1-associated protein was usually present perinuclearly or within the nuclei; ORF2-associated protein was chiefly restricted in the cytoplasm; the expression of ORF1-associated protein, but not ORF2, as well matched with TUNEL signal. Results of this study indicate that PBL are indeed susceptible to PCV2 infection and PCV2 is capable to replicate in dividing lymphocytes. This replication may play a role on PCV2-induced apoptosis in PBL. The result may explain in part the pathogenesis of PMWS.
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Chen, Hsu-Chung Gabriel, and 陳旭中. "Studies on the Vaccine Technology Developmentof Porcine Circovirus Type 2." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/40735549156225176387.
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博士國立臺灣大學
獸醫學研究所
101
Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus type 2 associated diseases (PCVAD). PCVAD causes huge economical losses in swine productions worldwide. Preventive methods of PCVAD include biosecurity, proper pig farm management, and vaccination. Up-to-date, there are four companies marketing three kinds of commercialized PCV2 vaccines which are PCV2 subunit vaccines, killed PCV1-2 vectored vaccine, and whole viral inactivated vaccine. Since PCV2 does not cause cytopathic effect (CPE) in PK-15 cells, thus detection of PCV2 has relied on immunofluorescence assays (IFA). In this thesis, creating an enhanced green fluorescent protein (EGFP) fused PCV2 could serve as a convenient diagnostic tool for measuring PCV2 titer or neutralizing antibodies. Another major purpose of this thesis was to enhance PCV2 vaccines through various developments in vaccine technology including the improvement of subunit vaccine, DNA vaccine, and full virus inactivated vaccine. In the fist study, construction and transfection of EGFP-fused PCV2 genome in pBluescript (pSK) vector and the recovery of the virus was described. When fusing EGFP downstream of five open reading frames (ORF) of PCV2, green fluorescent signals were observed in the nucleus of PK-15 cells only after PCV2 (ORF2)-EGFP/pSK transfection while PCV2 (ORF1)-EGFP/pSK, PCV2 (ORF3)-EGFP/pSK, PCV2 (ORF4)-EGFP/pSK, and PCV2(ORF5)-EGFP/pSK showed no fluorescent signals post-transfection. The presence of ORF2-EGFP fusion protein was demonstrated by dual signals of green fluorescence and anti-PCV2 antibodies conjugated with rhodamine in immunofluorescence assay (IFA). Furthermore, the released EGFP-fused PCV2 genome was quantified by real-time PCR for two more passages. In the second study, five different fragments of PCV2 ORF2 antigenic regions were cloned, over-expressed in E. coli, and immunized in rats to evaluate the immunogenicity of the PCV2 ORF2 recombinant proteins. Results showed that the ORF2 F2 fragment (residues 78-156) was the most immunogenic in terms of the antibody induction. Detoxified Pseudomonas exotoxin A (PE) and KDEL signal peptide were fused with F2 fragment at the N and C terminus, respectively. This study evaluated the application of using the binding and translocation domains of PE as a vehicle for PCV2 F2 fragment, resulting in the construction of a PE-F2-KDEL recombinant protein. F2 and PE-F2-KDEL recombinant proteins were intraperitoneally inoculated in mice, respectively. Results showed that PE-F2-KDEL induced significantly higher anti-PCV2 serum IgG antibodies than F2. Additionally, PE-F2-KDEL recombinant protein also stimulated significantly higher PCV2-specific IgG response compared to inactivated PCV2 whole virus antigen. These results showed that detoxified Pseudomonas exotoxin A could enhance the immune response of a PCV2 ORF2 recombinant protein. The third goal of the thesis was to construct and express a multiple function fusion protein to enhance intracellular delivery for PCV2 DNA vaccine. DNA vaccines are limited in clinical and practical uses due to its ineffective delivery. In this research study, the multiple function protein VP22-TmHU-PCV2.NLS was composed of cell-penetrating peptide originated from VP22 peptide of herpes simplex virus, DNA binding HU protein from Thermotoga maritima (TmHU), and a PCV2 nuclear localization signal (NLS). Firstly, as shown by the electrophoretic mobility shift assay (EMSA), VP22-TmHU (VT) and VP22-TmHU-PCV2.NLS (VTN) were able to bind to plasmid DNA (pEGFP-N1) effectively. Secondly, intracellular transport of pEGFP-N1 was observed by fluorescence microscopy and quantified by flow cytometry after transfection. VTN was successful in delivering pEGFP-N1 intracellularly but VT was not. Thirdly, when combined with Lipofectamine™, both VT and VTN enhanced the transfection rate from 25% with Lipofectamine™ alone up to 65%. Lastly, mice were injected intramuscularly with PBS, pcDNA3-ORF2, pcDNA3-ORF2 plus Lipofectamine™, pcDNA3-ORF2 plus VT, pcDNA3-ORF2 plus VT plus Lipofectamine™, pcDNA3-ORF2 plus VTN, and pcDNA3-ORF2 plus VTN plus Lipofectamine™. The highest level of antibodies raised against PCV2 ORF2 Cap protein was detected with pcDNA3-ORF2 plus VTN. Contrary to the in vitro results, VTN combined with Lipofectamine™ did not further enhance antibody level against PCV2 ORF2. In summary, the NLS of PCV2 ORF2 can enhance intracellular delivery of plasmid DNA. The last study of this thesis established a highly permissive and decontaminated cell line for growing porcine circovirus type 2 (PCV2). A porcine kidney-15 cell line (PK-15) contaminated with porcine circovirus type 1 (PCV1) was decontaminated by neutralizing with rabbit anti-PCV1 hyperimmune serum. Subsequently, by limiting dilution and cell subcloning, four PCV1-free monoclonal cells were grown to monolayers. Each cell subclones and PK-15 cell were infected with PCV2. The PKKC cell clone yielded up to 106.8 TCID50/ml at six days post-infection. In addition, PKKC was free of extraneous viral contamination and exhibited a cytopathic effect (CPE) to PCV2 at six days post-infection. The advantages of the PKKC cell are that it can grow a high PCV2 titer and exhibit CPE; therefore, it can be used for PCV2 cultivation, vaccine production, and diagnostic purposes.
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林郁宸. "Development of Porcine Circovirus type 2 and Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) subunit vaccine." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/67882811482427057094.
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