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.

La síndrome d'aprimament post-deslletament (SAPD) és considerada com una malaltia porcina d'origen multifactorial en la qual el Circovirus porcí tipus 2 (CP2) és l'agent infecciós essencial. L'objectiu de la present tesi doctoral era expandir el coneixement en l'epidemiologia de la infecció per CP2 i el SAPD a través de la realització d'estudis de cas-control a nivell de camp. De manera general, es va investigar la potencial influència de la genètica del CP2, el moment d'infecció del CP2, els anticossos contra CP2 derivats de la immunitat maternal i la resposta humoral dels porcs contra el CP2, en la presentació de la SAPD.En el primer estudi (estudi I) es van estudiar seqüències de PCV2 de porcs amb diferent condició clínica i patològica. Els resultats van confirmar l'existència de dos genogrups principals i es va proposar la definició de dos genotipus de CP2 (1 i 2). La metodologia suggerida per definir els genotipus està basada en la distribució de la relació p-distància/freqüència de les seqüències de CP2 conjuntament amb anàlisis filogenètics de CP2. Es va observar que el genotipus 1 era predominant en porcs de granges afectades per SAPD. Contràriament, totes les seqüències obtingudes de granges no afectades per la SAPD corresponien al genotipus 2. Així, es va suggerir que el genotipus 1 de CP2 podria ser potencialment més patogènic que el genotipus 2. Addicionalment, es va descriure la presència dels dos genotipus en el mateix moment en porcs individuals procedents de granges afectades per la SAPD.La present tesi doctoral es va desenvolupar dins del marc del projecte de la Unió Europea (UE) titulat Control de les malalties associades al Circovirus porcí (MACP): Cap a la Millora en la Qualitat i Seguretat Alimentàries (www.pcvd.org), la qual era finançada pel 6è programa marc de la UE. El consorci de la UE d'aquest porjecte va publicar la carta presentada com a addendum de l'estudi I. En aquesta carta es va donar suport a la definició de genotipus proposada, a la vegada que es va proposar la nomenclatura de genotipus a de CP2 (CP2a) i genotipus b (CP2b), corresponent als genotipus 2 i 1, respectivament, per tal d'evitar confusions amb la ja existent diferenciació entre PCV2 i PCV1. En el segon estudi (estudi II) es van comparar dues tècniques de PCR quantitativa (qPCR) de PCV2. Els resultats mostraven una associació lineal significativa entre les dues tècniques, i un biaix sistemàtic de 1.4 log10 copies of CP2 per mil·lilitre de mostra. Aquesta diferència indicava que la tècnica del laboratori danès generava un resultat sistemàticament més elevat que el generat a través de la tècnica del laboratori espanyol. A més, la tècnica del laboratori danès mostrava major sensibilitat que la tècnica del laboratori espanyol. En els treballs III i IV es van realitzar estudis longitudinals de tipus cas-control en granges afectades per la SAPD de Dinamarca i Espanya tot utilitzant dissenys similars. Es van observar patrons similars en la dinàmica d'infecció per CP2 tant a Espanya com a Dinamarca, amb un retard en l'edat de presentació de la SAPD a Espanya en comparació a Dinamarca. El diagnòstic individual de la SAPD es va confirmar, mitjançant tests laboratorials, en només la meitat dels porcs en els quals hi havia la sospita clínica. Globalment, els resultats van mostrar que la quantitat de CP2 incrementava concomitantment a la caiguda dels nivells d'anticossos maternals, assolint valors màxims de càrrega vírica en el moment d'aparició dels símptomes clínics. De manera interessant, la reacció de fase aguda (RFA) en porcs afectats per la SAPD s'observava paral·lelament a l'evolució de la virèmia per CP2, suggerint que el CP2 és el principal responsable de l'estat d'inflamació sistèmica que pateixen els porcs afectats per aquesta malaltia. Col·lectivament, els porcs afectats tenien quantitats de CP2 i concentracions de porc-MFA i HPT superiors en sang, excretaven càrregues víriques superiors tant per via nasal com a través de les femtes, i tenien nivells inferiors d'anticossos maternals enfront al CP2 que els porcs que no estaven afectats per la SAPD. Addicionalment, es va observar una menor resposta humoral en els porcs afectats per la SAPD provinents d'Espanya a les 11 setmanes de vida (abans de l'aparició dels símptomes clínics) i en el moment de la necròpsia, suggerint que aquesta circumstància era podria associar-se com a causa més que no pas una conseqüència de la malaltia. D'altra banda, la falta de sensibilitat i/o especificitat observades de les tècniques de qPCR i/o de serologia suggereixen que aquestes tècniques no poden substituir la histopatologia més la detecció de CP2 en teixits per l'establiment del diagnòstic individual de la SAPD. Malgrat això, els resultats indicaven que la qPCR podria ser potencialment útil per diagnosticar la SAPD a nivell poblacional. Addicionalment, els resultats obtinguts donaven suport a la idea que, malgrat que les PFA són marcadors inespecífics d'inflamació, aquestes proteïnes podrien ser marcadors útils de salut, esdevenint una eina potencialment útil per monitoritzar el desenvolupament de la SAPD en estudis epidemiològics o per la valoració de l'eficàcia de vacunes de CP2 a nivell de camp. / Postweaning multisystemic wasting syndrome (PMWS) is considered a multifactorial pig disease in which Porcine circovirus type 2 (PCV2) is the essential infectious agent. The present thesis aimed to expand the epidemiological knowledge on PCV2 infection and PMWS through the realization of case-control field studies. Mainly, the potential influence of PCV2 genetics, the timing of PCV2 infection, the PCV2 maternal derived humoral immunity and the pig humoral response against PCV2 infection in PMWS presentation were investigated.In the first study consisted in the sudy of PCV2 sequences obtained from pigs with different clinical and pathological conditions. Results further confirmed the existence of two main genogroups and the definition of two PCV2 genotypes (1 and 2) was proposed. The suggested methodology to define PCV2 genotypes is based on the p-distance/frequency distribution of PCV2 sequences together with PCV2 phylogenetic analyses. Genotype 1 was shown to be predominant within pigs coming from PMWS affected farms, while all sequences obtained from non-PMWS affected farms corresponded to genotype 2. Consequently, it was suggested that PCV2 genotype 1 might potentially be more pathogenic than PCV2 genotype 2. In addition, infection of single pigs from PMWS affected farms harbouring both genotypes at the same time was described. The present thesis was developed within the European Union (EU) project entitled Control of Porcine Circovirus Diseases (PCVD): Towards Improved Food Quality and Safety (www.pcvd.org), which was funded by the EU Sixth Framework Programme. The EU consortium on PCVD published the letter here presented as an addendum of study I. In this letter it was supported the genotype definition, but it was also proposed the nomenclature of PCV2 genotype a (PCV2a) and genotype b (PCV2b), corresponding to genotypes 2 and 1, respectively, in order to avoid potential confusions with the already existent PCV2 and PCV1. In the second study (Study II) two different real-time quantitative PCR (qPCR) assays were compared. Results showed a significant linear association between the assays, and a systematic difference of 1.4 log10 copies of PCV2 per millilitre of sample. This difference indicated that the assay from the Danish laboratory yielded a higher output than the assay from the Spanish laboratory. Moreover, the Danish assay had higher sensitivity than the Spanish one. In studies III and IV, longitudinal case-control studies were performed in PMWS affected farms from Denmark and Spain using similar designs. Similar PCV2 infection dynamic patterns were observed in Spain and Denmark, with a delay in PMWS age-presentation in Spain compared to the one in Denmark. Results showed that PCV2 load increased concomitantly to maternal antibody level waning, reaching the maximum viral load concurrently with the development of clinical signs. Interestingly, the acute phase response (APR) in PMWS affected pigs occurred in parallel to PCV2 viremia, suggesting that PCV2 is the main responsible for the systemic inflammatory status suffered by diseased pigs. As a collective, PMWS affected pigs harboured higher PCV2 loads and higher Pig-MAP and HPT concentrations in sera, shed higher viral loads through both nasal secretions and faeces, and had lower level of maternal antibodies against PCV2 than non-PMWS affected pigs. Furthermore, an impaired humoral response was observed in PMWS affected pigs from Spain at 11 weeks of age (prior to the appearence of clinical signs) and at the moment of necropsy, suggesting that this circumstance might be more a cause rather than a consequence of the disease. On the other hand, the lack of sensitivity and/or specificity observed from qPCR and/or serological techniques suggests that those techniques are not able to substitute histopathology plus detection of PCV2 in tissues for the individual PMWS diagnosis. However, results indicated that qPCR might potentially be a reliable technique to diagnose PMWS on a population basis. Additionally, obtained results supported the idea that although APPs are unspecific markers of inflammation, they might be useful indicators of health, becoming a potentially interesting tool to monitor PMWS development in epidemiological studies or in the assessment of the efficacy of PCV2 vaccines in the field.

The circovirus Beak and feather disease virus (BFDV) causes psittacine beak and feather disease (PBFD) that is characterised by a chronic disease process associated with feather abnormalities, beak deformities and eventual death in various species of birds in the order Psittaciformes. This disease is seen in captive and wild psittacine species in Australia and several other countries and is a significant threat to the survival of some endangered psittacine species. This thesis reports on genetic studies that have furthered the understanding of the diversity of BFDV present within Australia. These studies have optimised methods of detecting BFDV. They have also resulted in the production of an immunogenic and antigenic recombinant BFDV Capsid protein that could lead to alternate methods of producing viral antigen for serological tests and the development of a BFDV vaccine. To assess the optimal method of the detection of BFDV infection, feather and blood samples were submitted by referring veterinarians throughout Australia from psittacine birds tentatively diagnosed with PBFD or with a history of being in contact with PBFD-affected birds. These samples were examined by 3 procedures commonly used to detect BFDV infection: a polymerase chain reaction (PCR) assay and haemagglutination (HA) for the detection of virus, and haemagglutination inhibition (HI) tests for the detection of virus antibody in response to infection. Of the samples examined from 623 psittacine birds, the prevalence of BFDV DNA in feather samples detected by PCR was 18.85%. There was a strong correlation between PCR and HA testing of feather samples, although possible false-positive and false-negative PCR and HA results were obtained in some samples. Of the 143 birds that were PCR feather-positive only 2 had detectable HI antibody and these birds were also HA feather-negative, which suggests that they were developing immunity to recent infection. All birds with HI antibody were feather HA negative. Despite the rare occurrence of PBFD in cockatiels (Nymphicus hollandicus), 2 of the 13 samples collected from this species were PCR and HA positive indicating that this species can be infected with BFDV. Three studies were undertaken to further our understanding of the genetics of BFDV in Australian avifauna: sequence analysis of the BFDV detected in a grey cockatiel (Nymphicus hollandicus), a species normally considered resistant to infection with BFDV; analysis of the genome of BFDV present in lorikeets (Trichoglossus sp.) in Australia; and analysis of the genome of BFDV detected in endangered swift parrots (Lathamus discolor). Sequence analysis of the entire genome of the cockatiel BFDV isolate revealed that it clustered phylogenetically with 2 other viruses, one from a sulphur crested cockatoo (SCC1-AUS) and one from a Major Mitchell cockatoo (MMC-AUS), which suggests that this isolate from the grey cockatiel was not a cockatiel-specific biotype. Phylogenetic analysis of the ORF V1 of BFDV detected in 7 lorikeets demonstrated these 7 isolates clustered phylogenetically with other BFDV isolates obtained from Loriidae species elsewhere in the world and confirmed the presence of a loriid-specific genotype. Phylogenetic analysis of the sequence data generated from ORF V1 of virus detected in 2 endangered swift parrots provided evidence they were also infected with BFDV genotypes derived from other species of birds, one isolate clustering with viruses from a Loriidae genotype and the other with isolates derived from species of Cacatuidae and Psittacidae. As part of this research, a baculovirus expression system was successfully developed for the production of recombinant BFDV Capsid protein. Inoculation of this protein into chickens resulted in the development of HI antibody, which demonstrated its immunogenicity. When used as an antigen in HI tests it detected antibody in virus-infected birds, which demonstrated its antigenicity. This protein offers potential application as an antigen for the development of serological tests and as an immunogen for incorporation into vaccines for control of PBFD.

Zugl.: Berlin, Freie Univ., Diss., 2006 / Dateiformat: zip, Dateien im PDF-Format

Freie Universiẗat, Diss., 2006--Berlin. / Dateiformat: zip, Dateien im PDF-Format.

O presente estudo foi realizado com o objetivo de identificar genomas de vírus em fezes de lobos marinhos sul-americanos (Arctocephalus australis) e lobos marinhos subantárticos (Arctocephalus tropicalis), duas espécies de pinípedes encontradas no litoral do Rio Grande do Sul. Embora já existam estudos sobre esse tema em outras espécies de pinípedes, nas espécies aqui trabalhadas o tema permanece inexplorado. Amostras de fezes foram obtidas de vinte e um lobos marinhos sul-americanos e dois lobos marinhos subantárticos encontrados no litoral rio-grandense com indícios de morte recente, durante os meses de Junho e Julho de 2012. Através de técnicas de PCR e sequenciamento buscou-se identificar genomas de circovírus, adenovírus, morbilivírus, calicivírus e coronavírus. A amplificação de um fragmento do gene rep permitiu a identificação de prováveis circovírus em amostras de seis lobos marinhos sul-americanos. Análises filogenéticas revelaram que três dos seis segmentos são sugestivos de prováveis membros do gênero Cyclovirus. Os genes amplificados de outras duas amostras provavelmente correspondem a membros do gênero Circovirus. Uma das amostras deu origem a um segmento gênico que não apresenta similaridade com nenhum gênero já proposto da família Circoviridae. Além disso, foi possível detectar também fragmentos de genomas de adenovírus em duas amostras; estes apresentam alto grau de similaridade de nucleotídeos com amostras de adenovírus humano tipo C. Nenhum fragmento genômico indicativo da presença de morbilivírus, calicivírus ou coronavírus foi encontrado. Os resultados aqui obtidos sugerem a presença de circovírus, ciclovírus e adenovírus em populações de lobos marinhos encontrados na costa do Rio Grande do Sul. Estes achados reforçam a necessidade da ampliação do conhecimento a respeito da ocorrência de infecções virais nestas espécies. / This study was conducted with the objective of identifying genomes of viruses in feces of south american fur seals (Arctocephalus australis) and subantarctic fur seals (Arctocephalus tropicalis), two species of pinnipeds found on the coast of Rio Grande do Sul. Although there are studies about this topic in other species of pinnipeds, it remains unexplored in these two species. Stool samples were obtained from twenty-one south american fur seals and two subantarctic fur seals found in Rio Grande do Sul coastline with evidences of recent death, during the months of June and July 2012. PCR and sequencing techniques were utilized to identify circovirus, adenovirus, morbillivirus, calicivirus and coronavirus genomes. The amplification of a rep gene fragment allowed the identification of supposed circoviruses in samples of six south american fur seals. Phylogenetic analysis revealed that three of the six segments are suggestive of probable members of the genus Cyclovirus. The amplified genes from two other samples probably correspond to members of the genus Circovirus. One of the samples gave rise to a gene segment that has no similarity with any genera already proposed of the Circoviridae family. Furthermore, it was also possible to detect fragments of adenovirus genomes in two samples: these have a high degree of nucleotide similarity with a human adenovirus type C genomic fragment. No indication of the presence of morbillivirus, calicivirus and coronavirus genomes was found. The work reported here provide evidence for the occurrence of circoviruses, cicloviruses and adenoviruses in fur seal populations found in Rio Grande do Sul. These findings reinforce the need to expand the knowledge about the occurrence of viral infections in these species.

O presente estudo foi realizado com o objetivo de identificar genomas de vírus em fezes de lobos marinhos sul-americanos (Arctocephalus australis) e lobos marinhos subantárticos (Arctocephalus tropicalis), duas espécies de pinípedes encontradas no litoral do Rio Grande do Sul. Embora já existam estudos sobre esse tema em outras espécies de pinípedes, nas espécies aqui trabalhadas o tema permanece inexplorado. Amostras de fezes foram obtidas de vinte e um lobos marinhos sul-americanos e dois lobos marinhos subantárticos encontrados no litoral rio-grandense com indícios de morte recente, durante os meses de Junho e Julho de 2012. Através de técnicas de PCR e sequenciamento buscou-se identificar genomas de circovírus, adenovírus, morbilivírus, calicivírus e coronavírus. A amplificação de um fragmento do gene rep permitiu a identificação de prováveis circovírus em amostras de seis lobos marinhos sul-americanos. Análises filogenéticas revelaram que três dos seis segmentos são sugestivos de prováveis membros do gênero Cyclovirus. Os genes amplificados de outras duas amostras provavelmente correspondem a membros do gênero Circovirus. Uma das amostras deu origem a um segmento gênico que não apresenta similaridade com nenhum gênero já proposto da família Circoviridae. Além disso, foi possível detectar também fragmentos de genomas de adenovírus em duas amostras; estes apresentam alto grau de similaridade de nucleotídeos com amostras de adenovírus humano tipo C. Nenhum fragmento genômico indicativo da presença de morbilivírus, calicivírus ou coronavírus foi encontrado. Os resultados aqui obtidos sugerem a presença de circovírus, ciclovírus e adenovírus em populações de lobos marinhos encontrados na costa do Rio Grande do Sul. Estes achados reforçam a necessidade da ampliação do conhecimento a respeito da ocorrência de infecções virais nestas espécies. / This study was conducted with the objective of identifying genomes of viruses in feces of south american fur seals (Arctocephalus australis) and subantarctic fur seals (Arctocephalus tropicalis), two species of pinnipeds found on the coast of Rio Grande do Sul. Although there are studies about this topic in other species of pinnipeds, it remains unexplored in these two species. Stool samples were obtained from twenty-one south american fur seals and two subantarctic fur seals found in Rio Grande do Sul coastline with evidences of recent death, during the months of June and July 2012. PCR and sequencing techniques were utilized to identify circovirus, adenovirus, morbillivirus, calicivirus and coronavirus genomes. The amplification of a rep gene fragment allowed the identification of supposed circoviruses in samples of six south american fur seals. Phylogenetic analysis revealed that three of the six segments are suggestive of probable members of the genus Cyclovirus. The amplified genes from two other samples probably correspond to members of the genus Circovirus. One of the samples gave rise to a gene segment that has no similarity with any genera already proposed of the Circoviridae family. Furthermore, it was also possible to detect fragments of adenovirus genomes in two samples: these have a high degree of nucleotide similarity with a human adenovirus type C genomic fragment. No indication of the presence of morbillivirus, calicivirus and coronavirus genomes was found. The work reported here provide evidence for the occurrence of circoviruses, cicloviruses and adenoviruses in fur seal populations found in Rio Grande do Sul. These findings reinforce the need to expand the knowledge about the occurrence of viral infections in these species.

O presente estudo foi realizado com o objetivo de identificar genomas de vírus em fezes de lobos marinhos sul-americanos (Arctocephalus australis) e lobos marinhos subantárticos (Arctocephalus tropicalis), duas espécies de pinípedes encontradas no litoral do Rio Grande do Sul. Embora já existam estudos sobre esse tema em outras espécies de pinípedes, nas espécies aqui trabalhadas o tema permanece inexplorado. Amostras de fezes foram obtidas de vinte e um lobos marinhos sul-americanos e dois lobos marinhos subantárticos encontrados no litoral rio-grandense com indícios de morte recente, durante os meses de Junho e Julho de 2012. Através de técnicas de PCR e sequenciamento buscou-se identificar genomas de circovírus, adenovírus, morbilivírus, calicivírus e coronavírus. A amplificação de um fragmento do gene rep permitiu a identificação de prováveis circovírus em amostras de seis lobos marinhos sul-americanos. Análises filogenéticas revelaram que três dos seis segmentos são sugestivos de prováveis membros do gênero Cyclovirus. Os genes amplificados de outras duas amostras provavelmente correspondem a membros do gênero Circovirus. Uma das amostras deu origem a um segmento gênico que não apresenta similaridade com nenhum gênero já proposto da família Circoviridae. Além disso, foi possível detectar também fragmentos de genomas de adenovírus em duas amostras; estes apresentam alto grau de similaridade de nucleotídeos com amostras de adenovírus humano tipo C. Nenhum fragmento genômico indicativo da presença de morbilivírus, calicivírus ou coronavírus foi encontrado. Os resultados aqui obtidos sugerem a presença de circovírus, ciclovírus e adenovírus em populações de lobos marinhos encontrados na costa do Rio Grande do Sul. Estes achados reforçam a necessidade da ampliação do conhecimento a respeito da ocorrência de infecções virais nestas espécies. / This study was conducted with the objective of identifying genomes of viruses in feces of south american fur seals (Arctocephalus australis) and subantarctic fur seals (Arctocephalus tropicalis), two species of pinnipeds found on the coast of Rio Grande do Sul. Although there are studies about this topic in other species of pinnipeds, it remains unexplored in these two species. Stool samples were obtained from twenty-one south american fur seals and two subantarctic fur seals found in Rio Grande do Sul coastline with evidences of recent death, during the months of June and July 2012. PCR and sequencing techniques were utilized to identify circovirus, adenovirus, morbillivirus, calicivirus and coronavirus genomes. The amplification of a rep gene fragment allowed the identification of supposed circoviruses in samples of six south american fur seals. Phylogenetic analysis revealed that three of the six segments are suggestive of probable members of the genus Cyclovirus. The amplified genes from two other samples probably correspond to members of the genus Circovirus. One of the samples gave rise to a gene segment that has no similarity with any genera already proposed of the Circoviridae family. Furthermore, it was also possible to detect fragments of adenovirus genomes in two samples: these have a high degree of nucleotide similarity with a human adenovirus type C genomic fragment. No indication of the presence of morbillivirus, calicivirus and coronavirus genomes was found. The work reported here provide evidence for the occurrence of circoviruses, cicloviruses and adenoviruses in fur seal populations found in Rio Grande do Sul. These findings reinforce the need to expand the knowledge about the occurrence of viral infections in these species.

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

Doctor of Philosophy / Department 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.