Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Pestiviruses such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) are some of the most economically important livestock diseases in the world. The antigenic similarities between members of the pestivirus genus allow for both BVDV and CSFV to infect swine. Infections with heterologous pestiviruses in swine can interfere with diagnostic tests for CSFV. The identification of cross-reactive and cross-neutralizing epitopes between CSFV and BVDV for the development of improved diagnostics and vaccines that allow for the differentiation of infected animals from vaccinated animals (DIVAs) are necessary to accurately detect and control CSFV. The overall goal of this research was to identify epitope regions recognized by antibodies that can differentiate between CSFV and BVDV. The approach was to use serum neutralization assays to confirm the presence of neutralizing antibodies to BVDV in swine serum collected from animals immunized with one of three separate Alphavirus vaccine constructs: BVDV-1b, CSFV E2, and CSFV E[superscript]rns. Results showed that animals immunized with the Alphavirus BVDV-1b construct had high neutralizing titers against BVDV-1a and animals immunized with Alphavirus CSFV E2 and E[superscript]rns constructs had low, but detectable, neutralizing activity. Polypeptide fragments of CSFV and BVDV E2 were then expressed in E. coli and purified using affinity chromatography. Serum from a pig immunized with the CSFV E2 Alphavirus construct was tested against two fragments of CSFV E2, 2/4 and 4/4, and four fragments BVDV E2, 1/4, 2/4, 3/4, and 4/4, using western blot analysis. Reactivity to fragments CSFV E2 2/4 and 4/4 and BVDV E2 1/4 and 4/4 was observed. The results of this study identified CSFV amino acid positions 774 through 857 and BVDV amino acid positions 783 through 872 as the regions that contain the epitopes recognized by cross-reactive antibodies between BVDV and CSFV E2. These results provide more specific sequence regions to improve CSFV diagnostic assays and DIVA vaccines.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Classical swine fever virus (CSFV) is an enveloped, positive strand RNA virus, and member of the genus Pestivirus. It is a highly infectious and transmissible swine pathogen that threatens the global swine industry. The United States has been free of CSFV since 1977, however, monitoring the millions of domestic and feral pigs present in the US puts a significant strain on national surveillance efforts. There are no validated diagnostic techniques that can simultaneously sample multiple pigs (i.e. all pigs in a pen or barn). Similarly, there are no validated serological assays that can quickly test for CSFV without cross-reacting with other pestiviruses. The purpose of the first study was to establish a moderate CSFV-infectious model and determine how a single oral fluid sample from a pen of pigs can function as a diagnostic sample for detecting CSFV. Oral fluid (OF) and serum samples were collected from 10 pigs experimentally infected with CSFV Paderborn strain. Using RT-PCR, CSFV was detected in OF on 8 days post infection (dpi), and in the serum of one pig on 6 dpi. A single OF sample can, therefore, take the place of 10 serum samples to detect CSFV in a population. In a second study, monoclonal antibodies reactive to CSFV glycoproteins were generated in mice immunized with recombinant E2 and Erns antigens. Five E2-specific clones and two Erns-specific clones showed reactivity to CSFV-infected. Epitope mapping of the E2 clones showed that all reacted with the N-terminal portion of E2; a region highly variable among pestiviruses. Together with OF sampling, monoclonal antibodies can be used to develop new tools for improving CSF surveillance in large swine populations.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-20T13:40:24Z No. of bitstreams: 1 Marta Maria Nery Farias da Silva.pdf: 1335797 bytes, checksum: f15d589ae6541e086990082ab2a9ed97 (MD5) / Made available in DSpace on 2016-10-20T13:40:24Z (GMT). No. of bitstreams: 1 Marta Maria Nery Farias da Silva.pdf: 1335797 bytes, checksum: f15d589ae6541e086990082ab2a9ed97 (MD5) Previous issue date: 2012-02-20 / The classical swine fever virus causes a highly contagious disease in domestic and wild pigs, characterized by leading to great economic losses, for both large and small farming. The disease may present as acute, chronic, delayed, or persistent asymptomatic, which depend on the virulence of the strain, animal age, immune status, gestation period. The disease is notifiable and control measures are listed in the National Program to Swine Health from Ministry of Agriculture, Livestock and Supply. Brazil has an area considered as free of classical swine fever which is composed of the major part of states and non-free zone, which has the majority of states in the North and Northeast Brazil. Outside the area considered free of classical swine fever outbreaks continue to occur sporadically, but the proposal of the Ministry of Agriculture, Livestock and Supply is to eradicate the disease throughout the country. The diagnosis of infection may be done by viral isolation and virus identification in cell culture, wich is considered the gold standard, using the immunofluorescence and immunoperoxidase technics. Samples were analyzed by this methods in the National Agriculture and Livestock Laboratory of Pernambuco State, from 149 swines with clinical suspicion of classical swine fever during the period from 1999 to 2009, from the states of Pará and Amapá – North region, and Maranhão, Ceará, Rio Grande do Norte, Paraíba e Pernambuco of Northeast region, Among the swines analyzed, 51 were infected with the classical swine fever virus. The necropsial findings were mostly hemorragic, tipical of the classical form of the disease. The afected farms were in most low-level technificated. The results showed that the presence of positive animals is high, with the agent present in most states in the Northeast and North, and that the application of effective preventive measures in the field can reduce or prevent the emergence of cases and outbreaks of disease. / O vírus da peste suína clássica causa doença altamente contagiosa em suínos domésticos e selvagens, caracterizada por levar a grandes perdas econômicas, tanto para a suinocultura de grande porte como também para os pequenos produtores. A doença pode se apresentar de forma aguda, crônica, tardia, assintomática ou persistente, que dependem da virulência da cepa, idade do animal, estado imunológico, período de gestação. A doença é de notificação obrigatória, e as medidas de controle estão listadas no Programa Nacional de Sanidade Suidea do Ministério da Agricultura, Pecuária e do Abastecimento. Quanto à divisão sanitária atualmente adotada, o Brasil conta com uma zona considerada com livre de peste suína clássica que é constituída pelos principais estados produtores de suínos e pelas fronteiras de expansão da atividade e uma zona não livre, que engloba a maioria dos estados das regiões Norte e Nordeste do Brasil. Fora da área considerada livre focos de peste suína clássica continuam acontecendo de forma esporádica, porém a proposta do Ministério de Agricultura Pecuária e Abastecimento é erradicar a enfermidade em todo o território nacional. O diagnóstico de suspeitas da infecção pode ser feito através do isolamento e identificação viral em cultivos celulares, considerado o teste ouro, por meio das técnicas de imunofluorescência e imunoperoxidase. Foram analisadas por estas técnicas no Laboratório Nacional Agropecuário em Pernambuco amostras de órgãos e sangue de 149 suínos com suspeita de PSC, no período compreendido entre 1999 e 2009, provenientes dos estados do Pará e Amapá da região norte e Maranhão, Ceará, Rio Grande do Norte, Paraíba e Pernambuco da região nordeste. Dos suínos analisados, 51 estavam infectados com o vírus da peste suína clássica. Nos achados clínicos e de necropsia eram predominantes de alterações hemorrágicas, típicas da forma clássica da doença. As criações afetadas eram predominantemente de baixo nível de tecnificação. Os resultados obtidos evidenciaram que a presença de animais positivos é alta, estando o agente presente na maioria dos Estados das regiões Nordeste e Norte, e que a aplicação de medidas preventivas a campo de forma efetiva podem diminuir ou evitar o aparecimento de casos e focos da enfermidade.