Resumo: O Vírus da Diarréia Viral Bovina (BVDV) é um dos patógenos mais importantes na pecuária bovina em todo mundo, principalmente por desencadear manifestações clínicas relacionadas à esfera reprodutiva. A infecção em fêmeas gestantes pode resultar em abortamentos, reabsorções embrionárias, mumificações fetais, má formações e nascimento de bezerros fracos além do aparecimento de animais persistentemente infectados e imunotolerantes ao vírus, que são a principal fonte de infecção e disseminação da doença nos rebanhos. Atualmente, a complexidade do diagnóstico e consequentemente a patogenia, estão relacionados às diferenças genotípicas do agente. Por isso, a presente pesquisa teve como objetivo verificar a ocorrência dos genótipos BVDV-1 (Singer) e BVDV-2 (VS-253) em vacas, e respectivos fetos, abatidas em um frigorífico no Estado de São Paulo por meio da análise do soro sanguineo por meio da técnica de virusneutralização. No contexto geral, 52,51% (115/219) das vacas testadas foram reagentes, mas nenhum feto (0/219) reagiu na virusneutralização. Pela análise cruzada conforme a estirpe viral, observou-se que 42% (92/219) das vacas foram reagentes tanto para o genótipo BVDV-1 como para o genótipo do BVDV-2. Por outro lado 4,10% (9/219) reagiram apenas para o genótipo BVDV-1 e 6,39% (14/219) reagiram apenas para o genótipo do BVDV 2. Notou-se portanto que ambas as estirpes estão disseminadas nas regiões estudadas, fato que justifica o emprego de antígenos diferentes para evitar diagnóstico falso-negativo. Por fim, não foi observado qualquer alteração nos fetos que pudessem ser caracterizada como patologia da enfermidade. / Abstract: The Bovine viral diarrhea virus (BVDV) is one of the pathogens in bovine livestock worldwide most important mainly triggered by clinical manifestations related to the reproductive sphere. The infection in pregnant females may result in abortions, embryonic resorptions, fetal mummification, poor training, birth of weak calves in addition to persistently infected and virus immunotolerant animals, which are the main source of infection and spread of the disease. Currently, the complexity to diagnosis and consequently to the pathogenesis are related genotypic differences that he presents. Therefore, this research aimed to verify the occurrence of BVDV- 1 (Singer) and BVDV-2 (VS-253) genotypes in cows and their respective fetuses slaughtered in a abattoir at the state of São Paulo by analyzing the blood serum using virusneutralization technique. In the general context, 52.51% (115/219) of cows were reagents, but no fetus (0/219) reacted in virusneutralization. After a cross-examination we observed that 42% (92/219) of cows reacted for both BVDV-1 and BVDV-2 genotype. Furthermore 4,10% (9/219) reacted only to the genotype BVDV-1 and 6,39% (14/219) responded only to the genotype 2 of BVDV. It was noted therefore that both strains are widespread in the regions studied, which also justifies the use of different antigens to avoid false-negative diagnosis. Finally, there was no change in fetuses that could be characterized as a pathology of the disease. / Orientador: Samir Issa Samara / Coorientador: Fabio Carvalho Dias / Banca: José Gabriel Amoril / Banca: Sandra Possebon Gatti / Mestre

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Doctor of Philosophy / Department of Animal Sciences and Industry / Cassandra K. Jones / Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that devastated the United States swine industry. Because of its heat sensitivity, it was hypothesized that a pellet mill mimicking commercial thermal processing may mitigate PEDV infectivity. From the results, it was determined that a conditioning time of 30 sec or greater and temperatures above 54.4°C were effective point-in-time kill steps to inactive PEDV in a research setting. However, this does not prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step. To further explore this, various mitigation additives were evaluated to prevent or mitigate PEDV post-pellet contamination in swine feed and ingredients. Various additives were examined across 3 experiments and included mitigation additives of medium chain fatty acids (MCFA), organic acids (OA), essential oils (OA), formaldehyde based products, and sodium bisulfate. From Exp. 1, formaldehyde, medium chain fatty acids (MCFA), essential oils (EO), and organic acid (OA) each decreased detectable PEDV RNA compared to the control (P<0.05). Additionally, PEDV stability over time was influenced by matrix as the meat and bone meal and spray-dried animal plasma resulted in a greater (P<0.05) quantity of detectable PEDV RNA over 42 days compared to that of the swine diet and blood meal. In Exp. 2, the 1% MCFA inclusion was equally effective at mitigating PEDV as a commercially available formaldehyde product in the complete swine diet. To further explore the effects of MCFA against PEDV, Exp. 3 was conducted to evaluate lower inclusion levels of MCFA and fat sources containing MCFA. It was noted that formaldehyde, 1% MCFA (1:1:1: of caproic, caprylic, and capric acids), 0.66% caproic, 0.66% caprylic, and 0.66% capric acids enhance the RNA degradation of PEDV in swine feed as determined by a bioassay. The MCFA were also evaluated against Salmonella Typhimurium, Generic Escherichia coli, Enterotoxigenic Escherichia coli, and Campylobacter coli. It was noted that the efficacy of the MCFA varied between each bacteria species with caproic and caprylic being the most effective. Commercial developmental products were also tested and determined that Product A and B provided the lowest MIC values across Salmonella Typhimurium, Generic Escherichia coli, and Enterotoxigenic Escherichia coli (P < 0.05). Product A and B were further tested in an animal disease trial utilizing a strain of enterotoxigenic Escherichia. coli O149:K91: K88. From d 7 to 14, chlortetracycline, 1:1:1 blend, and Product B, all improved G:F compared to the control (P<0.05). This also led to chlortetracycline and Product B having an improvement (P<0.05) over the control diet from d 0 to 14. A treatment × day interaction for the enterotoxigenic E. coli plate scores was observed (P < 0.05), which occurred because of the decrease (P<0.05) in plate scores for Product B from d 1 to d 14 and an increase (P<0.05) in chlortetracycline from d 7 to 14. A decrease (P<0.05) in plasma urea nitrogen and haptoglobin was observed as time increased from d -2 to 14. In summary MCFA have shown to be an effect interventional mitigation strategy against PEDV and various bacteria.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

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.

Chlamydiae may exist at the site of infection in an alternative replicative form, called the aberrant body (AB). ABs are produced during a viable but non-infectious developmental state termed "persistence" or "chlamydial stress." As persistent/stressed chlamydiae: (i) may contribute to chronic inflammation observed in diseases like trachoma; and (ii) are more resistant to current anti-chlamydial drugs of choice, it is critical to better understand this developmental stage. We previously demonstrated that porcine epidemic diarrhea virus (PEDV) co-infection induced Chlamydia pecorum persistence/stress in culture. One critical characteristic of persistence/stress is that the chlamydiae remain viable and can reenter the normal developmental cycle when the stressor is removed. Thus, we hypothesized that PEDV-induced persistence would be reversible if viral replication was inhibited. Therefore, we performed time course experiments in which Vero cells were C. pecorum/PEDV infected in the presence of cycloheximide (CHX), which inhibits viral but not chlamydial protein synthesis. CHX-exposure inhibited PEDV replication, but did not inhibit induction of C. pecorum persistence at 24 h post-PEDV infection, as indicated by AB formation and reduced production of infectious EBs. Interestingly, production of infectious EBs resumed when CHX-exposed, co-infected cells were incubated 48-72 h post-PEDV co-infection. These data demonstrate that PEDV co-infection-induced chlamydial persistence/stress is reversible and suggest that this induction (i) does not require viral replication in host cells; and (ii) does not require de novo host or viral protein synthesis. These data also suggest that viral binding and/or entry may be required for this effect. Because the PEDV host cell receptor (CD13 or aminopeptidase N) stimulates cellular signaling pathways in the absence of PEDV infection, we suspect that PEDV co-infection might alter CD13 function and induce the chlamydiae to enter the persistent state.

No description available.

Bovine viral diarrhea virus (BVDV) is an important viral pathogen affecting all ages of cattle, resulting in significant economic losses worldwide. BVDV infection is associated with a diverse array of symptoms including gastrointestinal disorder, respiratory distress, fetal malformation, stillbirth, abortions, and mucosal disease (MD). Transplacental infections of fetuses between 42 and 125 days of gestation can result in immune-tolerance and the surviving fetuses become persistently infected (PI). PI animals are major reservoir of BVDV and it becomes problematic to control the disease. The objectives of this dissertation were to: 1) develop a cost-effective testing scheme to detect BVDV PI animals from exposed herds, 2) characterize two virulent BVDV-2 Mississippi isolates associated with severe hemorrhagic diseases, and 3) perform phylogenetic analysis based on sequences of 5'UTR, E2, and NS5B regions. First, we developed a BVDV testing scheme by combining pooled real-time RT-PCR with antigen capture enzyme-linked immunosorbent assay (ACE) to screen cattle herds. From positive pools individual positives were identified using ACE. Data from a three year period indicated that 92.94% PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'UTR of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. Second, two virulent BVDV isolates, M10-3432 and M10-5347, were successfully recovered from an adult beef breeding cow and feedlot calf respectively. When compared to the reference strain BVDV-2 125c, five and three unique amino acids in E2 regions were different from M10-5347 and M10-3432 respectively. Phylogenetic analysis of E2 region grouped both Mississippi isolates in BVDV-2a, a subtype containing high virulent strains. M10-3432 was clustered with high virulent strain 890 while M10-5347 was clustered with high virulent strain CD87. Third, we compared the phylogenetic analyses of BVDV based on the sequences of 5'UTR, E2, and NS5B at either nucleotides or amino acids level. Although slight differences were observed, the virulent BVDV isolates were consistently classified into BVDV-2a cluster regardless of region of sequences used. Furthermore, phylogenetic tree constructed using combined two or more regions had higher posterior probability and bootstrap value than phylogenetic trees constructed using a single region

Porcine epidemic diarrhea Virus (PEDV) is a virus effecting swine. It is the cause of disease that manifests with symptoms ranging from depression, to severe dehydration and death. Young piglets are particularly susceptible to the virus, which can reach mortality rates of 100%. Presence of the virus on a swine farm can therefore cause severe economic losses. Treatments currently exist for PEDV, but are mostly generated from the virus itself. There has recently been renewed interest in a vaccine that is made from a different source, which might help eliminate some of the side effects of those that currently exist on the market. This project outlines three experiments performed in animals. During the first experiment, a structural protein from the Hepatitis B virus was genetically altered to include important structural portions of PEDV. This new protein is generated in E. coli and purified. After purification, the protein assembles into a virus-like particle (VLP). VLPs are structural proteins of existing viruses that are expressed and assembled to mimic the virus. By doing so, the immune system recognizes the protein as a potential threat, and launches a response in the form of antibodies. Manipulations of the VLPs as describe herein allow the new vaccine to generate antibodies toward other diseases such as PEDV. Although all five of the vaccines used in the first experiment were able to generate appropriate antibodies, only two of them were effective at preventing PEDV from entering susceptible cells (virus neutralization). A second experiment, with three newly designed vaccines was therefore performed. This experiment, like the first, was successful in producing antibodies to several of the included PEDV protein sections, but none were able to neutralize the virus. These results led to a third experiment, during which further design improvements were made to the basic vaccine structure in an attempt to increase the neutralization capabilities of the vaccines. The results from the third experiment indicated that several changes to the vaccine increased the immune response to the structural portions of PEDV, providing a better overall vaccine candidate. This also led to the conclusion that one specific sequence from PEDV has a better ability to neutralize the virus than the other sections. / PHD

Bovine Viral Diarrhea Virus (BVDV) is a significant disease causing agent with major economic impact on the cattle industry, causing both productive and reproductive losses. One reason for its widespread distribution is that the majority of all BVDV infections occur without clinical signs, leaving most cases of BVDV undetected in cow herds. BVDV occur as cytopathic (CP) or non-cytopathic (NCP) biotypes, classified according to whether or not they produce visible changes in cell culture. CP BVDV biotype but not NCP biotype is implicated in the induction of apoptosis in vivo. The interaction of BVDV with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The pathogenesis of the disease caused by BVDV is complicated and interaction between BVDV and the host are poorly understood. The overall goal of this research is to identify mechanistic pathways that govern the outcome of BVDV infection in susceptible host cells. Specific aspects of this goal is to understand BVDV biotypes-induced changes on cellular proteome, cell death and survival mechanisms used by BVDV biotypes in apoptosis pathway, interactions of BVDV NS3 viral protein with host cellular proteins and how BVDV cell entry and infection interfere with an early step of professional antigen presentation, antigen uptake. The results of this work showed, for the first time, the successful use of proteomics in studying BVDV-host interactions in a comprehensive approach. Using the Gene Ontology and systems biology analysis we identified biotype-related differences in significant biological pathways and functions. Also, using a proteomics approach, we identified multiple critical cellular proteins that interact with CP NS3 viral protein at multiple stages of CP BVDV replication cycle. This project provides insight into the cellular pathways and functions involved in the viral cytopathogenicity of CP BVDV biotype. In addition, our data not only confirmed the previous observations on the critical involvement of the intrinsic pathway of apoptosis in CP BVDV infection, it also identified multiple mitochondrial and antioxidant proteins contributing to this pathway. Finally, we show that BVDV exploit selective antigen uptake mechanisms in professional antigen presenting cells monocytes during viral entry.