Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [113]-133).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 207-251).

Thesis (M.A. in veterinary science)--Washington State University, May 2009. / Title from PDF title page (viewed on Apr. 23, 2010). "Department of Veterinary Clinical Sciences." Includes bibliographical references (p. 83-89).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 131-157).

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: RNA packaging into HIV-1 Virions. Includes bibliographical references (152-174). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / "May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:28:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-07Bitstream added on 2014-06-13T19:16:12Z : No. of bitstreams: 1 pereira_mhc_me_botfmvz.pdf: 261210 bytes, checksum: 2050ad8bcfea0fcd04c28e04b116b34b (MD5) / Universidade Estadual Paulista (UNESP) / O objetivo deste estudo foi avaliar se vacinação contra doenças da reprodução melhora o desempenho reprodutivo de vacas de leite em lactação. Foram realizados quatro experimentos. O experimento 01 foi realizado em 38 fazendas que não utilizavam vacina contra Rinotraqueíte Infecciosa Bovina (IBR), Diarreia Viral Bovina (BVD) e Leptospirose, foram utilizadas 853 vacas Girolando que foram vacinadas (grupo tratado) ou não (grupo controle) no dia -11 (inicio do protocolo de IATF). A segunda dose da vacina foi realizada no dia 30 (diagnóstico de gestação). A vacina que foi utilizada é composta de amostras atenuadas quimicamente alteradas do vírus da IBR associada a amostras citopáticas e não citopáticas do vírus da BVD e culturas inativadas contra cinco sorotipos da Leptospira spp (canicola, grippotyphosa, hardjo, icterohaemorrhagiae e pomona). O experimento 02 foi realizado em 28 fazendas que não utilizavam vacina contra IBR, BVD e Leptospirose, foram utilizadas 287 vacas Girolando que foram pré vacinadas (grupo tratado) ou não (grupo controle) entre os dias -41 a -32, e a segunda dose da vacina foi realizada no dia -11 (inicio do protocolo de IATF). O experimento 03 foi realizado em 17 fazendas que não utilizavam vacina contra IBR/BVDV e Leptospirose, foram utilizadas 1680 vacas holandesas, sendo que vacas com mais de 28 dias em lactação foram vacinadas (grupo tratado) ou não (grupo controle), e a segunda dose da vacina foi realizada 14 dias após a primeira dose. As inseminações foram realizadas entre 15 a 135 dias após a segunda dose da vacina e as perdas de gestação foram avaliadas até 60 dias após a última IA. O experimento 04 foi realizado em 15 fazendas que utilizavam vacina contra IBR, BVD e Leptospirose, foram utilizadas 820 vacas Girolando que foram re-vacinadas... / The aim of this study was to evaluate the effect of vaccination against reproductive diseases on the reproductive performance of lactating dairy cows. The experiment 01 was performed in 38 farms that did not utilize vaccine against IBR/BVD and Leptospirosis, 853 Girolando cows were vaccinated (treated group) or not (control group) on day -11 (beginning of the TAI protocol). The second dose of vaccine was held on day 30 (preganancy diagnosis). The vaccine that was used consists of chemically altered live samples of the IBR virus associated with samples of cytpathic and non cytopathic BVD virus and inactivated cultures against five serotypes of Leptospira spp. (canicola, grippotyphosa, hardjo, icterohaemorrhagiae e pomona). The experiment 02 was performed in 28 farms that did not utilize vaccine against IBR/BVD and Leptospirosis, 287 Girolando cows were pre - vaccinated (treated group) or not (control group), between the days -41 to – 31, the second dose of vaccine was held on day -11 (beginning of the TAI protocol). The experiment 03 was performed in 17 farms that did not utilize vaccine against IBR/BVD and Leptospirosis, 1680 Holstein cows were used in this study, cows with more than 28 days in milk were vaccinated (treated group) or not (control group), the second dose of vaccine was performed 14 days after de first dose. The AI were performed between 15 and 135 days after the second vaccine dose and the pregnancy loss at 60 days after the last AI. The experiment 04 was performed in 15 farms that utilize vaccine against IBR/BVD and Leptospirosis, 820 Girolando cows were vaccinated (treated group) or not (control group) on day -11 (beginning of the TAI protocol). The pregnancy rate was evaluated in the experiments 1, 2 and 4 at 30 and 71 days after TAI, in experiment 03 the pregnancy...(Complete abstract click electronic access below)

Hantavirus é um gênero na família Hantaviradae, incluindo agentes polimórficos, envelopados e com genoma de RNA fita simples, polaridade negativa e trissegmentado. Os Hantavírus são zoonóticos e mantidos na natureza em reservatórios das ordens Rodentia, Soricomorpha e Chiroptera. No Brasil, foram descritos 8 Hantavírus e destes, 6 causam doença humana grave e de alta letalidade, a Síndrome Pulmonar e Cardiovascular, sendo dentre eles o Araraquara (ARQV) o vírus de ocorrência nas regiões de Cerrado do Sudeste (incluindo Ribeirão Preto) e Planalto Central. ARQV destaca-se por produzir a maior letalidade dentre todos os Hantavírus existentes e possuir como reservatório o roedor silvestre Necromys lasiurus que o transmite ao homem por inalação dos aerossóis contaminados de suas excretas. Neste trabalho, detectamos genoma de Hantavírus em tecidos de Necromys lasiurus e em tecidos de morcego Desmodus rotundus, ambos capturados no Nordeste do Estado de São Paulo. Destes animais, foram feitas tentativas de isolamento de Hantavírus a partir de amostras de tecido de Necromys lasiurus e Desmodus rotundus. Os procedimentos foram realizados em laboratório de nivel de biossegurança 3, onde fizeram-se as inoculações em cultura de células VERO E6, com detecção viral após uma única passagem de 4 dias. Desta forma, foi possível isolar um Hantavírus a partir de sobrenadante do lisado de coração do roedor, o que foi confirmado pela presença do genoma viral do isolado por RT-PCR do sobrenadante de cultura celular e dos antígenos virais nas células Vero E6, por imunofluorescência indireta e western blot. O processo de isolamento mostrou-se reprodutível, por 3 vezes, para o mesmo tecido do roedor. Tais resultados encorajam que esta metodologia para isolamento de Hantavírus deva ser experimentada por mais vezes. O Hantavírus isolado (provavelmente ARQV) deverá fornecer importantes informações sobre seu genoma completo, além de propiciar vários estudos futuros. / Hantavirus is a genus in the Hantaviradae family, including polymorphic agents, it is enveloped and with a single stranded RNA genome, negative and tri-polarity polarity. Hantaviruses are zoonotic and kept in nature in reservoirs of the orders Rodentia, Soricomorpha and Chiroptera. There are 8 hantaviruses recorded in Brazil, 6 of them cause severe human disease and high lethality, a Pulmonary and Cardiovascular Syndrome, among which Araraquara (ARQV) is the virus that occurs in the Southeastern Cerrado (including Ribeirão Preto) and Central Plateau. ARQV stands out for producing higher lethality among all existing hantaviruses and has as reservoir the wild rodent Necromys lasiurus, which transmits to humans by inhalation of the contaminated aerosols of their excreta. In this work, we detected the genome of hantavirus in Necromys lasiurus and Desmodus rotundus, bat tissues, both captured in the Northeast of the State of São Paulo. Of these animals, attempts were made to isolate hantavirus from tissue samples of Necromys lasiurus and Desmodus rotundus. The laboratory tests of biosafety level 3, where inoculations in culture of VERO cells E6 were done, with viral detection after a single passage of 4 days. Thus, it was possible to isolate hantavirus from the rodent heart lysate supernatant, which was confirmed by the presence of the viral genome of the isolate by RT-PCR of the cell culture supernatant and the viral antigens in the Vero E6 cells by Indirect Immunofluorescence and Western Blot. The isolation process was reproducible, 3 times, for the same tissue of the rodent. These results indicate that this methodology for hantavirus isolation should be tested more often. The isolated Hantavirus (ARQV), should provide important information about its complete genome, as well as providing several future studies.

Morbilliviruses belong to the family Paramyxoviridae of the Mononegavirale order of viruses. The Mononegavirale order contains viruses which contain negatively-polar, non-segmented and single stranded RNA genomes. This order contains some of most lethal pathogens known to the humankind. Ebola virus and Marburg virus are perhaps the most lethal human pathogens. Rinderpest virus, declared eradicated in 2011, was known to be the most significant cattle killer. Similarly the Canine distemper virus and Rabies virus, two topmost canine pathogens belong to this order. The L protein in the viruses of Morbillivirus genus harbours the viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome and also all the mRNA capping enzymes, viz. RNA 5’ triphosphatase, guanylyltransferase, RNA (guanine-7-)methyltransferase and RNA 5’ cap-dependent (2’-oxo-)methyltransferase. Moreover this protein can act as a protein kinase that can regulate the function of P protein which serves as a switch between transcription and replication. mRNA capping is necessary for the virus for the purpose of exploiting host cellular machinery towards viral protein synthesis. The Rinderpest virus L protein serves as a model to study the capping enzymes of Morbillivirus. RNA triphosphatase (RTPase), the first enzyme of the capping cascade had earlier been located on the L protein. The RTPase minimal domain on the L protein was identified earlier by sequence homology studies done with RTPase proteins of Baculovirus and Vaccinia virus and cloned. The bacterially expressed recombinant domain was shown to possess RTPase activity. The enzymatic activity was characterized and the RTPase was found to be a metal-dependent enzyme which is highly specific to capping viral mRNA. Further characterization of the domain revealed that the domain also possesses nucleotide triphosphatase (NTPase), tripolyphosphatase and pyrophosphatase activities. Two site-directed mutants in motif-A of the domain: E1645A and E1647A were also tested and were found to be essential for the RTPase and NTPase activity. It was also recognized through these mutant studies that the active sites of RTPase and NTPase activities are partially overlapping. Earlier work done with Vesicular stomatitis virus capping enzymes showed that the Rhabdoviridae family of viruses follow unconventional capping pathway utilizing an enzyme polyribonucleotidyltransferase (PRNTase) which transfers GDP to 5’-monophosphated RNA. Characterization of the RTPase activity which converts 5’-triphosphated RNA into 5’-diphosphated RNA is an evidence for the morbilliviruses utilizing the conventional eukaryotic capping cascade. The results show that Paramyxoviridae do not follow unconventional capping pathway for the mRNA capping as has been the paradigm in the past decade.

Murine hepatitis virus type 3 (MHV-3), a member of the coronavirus family, induces a response that varies with the age and genetic background of the host mouse strain. A/J mice are fully resistant to the virus, while Balbc/J are fully susceptible and C3HebFe/J are semi-susceptible, making it possible to predictably reproduce the major human responses to hepatitis viruses. Although there has been considerable discussion of viral pathology in the literature, there has been much less emphasis on pathogenesis. In the experiments described here, histological, biophysical, and immunological techniques have been used to define the processes and cells involved. Transmission electron microscopic observations have confirmed that Kupffer and endothelial cells of hepatic sinusoids show clear changes by 12 hrs post-infection (p.i.), which are more advanced than hepatocellular changes. No replicating virus was seen in altered hepatocytes up to 3 days p.i. Scanning electron microscopy demonstrated that areas of necrosis are focal in nature and at 2-3 days p.i. consist of small spherical areas without flow. In vivo microcirculatory studies confirm the localized nature of the lesion and have shown that red cell velocity can be recorded in individual sinusoids . Velocities were found to vary from zero within a lesion to a normal velocity of 69±31 um/sec over a distance of not more than 3 sinusoids. In-vivo microcirculatory studies also revealed the ability of macrophages to move upstream (against flow) in the hepatic sinusoids. Using fluorescein labelled antibodies to cell surface markers (Thy-1, Lyt-2, and L3T4) it was shown that no T-cells of any subset were present in the areas of hepatocellular necrosis. Furthermore, treatment with cyclosporine A, which would be expected to decrease necrosis due to cell mediated cytotoxicity, did not significantly alter the course of the disease. The only cells which increased in number in the liver post infection were cells of the monocyte/macrophage lineage (Mac 1+), which had increased twofold at 12 hrs (p<.025) p.i. and to greater than twenty fold (p<.005) by 3 days p.i. Resistance in the A/J strain did not reflect an inability of the immunocompetent cells to present and respond to viral antigen. It was demonstrated that MHV-3 infected macrophages from resistant A/J mice are better able to stimulate proliferation of allogeneic and syngeneic lymphocytes than those from the sensitive Balb/cJ strain. In contrast, MHV-3 infection caused a significant enhancement of chemiluminescence from Balb/cJ macrophages, which did not occur in A/J animals. In vivo studies demonstrated a significant increase in free radical reaction products, including conjugated dienes (of long chain free fatty acids and aldehydes), thiobarbituric acid reactive substances, and lipid soluble fluorescent products between 12-72 hours p.i. with MHV-3 in the livers of susceptible Balb/cJ strain mice. All of these are products of oxidative cleavage of cellular and membrane polyunsaturated fatty acids, and result from the action of oxygen free radicals. Free radical inhibitors, or quenchers of free radical reaction products, were able to significantly reduce the liver necrosis in the susceptible mouse strain following infection. Radioimmune assays for antibody to MHV-3 have confirmed the presence of preformed antibodies to (or cross-reactive with) MHV-3 in the sera of both susceptible and resistant mice, pre and post-infection. Immunofluorescent labelled antibodies have also been used to demonstrate the presence of IgG deposits in the sinusoids of the liver both pre and post infection. This suggests the possibility that these mice have been infected with a non-virulent MHV strain prior to these experiments. From these studies, we conclude that the hepatic injury caused by MHV-3 infction in Balb/cJ mice is mediated predominantly by fixed and migratory cells of the mononuclear phagocytic series. Susceptibility and resistance are related to strain dependant differences in the response of macrophages (and Kupffer cells) to infection, and include the release of procoagulant activity (previously shown) and reactive oxygen radicals (and possibly other macrophage activation products such as PAF) that act together to induce hepatocellular necrosis. Preformed non-neutralizing antibody and an intact complement cascade may enhance viral uptake and activation of macrophages in the Balbc/J mice. Resistance to necrosis may be enhanced by a genetic deficiency of C5 in the A/J mice, preventing the formation of the membrane attack complex and hence complement dependant cell lysis, or macrophage activation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate