Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies. / AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

The fusion protein of the Newcastle Disease Virus (NDV) contains three hydrophobic domains. To explore the topogenic signals of these domains, mutants were constructed in which each of the hydrophobic domains was deleted. The membrane insertion and topology of these proteins was characterized in a wheat germ cell-free translation system supplemented with canine microsomal membranes. The results indicated that the first 13 amino acids of the fusion protein are necessary to confer translation inhibition by SRP. Translocation of the nascent chains containing all or part of the first hydrophobic sequence resulted in the appearance of a species of higher molecular weight consistent with glycosylation of at least four of the five potential N-linked glycosylation sites. When glycosylation was inhibited with a glycosylation competitor peptide, signal sequence cleavage was detected. Protease digestion of mutants missing the C-terminal hydrophobic domain indicated that the C-terminus has stop transfer activity. A comparison of membrane insertion of the wild-type fusion protein to that of a mutant missing the second hydrophobic domain, the fusion sequence, indicated that the fusion domain has stop-transfer activity when synthesized in vitro. Furthermore, the fusion domain shows little signal sequence activity when positioned near the amino terminus of the fusion protein. The fusion protein has a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit. In order to determine the role that the conserved leucines have for the oligomeric structure and biological activity of the NDV fusion protein, the heptadic leucines at positions 481,488, and 495 were changed individually and in combination to an alanine residue. Whereas single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein although cell surface expression of the mutants and sedimentation in sucrose gradients was similar to that of the wild type. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain resulted in secretion of an oligomeric structure. These results indicate that the conserved leucines do not play a role in oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative substitutions of a serine-to-alanine (position 473), glutamic acid-to-lysine (position 482) or an asparagine-to-lysine (position 485), the fusogenic ability of the protein was not significantly disrupted. A phenylalanine residue is at the amino terminus of the F1 protein of all paramyxovirus fusion proteins with the exception of the avirulent strains which have a leucine residue in this position. To explore the role of this phenylalanine in the fusion activity of the protein, this residue was changed to leucine (F117L) or to glycine (F117G) by site-specific mutagenesis while maintaining the cleavage site sequence of virulent strains of NDV. Whereas both the wild-type and the F117G proteins were proteolytically cleaved and F1 was detected, the leucine subsitution abolished cleavage. When co-expressed with the HN protein, the fusion protein with either a phenylalanine and glycine residue at position 117, but not a leucine, was shown to stimulate membrane fusion. However, incubation in trypsin activated the fusion activity of the F117L protein. Thus the presence of a leucine at position 117 of the precursor sequence blocks cleavage, but not fusion acitivity, and indicated that the phenylalanine at the amino terminus of the F1 subunit is not conserved for the fusion activity of the protein.

There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.

Definitive evidence that natural killer (NK) cells mediate an antiviral effect in vivo was obtained using murine cytomegalovirus (MCMV) as a model system. Adoptive transfer studies using a variety of physical and immunochemical techniques to enrich and deplete NK cell activity showed that the cell population capable of mediating resistance (as assayed by enhanced survival and reduction in spleen virus titers) had the phenotype of an NK cell: a nylon wool nonadherent, asialo GM1+, NK 1.2+, ly 5+, Thy-1-, Ia-, low-density lymphocyte. Adoptive transfer of IL-2-dependent cloned NK cells (but not T cells) also provided resistance. NK cells did not provide resistance to lymphocytic choriomeningitis virus (LCMV). Selective depletion of NK cell activity by injection of mice with antibody to anti-asialo GM1 lowered resistance to MCMV, mouse hepatitis virus, and vaccinia virus but not to LCMV. NK cell depletion resulted in up to 1000-fold increases in spleen and liver virus titers, correlating with more severe pathology in these organs. NK cells were found to have antiviral effects early (0-3 days) but not late (6-9 days) postinfection. NK cell depletion resulted in markedly increased MCMV-induced suppression of T cell function, which is probably responsible for the delayed clearance of virus seen in these mice. NK cell depletion resulted in increased virus synthesis during persistent MCMV infection, but had no effect on the course of persistent LCMV infection, despite elevated NK cell and interferon (IFN) levels found in these LCMV-infected mice. The reason why NK cells play a role against MCMV but not LCMV infection was not due to differences in NK cells induced by these 2 viruses, but more likely due to target cell susceptibility. IFN pretreatment of MCMV-infected cells failed to protect them against NK cell-mediated lysis, whereas uninfected and LCMV-infected cells were almost totally protected. These IFN-pretreated, LCMV-infected cells were not resistant to cell-mediated lysis in general, as this treatment increased their sensitivity to virus-specific T cell-mediated lysis by 2- to 3-fold. This enhanced sensitivity to lysis correlated with increased surface expression of H-2 antigens, but not viral antigens. In summary, these studies provide compelling evidence that NK cells can mediate antiviral effects in vivo, and provide some insights into their mode of action and consequences of their disfunction.

Although it is unclear which cellular factor(s) is responsible for the genetic instability associated with initiating and sustaining cell transformation, it is known that most, if not all, cancers have mutations that inactivate the Rb-mediated growth control pathway. We show here that acute inactivation of Rb by RNA interference or expression of the E7 viral oncoprotein from human papillomavirus (HPV), and the resultant deregulation of one E2F family member, E2F1, leads to DNA double strand break (DSB) accumulation. These DSBs occur independent of apoptosis induction, and activation of ATM, NBS1, p53, or MAD2, and generation of reactive oxygen species (ROS). Although ROS accumulation is associated with DSBs generated from the mis-expression of another nuclear oncoprotein, c-MYC, we find that E2F1 does not contribute to c-MYC associated DSBs, indicating that the DSBs associated with these oncoproteins arise through distinct pathways. However, we find that small changes in E2F1 levels by inhibition of c-MYC transactivated microRNAs known to limit E2F1 protein expression, lead to DSB accumulation. These results suggest that despite the DSBs arising by different mechanisms, c-MYC assists in the regulation of E2F1-associated DSBs. We also find elevated levels of E2F1-associated DSBs in Rb mutated cancer cell lines in the absence of an exogenous DSB stimulus. These basal, E2F1-associated DSBs are substantially lower in Rb wildtype cancer cell lines that have p16ink4 inactivated or express HPV E7. However, we show that we can manipulate DSB levels in these cancer cell lines by modulating Rb and E2F1 activity and suggest that these results may be extended to breast tumor organ culture. Thus, Rb status is key to regulating both the proliferation promoting functions associated with E2F and for preventing DNA damage accumulation if E2F1 becomes deregulated. Taken together, these data suggest that loss of Rb creates strong selective pressure, via DSB accumulation, for inactivating p53 mutations and that E2F1 might contribute to the genetic instability associated with transformation and tumorigenesis.

Made available in DSpace on 2015-10-06T13:02:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-12. Added 1 bitstream(s) on 2015-10-06T13:19:26Z : No. of bitstreams: 1 000847194.pdf: 1197011 bytes, checksum: 7209fde66f8374f6f0f630a8a112ded9 (MD5) / Herpesvirus bovino (BoHV-5) é um alfaherpesvirus que causa meningoencefalite não-supurativa preferencialmente em bovinos jovens. Esta enfermidade ocorre naturalmente em surtos ou casos isolados, apresentando baixa morbidade e alta letalidade. A epidemiologia, as características anatomopatológicas e o diagnóstico do BoHV-5 são bem conhecido. No entanto, as interações moleculares entre a células hospedeiras e o BoHV-5 são pobremente compreendidas. Técnicas moleculares como a PCR quantitativa e o sequenciamento de RNA (RNA-seq) são importantes ferramentas para o estudo de interações entre vírus e células hospedeiras. Neste estudo células MDBK foram infectadas pelo BoHV-5, cepa SV507-99 e o mRNA extraído em 0, 6, 12, 18, e 24 horas após a infecção (pi) foi utilizado para avaliar a expressão de genes do vírus e da célula hospedeira por qPCR e o mRNA de 1h, 6h e 24h pi foram utilizado no estudo por RNA-seq. Células MDBK não infectadas foram usadas como controle. A qPCR demonstrou que os três genes do BoHV-5 estudados (bICP0, UL9 e US4) apresentaram perfil de expressão semelhante nos diferentes momentos após infecção e que o gene bovino GAPDH teve sua expressão diminuída pela infecção viral. Ao menos 900 genes foram diferencialmente expressos para cada momento de infecção na análise por RNA-seq. Estes genes estavam principalmente relacionados a vias celulares de controle de ciclo celular, produção de interleucinas, quimiotaxia para células inflamatórias, reparo e dano ao DNA, resposta a vírus, apoptose, processo oxidativo, e ubiquitização de moléculas. Estes resultados representam novos conhecimentos sobre a interação entre o BoHV-5 e as células bovinas e podem representar um ponto de partida para novas pesquisas e melhor compreensão da patogenia deste vírus / Bovine herpesvirus 5 (BoHV-5) is an Alphaherpesvirus that causes nonsuppurative meningoencephalitis mainly in young cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Besides BoHV-5 epidemiology, pathological findings and diagnosis were well known, the molecular interactions between host cell and BoHV-5 are poorly understood. Molecular biology techniques such as quantitative PCR (qPCR) and RNA sequencing (RNA-seq) are important tools to study virus and host cell interactions. In this study we infected MDBK cells and use the extracted mRNA at 0, 6, 12, 18 and 24h post infection (pi) to analyse BoHV-5 (bICP0, UL9 e US4) and host cell gene (GAPDH) expression using qPCR and 1h, 6h and 24h pi in the RNA-seq study. Mock-infected cells were used to control purpouse. The qPCR releveled that the three BoHV-5 genes showed the same expression behavior during the infection and the GAPDH gene were down-regulated in the infected group. At least 900 genes were differentially expressed during BoHV-5 in each moment analysed by RNA-seq. These genes were up- or down-regulated and mainly associated with cell cycle, interleukin production, inflammatory cells chemotaxis, DNA damage and repair, response to virus, apoptosis, oxidation-reduction process, and ubiquitination pathways. The results demonstrate new insights about BoHV-5 and bovine cells interactions and could be a starting point to new researches and to better understand the pathology of this virus

Made available in DSpace on 2014-06-11T19:32:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-05Bitstream added on 2014-06-13T19:22:37Z : No. of bitstreams: 1 souza_wj_dr_jabo.pdf: 513241 bytes, checksum: 3c1539fc3f14169cf4056ce94931dea2 (MD5) / Este trabalho teve como objetivo analisar parâmetros clínicos, alterações sorológicas e qualidade do sêmen de touros jovens infectados experimentalmente com o herpesvírus bovino tipo 1 (BoHV-1) entre os dias 0 e 49 pós-inoculação (PI), com o intuito de caracterizar as manisfestações que direcionam o diagnóstico, nas diversas fases de infecção. Neste experimento foram utilizados 12 touros com aproximadamente 30 meses de idade. Seis animais foram inoculados com a amostra padrão Nebraska do BoHV-1 pelas vias intranasal e intraprepucial com 10 mL de suspensão viral (107,0TCID50/mL) repartidos 5 mL em cada via. Os outros seis animais ficaram como grupo controle. Todos os touros inoculados com o BoHV-1 apresentaram edema e hiperemia no prepúcio entre o terceiro e o décimo sexto dia pi, dos quais dois também apresentaram exsudato nasal, detectado entre o terceiro e o oitavo dia PI. Nenhum dos animais do grupo controle manifestou sinais clínicos da doença. Por meio da técnica de virusneutralização (VN) foram detectados títulos de anticorpos com valores entre 4 e 512 em todos os animais inoculados, no período entre 14º e 49º dia PI. Em todos os animais inoculados foi constatada a presença de material genético do BoHV-1 nas amostras de sêmen analisadas por meio da técnica da reação em cadeia pela polimerase (PCR). Em quatro desses animais foi detectado o material genético viral aos 7,14 e 21 dias PI e em dois animais o material genético do vírus foi identificado por mais uma semana, ou seja, até o 28º dia PI. Todos os animais do grupo controle apresentaram resultados negativos nas técnicas de VN e PCR. Em relação à qualidade do sêmen, não foram observadas diferenças estatísticas (p≥0,05) entre o grupo de touros controle e o grupo de touros inoculados experimentalmente com o BoHV-1, por meio das... / This study aimed to analyze clinical parameters, serological alterations and quality from the semen of young bulls experimentally infected with type 1 bovine herpes virus (BoHV-1) between days 0 and 49 post-inoculation (P. I.), in order to characterize the manifestations that orientate diagnosis, in several phases of infection. Twelve bulls were used in this experiment, all being approximately 30 months old. Six animals were inoculated with the Nebraska standard sample of BoHV-1 through intranasal and intrapreputial routes, each with 10 ml of the viral suspension (total dosage of 107.0TCID50/ml) divided in 5 ml for each route. The other six animals were used as a control group. All bulls inoculated with BoHV-1 presented edema and hyperemia on the foreskin between the third and sixteenth days after the inoculation, from which two animals also presented nasal exudate, detected between the third and eighth days P. I. No animals from the control group manifested any clinical signs characteristic of the infection caused by BoHV-1. By means of the viral neutralization (VN) technique, antibody titers with values ranging from 4 to 512 were detected in all inoculated animals between days 14 and 49 after the inoculation. In all inoculated animals, genetic material of BoHV-1 was present in semen samples analyzed by means of the polymerase chain reaction (PCR) technique. In four of these animals, viral genetic material was detected on days 7, 14 and 21 P. I. and in two animals the genetic material of the virus was identified for one more week, until day 28 after the infection. All animals in the control group showed negative results for the previously mentioned VN and PCR techniques. Considering the quality of semen samples from experimental bulls, no statistical differences were observed (p ≥ 0.05) between the control group and the group... (Complete abstract click electronic access below)

Made available in DSpace on 2014-08-13T14:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-17Bitstream added on 2014-08-13T18:00:23Z : No. of bitstreams: 1 000760131_20150710.pdf: 238404 bytes, checksum: d603c88625d587b452adf4dd0eb7962f (MD5) Bitstreams deleted on 2015-08-03T12:21:13Z: 000760131_20150710.pdf,. Added 1 bitstream(s) on 2015-08-03T12:22:26Z : No. of bitstreams: 1 000760131.pdf: 2193129 bytes, checksum: 28172a25b2901b04864665b4d4b50f8b (MD5) / O herpesvírus canino (HVC-1) causa doença infecto-contagiosa que acomete cães em todo o mundo. É considerado o responsável por causar problemas reprodutivos, respiratórios, oculares, alterações neurológicas podendo levar à morte neonatos e adultos imunossuprimidos. Assim, o presente trabalho teve como objetivos isolar o HVC-1 a partir de amostras biológicas, padronizar uma técnica diagnóstica para detecção do herpesvírus canino, verificar a presença de cães infectados no estado de São Paulo e comparar 3 modificações na técnica de diluição limitante em relação à quantificação molecular pela qPCR (quantitative Polymerase Chain Reaction). Para isso, foram coletadas 139 amostras sangue, fragmentos de órgãos de 12 neonatos que foram a óbito e 5 amostras de suabes de secreção genital, de animais provenientes de canis e domicílios. Após a obtenção do DNA das amostras colhidas, foram submetidas à qPCR e snPCR (seminested Polymerase Chain Reaction) para o teste de detecção do HVC-1. Foi encontrada positividade em nove foram amostras de sangue e em fragmentos de órgãos de um animal, as amostras de secreções genitais foram todas negativas. O isolamento viral foi realizado a partir de fragmento de rim. As células infectadas apresentaram efeitos citopáticos compatíveis com as do HVC-1. As amostras positivas foram sequenciadas apresentando identidade de 100% com o HVC-1 (Genbank ™: X75765). As técnicas moleculares para a detecção do agente mostraram-se sensíveis e específicas, tanto a snPCR como a qPCR. Nessas técnicas foi possível detectar até 15,35 cópias/ μL de DNA de amostras de sangue e 1,15 cópias/μg de tecido. O uso de técnicas moleculares possibilitou a detecção de infecções ativas por HVC-1 com ótimo limiar de detecção. No entanto, mais estudos são necessários para conhecer a epidemiologia da infecção do HVC-1 na população canina no Brasil / The canine herpesvirus (CHV-1) causes contagious disease affecting dogs worldwide. It is considered the responsible for cause reproductive problems, respiratory, eyes, neurologic changes may lead to death newborns and immunocompromised adults. Therefore, our study aimed to isolate CHV-1 from biological samples, standardize a diagnostic techniques for the detection of canine herpesvirus, verify the presence of infected dogs in São Paulo state and compare three modifications in the technique of limiting dilution in relation to molecular quantification to qPCR (by quantitative PCR to determine the viral titer). Thus, we collected 139 blood samples, organ´s fragments from 12 neonates death and 5 genital secretions samples with swabs of animals from kennels and households. After obtaining the DNA from samples were accomplished the qPCR (quantitative PCR) and snPCR (seminested PCR) assay for detection of CHV-1. It was found positivity in nine blood sample and organ´s fragments of an animal, the genital secretions samples were all negative. The viral isolation was performed from kidney. The infected cells showed cytopathic effects compatible with the CHV-1. Positive samples were sequenced showed 100% identity with the CHV-1 (Genbank™: X75765). The molecular techniques for detection agent were sensitive and specific, both the snPCR as the qPCR. In these techniques was enabled to detect DNA 15.35 copies/mL of blood samples and 1.15 copies/μg of tissue. The use of molecular techniques allowed the detection active infections of of HCV-1 with optimal detection limit. More studies are needed to understand the infection epidemiology of CHV-1 in the canine population in Brazil

Orientador: Samir Issa Samara / Coorientador: Carlos Frederico Martins / Coorientador: José Robson Bezerra Sereno / Banca: Luis Antonio Mathias / Banca: Iveraldo dos Santos Dutra / Banca: Karen Martins Leão / Banca: Bruna Alexandrino / Resumo: Este trabalho teve como objetivo analisar parâmetros clínicos, alterações sorológicas e qualidade do sêmen de touros jovens infectados experimentalmente com o herpesvírus bovino tipo 1 (BoHV-1) entre os dias 0 e 49 pós-inoculação (PI), com o intuito de caracterizar as manisfestações que direcionam o diagnóstico, nas diversas fases de infecção. Neste experimento foram utilizados 12 touros com aproximadamente 30 meses de idade. Seis animais foram inoculados com a amostra padrão Nebraska do BoHV-1 pelas vias intranasal e intraprepucial com 10 mL de suspensão viral (107,0TCID50/mL) repartidos 5 mL em cada via. Os outros seis animais ficaram como grupo controle. Todos os touros inoculados com o BoHV-1 apresentaram edema e hiperemia no prepúcio entre o terceiro e o décimo sexto dia pi, dos quais dois também apresentaram exsudato nasal, detectado entre o terceiro e o oitavo dia PI. Nenhum dos animais do grupo controle manifestou sinais clínicos da doença. Por meio da técnica de virusneutralização (VN) foram detectados títulos de anticorpos com valores entre 4 e 512 em todos os animais inoculados, no período entre 14º e 49º dia PI. Em todos os animais inoculados foi constatada a presença de material genético do BoHV-1 nas amostras de sêmen analisadas por meio da técnica da reação em cadeia pela polimerase (PCR). Em quatro desses animais foi detectado o material genético viral aos 7,14 e 21 dias PI e em dois animais o material genético do vírus foi identificado por mais uma semana, ou seja, até o 28º dia PI. Todos os animais do grupo controle apresentaram resultados negativos nas técnicas de VN e PCR. Em relação à qualidade do sêmen, não foram observadas diferenças estatísticas (p≥0,05) entre o grupo de touros controle e o grupo de touros inoculados experimentalmente com o BoHV-1, por meio das... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to analyze clinical parameters, serological alterations and quality from the semen of young bulls experimentally infected with type 1 bovine herpes virus (BoHV-1) between days 0 and 49 post-inoculation (P. I.), in order to characterize the manifestations that orientate diagnosis, in several phases of infection. Twelve bulls were used in this experiment, all being approximately 30 months old. Six animals were inoculated with the Nebraska standard sample of BoHV-1 through intranasal and intrapreputial routes, each with 10 ml of the viral suspension (total dosage of 107.0TCID50/ml) divided in 5 ml for each route. The other six animals were used as a control group. All bulls inoculated with BoHV-1 presented edema and hyperemia on the foreskin between the third and sixteenth days after the inoculation, from which two animals also presented nasal exudate, detected between the third and eighth days P. I. No animals from the control group manifested any clinical signs characteristic of the infection caused by BoHV-1. By means of the viral neutralization (VN) technique, antibody titers with values ranging from 4 to 512 were detected in all inoculated animals between days 14 and 49 after the inoculation. In all inoculated animals, genetic material of BoHV-1 was present in semen samples analyzed by means of the polymerase chain reaction (PCR) technique. In four of these animals, viral genetic material was detected on days 7, 14 and 21 P. I. and in two animals the genetic material of the virus was identified for one more week, until day 28 after the infection. All animals in the control group showed negative results for the previously mentioned VN and PCR techniques. Considering the quality of semen samples from experimental bulls, no statistical differences were observed (p ≥ 0.05) between the control group and the group... (Complete abstract click electronic access below) / Doutor

Orientador: Daniel Junior de Andrade / Banca: Marineide Rosa Vieira / Banca: Renato Beozzo Bassanezi / Resumo: O ácaro Brevipalpus yothersi Baker é vetor da leprose dos citros, principal doença viral da citricultura mundial. Informações sobre a biologia de B. yothersi são essenciais para compreender a dinâmica populacional do ácaro no campo e inferir se mudanças no manejo do pomar em função da região pode alterar a biologia do ácaro. O objetivo do trabalho foi determinar a biologia e elaborar a tabela de vida de fertilidade de B. yothersi coletados em diferentes regiões citrícolas do estado de São Paulo. Os experimentos foram realizados no Laboratório de Acarologia, pertencente à Faculdade de Ciências Agrárias e Veterinárias - FCAV/UNESP, Jaboticabal - SP. Os ácaros foram coletados em pomares cítricos das regiões de Barretos, Jales e Santa Cruz do Rio Pardo, posteriormente, em laboratório, foram multiplicados em frutos de laranja. Os parâmetros biológicos avaliados foram duração das fases de desenvolvimento, oviposição, período de incubação, viabilidade dos ovos, longevidade, taxa líquida de reprodução (Ro), tempo médio de geração (T), taxa intrínseca de crescimento populacional (rm) e taxa finita de crescimento populacional (λ). Estes parâmetros foram avaliados em dois experimentos, o primeiro consistiu na biologia de B. yothersi em frutos isentos de resíduos de produtos fitossanitários à 23±1ºC e o segundo sob frutos com resíduo de espirodiclofeno à 25±1ºC. As observações foram realizadas diariamente, pela manhã e ao fim da tarde. A duração do desenvolvimento, longevidade, período d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The mite Brevipalpus yothersi Baker is the vector of the citrus leprosis, major viral disease of citrus worldwide. Information about B. yothersi's biology are essential to understanding the population dynamics of the mite in the field and infer whether changes in orchard management by region can change the mite biology. The objective was to determine the biology and prepare the fertility life table of B. yothersi collected in different citrus regions of São Paulo state. The experiments were performed in Acarology Laboratory, belonging to the Faculty of Agricultural and Veterinary Sciences - FCAV/UNESP, Jaboticabal - SP. The mites were collected in citrus orchards in the regions of Barretos, Jales and Santa Cruz do Rio Pardo, later in the laboratory were multiplied in orange fruits. The biological parameters assessed were duration of the stages of development, oviposition, incubation period, egg viability, longevity, net reproductive rate (Ro), mean generation time (T), intrinsic rate of increase (rm) and finite rate increase (λ). These parameters were evaluated in two experiments, the first consisted the biology of B. yothersi in fruits free of residues of pesticides at 23 ± 1°C and the second consisting of the biology of B. yothersi under fruit with spirodiclofen residue at 25 ± 1°C . The observations were performed daily, in the morning and in the afternoon. The duration of the development, longevity, pre-oviposition period, oviposition rate and number of B. yothersi eggs s... (Complete abstract click electronic access below) / Mestre