Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral and anti-fungal properties, but the exact mechanism of these proteins still need to be elucidated.The mechanism of resistance however, appears to be independent of the pathogen. For resistance the RIP terminates virus infected plant cells and stops the reproduction and spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide range of viruses. The ultimate goal of the larger project of which this forms part is the development of virus resistant plants. To monitor the expression of a RIP in a transgenic plant a detection method had to be developed. Antibody detection of the RIP was decided upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus (carnation), was used and expressed in bacterial and insect expression systems. The bacterial expression experiments were done using the pET expression system in BL21(DE3)pLysS cells. The expression in this system yielded recombinant protein at a very low concentration. Expression experiments were also performed in insect tissue culture with the baculovirus vector BAC-TO-BAC™.With this system the expression was also too low to be used for the production of antibodies. A Dianthin 30 specific peptide was then designed and then produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin 30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this detection method was effective to monitor the expression in plants, tobacco plants were transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant expression vector. The putative transformed plants were analysed with peR and Southern blots. / AFRIKAANSE OPSOMMING: Tans word Ribosomale-inaktiverende proteïene (RIPs) geklassifiseer as rRNA N-glikosidase wat ook polinukleotied: adenosien glikosidase aktiwiteit bevat. Daar word geglo dat RIPs anti-virale en anti-fungus eienskappe bevat, maar die meganisme van beskerming word nog nie ten volle verstaan nie. Dit is wel bewys dat die meganisme van weerstand onafhanklik is van die patogeen. Virus geinfekteerde plantselle word deur die RIP gedood om die voortplanting en verspreiding te bekamp en sodoende word weerstand bewerkstellig. Transgeniese plante wat dan 'n RIP bevat sal dus weerstandbiedend wees teen 'n wye spektrum virusse. Die hoofdoel van die breër projek, waarvan die projek deel uitmaak: is die ontwikkeling van virusbestande plante. Om die uitdrukking van die RIP in die transgeniese plante te kontroleer, moes 'n deteksie metode ontwikkel word. Die mees koste effektiewe deteksie metode is met teenliggame. Die RIP, Dianthin 30 from Dianthus caryophyllus (angelier) was gebruik vir uitdrukking in bakteriele- en insekweefselkultuur. Die bakteriele uitdrukkingseksperimente was gedoen met die pET uitdrukkings sisteem III BL21(DE3)pLysS selle. Die uitdrukking in die sisteem het slegs rekombinante proteïene gelewer in uiters lae konsentrasies. Uitdrukkingseksperimente was ook gedoen in insekweefselkultuur met die baculovirus vektor BAC-To- BACTM. Met die sisteem was die uitdrukking ook veels te laag om bruikbaar te wees vir die produksie van teenliggame. Daar is toe 'n peptied ontwerp wat Dianthin 30 kan verteenwoordig vir die produksie van teenliggame. Die teenliggame is getoets teen Dianthus caryophyllus proteïene. Om vas te stel of die deteksiemetode wel die uitdrukking van Dianthin 30 sal kan monitor, is tabak ook getransformeer met Dianthin 30. Die transformasies is gedoen met die hulp van Agrobacterium tumefaciens en die pART27 plant uitdrukkings vektor. Die plante is getoets met die polimerase ketting reaksie en Southern klad tegnieke.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is one of the most economic important viral diseases affecting grapevine. Grapevine leafroll associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of the leafroll associated virus family. To prevent the spread of GLD, management strategies such as rogueing and insect vector control are required to limit crop losses. Alternative control strategies based on genetic modification of the grapevine genome, such as pathogen-derived resistance (PDR), is proven to be effective in conferring resistance to several viruses. Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs (amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host and the development of an amiRNA-mediated silencing validation system. In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and #17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus titres of all grafted plants were quantified relative to two reference genes using RT-qPCR. Results were evaluated by comparing the relative virus titre of each transgenic plant line to that of the non-modified control plant line. Results showed that resistance levels of plant line #3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more susceptible to the virus. The second part of the study was the construction and validation of amiRNAs targeting GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were quantified to determine the silencing efficiency of the amiRNAs. Results showed that the amiRNAs were successful in silencing the GFP target construct, however, they were not specific in silencing exclusively their corresponding target. These amiRNA constructs are ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD infected grapevines. / AFRIKAANSE OPSOMMING: Wingerd rolblaar siekte (GLD), wat veroorsaak word deur die lede van die familie Closteroviridae, is een van die ekonomies mees belangrike virus siektes van wingerd. Grapevine leafroll-associated virus 3 (GLRaV-3), van die genus Ampelovirus, is die mees wydverspreide lid van die rolblaar geassosieerde virus familie. Om die verspreiding van GLD te voorkom, is bestuur strategieë, soos die verwydering van geïnfekteerde plante en insekvektor beheer, ’n vereiste om oes verliese te beperk. Alternatiewe beheer strategieë gebaseer op genetiese modifikasie van die wingerdgenoom, soos patogeen-afgeleide weerstand (PDR), is bewys om effektief te wees in die verlening van weerstand teen verskeie virusse. Daarom was die fokus van hierdie studie om patogeen-afgeleide weerstand strategieë vir GLRaV-3 te evalueer met behulp van die volgende twee benaderings; 1) die evaluering van transgeniese plante wat 'n disfunksionele GLRaV-3 hitte-skok proteïen 70 homoloog (HSP70h) uitdruk, ten einde weerstand te verleen teen GLRaV-3, en 2) die konstruksie van kunsmatige mikroRNAs (amiRNAs) om te gebruik as 'n instrument vir die ondrukking van spesifieke genoomvolgordes van GLRaV-3 in die wingerd gasheer en die ontwikkeling van ’n stelsel om amiRNA-bemiddelde onderdrukking te bevestig. In die eerste deel van hierdie studie, is ses transgeniese plant lyne (plant lyne # 1, # 3, # 9, # 14, # 15 en # 17) sowel as 'n nie-gemodifiseerde gesonde plant lyn, geïnokuleer met GLRaV- 3 deur enting van ogies van elk op GLRaV-3 besmette plantmateriaal. Na ongeveer vyf maande, is GLRaV-3 virus konsentrasies van alle ingeënte plante gekwantifiseer relatief tot twee verwysing gene deur gebruik te maak van tru-transkripsie kwantitatiewe PCR (RTqPCR). Resultate is geëvalueer deur die relatiewe virus konsentrasie van elke transgeniese plant lyn te vergelyk met dié van die nie-gemodifiseerde kontrole lyn. Resultate het getoon dat weerstand vlakke van plant lyn # 3 beduidend verbeter is (> 99%) en merkwaardig is plant lyn # 14 bewys om meer vatbaar vir die virus te wees. Die tweede deel van die studie was die konstruksie en bevestiging van kunsmatige mikroRNAs (amiRNAs) wat GLRaV-3 genoomvolgordes teiken. Twee 21 nt streke van GLRaV-3 is suksesvol geïnkorporeer in die ruggraat van wingerd mikroRNA vvi167b. Verder is teiken konstrukte ontwikkel deur die inkorporering van ooreenstemmende GLRaV-3 teiken genoomvolgordes in die 3'UTR (3’ ongetransleerde area) van 'n groen fluoressensie proteïen (GFP) geen. Daarna is die teiken konstrukte gesamentlik geïnfiltreer met die gekonstrueerde amiRNA in Nicotiana benthamiana en GFP uitdrukkingsvlakke is gekwantifiseer deur die onderdrukkingsdoeltreffendheid van die amiRNAs te bepaal. Resultate het getoon dat die amiRNAs suksesvol was in die onderdrukking van die GFP teiken konstruk, maar hulle was egter nie-spesifiek in die eksklusiewe onderdrukking van die ooreenstemmende teiken. Hierdie amiRNA konstrukte is ideaal vir verdere virus studies om die doeltreffendheid van GLRaV-3 onderdrukking in GLD besmette wingerdstokke te bepaal.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-04-07Bitstream added on 2014-06-13T20:18:28Z : No. of bitstreams: 1 000754808.pdf: 1123345 bytes, checksum: 945c6c9f352406de0091406c4f189c94 (MD5) / A mandioquinha-salsa (Arracacia xanthorrhiza Bancroft) é uma hortaliça originária da região andina, Venezuela, Colômbia, Equador, Peru e Bolívia. A família das apiáceas compreende também outras hortaliças importantes, como cenoura, salsa, coentro, aipo, dentre outras. A mandioquinha-salsa é propagada de forma vegetativa através dos propágulos, e isso pode favorecer a ocorrência de um número considerável de patógenos que causam degenerescência, principalmente os vírus. Está comprovado que alguns vírus são fator limitante em culturas de propagação vegetativa. Portanto, o presente trabalho teve como objetivos avaliar por meio de métodos sorológico e molecular 241 amostras oriundas de diferentes localidades, a fim de fazer um estudo de ocorrência e predominância das espécies virais e verificar a variabilidade biológica dos isolados encontrados por meio de transmissão por extratos vegetais. A detecção foi realizada através de Enzyme linked immunossorbent assay (ELISA) utilizando antissoro comercial anti-poty para o gênero Potyvirus. Para a análise da variabilidade, foram desenhados 2 oligonucleotídeos para amplificar a região codificadora da proteína capsidial para o gênero Cheravirus, e para a detecção de potyvirus e nepovirus, oligonucleotídeos foram obtidos da literatura. No estudo foi possível detectar a espécie Arracacha motlle virus do gênero Potyvirus, coletadas nos estados de Goiás, Minas Gerais, Brasília e Paraná. Os demais vírus testados não foram identificados em nenhuma das amostras analisadas. Além desses testes de identificação, ainda foi realizada microscopia eletrônica de transmissão em seis amostras, das quais duas foi possível a identificação de potyvirus. O teste de gama de hospedeiras foi realizado e mostrou que o AMoV tem gama de hospedeiras bastante restrita, sendo possível infectar somente três de nove espécies testadas... / Arracacha (Arracacia xanthorrhiza Bancroft) is a vegetable crop of the Andean region, formed by Venezuela, Colombia, Ecuador, Peru and Bolivia. The Apiacea family also have other important vegetable crops such as carrots, parsley, cilantro, celery, among others. Arracacha is propagated vegetatively and this can favor the occurrence of a considerable number of pathogens that cause degeneration, especially viruses. Some viruses are a limiting factor in vegetatively propagated crops. Therefore, the present study aimed to evaluate by serological and molecular methods 241 samples from different localities in order to verify the occurrence and predominance of the viruses species and verify the biological variability by sap inoculation. The detection was performed using enzyme-linked immunossorbent assay (ELISA) technique, using commercial antiserum against-potyvirus. For variability analysis, primers were designed to amplify the coding region of the coat protein for the 4 genus Cheravirus, and oligonucleotides were obtained from literature for the detection of the genus potyvirus and nepovirus, . It was possible to detect Arracacha mottle virus, from genus Potyvirus, in the states of Goiás, Minas Gerais, Brasilia and Paraná. The others viruses tested were not identified in the collected samples. Six samples were analysed by electron microscopy, and the potyvirus were identified only in two samples. The host range was restricted, infecting only three species of nine tested. The nucleotide identity ranged from 96% to 99%, between collected samples and sequence from GenBank (acess DQ925486), showing low genetic variability among them

Infection with human immunodeficiency virus type 1 (HIV-1) results in a wide range of immunologic and hematopoietic abnormalities. The overall goal of this dissertation was directed toward obtaining a better understanding of the interactions of HIV-1 and myeloid cells in relation to the pathogenesis of AIDS. The human myelomonocytic cell line, HL-60, was used as a model system to determine if HIV-1 infects myeloid progenitor cells and subsequently, if infection affects their differentiation. HL-60 cells and the human prototypic T cell line, H9 were infected with three different HIV-l isolates (IIIB, PM213, and NL4-3) which are known to infect T cells. All three isolates productively infected both H9 and HL-60 cells; however, HIV-1 antigen expression and cytopathicity was delayed by approximately 15 days in infected HL-60 cells compared H9 cells. To examine the effect of HIV-l infection on myeloid differentiation, chronically infected HL-60 cells and clonal lines derived from them were induced to differentiate into either granulocytes by treatment with dimethyl formamide (DMF) or into monocytes by treatment with phorbol l2-myristate 13 acetate (PMA). By both cellular morphology and function, approximately the same percentage of treated, HIV-infected HL-60 cells differentiated into either granulocytes or monocytes as treated, control HL-60 cells. Taken together, these results indicate that HIV-1 infection does not affect the morphological or functional differentiation of HL-60 cells. In an effort to understand the differences in the regulation of HIV-l infection in myeloid versus T cells, the life cycle of NL4-3 was examined in HL-60 cells and H9 cells. Initially, NL4-3 replication was restricted in HL-60 cells compared to H9 cells. This restriction was overcome 15 days after infection by the generation of a viral isolate, NL4-3(M). NL4-3(M), harvested during the lytic phase of NL4-3 infection of HL-60 cells, caused cell death approximately 8 days after infection in both H9 and HL-60 cells. Although measurements of viral entry kinetics demonstrated that the timing of entry of NL4-3 and NL4-3(M) in HL-60 cells and NL4-3 in H9 cells was similar, a quantitative polymerase chain reaction (PCR) analysis of newly reverse transcribed NL4-3 DNA in H9 and HL-60 cells revealed that NL4-3 infected H9 cells and NL4-3(M) infected HL-60 cells contain consistently higher amounts of newly reverse transcribed DNA than NL4-3 infected HL-60 cells. The delay in NL4-3 replication in HL-60 cells was further amplified by inefficient spread of the virus throughout the HL-60 culture as measured by RNA production and DNA integration suggesting that another step in the viral life cycle after reverse transcription was also restricted. These results suggest that the efficiency of NL43 replication in HL-60 cells is restricted at several steps in the viral life cycle. Further, these restrictions are overcome by the generation of a viral variant, NL4-3(M), which efficiently replicates in myeloid cells. The tropism of NL4-3(M) was further characterized by testing its growth in monocyte-derived macrophages (MDM). Unlike NL4-3, NL4-3(M) productively infected MDM cultures. The ability of NL4-3(M) to infect macrophages was conferred by the envelope gene. This was demonstrated by the ability of the recombinant virus, NL4-3envA, which contains the envelope of NL4-3(M) in the context of the NL4-3 genome, to infect and replicate in MDM cultures. The envelope gene of NL4-3(M), however, did not confer ability to rapidly kill HL-60 cells. Together, these findings demonstrate that viral determinants controlling entry into MDM are different trom the determinants controlling the cytopathic phenotype in HL-60 cells.

Murine leukemia viruses (MuLV) are retroviruses that play important roles in the study of oncogenes, integration, transcriptional regulation and gene therapy. Mink cell focus-inducing (MCF) viruses are polytropic MuLVs that by definition infect cells from a wide variety of species. Their ability to infect human cells and their utility as gene therapy vectors were not well characterized. To address this issue, primary and immortalized human cells were tested for their ability to be infected by MCF packaged defective vectors as well as replication competent MCF virus. A new packaging cell line, called MPAC, was created to package defective retroviral vectors in virus particles with envelope proteins derived from a Moloney mink cell focus-inducing (Mo-MCF) virus. The cellular tropism of MPAC-packaged retroviral vectors was the same as replication competent MCF viruses. Testing various established cell lines showed some human cell lines could be infected with MPAC-packaged vectors while others cannot. In addition, I show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication. This indicates that some human cells express a protein on their surface that acts as a receptor for MCF viruses and allows MCF viral entry. In addition, the human cells that express a receptor for MCF viral entry did not show any further block to viral replication. An important determinant in the pathogenic phenotype of MCF 247 has been mapped to the enhancer region of the retroviral long terminal repeat (LTR). Recombination of endogenous genetic elements with the 3' portion of envoccurs and incorporates unique LTR sequences. Most strongly pathogenic MCF viruses have a duplication of the enhancer element found in the LTR. AKR mice are an inbred strain of mice that develop spontaneous T-cell lymphomas between 6 and 12 months of age. 12-25 % of MCF induced early lymphomas of AKR mice show MCF viral integration's near c-myc in an opposite transcriptional orientation. A replication competent MCF virus containing a bacterial amber suppressor tRNA gene (supF) was used to investigate the changes in the enhancer region following injection of MCF containing one enhancer in the LTR. Newborn AKR mice were injected with the supF tagged replication competent virus and observed for signs of leukemia development (ruffled fur, lethargy, and tumor development). When these signs were detected, the animals were sacrificed and DNA was prepared from the isolated tumors. Thirty-one tumors DNA were analyzed for the presence of supF tagged virus and rearrangement of the c-myc locus. Nine supF tagged proviral LTRs integrated near c-myc from four animals were PCR amplified, sequenced, and/or cloned. All of the enhancer elements analyzed were derived from proviruses that integrated in a reverse orientation with respect to c-myc locus. Two of the isolated enhancer elements contained only a few base changes whereas the majority contained duplications of different sizes that encompassed different transcription factor binding sites. The duplicated enhancer regions contained duplications from 82-134 bp in length. One tumor contained a proviral enhancer with only 5 bp changes relative to the injected virus. This suggests that the enhancers need only a few specific base changes relative to the injected virus to accelerate leukemogenesis. The other three tumors contained proviral enhancers with various size duplications and additional transcription factor binding sites. These data suggest that the injected virus is not pathogenic unless the enhancer region is altered. One proviral integration site encompassing a duplicated enhancer region and 139 bp of the c-myc gene locus was PCR amplified, cloned and sequenced. A search of the current transcription factor database (Transfac 3.3) showed no known transcription factor binding site sequences were created at the junction of the enhancer duplications. The common motif of LVb, core NF-1, and GRE transcription factor binding sites, described by Golemis at al (57), was conserved throughout the isolated enhancers. Most of the enhancer elements contained additional NF-кB and/or GRE sites in close proximity to the conserved LVb-core region. These results support the hypothesis that additional NF-кB and/or GRE binding sites cooperatively interact with the conserved GRE-NF-1-LVb-core motif in c-myc induced leukemogenesis. In addition, two unique families of enhancer duplications were identified. The two families contained enhancers isolated from different tumors that displayed sequence homology and transcription factor binding site organization unique to each group.

Made available in DSpace on 2015-12-10T14:24:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-07. Added 1 bitstream(s) on 2015-12-10T14:30:35Z : No. of bitstreams: 1 000851956.pdf: 1446006 bytes, checksum: d0fd0cae1f3cd364c6ad457c27e8870b (MD5) / A cultura do pimentão (Capsicum annuum L.) tem elevada importância econômica e social para o Equador. Vários fatores têm contribuído para a baixa produtividade dessa cultura no país e dentre eles destacam-se as doenças de diversas etiologias. Poucas informações sobre a ocorrência de doenças nessa cultura estão disponíveis nas para as condições equatorianas. Neste trabalho foi realizado levantamento da ocorrência da doença pata seca e de viroses na cultura do pimentão, em plantios comerciais, durante anos de 2013 e 2014; a identificação dos agente(s) causal(ais) da pata seca; avaliações do eficácia da pulverização da aplicação de produtos fitossanitários (sulfato de cobre + fosetil alumínio; iprodione + clorotalonil; Trichoderma harzianum + T. koingii) em condições de campo, no controle da doença nos híbridos Nathalie e Quetzal e a sensibilidade in vitro do(s) patógeno(s) dessa doença a fungicidas (tebuconazole, benomil, clorotalonil, fosetil alumínio, iprodione, metalaxil + propanocarb e tebuconazole). A pata seca foi constatada em um 79,2% das propriedades, com incidência entre 5% a 53,6%, em quanto as viroses, foram detectada em 62,5% das propriedades, com incidência entre 9,6% a 61,2%. Os fungos S. clerotium rolfsii e Fusarium spp. foram isolados com maior freqüência das plantas com sintomas de pata seca. Entretanto, S.rolfsii foi mais virulento, sendo considerado o principal agente causal da doença pata seca. Não foi observada diferença dos produtos fitossanitários, aplicados no colo das plantas, na incidência de pata seca, nos dois ensaios realizados em campo. A aplicação de iprodione + clorotalonil propiciou uma maior quantidade de frutos comerciais nos dois ensaios e uma maior produção em um dos ensaios. Os Isolados de S. rolfsii apresentaram maior sensibilidade aos fungicidas tebuconazole, benomil e iprodione, ... / Sweet pepper (Capsicum annuum L.) has high economic and social importance for Ecuador. Several factors have contributed to the low productivity of this crop in the country and among them there are the diseases of different etiologies. Little information on the occurrence of diseases in this culture is available to the Ecuadorian conditions. This study was carried out survey of the occurrence of the disease pata seca and viruses in sweet pepper in commercial plantations, during the years 2013 and 2014; the identification of the agent (s) causal (es) of pata seca; evaluations of the effectiveness of spray application of pesticides (copper sulfate + fosetyl aluminum, iprodione + chlorothalonil; Trichoderma harzianum + T. koingii) in field conditions in controlling the disease in hybrid Nathalie and Quetzal and in vitro sensitivity ( s) pathogen (s) of the disease to fungicides (tebuconazole, benomyl, chlorothalonil, fosetyl aluminum, iprodione, metalaxyl + propanocarb and tebuconazole). The pata seca was found in a 79.2% of the properties, with an incidence of 5% to 53.6%, as in the viruses were detected in 62.5% of the properties, with an incidence of 9.6% to 61.2%. The fungus Sclerotium rolfsii and Fusarium spp. were isolated more frequently plants with symptoms of pata seca. However, S. rolfsii was more virulent, being considered the main causative agent of the disease pata seca. There was no difference of pesticides applied in the base of the plants, the incidence of pata seca in the two trial carried out in the field. The application of iprodione + chlorothalonil provided a larger amount of fruits in the two ...

Made available in DSpace on 2014-06-11T19:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-18Bitstream added on 2014-06-13T19:24:10Z : No. of bitstreams: 1 000754099.pdf: 1729116 bytes, checksum: 2b7d02470d50c8efac136fc81e002d7e (MD5) / Espécies de vírus dos gêneros Potyvirus, Carlavirus e Allexivirus são comumente encontradas na cultura do alho (Allium sativum L.) e em decorrência da sua propagação vegetativa, há o acúmulo de vírus de um ciclo para outro, formando um complexo entre espécies dos gêneros citados. Uma das principais causas da redução da produtividade ocorre devido à infecção por vírus, e a termoterapia associada à cultura de tecido pode ser um método eficiente para limpeza clonal e obtenção de alho semente livre de vírus. O estudo da degenerescência do alho livre de vírus é, portanto, importante para se conhecer o número de ciclos em que o alho semente, inicialmente sadio, pode ser multiplicado no campo, sem que ocorra a redução da produtividade. Neste estudo foi verificado que após três anos a produtividade foi 13,75% superior ao alho 100% infectado por vírus. Os bulbilhos aéreos também podem ser utilizados para a multiplicação de sementes e tem como vantagem o baixo custo quando comparado à utilização de bulbos provenientes da cultura de tecido. Entretanto, essa técnica só se torna viável com a utilização de matrizes isentas de vírus, pois a transmissão de vírus para os bulbilhos aéreos, provenientes de matrizes infectadas, podem chegar a 83,33% no caso dos potyvirus, 20% para carlavirus e 70% para allexivirus, segundo dados obtidos neste trabalho. Realizar uma diagnose precisa dos vírus que infectam o alho torna-se essencial para que não ocorram falhas durante a indexação. Pôde-se observar que para a detecção dos potyvirus, utilizando o teste de ELISA indireto, deve-se utilizar folhas novas, entre 21 e 63 dias após o plantio, pois a concentração viral nesse período é a mais elevada. Diferentes técnicas como o DIBA colorimétrico, DIBA quimioluminescente, ELISA e PCR em tempo real foram avaliadas para detecção do LYSV, aos 21 dias após a inoculação... / Virus species of genera Potyvirus, Carlavirus and Allexivirus are commonly infecting garlic plants (Allium sativum L.). Due to the vegetative propagation, the accumulation of viruses might occur from one cycle to another. The infection of viruses might induce yield losses, and the technique of thermotherapy associated with meristem tip culture is an efficient method to obtain virus-free seeds. The study of degeneration of seeds free of virus is important to know the number of cycles in which garlic may be multiplied in the field without reduction of productivity. In this study it was observed that after three years, the productivity increased 13,75% compared to 100% infected seeds. Currently, aerial bulbils may be used for multiplication of seeds with low cost, compared to meristem tip culture. However, this is a viable technique only if the matrix is free of viruses, because the transmission to aerial bulbils, from infected plants can reach 83.33 % for potyviruses, 20% for carlavirus and 65% for allexivirus, data obtained in this work. An accurate diagnosis in viruses that infect garlic is important to avoid mistakes during indexing material. It was 4 observed that the best time to detect potyvirus, through ELISA test is between 21 and 63 days after planting on young leaves, as the virus concentration is higher in this period. Whem using different techniques, such as the colorimetric DIBA, chemiluminescent DIBA, ELISA, and real-time PCR, it was possible to perform the detection of LYSV 21 days after inoculation. ELISA test detected LYSV only at 21 days after inoculation, whereas using real time PCR, in 5 days after inoculation it was possible to detect the virus due to its high sensitivity. The other tests also detected the virus 21 days after infection

Made available in DSpace on 2015-05-14T16:53:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-16Bitstream added on 2015-05-14T16:58:54Z : No. of bitstreams: 1 000828431.pdf: 2597564 bytes, checksum: d2a5d2090c67ea6f9db3f644cfef982f (MD5) / Realizou-se levantamento sorológico dos seguintes vírus respiratórios Herpesvírus Bovino 1 (BoHV-1), Vírus da Diarreia Viral Bovina (BVDV), Vírus da Parainfluenza Bovina Tipo 3 (bPIV-3) e Vírus Respiratório Sincicial Bovino (BRSV) em bovinos da raça Holandesa variedade Preta e Branca (HPB), criados no estado do Paraná. Colheram-se 714 amostras de sangue da veia jugular de bovinos, não vacinados para os agentes estudados, com mais de seis meses de idade, de 26 propriedades leiteiras, distribuídas em 17 municípios do estado do Paraná. Paralelamente à colheita, aplicou-se um questionário para cada propriedade estudada, com a finalidade de avaliar os fatores de risco associados aos vírus examinados. As amostras sorológicas foram submetidas ao teste de diagnóstico de virusneutralização no Laboratório de Viroses de Bovinos do Instituto Biológico de São Paulo. Foram observadas prevalências de 22,3%; 35,8%; 80,4% e 93,5% de bovinos sororreagentes e 65,3%, 88,5%, 96,1% e 100% das propriedades reagentes para BoHV-1, BVDV, bPIV-3 e BRSV, respectivamente. A coinfecção que ocorreu com maior prevalência foi a bPIV-3 e BRSV (37,1%). Após análise de regressão logística, os fatores de risco associados ao BoHV-1 foram: bovinos com idade maior que 48 meses (OR=15,012; IC95% 7,500 – 30,051), área da propriedade 26 a 50 hectares (OR=11,328; IC95% 2,482 – 51,697), lotação maior de três bovinos por hectare (OR=1,026; IC95% 0,370 – 2,847), percentual de entrada maior que 10% de bovinos (OR=52,520; IC95% 13,269 – 207,876); para o BVDV foram: bovinos com idade maior que 48 meses (OR=4,407; IC95% 2,456 – 7,906), região Sudoeste (OR=52,388; IC95% 4,629 – 5892,873), sistema de produção semi-intensivo (OR=1,333; IC95% 0,261 – 6,815), propriedade com número de animais menor ou igual a 50 bovinos (OR=16,682; IC95% 3,218 – 86,481), percentual de entrada maior que 10% de bovinos (OR=17,56; IC95% 7,613 – 40,506) e ... / A serological survey of the following respiratory viruses: Bovine Herpesvirus 1 (BoHV-1), Bovine Viral Diarrhea Virus (BVDV), Bovine Parainfluenza Virus Type 3 (bPIV-3) and Bovine Respiratory Syncytial Virus (BRSV) in Holstein cattle black and White (HPB), raised in Paraná state was performed. 714 blood samples were collected from the jugular vein of cattle not vaccinated for the studied agents, over six months of age, from 26 dairy herds, distributed in 17 municipalities of Paraná state. Alongside the collection, a questionnaire was used for each property studied in order to assess the risk factors associated with the viruses examined. The serum samples were subjected to the diagnosis test of virus neutralization at the Laboratory of Viral Diseases of Cattle of the Biological Institute of São Paulo. Frequencies of 22.3%; 35.8%; 80.4% and 93.5% of seropositive cattle were observed and 65.3%, 88.5%, 96.1% and 100% of the seropositive properties BoHV-1, BVDV, bPIV-3 and BRSV, respectively. The co-infection that occurred most frequently was the bPIV-3 and BRSV (37.1%). After logistic regression analysis, risk factors associated with BoHV-1 were cattle with more than 48 months of age (OR = 15.012; 95% CI 7.500 to 30.051), property area 26-50 hectares (OR = 11.328; 95% CI 2.482 – 51.697), stocking higher than three animals per hectare (OR = 1.026; 95% CI 0.370 to 2.847), cattle entry percentage higher than 10% (OR = 52.520 entry; 95% CI 13.269 to 207.876); for BVDV were cattle older than 48 months (OR = 4.407; 95% CI 2.456 to 7.906), Southwest region (OR = 52.388; 95% CI 4.629 to 5892.873), semi-intensive production system (OR = 1.333; 95% CI 0.261 to 6.815),property with fewer animals than or equal to 50 (OR = 16.682; 95% CI 3.218 to 86.481), a percentage of cattle entry higher than 10% (OR = 17.56; 95% CI 7.613 – 40.506) and neighboring pasture sharing (OR = 9.148; 95% CI 3.751 to 22.310); for bPIV-3 cattle older than 48 months ...

Made available in DSpace on 2015-08-20T17:10:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-20. Added 1 bitstream(s) on 2015-08-20T17:25:55Z : No. of bitstreams: 1 000837195_20150920.pdf: 65943 bytes, checksum: 3d08551c973f38fe482173b18c355947 (MD5) Bitstreams deleted on 2015-09-21T13:18:54Z: 000837195_20150920.pdf,. Added 1 bitstream(s) on 2015-09-21T13:19:51Z : No. of bitstreams: 1 000837195.pdf: 1333023 bytes, checksum: 03e8d0fbb6f677497c15b782303ed41d (MD5) / O herpesvírus caprino tipo 1 (CpHV-1) e o herpesvírus bovino tipo 1 (BoHV-1) acarretam consideráveis perdas econômicas para o produtor, uma vez que determinam transtornos reprodutivos na espécie hospedeira. A escassez de estudos sobre esses vírus em plantéis caprinos brasileiros direcionaram o presente estudo que objetivou verificar se indícios da infecção por esses vírus influenciam a eficiência reprodutiva, por meio de uma análise precedente dos parâmetros reprodutivos descritos na escrituração zootécnica apresentados por animais positivos e negativos no exame pontual de um rebanho caprino com histórico de problemas reprodutivos no município de Monte Santo de Minas (MG), além de proceder pesquisa de ocorrência de anticorpos neutralizantes contra o BoHV-1 e o CpHV-1 pelo teste de virusneutralização (VN) em rebanhos caprinos em três ambientes diferentes: estado de São Paulo, Zona da Mata Mineira (MG) e uma propriedade no município de Monte Santo de Minas (MG). Assim, verificou-se que nos rebanhos do estado de São Paulo e da Zona da Mata Mineira não havia indícios de circulação viral recente de qualquer das espécies virais estudadas. Por outro lado, no rebanho de Monte Santo de Minas (MG) houve evidências da circulação viral recente especificamente para o CpHV-1, mas não para o BoHV-1. A análise estatística mostrou que houve mais chances dos animais serem positivos para o BoHV-1 no rebanho de Ipiguá (SP), e para o CpHV-1 no rebanho de Monte Santo de Minas (MG) e que, apesar do não isolamento, tanto BoHV-1 quanto CpHV-1 estão presentes entre rebanhos caprinos nos estados de São Paulo e de Minas Gerais. Ademais, a associação das condições sanitárias caracterizadas pela análise sorológica com detecção de alterações de parâmetros reprodutivos específicos no rebanho de Monte Santo de Minas (MG) direcionam o diagnóstico da infecção pelo CpHV-1 como... / The caprine herpesvirus type 1 (CpHV-1) and the bovine herpesvirus type 1 (BoHV-1) cause considerable economic losses for the producer, since they determine the host species reproductive disorders. The scarcity of studies about theses virus in brazilian goat herds directed this study that aimed to verify if infection evidence by them influence reproductive efficiency, carrying out the previous analysis of reproductive parameters of the zootechnical bookkeeping showed by negative and positive animals on the pontual assay of a goat herd with reproductive problems, in the municipality of Monte Santo de Minas, by research of the occurrence of neutralizing, besides research of the occurrence of neutralizing antibodies against BoHV-1 and CpHV-1, using virusneutralization test (VN), in goat herds in three different environments: São Paulo State, Zona da Mata Mineira (Minas Gerais State) and in a farm in the municipality of Monte Santo de Minas (Minas Gerais State). Thus, it was found that in São Paulo State and in Zona da Mata Mineira (MG) there was no evidence of recent viral circulation of any of the genotypes studied. On the other hand, in Monte Santo de Minas farm there were evidences of recent viral circulation specifically for CpHV-1, but not for BoHV-1. The statistical analysis showed that there were more chances of the animals were positive for BoHV-1 in Ipiguá herd (SP), and for CpHV-1 in Monte Santo de Minas (MG) herd and, despite not isolated both BoHV-1 and CpHV-1 are present in got herds of São Paulo and Minas Gerais herds. In addition, the association of health conditions characterized by serological analysis with detection of specific changes in reproductive parameters in Minas Gerais (MG) goat directs the diagnosis of infection by CpHV-1 as the cause of the problems presented by the herd showing that the presence of this virus causes reproductive problems in ...

Orientador: Samir Issa Samara / Coorientador: Edviges Maristela Pituco / Banca: Maria da Glória Buzinaro / Banca: Bruna Alexandrino / Banca: Moacir Marchiori Filho / Banca: Fernanda Senter Magajevski / Resumo: O herpesvírus caprino tipo 1 (CpHV-1) e o herpesvírus bovino tipo 1 (BoHV-1) acarretam consideráveis perdas econômicas para o produtor, uma vez que determinam transtornos reprodutivos na espécie hospedeira. A escassez de estudos sobre esses vírus em plantéis caprinos brasileiros direcionaram o presente estudo que objetivou verificar se indícios da infecção por esses vírus influenciam a eficiência reprodutiva, por meio de uma análise precedente dos parâmetros reprodutivos descritos na escrituração zootécnica apresentados por animais positivos e negativos no exame pontual de um rebanho caprino com histórico de problemas reprodutivos no município de Monte Santo de Minas (MG), além de proceder pesquisa de ocorrência de anticorpos neutralizantes contra o BoHV-1 e o CpHV-1 pelo teste de virusneutralização (VN) em rebanhos caprinos em três ambientes diferentes: estado de São Paulo, Zona da Mata Mineira (MG) e uma propriedade no município de Monte Santo de Minas (MG). Assim, verificou-se que nos rebanhos do estado de São Paulo e da Zona da Mata Mineira não havia indícios de circulação viral recente de qualquer das espécies virais estudadas. Por outro lado, no rebanho de Monte Santo de Minas (MG) houve evidências da circulação viral recente especificamente para o CpHV-1, mas não para o BoHV-1. A análise estatística mostrou que houve mais chances dos animais serem positivos para o BoHV-1 no rebanho de Ipiguá (SP), e para o CpHV-1 no rebanho de Monte Santo de Minas (MG) e que, apesar do não isolamento, tanto BoHV-1 quanto CpHV-1 estão presentes entre rebanhos caprinos nos estados de São Paulo e de Minas Gerais. Ademais, a associação das condições sanitárias caracterizadas pela análise sorológica com detecção de alterações de parâmetros reprodutivos específicos no rebanho de Monte Santo de Minas (MG) direcionam o diagnóstico da infecção pelo CpHV-1 como... / Abstract: The caprine herpesvirus type 1 (CpHV-1) and the bovine herpesvirus type 1 (BoHV-1) cause considerable economic losses for the producer, since they determine the host species reproductive disorders. The scarcity of studies about theses virus in brazilian goat herds directed this study that aimed to verify if infection evidence by them influence reproductive efficiency, carrying out the previous analysis of reproductive parameters of the zootechnical bookkeeping showed by negative and positive animals on the pontual assay of a goat herd with reproductive problems, in the municipality of Monte Santo de Minas, by research of the occurrence of neutralizing, besides research of the occurrence of neutralizing antibodies against BoHV-1 and CpHV-1, using virusneutralization test (VN), in goat herds in three different environments: São Paulo State, Zona da Mata Mineira (Minas Gerais State) and in a farm in the municipality of Monte Santo de Minas (Minas Gerais State). Thus, it was found that in São Paulo State and in Zona da Mata Mineira (MG) there was no evidence of recent viral circulation of any of the genotypes studied. On the other hand, in Monte Santo de Minas farm there were evidences of recent viral circulation specifically for CpHV-1, but not for BoHV-1. The statistical analysis showed that there were more chances of the animals were positive for BoHV-1 in Ipiguá herd (SP), and for CpHV-1 in Monte Santo de Minas (MG) herd and, despite not isolated both BoHV-1 and CpHV-1 are present in got herds of São Paulo and Minas Gerais herds. In addition, the association of health conditions characterized by serological analysis with detection of specific changes in reproductive parameters in Minas Gerais (MG) goat directs the diagnosis of infection by CpHV-1 as the cause of the problems presented by the herd showing that the presence of this virus causes reproductive problems in ... / Doutor