A high-throughput sequencing pipeline to characterize Citrus tristeza virus isolates was developed. Three alternative viral templates (total RNA, double-stranded RNA and virus particles) were first tested on a single, previously characterized GFMS12 sub-isolate for their enrichment qualities, and combined with random RT-PCR amplification were subjected to Illumina paired-end sequencing. Double-stranded RNA was found to be most useful and was selected for further characterization of additional isolates (glasshouse-kept and field-derived). A novel South African genotype, named CT-ZA3 was assembled de novo and shown to be the dominant component in all GFMS12 sub-isolates tested. Genotype distributions within field-derived isolates collected from commercial orange (Citrus sinensis) orchards revealed a mixed infection status, dominated by a resistance breaking (RB)-like component (Tai-SP) coupled with a minor, VT-like (mild) (Kpg3) component. Based on read mapping patterns from field isolates, it is further suggested that two previously unknown recombinants may be present: a SP/Kpg3 and HA16-5/Kpg3 combination. This study underlined the effectiveness of next-generation sequencing for genotype discovery as well as whole-genome characterization of CTV isolates to a level of detail previously unreachable with classical methods such as SSCP and Sanger sequencing of multiple clones. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / unrestricted

The aim of this study was to characterize strains used in the cross-protection scheme in South Africa by establishing Polymerase Chain Reaction (PCR) systems aimed at differentiating the strains by targeting the conserved p23 gene and the variable 5' half of the Citrus tristeza virus (CTV) genome. Two cross-protecting sources GFMS 12 and GFMS 35; and eight single aphid sub-isolates were tested and classified into strain types or genotypes. An oligonucleotide microarray system was developed to differentiate T30 and T36 strains of CTV. The establishment and development of such tests will enable the South African Citrus Industry to better select mild strains for cross-protection and determine which strains are present in citrus growing areas so as to better understand the dynamics of the disease. The first aim was to characterise the p23 gene of possible mild-strain cross-protection isolates in South Africa (RSA) and compare them to known isolates worldwide. Isolates were amplified with bi-directional RT-PCR, sequenced and phylogenetic analysis performed. The predicted amino acid sequences were compared for areas of possible variability for further strain differentiation. A bi-directional PCR developed by Sambade et al. (2003) was established that targets differences in amino acid positions 78-80 of the p23 gene and allows discrimination of isolates into mild, atypical and severe groups. The group designations of RSA isolates 390-3 and 390-5 were atypical; 390-4, 389-4 and 389-3 were mild; GFMS 35 had mild and atypical isolates; GFMS12, 12-7 and 12-9 had mild and severe isolates and; 12-5 was severe. The three main clusters on the phylogenetic tree confirmed the group designations of these isolates. Isolates in the atypical group were more diverse than ones in the mild or severe groups. There were 53 polymorphic sites within the amino acid sequences of p23 gene of the RSA and reference isolates, of which 4 distinct regions showed variability. The amino acid region 78-80 was confirmed as being very useful in grouping these isolates as mild, severe or atypical. The PCR system was robust, reproducible and has potential in the RSA Citrus industry as a screening tool in selecting mild strains for cross-protection and in detecting mixed strains in isolates. The secondary aim was to establish the 23 primer pair PCR system developed by Hilf et al. (2000) to differentiate isolates as T36, T30, VT or T3 genotypes. Each isolate was tested with RT-PCR using 23 individually optimised genotype specific primer sets (Hilf et al., 2000). The most common genotype detected was T30 and the least common was T3. The GFMS 35, T30 plant and 389-3 isolates had a homogenous T30 genotype profile and isolate 12-5 had a VT genotype profile. The 389-4, 390-3 and 390-4 isolates had a predominantly T30 genotype profile and isolates 12-7 had a predominantly VT genotype profile. Isolate GFMS 12 had a mixed genotype profile indicative of a mixed infection while isolates 390-5 and 12-9 appeared to have mixed genotypes of VT, T30 and T36. Isolates 390-3 390-4 and 390-5 had no amplification within regions 4-7 and appear to be highly variable isolates or possible recombinants. The T3 genotype specific markers were found in region 2 of a few isolates and could be a cross-reacting primer set to the T3o genotype. It is useful for homogenous strains in determining the genotypes, molecular marker information, possible variability or recombination and for approving isolates for mild strain cross-protection. Potential drawbacks of the system include non-amplification of regions; cross-reacting primers; difficulty in optimising; and secondary structures. It was difficult to objectively draw conclusions if an isolated had mixed genotypes since mixed genotype amplifications were not consistently found in all regions targeted. The third aim was to develop an oligonucleotide (oligo) microarray system to differentiate mild T30 and severe T36 strains. The 5' half of the CTV genome was Cy3 5'-end labelled and amplified. Oligos were designed to be T36-strain specific with a Tm above 60 °C and if possible a GC content above 65 %; and differed in amount and position of mismatches to strain T30. A standard operating protocol was set up by testing different labelling methods, hybridization mixes and washing steps. The array was tested using individual T30 and T36 strains as templates at 42, 52 and 60 °C. Experimental variation was quantified and normalised. The secondary structures of the hybridizing amplicons were determined by mfold (Zuker et al., 2003). Some oligos were specific at 42 °C and others at 52 °C. The hybridization allowed a clear differentiation of strain T36 with 13 of the T36-specific oligos at their optimal hybridization temperature. A few oligonucleotides showed cross-hybridization to strain T30 and were not used in further analysis. Oligonucleotides with 21 % or more mismatches were successful oligos, whereas ones that had 18 % or fewer mismatches had cross-hybridization. Some oligos were modified to include Locked Nucleic Acid (LNA) instead of DNA in an attempt to increase specificity with two of them having increased specificity compared to the unmodified DNA oligonucleotides. The successful differentiation by hybridization to strain specific oligos opens paths for highly parallel, yet specific assays for strain differentiation of CTV strains and a more thorough insight into the future strains circulating in RSA. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Orientadores: Dagmar Ruth Stach-Machado, Marcos Antonio Machado / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T17:36:17Z (GMT). No. of bitstreams: 1 Peroni_LuisAntonio_D.pdf: 5677765 bytes, checksum: 2a4cb6ec8aba9b1ec6655c771981377f (MD5) Previous issue date: 2008 / Resumo: Este trabalho visou efetuar a identificação dos epitopos das prottnas recombinantes CB-22 e CB-104 do capsídeo do Cítrus tristeza vírus (CTV), reconhecidos pelos anticorpos monoclonais (MAb) produzidos por Stach-Machado e colaboradores (2000). Assim, os epitopos reconhecidos pelos MAbs 30.G.02 e 37.G.ll, correspondem às seqüências de aminoácidos NLHIDPTLI e TQQNAALNRDLF, localizadas nas posições 32 a 40 e 50 a 61 das proteínas recombinantes CB-22 e CB-104. O epitopo reconhecido pelo MAb 39.07 que apresenta especificidade apenas para a CB-104, homóloga aos isolados severos do CTV corresponde a seqüência TDVVFNSKGIGN, localizados na posição 120 a 131 da proteína. Estes dados corroboram com os ensaios de ELlSA, confirmando o MAb 30.G.02 como um anticorpo universal, atuando quer como anticorpo de captura ou de detecção em ELlSA Sandwich, uma vez que, seu epitopo é conservado em 87.2% dos isolados de CTV. O MAb 37.G.ll deve ser adotado apenas como anticorpo de detecção pois reconhece uma seqüência encontrada em 62,6% dos isolados, permitindo uma identificação ampla, sem efetuar a diferenciação de estirpes. O MAb 39.07 foi classificado como "forte", pois seu epitopo está presente em apenas 19,7% das seqüências, sendo estas provenientes de isolados fortes do CTV de todo o mundo. A utilização de peptídeos lineares não permitiu a identificação e caracterização do epitopo do MAb 1C.04-12, confirmando dados preliminares de nosso laboratório que permitiram inferir que o mesmo reconhece um epítopo conformacional. O conhecimento das características estruturais da proteína do capsídeo viral possibilita obtenção de informações relevantes quanto às suas propriedades biológicas, como a participação na organização e montagem da partícula viral, proteção do material genético, interação com o inseto-vetor e com a planta hospedeira e também, pela "movimentação" da partícula viral no processo de infecção na planta. Considerando que até a presente data não existem dados disponíveis no Protein Data Bank (PDB) sobre a estrutura tridimensional da proteína do capsídeo do CTV, efetuamos o estudo estrutural da CB-22, utilizando Dicroísmo Circular (CD) e Difração de Raios X a Baixo Ângulo (SAXS). A análise dos dados de CD mostrou um espectro característico de proteínas cuja estrutura secundária é predominantemente formada por a-hélices, sendo este resultado coerente com a estrutura secundária predita pelo software PSIPRED. Os estudos de SAXS revelaram uma proteína altamente oligomerizada com estruturas formadas por subunidades, que variam de acordo com a concentração da mesma em solução. Este resultado, embora tenha impossibilitado a resolução e modelagem da estrutura secundária da CB-22, demonstrou que esta proteína recombinante mantém a função da proteína nativa, responsável pela formação do capsídeo vira!. Assim sendo, podemos postular a sua utilização como uma molécula carreadora, podendo ser aplicada em protocolos de vacinação ou no transporte dirigido de ácidos nucléicos ou proteínas. Por fim, este trabalho estabeleceu diagnósticos imunomoleculares como Imunocaptura-PCR (IC-PCR) e Imuno-PCR (I-PCR) para a detecção da bactéria gramnegativa Xylella jastid[osa, agente etiológico da Cvc. Os ensaios imunomoleculares apresentaram o limite de detecção muito superior aos diagnósticos utilizados na rotina como ELlSA e PCR, sendo o limite de detecção do IC-PCR e do I-PCR de 103 a 101 células, respectivamente. Portanto, estes métodos são uma alternativa viável para efetuar o diagnóstico da CVC e também em estudos epidemiológicos, com a vantagem de eliminar as etapas de extração e concentração do material genético bacteriano, permitindo um diagnóstico acurado dessa doença / Abstract: This work aims to carry out the identification of epitopes of the Citrus tristeza virus (CTV) recombinant coat protein (CB-22 and CB-l04) which are recognized by four monoclonal antíbodies (MAb) produced by Stach-Machado et aI. (2000). MAb 1C.04-12 recognizes CB-22 protein, homologous to CTV isolates that induce weak symptoms in indicators plants; while MAb 39.07 reacts to CB-l04 protein, homologous to severe CTV isolates and, MAbs 30.G.02 and 37.G.ll recognize both proteins. MAb 30.G.02 recognized the sequence formed by amino acids NLHIDPTLI, located at positions 32 to 40, while MAb 37.G.ll recognized the amino acids from 50 to 61, whose sequence is TQQNAALNRDLF. On the other hand, the MAb 39.07 recognized the sequence TDVVFNSKGIGN, located at positions 120 to 131 in CB-l04 protein. MAb 30.G.02 was considered a Universal antibody, once its epitope is conserved in 87.2% of CTV isolates, and can be applied as a coating antibody or even as a detection antibody in ELlSA Sandwich assays. The epitope of MAb 37.G.ll is conserved in 62.6% of sequences and, can be applied as detection antibody, allowing a quick and wide range screening but without strains differentiation. Finally, MAb 39.07 was classified as "Severe" antibody, once its epitope is present at only 19.7% of CTV sequences, which were derived frem severe worldwide CTV isolates. The technique of linear peptides did not allow the epitope determination of MAb 1C.04-12, since this antibody may recognize a conformational epitope in the CB-22 tridimensional structure, according to the early data from our laboratory showing that this MAb did not recognize the CB-22 under denaturating conditions, confirming the conformational nature of its eptiope. The knowledge about structural features of viral coat protein enables acquiring relevant information about biological properties of this protein. The coat protein plays an essential role in the organization and assembly of viral particle, acts in genetic material protection and, probably is involved in the interaction with insect-vector and with host plant, being responsible for the movement of viral particle in the infection processo However, there are not any data in Protein Data Bank (PDB) about the secondary and tertiary structures of CTV coat protein, and so, this work carried out a structural study about CB-22 by Circular Oichroism Spectroscopy (CO) and Small-Angle X-ray Scattering (SAXS). The CO data analysis demonstrated a spectrum characteristic of proteins whose secondary structure is predominantly formed by a-helixes, and these results are coherent of predicted structure by PSIPREO software. The SAXS data revealed a highly oligomeric protein comprising many subunits whose quantities are dependent and vary according to the protein concentration in solution. These data, although had impaired the modeling and resolving of CB-22 secondary structure, demonstrated that this recombinant protein maintain the function of native protein, responsible for the assembly of CTV capsid, and this structure without bacterial ONA is also called virus-/ike, which allows to postulate its usefulness as nucleic acid ca rrier in vaccination protocols. Finally, the third objective of this work was the establishment of immunomolecular diagnosis to gram-negative bacterium, Xy/ella fastidiosa, which causes Citrus Variegated Chlorosis in Brazil. We carried out two immunomolecular assays, like !mmmuno,Çapture PCR and !mmuno-PCR for direct detection of X. fastidiosa without ONA isolation. Whereas the reactivity of ELlSA and PCR ranged from 106 to 104 bacterial cells, the IC-PCR sensitivity was up to 103 and the detection limit of I-PCR was up to 101 bacterial cells. Therefore, ICPCR and I-PCR assays provide an alternative for quick and very sensitive rnethods to screening X. fastidiosa, with the advantage of not requiring any concentration or ONA purification steps while still allowing an accurate diagnosis of Cvc / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

Citrus tristeza virus (CTV), an economically important graft-transmissible pathogen of citrus, causes major global declines in citrus production. In the commercial citrus of the Lower Rio Grande Valley of Texas (LRGV), where red grapefruit on tristeza-decline sensitive sour orange rootstocks predominate, incidence of CTV is low. The efficient CTV vector, the brown citrus aphid (BrCA, Toxoptera citricida Kirkaldy) is now established in Mexico and Florida, thus information is needed on the severity of CTV, CTV aphid transmission and the performance of transformed citrus towards CTV before T. citricida arrives in Texas so that appropriate management strategies can be selected. Biological indexing and molecular typing were performed on fifteen Texas CTV isolates. The majority of the CTV isolates tested contained the most severe CTV types known. In Florida, T. citricida were fed on crude CTV preparations in vitro and could transmit CTV to virus-free receptor plants with two CTV isolates, whereas a more highly purified CTV preparation from one CTV isolate was not transmitted by T. citricida. There were no differences in the majority of treatments in infectivity neutralizations using three CTV-derived antibodies (p25, p27 and p20). CTV p20 antibodies significantly enhanced the occurrence of CTV transmission in one test. The CTV genome of isolate H33 was sequenced using 'shot gun' methods. The H33 major component and H33 minor components were phylogenetically compared to the six other full-length CTV sequences. An untranslatable CTV coat protein gene was genetically transformed into the genome of the Texas commercial Rio Red grapefruit variety, and fifty-two independent transgenic lines were produced. CTV challenge responses by the transgenic lines were variable. Individual plants could be identified which had low virus titers by ELISA detection, a temporal decrease in virus titer, or a delay in virus titer accumulation. Comparing all wild types to all transgenic lines over every assessment revealed significant decreases in virus titer in the transgenic lines compared to that of the wild type. An RNA entity with similarities to marafiviruses was identified in a CTV infected plant. The entity appears non-graft transmissible to citrus, and non-mechanically transmissible to a range of herbaceous species.

Orientador: Mônica Alonso Cotta / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-17T12:15:05Z (GMT). No. of bitstreams: 1 Moreau_AlbertoLuisDario_D.pdf: 4972430 bytes, checksum: 23390541e98ea8174b689428392b8457 (MD5) Previous issue date: 2011 / Resumo: Na primeira parte deste trabalho, nós investigamos o processo de crescimento de um biofilme de bactérias (Xylella fastidiosa) inoculadas sobre lamínulas de vidro. O tamanho e a distância entre os biofilmes foram estudados por imagens de microscopia óptica; e uma análise fractal foi realizada usando conceitos de escala e imagens de AFM. Observamos que biofilmes diferentes mostram características fractais semelhantes, embora as variações na morfologia possam ser identificadas para diferentes estádios de crescimento do biofilme. Dois tipos de padrões estruturais são identificados através da dimensão fractal (Df) sugerindo que o crescimento do biofilme pode ser entendido como o modelo de Eden nos estágios de formação e no final, enquanto para o estágio de maturação aparecem evidências do modelo DLA (diffusion-limited aggregation). Estes resultados foram correlacionados à formação da matriz do biofilme que pode dificultar a difusão dos nutrientes e por isso criar condições para um crescimento DLA. Ainda com o AFM, fizemos medidas de espectroscopia de força para estudar a interação específica entre antígeno-anticorpo relacionados ao vírus CTV (citrus tristeza virus). Para tanto foi realizado o estudo da imobilização deste material biológico nas superfícies da ponta do AFM, e nos substratos planos de Si e InP. Usamos para isto imagens topográficas de AFM, imagens de microscopia eletrônica e ensaios imunoquímicos de ELISA; com isso pudemos confirmar que tanto o antígeno quanto o anticorpo foram imobilizados e que eles continuavam em seus estados nativos. Com as medidas de espectroscopia de força, detectamos que a diferença de força entre as interações específicas (antígeno-anticorpo) e não-específicas (antígeno-antígeno) foi de aproximadamente 60%. Utilizamos a mesma rotina de preparação em substrato de InP para o desenvolvimento de um biossensor baseado no funcionamento de um transistor FET (field-effect transistor). Os anticorpos foram imobilizados na superfície do semicondutor, enquanto doses de antígenos livres eram adicionadas a uma célula líquida que mantinha contato com esta superfície. Com curvas de corrente vs tensão em regiões lineares do dispositivo, estudamos a resposta, a sensibilidade e a especificidade do sensor, obtendo resultados promissores indicando viabilidade no desenvolvimento e no uso do mesmo. Por fim, estudamos a durabilidade de pontas de AFM com CNT (carbon nanotube) encapsulados com carbono amorfo numa amostra padrão semicondutora de pontos quânticos, além de estudar a sua estabilidade em meio líquido. Duas pontas foram estudadas, uma fabricada pelo nosso grupo e outra comercial, fornecida pela empresa americana CDI (Carbon Design Inovation). Utilizamos ferramenta de FT (fourier transform) para o estudo da resolução das pontas em imagens topográficas de AFM, e verificamos que ambas as pontas duraram mais de 400 imagens tendo uma perda da resolução de ~ 7% no ar e de ~ 5% ¿ em relação às medidas a seco ¿ em meio líquido, mostrando a viabilidade de seu uso no estudo de amostras biológicas / Abstract: We have investigated the growth process of Xylella fastidiosa biofilms inoculated on glass. The size and the distance between biofilms were analyzed by optical microscopy images; a fractal analysis was carried out using scaling concepts and Atomic Force Microscopy (AFM) images. We observed that different biofilms show similar fractal characteristics, although morphological variations can be identified for the different biofilm stages. Two types of structural patterns are suggested from the observed fractal dimensions Df: and suggest that the biofilm growth can be understood as an Eden model in the initial and final stages, while diffusion-limited aggregation (DLA) seems to dominate the maturation stage. Changes in the correlation length parallel to the surface were also observed; these results were correlated to the biofilm matrix formation which can hinder nutrient diffusion and thus create conditions to drive DLA growth. Atomic force spectroscopy was used as a method to investigate the specific antibody-antigen binding using proteins of the CTV (citrus tristeza virus). Subsequently, the chemical processes of covalent immobilization of the used biomolecules to different solid supports ¿ Si3N4 AFM Tips, Si and InP plane substrates ¿ was developed and characterized. The verification of the chemical binding process by different methods like AFM topography experiments, electron microscopy and ELISA immunochemical assays showed a successful immobilization of the biomolecules and that they were still in their native state. The analysis of the spectroscopic force data showed a significant difference in binding force between the specific antibody-antigen complexes and the non-specific controls (approximately 60%). The same chemical immobilization process was used to bind biomolecules to solid InP supports to develop a biosensor based on the field-effect transistor (FET) principle. The same antibodies, as used in atomic force experiments, were immobilized covalently to the semiconductor surface. The antibody-antigen complexation by adding the specific antigens to the functionalized surface was detected through changes in the current vs tension curves of semiconductor sample. The characterization of the electrochemical response, the molecular sensibility and the specificity of this biosensor showed a suitable method to detect specific molecular binding events. Further, the durability and stability of AFM tips with bonded carbon nanotubes with encapsulated amorphous carbon was studied on a standard semiconductor sample with quantum dots in air and liquid environment. Two different CNT AFM-tips were studied within this study in acoustic mode. One was assembled in our group and the other tip was provided by the american CDI (Carbon Design Innovation) company. The topographic AFM images were analyzed via FT (Fourier Transform) to study the spatial resolution of these tips. In air, both types of CNT tips showed a minor resolution decrease of approximately 6% after 400 topographic scans. In liquid environment an overall 10% lower spatial resolution of the tips was observed compared to the resolution in air which nevertheless makes these CNT AFM-tips suitable for experiments with native biological samples / Doutorado / Biofísica / Doutor em Ciências

La tristeza de los cítricos es una de las enfermedades de mayor importancia en el cultivo de los cítricos. Dada la imposibilidad práctica de contener su dispersión y evitar su evolución hacia formas más virulentas, el control de los daños mediante protección cruzada o mediante resistencia mediada por genes del patógeno utilizando plantas transgénicas requiere un conocimiento detallado de las interacciones CTV-cítricos. Para este estudio se inocularon cuatro aislados con distintas características patogénicas en lima Mexicana (LM), Citrus macrophylla (CM), naranjo dulce (ND) y naranjo amargo (NA). La estimación de la carga viral mediante ELISA y RT-PCR cuantitativa a tiempo real en la primera brotación y al cabo de 9 meses, mostró que NA ofrece una resistencia inicial a la invasión por CTV. Asimismo, sugirió que la intensidad de los síntomas inducidos por CTV no era una consecuencia directa de la acumulación viral. El análisis de la actividad replicativa de las distintas combinaciones aislado/huésped reveló una cinética de acumulación viral paralela a la actividad replicativa. Por otro lado, mientras en los huéspedes susceptibles se detectaron siRNAs de CTV en la primera brotación, la activación del silenciamiento en NA fue mucho más tardía indicando que la resistencia inicial a la acumulación viral en este huésped no era consecuencia del silenciamiento. Posteriormente, se analizó en más detalle el tipo de limitaciones que ofrecía NA a la invasión sistémica de CTV observándose que éste presentaba una limitación al movimiento viral. El virus se detectó inicialmente en la raíz y el nivel de acumulación del mismo afectaba la posterior carga viral en la copa, de forma variable según los aislados, así para el aislado T385 la raíz actuaría como reservorio viral. Finalmente, el empleo de la tecnología de las micromatrices de cDNA permitió analizar a nivel transcriptómico la respuesta del NA al inicio de la infección por CTV y confirmar las hipótesis formuladas anteriormente. / Comellas Serra, M. (2009). Estudio de la interacción entre naranjo amargo y el virus de la tristeza de los cítricos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/7323 / Palancia