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Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T21:03:41Z (GMT). No. of bitstreams: 1 Gregoracci_GustavoBueno_M.pdf: 4620017 bytes, checksum: f7feaaf632175abbe340a9d82f379a0c (MD5) Previous issue date: 2006 / Resumo: Os bacteriófagos, vírus que infectam procariotos, são as entidades biológicas mais numerosas do globo. Sua abundância desencadeou revisões em diversos campos como genética, evolução e ecologia, e os fagos atualmente são reconhecidos como importantes vetores para o fluxo de matéria, energia e informação em ambientes naturais. Contudo, há uma enorme diferença entre a quantidade de bacteriófagos estimada no globo (mais de 1030 partículas virais) e a quantidade descrita na literatura (em torno de 5300 amostras). Esta amostragem restrita reflete complicações para estudos de diversidade viral e demonstra as limitações de nosso conhecimento sobre estes vírus. Além disso, a emergência de bactérias resistentes a múltiplos antibióticos implica em uma necessidade de novas práticas terapêuticas, e os fagos representam uma alternativa digna de estudos aprofundados. Assim sendo, este trabalho objetivou o isolamento, a partir de esgoto, e a caracterização biológica de bacteriófagos líticos contra patógenos humanos, visando ampliar a amostragem destes vírus e selecionar novos possíveis agentes terapêuticos. A caracterização envolveu análises morfológica, genética e de especificidade de hospedeiros. As metodologias adaptadas de isolamento e caracterização permitiram a análise de mais de 20 bacteriófagos, todos pertencentes à ordem Caudovirales, bem como de uma proposição para a classificação taxonômica dos isolados. Merece destaque especial o isolamento de três fagos contra Chromobacterium violaceum, feito inédito na literatura, bem como estudos sobre a suscetibilidade deste organismo a diversos bacteriófagos isolados. Por fim, ensaios biológicos sugerem estudos posteriores sobre a aplicação de um destes fagos, denominado Shfl1, para a terapia de Shigella flexneri em infecções humanas, mas são incertos quanto ao potencial do mesmo para contenção deste patógeno no processamento de esgoto comum / Abstract: Bacteriophages, viruses that infect prokaryotic hosts, are the most numerous biological entities of the world. Their abundance implied in reviews in several areas of knowledge, like genetics, evolution and ecology, and phages are now recognized as important vectors in the flux of matter, energy and information in natural environments. However, there is a huge difference between the estimated number of bacteriophages in the world (more than 1030 viral particles) and the quantity described in the literature (around 5300 samples). This restricted sampling reflects in complications to studies of viral diversity and demonstrates our limited knowledge regarding these viruses. Furthermore, the emergence of bacteria resistant to multiple antibiotics implies in a need for new therapeutical approaches, and phages represent an alternative worthy of additional studies. As being so, our purpose in this study was to isolate, from raw sewage, and to biologically characterize lytic bacteriophages, intending to extend these viruses¿ sampling and to select possible new therapeutic agents. Characterization involved morphological, genetic and host range analysis. The adapted protocols for isolation and characterization permitted the analysis of more than 20 bacteriophages, all belonging to the Caudovirales order, as well as a tentative taxonomic classification of the samples. Other distinctive results include the isolation of three bacteriophages against Chromobacterium violaceum, previously unknown in the literature, as well as the study about the susceptibility of this organism to several isolated phages. Moreover, biological assays suggested further studies about the application of one of these phages, named Shfl1, in the treatment of Shigella flexneri infections in humans, but were uncertain about the potential of the same virus to restrain this pathogen during sewage processing / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

Rotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.

Vaccines directed against the influenza virus become ineffective due to continuous mutation. An alternative approach might be to control replication at the genomic level by enzymatic methylation of the polymerase genes. Hence in this study, a method to locate and successfully isolate the H1N1 influenza A polymerase genes was investigated. The virus was cultured in chick embryos via the allantoic route using aseptic techniques. Following incubation, the allantoic fluid was isolated and washed to remove any contaminating blood cells. The allantoic fluid was checked for fungal and bacterial contamination using the blood agar test and the presence of the virus was established by the haemagglutination titration test. Viral particles were pelleted by ultracentrifugation. Electron microscopy verified the morphology and size of these viruses while immunofluorescence studies, using a monoclonal antibody, confirmed the influenza A strain. The ribose test verified the presence of RNA in the samples. Purified viral pellets were pooled and homogenised in buffer containing guanidine thiocyanate, mercaptoethanol and sarkosyl. The samples were incubated on ice before mechanical disruption of the virus. Viral RNA was isolated from the upper aqueous layer after a standard phenol/chloroform extraction procedure. RNA was quantified spectrophotometrically and purity assessed initially by the absorbance ratio readings at 260/280 nm. Electrophoresis of the RNA samples was performed together with RNA molecular weight markers on a 1.5% formamide agarose gel. Five bands were identified and the band containing the polymerase genes was size selected, located and excised. Purification of the polymerase genes from the agarose was achieved by using the BIO 101 RNAid kit. The three isolated polymerase RNAs were reverse transcribed using the Boehringer Mannheim cDNA synthesis kit. The results indicate that the H1N1 influenza virus was successfully grown and isolated from chick embryos. Absence of contamination and verification of viral presence at different stages of the study were indications that asepsis was successfully achieved. The RNA obtained was sufficient and suitable for cDNA synthesis. This cDNA may now be used for further molecular analysis and subsequent DNA methylation studies. Further, transfection studies may then be performed to determine, if any, the the expression of methylated and unmethylated cDNA. / Thesis (M.Med.)-University of Natal, 1992.

Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T13:06:28Z (GMT). No. of bitstreams: 1 Carvalho_RodrigoFranco_D.pdf: 1262824 bytes, checksum: 9c044e92e7146ea5bb090e025e656061 (MD5) Previous issue date: 2005 / Resumo: O herpesvírus eqüino do tipo 1 (EHV-1) é um membro da subfamília Alfaherpesvirinae, implicado no surgimento de distúrbios respiratórios, reprodutivos e nervosos em cavalos. A principal forma de contaminação dos animais é através do contato direto com secreções contaminadas pelo vírus. No eqüino, a disseminação do vírus ocorre pela transposição da infecção respiratória a outros órgãos e sistemas através da corrente sanguínea. Pouco se sabe sobre a ocorrência do EHV-1 no Brasil. Dessa forma, este estudo teve por objetivo o isolamento do EHV-1 a partir de material biológico e produção e análise de dados moleculares de isolados brasileiros de EHV-1. Durante este estudo, foi realizado o isolamento de uma amostra de EHV-1 a partir da inoculação de material clínico em células de derme eqüina (ED). Este isolado foi diagnosticado como EHV-1 através da reação em cadeia da polimerase (PCR) para o gene da timidina quinase (tk). Neste trabalho, foram também realizados os seqüenciamentos de fragmentos de PCR derivados do isolado aqui descrito, de uma outra amostra brasileira de EHV-1 e de duas amostras estrangeiras do vírus para análise filogenética. A análise comparativa entre seqüências permitiu inferências sobre o nível de divergência entre os vírus estudados, além da listagem de seqüências regulatórias para atividade gênica em um sítio do genoma localizado próximo ao gene tk. Na região genômica reportada foram contextualizadas ao menos três genes (ORF 38, ORF 37 e ORF 36). Os dados levantados com o seqüenciamento de amostras de EHV-1 de origens geográficas distintas (Brasil, Europa e América do Norte) não mostraram divergências, o que pode estar associado a um processo seletivo constritivo, que impediria a fixação de novas mutações naquela região. A ausência de divergências também pode estar associada à importância dessa região na regulação gênica do EHV-1. Também é um indicativo para a fidelidade dos mecanismos de replicação envolvidos na síntese do DNA viral, o que sugere a importância da região estudada na regulação da expressão gênica do EHV-1 / Abstract: Equine Herpesvirus Type 1 (EHV-1) is a member of Alphaherpesvirinae subfamily implicated with abortions, respiratory and neurological disturbs in horses. The principal mode of viral transmission is through close contact virus-containing secretions of infected horses. Systemic pathogenesis in which this virus is implicated combines primary respiratory infection and spread of viral particles through the circulatory/lymphatic system. Until today, there are only few studies involving the isolation of this virus in Brazil. Thus, the main goal of this study was the isolation of EHV-1 from biological material and the production and analysis of molecular data derived from Brazilian EHV-1 isolates. Clinical samples were screened by inoculation into Equine Dermis (ED) cells monolayers, searching for the characteristic citopathic effect produced by EHV-1. Inoculation of one tissue sample has presented a suggestive citopathic effect. Re-inoculation of the original tissue homogenate in a second, independent experiment reproduced the same positive result. Following these observations, infection agent diagnostic was done by PCR for thymidine kinase (tk) gene. The results demonstrated that sample was EHV-1 positive. In this work, it was done either the sequencing of PCR fragments derived from two Brazilian and two foreign samples of EHV-1 for filogenetic and genomic analyses purposes. It was assigned at least three Open Reading Frames contexts (ORF 38, ORF 37, ORF 36). The data do not show genetic variation between sequences. The high level of genetic conservation for this region, despite the distinct geographic origins (Brazil, Europe and North America) of EHV-1 samples studied, indicates a strong selection process against the fixation of new mutations. It also highlights a high level of fidelity for DNA replication and strongly suggests the importance of the studied region for EHV-1 gene regulation / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Tomato spotted wilt virus (TSWV), a Tospovirus, is one of the ten most economically destructive plant viruses worldwide, causing losses exceeding one billion U.S. dollars annually on several crops. In South Africa (SA), TSWV has become an important virus in many economically important crops. The main objective of this research project was to isolate, identify and characterise TSWV isolates occurring in SA. A review of current literature assembled background information on TSWV molecular biology, epidemiology, transmission, detection and control. A TSWV isolate infecting pepper (Capsicum sp.) occurring in KZN was isolated and partially characterised. The virus was positively identified as TSWV using the enzyme-linked immunosorbent assay (ELISA) and the presence of typical necrotic TSWV symptoms on Nicotinia rustica L. Symptomatic leaves were harvested and the virus was partially purified using standard procedures. Under the transmission electron microscope (TEM), typical quasi-spherical and dumbbell-shaped particles of 80-100nm in diameter were observed in negatively stained preparations of both crude and purified virus samples. In negatively stained ultra-thin virus infected leaf sections, an abundance of mature viral particles (100nm) housed in the cisternae of the endoplasmic reticulum (ER) were observed among typical viroplasm inclusions (30nm) and hollow tubules (200-300nm). A viral protein migrating as a 29kDa band, which corresponds to the TSWV nucleocapsid (N) protein, was observed after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Total plant RNA, isolated from N. rustica displaying typical symptoms was subjected to reverse-transcription polymerase chain reaction (RT-PCR) using .primers specific to the nucleocapsid (N) gene. An expected 760bp product was amplified. The results obtained in this study confirm the presence of TSWV in infected pepper plants from KZN. The genetic diversity of TSWV isolates occurring in SA was examined. The nucleocapsid (N) gene sequences of six SA TSWV isolates originating from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were determined and used in a phylogenetic tree comparison with TSWV isolates occurring in different geographical locations in the world. Nucleotide sequence comparisons of the N gene revealed high levels of similarity between the SA isolates and TSWV isolates from Asia and Europe. SA isolates showed a high degree of sequence similarity (99-100%) which was reflected in their distinct clustering pattern. The resistance of tomato (Lycopersicon escuJentum Mill.) plants with natural and transgenic resistance against mechanical inoculation with TSWV isolates occurring in SA was evaluated. The Stevens cultivar which has natural resistance conferred by the Sw-5 gene and the transgenic 13-1 line, which expresses the nucleocapsid (N) protein gene of the TSWV-BL isolate, was used as test cultivars. Plants were assessed for TSWV resistance using a disease severity rating scale and measurements of virion accumulation levels (A405nm). There were no significant differences among the reactions produced by the six TSWV isolates on the test plants. Although both plants were susceptible to the SA TSWV isolates by exhibiting similarly high viral accumulation levels, the transgenic tomato line showed milder disease severity compared to the natural resistant cultivar. Results suggest that transgenic resistance is a more effective approach in the control of TSWV in SA. The information generated in this study will be useful in formulating effective control measures using genetic engineering approaches for this economically important virus. Such approaches will be used as a tool to make strategic decisions in an integrated control programme for ISWV. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.