Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

Assessment of recycled concrete aggregate for road base and sub-base

Population increase and economic developments can lead to construction as well as demolition of infrastructures such as buildings, bridges, roads, etc and used concrete is the main waste product of them. Recycling of waste concrete to obtain the recycled concrete aggregates (RCA) for base and/or sub-base materials in road construction is a foremost application to be promoted to gain economical and sustainable benefits. As the mortar, bricks, glass and asphalt present in different constituents in RCA, it exhibits inconsistent properties and performance. In this study, six different types of RCA samples were subjected classification tests such as particle size distribution, plasticity, compaction test and California Bearing Ratio (CBR). Results were compared with those of the standard road materials used in Queensland, Australia and found that ‘RM1-100/RM3-0’ and ‘RM1-80/RM3-20’ samples are sitting in the margin of the minimum required specifications of base materials while others are lower than that.

Laboratory study on the performance of recycled concrete aggregates blended with reclaimed asphalt pavement as pavement granular material

Production of recycled concrete aggregates (RCA) from construction and demolition (C&D) waste has become popular all over the world since the availability of land spaces are limited to dispose. Therefore it is important to seek alternative applications for RCA. The use of RCA in base and sub-base layers in granular pavement is a viable solution. In mechanistic pavement design, rutting (permanent deformation) is considered as the major failure mechanisms of the pavement. The rutting is the accumulation of permanent deformation of pavement layers caused by the repetitive vehicle load. In Queensland, Australia, it is accepted to have the maximum of 20% of reclaimed asphalt pavement (RAP) in RCA and therefore, it is important to investigate the effect of RAP on the permanent deformation properties of RCA. In this study, a series of repeated load triaxial (RLT) tests were conducted on RCA blended with different percentage of RAP to investigate the permanent deformation and resilient modulus properties of RCA. The vertical deformation and resilient modulus values were used to determine the response of RCA for the cyclic loading under standard pressure and loading conditions.

Assessment of recycled concrete aggregates as a pavement material

Population increase and economic developments can lead to construction as well as demolition of infrastructures such as buildings, bridges, roads, etc resulting in used concrete as a primary waste product. Recycling of waste concrete to obtain the recycled concrete aggregates (RCA) for base and/or sub-base materials in road construction is a foremost application to be promoted to gain economical and sustainability benefits. As the mortar, bricks, glass and reclaimed asphalt pavement (RAP) present as constituents in RCA, it exhibits inconsistent properties and performance. In this study, six different types of RCA samples were subjected classification tests such as particle size distribution, plasticity, compaction test, unconfined compressive strength (UCS) and California bearing ratio (CBR) tests. Results were compared with those of the standard road materials used in Queensland, Australia. It was found that material type ‘RM1-100/RM3-0’ and ‘RM1-80/RM3-20’ samples are in the margin of the minimum required specifications of base materials used for high volume unbound granular roads while others are lower than that the minimum requirement.

Effects of moisture content on resilient properties of recycled concrete aggregates (RCAs)

In pavement design, resilient modulus of a pavement material is one of the key design parameters. Resilient modulus of a granular pavement material can be measured using repeated load Triaxial (RLT) test or estimated using empirical models. For conventional granular pavement materials, a significant amount of resilient modulus data and empirical models to estimate this key design parameter are available. However, RCA is a relatively new granular pavement material and therefore no such data or empirical models are available. In this study, a number of RLT tests were conducted on RCA sample to investigate the effects of moisture content on its resilient modulus (Mr). It was observed that the resilient modulus of RCA increased with a number of loading cycles but decreased as the moisture content was increased. Further, using RLT test results, empirical models to estimate the resilient modulus of RCA were enhanced and validated.

Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV

Eleven new human polyomaviruses have been recently discovered, yet for most of these viruses, little is known of their biology and clinical impact. Rolling circle amplification (RCA) is an ideal method for the amplification of the circular polyomavirus genome due to its high fidelity amplification of circular DNA. In this study, a modified RCA method was developed to selectively amplify a range of polyomavirus genomes. Initial evaluation showed a multiplexed temperature-graded reaction profile gave the best yield and sensitivity in amplifying BK polyomavirus in a background of human DNA, with up to 1 × 10(8)-fold increases in viral genomes from as little as 10 genome copies per reaction. Furthermore, the method proved to be more sensitive and provided a 200-fold greater yield than that of random hexamers based standard RCA. Application of the method to other novel human polyomaviruses showed successful amplification of TSPyV, HPyV6, HPyV7, and STLPyV from low-viral load positive clinical samples, with viral genome enrichment ranging from 1 × 10(8) up to 1 × 10(10). This directed RCA method can be applied to selectively amplify other low-copy polyomaviral genomes from a background of competing non-specific DNA, and is a useful tool in further research into the rapidly expanding Polyomaviridae family.

Campagne Chalci 78-01 (30/01/78 - 12/02/78): résultats des chalutages

The aim of this campaign was the trawl exploration of the Ivoirian continental shelf at the depth of 10 to 120 metres.

Prevalência da síndrome metabólica em adolescentes de 06 Escolas na Cidade de São Luís/MA Brasil

Na população pediátrica tem aumentado a prevalência da Síndrome Metabólica. No Brasil, poucos estudos foram realizados em relação a sua prevalência, critérios de diagnósticos, principalmente no Norte/Nordeste. Apesar de não termos uma definição oficial para a Síndrome Metabólica em adolescentes, utilizamos critérios da NCEP ATP III modificados que consistiram em: Obesidade Abdominal >= 90 percentil, HDL-colesterol <= 40 mg/dl, Triglicerídeo > = 110 mg/dl, Glicemia em Jejum >= 100 mg/dl, Pressão Arterial >= 90 percentil ajustável para idade e gênero. Participaram deste estudo 468 adolescentes sendo 168 (40,2%) do gênero masculino e 280 (59,8%) do gênero feminino de 06 (seis) escolas públicas e particulares, da cidade de São Luís/MA, durante o ano de 2012. A prevalência da Síndrome Metabólica foi de 12,2% sendo 30 pacientes do gênero masculino e 27 do gênero feminino. Houve uma predominância no gênero masculino e a faixa etária mais acometida foi 16-17 anos, seguido da faixa de 13-14 anos. Em relação aos componentes da Síndrome Metabólica, a Hipertensão Arterial foi o componente dominante (100% dos casos), seguida do HDL-colesterol diminuída (94,7%), obesidade (79%), triglicerídeos (71,9%), proteína C reativa média e alto risco em 28,1% dos casos, e glicemia em jejum alterada não foi encontrada em nenhum caso, porém, evidenciamos HOMA-IR alterado em 75,4% dos casos. Apesar de termos apena 01 referência na literatura brasileira na padronização sobre a medida da circunferência abdominal, foi realizado a sua medida na população estudada e a sua relação com a altura. Utilizamos como ponto de corte >= 0,5 para avaliar a adiposidade visceral nos pacientes estudados com Síndrome Metabólica encontramos relação Circunferência Abdominal e Altura aumentada em 66,7% dos casos. Em relação aos antecedentes mórbidos familiares dos adolescentes portadores de Síndrome Metabólica, a prevalência de obesidade foi de 22,8%, seguido de diabetes e hipertensão arterial em 17,5%, diabetes, hipertensão arterial e obesidade foi de 15,8%.

Manoel Bomfim e Juliano Moreira: Aproximações e oposições ao racismo científico na Primeira República

Este trabalho analisa produções de Manoel Bomfim (1868-1932) e Juliano Moreira (1873-1933) com relação ao tema da raça e miscigenação, no contexto das primeiras décadas do período republicano brasileiro. Os artigos e livros consultados variam entre os anos de 1905 e 1931. O objetivo é compreender como se dava, para ambos, numa perspectiva comparativa, a relação entre as três raças formadoras da nacionalidade brasileira, a intensa mestiçagem e o progresso do país nos âmbitos social, político e econômico. A análise apresenta uma visão das linhas teóricas das Faculdades de Medicina da Bahia e do Rio de Janeiro por sua importância como centros divulgadores do saber médico institucionalizado no país, aos quais estiveram vinculados os autores que constituem o objeto da pesquisa. De igual forma, considera, ainda, as recentes produções acerca do conceito de intelectual homens letrados que atuaram publicamente entre o final do século XIX e o início do XX e as propostas de leitura e aplicação das teorias raciais no Brasil no seio deste grupo, bem como seus discursos sobre identidade nacional e imigração estrangeira, sob o viés da eugenia da raça brasileira

Mechanical and durability properties of concretes containing recycled lime powder and recycled aggregates

According to a recent report by the European Commission, within the European Union, the construction and demolition wastes come to at least 450 million tons per year. Roughly 75% of the waste is disposed to landfill, despite its major recycling potential. The bulk constituents of demolition debris are concrete (50-55%) and masonry (30-40%) with only small percentages of other materials such as metals, glass and timber. In Cyprus, at present, recycling of waste materials is practically inexistent and almost the entire demolition waste products are disposed in landfill sites, with all possible economic, technical and environmental impacts. This research paper presents the evaluation and the effective reuse of waste construction materials, such as recycled lime powder (RLP) and recycled concrete aggregates (RCA), disposed to landfill sites in Cyprus, due to the lack of a lucid recycling policy and knowledge. Results show that both RLP and RCA have the potential to produce good quality and robust concrete mixtures both in terms of mechanical and durability performance. © 2013 Elsevier B.V. All rights reserved.

Generating Options for Active Risk Control (GO-ARC): introducing a novel technique.

BACKGROUND: After investing significant amounts of time and money in conducting formal risk assessments, such as root cause analysis (RCA) or failure mode and effects analysis (FMEA), healthcare workers are left to their own devices in generating high-quality risk control options. They often experience difficulty in doing so, and tend toward an overreliance on administrative controls (the weakest category in the hierarchy of risk controls). This has important implications for patient safety and the cost effectiveness of risk management operations. This paper describes a before and after pilot study of the Generating Options for Active Risk Control (GO-ARC) technique, a novel tool to improve the quality of the risk control options generation process. OUTCOME MEASURES: The quantity, quality (using the three-tiered hierarchy of risk controls), variety, and novelty of risk controls generated. RESULTS: Use of the GO-ARC technique was associated with improvement on all measures. CONCLUSIONS: While this pilot study has some notable limitations, it appears that the GO-ARC technique improved the risk control options generation process. Further research is needed to confirm this finding. It is also important to note that improved risk control options are a necessary, but not sufficient, step toward the implementation of more robust risk controls.

清洗后硅片表面的电子结构

介绍了一种含表面活性剂和螯合剂的新型半导体清洗剂和清洗工艺。利用红外吸收谱、X射线光电子谱和原子力显微镜等，把它和标准RCA清洗工艺的清洗效果做了比较。测试结果表明，经清洗过的硅片表面主要是由硅、氧和碳三种元素组成，它们分别以Si-O健、C-O健和Si-C键的形式存在。两种清洗技术都在硅片表面产生氧化硅层，在硅片表面都存在有机碳污染，但新型半导体清洗工艺产生的有机碳污染少于标准RCA清洗。在对硅片表面的粗糙化影响方面，新型半导体清洗技术清洗明显优于标准RCA清洗技术。

用含表面活性剂和螯合剂的清洗液清洗硅片的研究

目前半导体工业生产中普遍采用的清洗技术是RCA清洗技术。文中介绍了一种含表面活性剂和螯合剂的新型半导体清洗剂和清洗技术，并利用X射线光电子谱和原子力显微镜等测试方法，分别比较了用两种清洗技术清洗过的硅片表面。测试结果表明，它们的去污效果基本相当。但对硅片表面的粗糙化影响方向，新型半导体清洗技术优于标准RCA清洗技术。