CICYT (BIO-093-0600-C04-03); Generalitat Valenciana (GV-1170/93)

CICYT (PB95-0695)

No description available.

Estudos sobre a diversidade de Bacteria em solos de mangue (Brasil) e marisma (Espanha) são escassos. A vegetação de mangue, composta por espécies como Spartina alterniflora, Rhizophora mangle, Avicennia schaueriana e Laguncularia racemosa, pode ser um dos fatores que determinam a estruturação das comunidades de procariotos. Determinações das estruturas das comunidades e de diversidade de Bacteria podem ocorrer em função das diferentes condições físico-químicas dos solos, refletindo na configuração dos processos biogeoquímicos. O objetivo deste trabalho foi avaliar a variação das estruturas das comunidades de Bacteria e Archaea, bem como a diversidade, em solos de mangue e marisma utilizando DGGE e sequenciamento parcial do rDNA 16S. As estruturas das comunidades de procariotos apresentaram variações em função de condições de vegetação. Proteobacteria e Bacteroidetes estão presentes em todos os solos estudados. A comunidade de Bacteria destes ambientes é dominada por Proteobacteria. Vários dos táxons detectados estão relacionados com ciclos biogeoquímicos importantes para os ambientes estudados. As estimativas não-paramétricas de riqueza de espécies (ACE e Chao1) mostram que solos de mangue e marisma podem conter milhares de espécies de bactérias. As comunidades de Bacteria dos solos de mangue e marisma são significativamene diferentes. Na camada mais superficial do sedimento de mangue predomina Euryarchaeota metanogênicas enquanto que na camada mais profunda predomina Crenarchaeota. Bactérias das ordens Desulfobacterales, Desulfovibrionales e Desulfuromonales podem estar relacionadas com a atividade de sulfato-redução e formação de pirita na camada anaeróbia do perfil de solo de marisma. De uma maneira geral, pode-se concluir que a diversidade e estrutura das comunidades de procariotos de ambientes estuarinos pode variar em função da vegetação estabelecida e do tipo de ambiente. Adicionalmente, solos de mangue e marisma possuem grande diversidade de procariotos, grande parte da qual é desconhecida, podendo representar elevado potencial genético para utilização biotecnológica. / The bacterial diversity in mangrove (Brazil) and marisma (Espanha) soils are largely unknown. Bacterial communities participate in biogeochemicals processes that occurs in soils of estuarine ecosystems. Determinations of the bacterial communities structures and diversity can occur in function of different physico-chemical conditions, reflecting in the biogeochemical processes. The aim of this work was to evaluate the variation of bacterial an archaeal communities structures utilizing DGGE and partial sequencing of 16S rDNA. Bacterial community structures showed more similarity between repetitions samples than the areas under different vegetation. Phylogenetic afiliation shows that several sequences were not clamped into known phyla. Proteobacteria prevails in bacterial communities of mangrove and marisma soils. Several taxa detected are associated to important biogeochemical cycles that occur in estuarine ecosystems. Analysis of species richness showed that mangrove and marisma soils can contain 200 to 6000 species of bacteria. Methanogenic Euryarchaeota was found specially in the upper sample of mangrove sediment analysed whereas the Crenarchaeota was found specially in the lower. Based on the data obtained, it can be concluded that the vegetation is one of the factors affecting the structure of bacterial and archaeal communities in mangrove soils. Additionaly, the effects of edafic factors and seasonal variations have to be considered as determining the prokaryotic community sctuctures, and bacterial and archaeal communities can respond independently to the factors that determine their community structures. Bacterial diversity can vary with the studied estuarine ecosystem. Studies are necessary concerning to diversity of Bacteria, it variation and correlation with biogeochemical process in the mangrove and marisma soils. These soils show a great diversity of bacteria, much of than unknown, which represent a great genetic potential to the biotechnology.

Bifenilas policloradas (PCBs) são compostos de difícil degradação presentes na composição de ascarel, muito utilizado como fluidos dielétricos e isolantes. Neste contexto, a presente pesquisa teve como objetivo avaliar a diversidade de microrganismos em biofilmes de reatores anaeróbios na presença de PCB empregando Métodos de Microbiologia de Anaeróbios Estritos e de Biologia Molecular. Em reator anaeróbio horizontal de leito fixo (RAHLF), alimentado com etanol, formiato, Triton X-100 (0,1%) e ascarel (1 mL/L), operado com tempo de detenção hidráulica (TDH) de 24 horas, foi retirado a comunidade microbiana do biofilme da espuma de poliuretano. Os grupos microbianos encontrados por meio da clonagem e sequenciamento do gene RNAr 16S para o domínio Bacteria foram relacionados aos filos Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% de similaridade e Methylobacillus, 98% de similaridade), Firmicutes (Clostridium, 97% de similaridade, Syntrophomonas, 100% de similaridade e Sporomusa com 100% de similaridade), Synegistetes (Synergistes, 98% de similaridade), Spirochaetes (Leptonema illini, 98% de similaridade), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi e Armatimonadetes. Além disso, como nesse biofilme foram identificadas bactérias redutoras de ferro, procedeu-se a sua quantificação por meio da técnica de tubos múltiplos (NMP, Número Mais Provável) obtendo 5,26 x \'10 POT.12\' NMP/g STV de bactérias redutoras de ferro. Ensaio em batelada foi realizado separadamente sob duas condições: (1) metanogênica e (2) ferro redutora. Em ambas as condições foram adicionadas aroclor 1260 (PCB). Os reatores, sob condição metanogênica, foram alimentados com meio de cultivo Angelidaki e substratos orgânicos (formiato e etanol), além de aroclor 1260 (0,2 \'mü\'g/L). Para simular a condição redutora de ferro foi acrescido ao meio de cultura Angelidaki, EDTA férrico (1,86 g/L). A produção de metano, na presença de aroclor 1260 foi de 3,8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. A presença de bactérias ferro redutoras foi confirmada indiretamente pela taxa média de redução férrica (90%) nos reatores em batelada, após 60 dias de operação. Por meio de PCR/DGGE, elaborou-se um dendograma das amostras deste ensaio em batelada (metanogênico e redutor de ferro) comparativamente com as do reator RAHLF (biofilme presente na parede do reator e no material suporte). Os reatores em batelada apresentaram similaridade entre si de 79% e 92% para os domínios Bacteria e Archaea, respectivamente. As amostras do reator RAHLF foram 80% (Bacteria) e 96% (Archaea) similares. A existência de bactérias degradadoras de PCB, bem como, bactérias redutoras de ferro no biofilme anaeróbio contribuiu com informações sobre o consórcio microbiano e sua diversidade. / Polychlorinated biphenyls (PCBs) are compounds of difficult degradation, a component of askarel, which were used widely as coolants and lubricants. Hence, this study evaluated the diversity of microorganisms in the presence of PCBs in anaerobic reactors. For such, methods as Strict Anaerobic Microbiology and Molecular Biology were employed. The microbial community of the biofilm, developed in a fixed horizontal bed anaerobic reactor (RAHLF), was studied using the technique of cloning and sequencing of RNAr 16S gene for the Bacteria domain. The reactor had immobilized cells in polyurethane foam with ethanol and formate as a carbon source, Triton X-100 (0.1%) and polychlorinated biphenyls (1 mL/L), and operated with 24 hours HRT. The microbial groups found in this biofilm were related to phyla Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% similarity and Methylobacillus, 98% similarity), Firmicutes (Clostridium, 97% similarity Syntrophomonas, and 100% similarity with Sporomusa 100% similarity), Synegistetes (Synergistes, 98% similarity), Spirochaetes (Leptonema Illini, 98% similarity), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi and Armatimonadetes. Furthermore, as bacteria that reduce iron were found, we proceeded the quantification by the multiple tube method (MPN) for this group, obtaining 5.26 x \'10 POT.12\' MPN/g STV of iron-reducing bacteria. The batch reactors evaluated the growth of microorganisms in two condictions: (1) methanogenic e (2) iron reduction, both had the presence of PCBs (Aroclor 1260). The reactor, under methanogenic condition, was fed with synthetic substrate Angelidaki, ethanol and formate, used as carbon source, and aroclor 1260 (0.2 \'mü\'g /L). To simulate the condition of iron reducing, the same synthetic substrate was supplemented with ferric EDTA (1.86 g/L). The production of methane in the presence of aroclor 1260, was 3.8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. The presence of iron reducing bacteria, after 60 days, was confirmed indirectly by the average rate of iron ferric reduction (90%). Filogenetics analysis (PCR/DGGE) compared the samples of this batch reactor - methanogenic and reduction of iron ferric -, with the samples of RAHLF - the biofilm in the reactor wall and the support material. The two condictions in batch reactors showed similarity of 79% and 92% respectively for the Bacteria and Archaea domain. Therefore, both samples of RAHLF showed 80% (Bacteria) and 96% (Archaea) of similarity. In other words, more similarity were presented due configuration of the reactor as well as the type of PCB added. As a result, the existence of PCBs degrading bacteria and iron-reducing bacteria in anaerobic biofilm, provided informations about the microbial consortium and its diversity in the presence of PCB.

Alguns ecossistemas chamam atenção devido à particular combinação de condições ambientais únicas, que resultam na evolução de espécies capazes de colonizar estes ambientes. Os manguezais compõem um bioma composto por espécies de plantas, animais e microrganismos que interagem neste ambiente, que tem como principal característica a interface entre o continente e o oceano em regiões intertropicais. Nosso objetivo foi acessar via sequenciamento massivo de DNA e RNA (via Illumina HiSeq 2000) o perfil taxonômico e funcional da comunidade microbiana de quatro manguezais com distintos níveis de contaminação. As sequências foram analisadas na plataforma MG-RAST, para a análise taxonômica foi utilizado BlastN contra a base de dados RDP ou M5NR, enquanto que a análise funcional foi baseada na comparação por BlastX das sequências obtidas com as disponíveis no banco de dados M5RNA e M5Nr. Na análise funcional, as sequências classificadas foram ainda integradas na classificação hierárquica SEED (subsystems), KEGG (Kyoto Encyclopedia of Genes and Genomes) e COG (Clusters of Orthologous Group). No total, foram obtidas 682 milhões de sequências válidas ( 88,2, 303,1 e 290,9 milhões a partir das análises de DNA, RNA total e RNA purificado, respetivamente). Estas indicaram como grupos taxonômicos mais abundantes as classes Gammaproteobacteria e Deltaproteobacteria (dentro do domínio Bacteria), e Methanomicrobia e Methanobacteria (dentro do domínio Archaea). Além destas observações, alterações na representação de determinados grupos quando as análises de DNA e RNA são comparadas. Em relação a classificação funcional das sequências, foi possível observar uma similaridade no número de funções encontradas nos diferentes manguezais, mas uma maior quantidade de sequências anotadas foi observada, como esperado, na análise de DNA. De maneira mais detalhada, o estudo das funções relacionadas a transformação de nitrogênio e enxofre indicou que há uma correlação entre a abundância de sequências de um referido gene na análise metagenômica, e sua correspondente quantidade dentro dos grupos de dados de metatranscriptômica. Os grupos microbianos mais representados nestes ciclos foram Deltaproteobacteria e Gammaproteobacteria, atuantes principalmente nos processos de fixação de nitrogênio atmosférico (N2), desnitrificação e redução de sulfato, enquanto que para oxidação do enxofre as classes mais frequentes foram Gammaproteobacteria, Betaproteobacteria e Epsilonproteobacteria. De maneira geral, este estudo fornece indícios sobre a atividade microbiana nos manguezais, e indica que as frequentemente correlações observadas entre os resultados da análise metagenômica e metatranscriptômica, porém essas correlações se tornam menos evidentes quando analisamos em nível de ordem ou em níveis mais específicos. / Some ecosystems call particular attention due to unique combination of environmental conditions that result in evolution of species capable of colonizing these environments. Mangroves constitute a biome composed of species of plants, animals and micro-organisms that interact in this environment, whose main characteristic is the interface between the continent and the ocean in tropical areas. Our goal was to access via massive sequencing of DNA and RNA (via Illumina HiSeq 2000) the taxonomic and functional profile of the microbial community four mangroves with different levels of contamination. The sequences were analyzed on MG-RAST platform for taxonomic analysis was performed using BLASTN against the RDP database or M5NR, while the functional analysis was based on comparison of the sequences obtained by BlastX available on M5RNA with the database and M5Nr . In functional analysis, the sequences were classified yet integrated into the hierarchical classification SEED (subsystems), KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Group). A total of 682 million valid sequences were obtained (88.2, 303.1 and 290.9 million from DNA analyzes, purified total RNA and RNA, respectively). These indicated as the most abundant taxa Gammaproteproteobacteria and Deltaproteobacteria classes (within the domain Bacteria), and Methanomicrobia and Methanobacteria (within the domain Archaea). In addition to these observations, changes in the representation of particular groups when analyzes of DNA and RNA are compared. Regarding the functional classification of the sequences was observed in the number of a similarity functions found in different mangrove, but a greater amount of annotated sequences was observed, as expected, the analysis of DNA. In more detail, the study of the functions related to transformation of nitrogen and sulfur indicated that there is a correlation between the abundance of sequences of said gene in metagenomic analysis, and its corresponding quantity within the metatranscriptômica data groups. The microbial groups were over-represented in these cycles and Deltaproteobacteria Gammaproteobacteria mainly active in nitrogênioatmosférico fixation processes (N2), the denitrification and sulfate reduction, while for sulfur oxidation were the most frequent class Gammaproteobacteria, and Betaproteobacteria Epsilonproteobacteria. Overall, this study provides evidence of microbial activity in the mangroves, and often indicates that the correlations observed between the results of metagenomics and metatranscriptômica analysis, but these correlations become less evident when we look at order level or other specific levels.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-09-24T20:12:44Z No. of bitstreams: 2 hoffmann_doctoralThesis_2013_final.v4_forPrint.pdf: 2888260 bytes, checksum: 4331871b2fa71a10777e85a507ba14c8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-24T21:00:52Z (GMT) No. of bitstreams: 2 hoffmann_doctoralThesis_2013_final.v4_forPrint.pdf: 2888260 bytes, checksum: 4331871b2fa71a10777e85a507ba14c8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-24T21:00:52Z (GMT). No. of bitstreams: 2 hoffmann_doctoralThesis_2013_final.v4_forPrint.pdf: 2888260 bytes, checksum: 4331871b2fa71a10777e85a507ba14c8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-08-15 / A dieta influencia a saúde sendo uma fonte de nutrientes e toxinas, e por moldar a composição de populações microbianas residentes no corpo humano. Estudos prévios começaram a mapear as associações entre a dieta e bactérias e vírus do microbioma intestinal humano. Este trabalho investiga as associações entre a dieta e populações arqueanas e fúngicas, tomando vantagem de amostras oriundas de 98 indivíduos bem caracterizados, e integra esses dados novos com o conhecimento corrente relacionado a bactérias e o microbioma intestinal humano. A dieta foi quantificada utilizando questionários que acessam a dieta usual e recente, e arquêas e fungos foram caracterizados usando genes marcadores obtidos de amostras fecais através e sequenciamento de DNA em larga escala de última geração. Foram encontrados 66 gêneros de fungos, geralmente com uma presença mutuamente exclusiva dos filos Ascomycota e Basidiomycota. Quanto as arquêas, Methanobrevibacter foi o gênero mais prevalente, presente em 30% das amostras. Diversas outras arquêas foram detectadas com abundancia e frequência mais baixa. Uma miríade de associações foi observada entre fungos e arquêas e a dieta, entre fungos e arquêas, e entre estes e linhagens bacterianas. Metanobrevibacter e Candida foram positivamente associados com uma dieta rica em carboidratos, e negativamente com dietas ricas em amino acidos, proteínas e ácidos graxos. Dados publicados previamente enfatizam que a estrutura das populações bacterianas no intestino são primariamente com hábitos alimentares de longo prazo, porém, uma abundancia alta de Candida foi fortemente associada com a ingestao recente de carboidratos. A abundância de Methanobrevibacter foi associada tanto com a ingestão usual ou recente de carboidratos. Estes resultados confirmam estudos direcionados anteriores e provém varias novas associações a serem consideradas quando modelando os efeitos da dieta no microbioma intestinal e a na saúde humana. / Diet influences health as a source of nutrients and toxins, and by shaping the composition of resident microbial populations. Previous studies have begun to map out associations between diet and the bacteria and viruses of the human gut microbiome. This work investigates associations of diet with fungal and archaeal populations, taking advantage of samples from 98 well-characterized individuals, and integrates this novel data with the current knowledge regarding the bacteria of the human gut microbiome. Diet was quantified using inventories scoring both long-term and recent diet, and archaea and fungi were characterized by deep sequencing of marker genes in DNA purified from stool. For fungi, we found 66 genera, with generally mutually exclusive presence of either the phyla Ascomycota or Basiodiomycota. For archaea, Methanobrevibacterwas the most prevalent genus, present in 30% of samples. Several other archaeal genera were detected in lower abundance and frequency. Myriad associations were detected for fungi and archaea with diet, with each other, and with bacterial lineages. Methanobrevibacter andCandida were positively associated with diets high in carbohydrates, but negatively with diets high in amino acids, protein, and fatty acids. Previously published data emphasized that bacterial population structure was associated primarily with long-term diet, but high Candida abundance was most strongly associated with the recent consumption of carbohydrates. Methobrevibacter abundance was associated with both long term and recent consumption of carbohydrates. These results confirm earlier targeted studies and provide a host of new associations to consider in modeling the effects of diet on the gut microbiome and human health.

No description available.

‘Aigarchaeota’, a deeply branching lineage in the domain Archaea with no cultivated representatives, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. The ‘Aigarchaeota’ consists of at least nine proposed genus-level groups that have been confirmed via 16S rRNA sequencing, with ‘Aigarchaeota’ Group 1 (AigG1) being the focus of this study. Based on cultivation-independent genomic data available from several AigG1 members in Great Boiling Spring (GBS), NV, and Yellowstone National Park, 22 different types of growth media were designed and tested for their ability to support growth of AigG1. One of these cultures, G1-10, was found to contain AigG1 at ~5% abundance, as well as other novel thermophilic microbial groups including a new species of the genus Pyrobaculum, members of the candidate phyla ‘Calescamentes’ and ‘Fervidibacteria’, and the novel archaeal lineage NAG1 (‘Geoarchaeota’). To attempt to obtain pure cultures of AigG1 and other novel thermophiles, a single-cell sorting system using an optical trap and a microfluidic device was constructed. The system was validated by sorting E. coli cells, which demonstrated that single, viable cells could be reliably obtained. Using this single cell sorting device on the G1-10 culture, a pure culture of a member of the genus Pyrobaculum was obtained, which was shown to represent a distinct species in this phylum by whole genome sequencing and in silico DNA-DNA hybridization. Additionally, a pure culture of the first representative of the candidate phylum ‘Fervidibacteria’ from an enrichment culture derived from G1-10. Additionally, to aid in morphology-based sorting of AigG1 and stable isotope labeling studies, fluorescence in situ hybridization (FISH) based on catalyzed reporter deposition (CARD-FISH) were developed and an AigG1-specific probe was tested. CARD-FISH was successfully used to detect AigG1 in both the G1-10 culture and in natural sediment samples from GBS. Stable isotope labeling incubations were performed with a variety of 13C-labeled substrates (bicarbonate, amino acids, sugars, and short chain fatty acids) on GBS sediments and G1-10 culture samples, and CARD-FISH was used to specifically detect AigG1 in the fixed samples. Nanometer-scale secondary-ion mass spectrometry (nano-SIMS) will then be used to determine whether AigG1 was capable of taking up the different carbon substrates tested. Overall, the results and accomplishments from this project and follow up nano-SIMS analysis will allow a better understanding of the metabolic potential of AigG1 and will aid future efforts to attempt to obtain pure cultures of this novel lineage.

L'étude des virus d'archaea, la manière dont ils sont capables d'infecter leurs hôtes et éventuellement de réaliser le transfert de certains gènes est d'intérêt pour mieux comprendre les mécanismes moléculaires qui ont permis le brassage de l'information génétique dans le phylum des archaea. Notre modèle d'étude est le fusellovirus SSV1 qui infecte certaines souches du genre Sulfolobus, dont Sulfolobus shibatae et Sulfolobus solfataricus. Le cycle viral de SSV1 est actuellement très peu connu, mais comprend l'intégration du génome viral dans celui de l'hôte à un site spécifique et la production de particules virales, sans lyse cellulaire, lors d'irradiation UV des cultures infectées. Nous avons initié l'étude de ce virus en analysant les propriétés biochimiques de son intégrase. Nous avons ainsi montré que l'intégrase virale était un membre à part entière de la famille des tyrosine recombinases, une classe de recombinases spécifiques de site que l'on trouve chez les procaryotes eubactériens mais également chez les eucaryotes. L'étude biochimique de l'intégrase de SSV1 nous a permis de mettre en évidence le caractère hybride du mécanisme de recombinaison spécifique de site chez les archaea. En effet, si l'organisation des sites de recombinaison est similaire à celle de systèmes phagiques eubactériens, celle du site actif de la recombinase est de type eucaryote, puisqu'il est assemblé à l'interface de deux protomères fournissant chacun des résidus intervenant dans la catalyse. Nous continuerons l'étude de l'organisation spatiale de ce système mosaïque en cristallisant l'intégrase sur un site synthétique. Une autre originalité du système archaéen est que le gène de l'intégrase est disrupté lors de l'intégration du génome viral dans celui de son hôte. Cette particularité, conservée chez tous les fusellovirus séquencés à ce jour, ouvre de nombreuses hypothèses quant au rôle de la recombinaison spécifique de site dans leur cycle réplicatif. La compréhension du mode de réplication, du maintien et de la mobilisation de ces virus lors de signaux environnementaux tels que l'irradiation UV est essentielle non seulement pour évaluer leur rôle dans la plasticité des génomes d'archaea, mais également pour leur utilisation future comme outils génétique chez leurs hôtes naturels, les Sulfolobales.<br />Nous exploiterons les résultats obtenus in vitro pour évaluer le rôle de l'intégration dans le maintien du virus sous forme stable, en replaçant des mutations inactivant soit l'intégrase soit le site viral de recombinaison dans le génome de SSV1. Le devenir de ces virus recombinants réintroduits dans Sulfolobus solfataricus sera évalué et nous permettra de déterminer si l'intégration du génome viral dans celui de son hôte est essentiel au maintien et à l'amplification du virus. Une analyse biochimique réciproque consistera à déterminer si une forme tronquée de l'intégrase (correspondant au produit de la disruption intégrative) est fonctionnelle pour le processus d'excision. La directionalité des évènements d'intégration et d'excision peut reposer soit sur la forme active de la recombinase (tronquée ou non) soit, et de manière non exclusive, par l'intervention de protéines accessoires fournies soit par l'hôte soit par le virus. L'identification de ces partenaires éventuels sera réalisée en utilisant des approches biochimiques classiques (co-immunoprécipitation, affinité, séquence peptidique) dans différentes conditions de croissance induisant ou non la production virale. Les résultats seront confrontés aux informations obtenues par les analyses transcriptomiques des effets des radiations réalisées sur Sulfolobus mais également Thermococcus ou Pyrococcus. L'analyse du pool de gènes induits lors d'une irradiation devrait contribuer à l'identification des facteurs de l'hôte intervenant dans la production virale en réponse au stress.<br />Outre le rôle de l'intégration dans le cycle viral, nous évaluerons dans une approche plus globale le rôle des différentes protéines codées par le virus. En effet, sur les 34 protéines potentiellement produites par SSV1 seules 4 ont une fonction identifiée. Par ailleurs, l'analyse comparative des différents génomes de fusellovirus montre que seules 18 ORFs (dont l'intégrase) sont communes à tous ces virus, suggérant que les protéines correspondantes assurent les fonctions minimales essentielles au développement viral. Chacune de ces ORFs sera délétée par LI-PCR. Cette stratégie devrait nous permettre de nous affranchir d'effets secondaires transcriptionnels liés à l'organisation polycistronique du génome viral. L'effet de l'inactivation de chaque ORF sera évalué en prenant en compte différentes étapes du développement viral (stabilité du génome dans la cellule hôte, production de particules virales, infectivité...). Nous espérons ainsi définir la fonction de ces protéines qui n'ont pour l'heure aucun homologue dans le vivant.