Les zoonoses constituent plus des deux tiers des pathologies virales qui concernent l’homme. Le développement et la démocratisation des outils de métagénomique en font de bons outils d’inventaire et de surveillance de virus potentiellement émergents.Dans un premier temps j’ai développé et validé un protocole expérimental de purification des viromes à ARN qui permettait le maintien de l’infectivité des particules virales. Ce protocole a ensuite été appliqué pour caractériser les communautés virales d’arthropodes hématophages et de prélèvements de faune sauvage. J’ai par la suite réalisé l’inventaire des communautés virales de viande de singe fumée illégalement importée en France et confisquée par les douanes, qui a révélé la présence de nombreux bactériophages, dont certains pourraient infecter des bactéries potentiellement pathogènes pour l’homme.Enfin j’ai caractérisé les communautés virales de culicoïdes collectés au Sénégal, ce qui a permis de mettre en évidence la présence de nombreux virus géants à ADN infectant les amibes. Le séquençage des viromes à ARN a quant à lui révélé la présence d'un certain nombre d'arbovirus qui pourraient constituer un risque d’émergence pour la santé humaine. Du fait de nombreux facteurs intrinsèques et extérieurs à l’agent infectieux, la prédiction des futures émergences de virus zoonotiques est très compliquée voire utopique, mais elle reste un challenge crucial et d’actualité. La stratégie de réalisation d’inventaires des communautés virales présentes dans les différents acteurs des cycles de transmission zoonotique est un premier pas indispensable dans la connaissance des risques potentiels d’émergence en population humaine. / Zoonoses are responsible of more than two thirds of human viral infections. The development of high-throughput sequencing tools and their application in metagenomics allow inventorying the viral communities of various reservoirs in order to detect the emergence of viruses before their infection to humans. In this context, I characterized the viral communities of simian bushmeat illegally imported into France and of Culicoides biting midges, recognized vectors of several viruses of human and veterinary medicine importance. I have first developed a protocol for the purification of RNA viromes which allowed maintaining the infectivity of viral particles. This protocol was subsequently applied to characterize viral communities of bloodsucking arthropods and wildlife samples. In a second part I realized the inventory of viral communities of smoked simian bushmeat illegally imported into France and confiscated by the French customs. This study revealed the presence of a wide diversity of bacteriophages, in which some of them could infect bacteria potentially pathogenic for humans.Finally I characterized the viral communities of Culicoides biting midges collected in Senegal, which revealed the presence of sequences related to several giant DNA viruses infecting amoeba. Sequencing of the RNA virome revealed the presence of several arboviruses that could constitute a risk of emergence of zoonoses for humans.The prediction of future emerging zoonotic viruses is very difficult, if not impossible. However the characterization of viral communities present in the different actors of zoonotic transmission cycle is a first step to evaluate potential risks of transmission to humans.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-04-19T15:30:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE KAYNARA CECILIA NERY RABELO.pdf: 2652314 bytes, checksum: e260b3caf6e8bee65596000a545c79d1 (MD5) / Made available in DSpace on 2016-04-19T15:30:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE KAYNARA CECILIA NERY RABELO.pdf: 2652314 bytes, checksum: e260b3caf6e8bee65596000a545c79d1 (MD5) Previous issue date: 2015-06-30 / Insetos e outros artrópodes quando em locais de crime podem servir como vestígio criminal. O advento da genética forense pode auxiliar na obtenção do DNA humano a partir destes insetos, podendo relacionar o suspeito a cena do crime. Desta forma, esta pesquisa objetivou a obtenção e a comparação do perfil genético humano extraído de mosquitos hematófagos com diferentes metodologias para a extração de DNA, analisando os seguintes fatores: variação temporal para obtenção dos perfis genéticos após a hematofagia; a obtenção e a comparação dos perfis de DNA humano do sangue proveniente do trato digestivo dos mosquitos hematófagos com as amostras referências (saliva) de voluntários; influência da amônia, ácido lático e tipo sanguíneo, além da temperatura corporal dos voluntários e a relação na atratividade dos mosquitos e consequente obtenção do material genético; avaliou-se também a mistura de perfis genéticos provenientes de um único mosquito e o intervalo temporal após a hematofagia. Para a análise da comparação das extrações foi utilizado o kit DNA IQTM ,a resina Chelex® 100 e extração com NaOH; e para as outras variáveis em estudo utilizou-se somente o DNA IQTM. A quantificação foi realizada com o Quantifiler® Duo e a amplificação com o kit AMPFlSTR Identifiler® Plus® PCR, que analisou 15 loci STR e amelogenina. A quantificação para o estudo das misturas de DNA nos mosquitos foi realizada com PowerPlex 16HS System. Os dados foram analisados através do programa estatístico PATCAN v. 1.2 software e para a análise das misturas foi utilizado o programa DNA MIX v. 3.2 software. Os resultados demonstraram que o uso do DNA IQTM foi melhor quando comparado a resina Chelex® 100, com obtenção de perfis viáveis em até 72h após a refeição sanguínea. Não foi obtido perfil de DNA quando utilizado NaOH. Os resultados demonstraram também uma confrontação positiva entre o sangue encontrado no trato digestivo dos mosquitos e o material genético cedido pelos voluntários, como amostra referência. As análises bioquímicas demonstraram que o tipo sanguíneo com maior número de obtenção de perfil genético foi o tipo O; além disso, foi constatado valores de normalidade para o exame de lactato, mas para a análise de amônia foi obtido DNA também com valores maiores que o padrão de referência para este tipo de exame, tanto em homens quanto em mulheres. Houve obtenção de DNA nas temperaturas corporais registradas entre 36ºC a 37º C. Foi observado também que mistura de DNAs humano pode ser detectado a partir de um único mosquito hematófago. Desta forma, os resultados demonstraram que os mosquitos hematófagos quando encontrados em cenas de crimes tem efetivo valor forense. / Insects and others arthropods can be used as traces when located in the crime scene. The advent of forensic genetic can assist in obtaining human DNA from these insects, relating the suspect to the crime scene. So, this research aimed the obtainment and the comparison of the human gene profile extracted for the hematophagous mosquitoes with different methodologies to the DNA extraction, analyzing the following factors: temporal variation to the obtainment of the genetic profile after the hematophagy; obtainment and comparison of the human gene profile from the hematophagous mosquitoes´ digestive tract with the volunteers´ samples (saliva); influence of ammonia, lactic acid and blood type, besides the volunteers’ body temperature and the relation on mosquitoes´ attractiveness and genetic material obtainment; the compound of the genetic profile from one mosquito and temporal intermission after hematophagy was also evaluated. DNA IQTM, resin Chelex® 100 and extraction with NaOH was used to the analyses of the extract comparison. The quantification was held with Quantifiler® Duo and the amplification with AMPFlSTR Identifiler® Plus® PCR kit, that analyzed 15 loci STR and amelogenin. The quantification to study the compound of DNA in the mosquitoes was held with PowerPlex 16HS System. Data were analyzed through statistic program PATCAN v. 1.2 software and to analyze the profiles mixtures DNA MIX v. 3.2 software program was used. The results showed that the use of DNA IQTM was typical when compared with Chelex® 100, and success in gene amplification with obtainment of viable profiles up to 72 hours after blood meal. DNA profile was not obtained when used NaOH. The results also showed a positive confrontation between blood found in the mosquitoes´ digestive tract and the material assigned by the volunteers, with reference sample. Biochemical analysis demonstrated that the blood type with bigger obtainment number obtained profile gene was type O; besides that the human DNA profiles were achieved from hematophagous mosquitoes when compared with the correspondent biochemical analysis of the volunteer found normal values to lactate exam, but to ammonia analysis was obtained DNA with higher values than the reference standard to this type of exam, in both gender. There was DNA obtainment from body temperatures registered between 36°C to 37°C. It was also observed that human DNA compound can detected through a only single hematophagous mosquito. With that the results showed hematophagous mosquitoes when found in the crime scene have effective forensic value.

Since ticks are classified as hematophagous ectoparasites, the primary feeding event involves a bloodmeal on a vertebrate host. Such activities facilitate the ingestion of microorganisms which may be detrimental to the survival of a tick. It is observed, however, that ticks are able to survive such invasion by microorganisms and in several cases, facilitate the transmission of pathogens, while themselves remaining unaffected. This phenomenon is attributed to the innate immune system of ticks. The focus of this project is on stimulus-induced immunoreactive peptides known as antimicrobial peptides. In chapter 2, an attempt was made to identify a homolog of the anti Gram-positive and bacteriostatic peptide microplusin, in the salivary glands of the argasid tick Ornithodoros savignyi. It was reported previously that tissue and life stage specific expression of this transcript occurs in the fat body of adult, fully fed, female Rhipicephalus (Boophilus) microplus ticks. The positive control used for this study was unsuccessful due to the incorrect tissue and life stage of R. (B.) microplus ticks. No significant homolog was identified due to the possible existence of stringent regulation of expression as well as differences in the induction stimuli between argasid and ixodid ticks. Lysozyme catalyzes the cleavage of the β-1,4 glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine of the peptidoglycan layer of bacterial cell walls affording the molecule antibacterial activity. In argasid ticks, lysozyme was observed to be induced by feeding. In chapter 3, an attempt was made to elucidate the O. savignyi homolog of the O. moubata lysozyme molecule. The partial sequence obtained revealed the presence of a lysozyme homolog in O. savignyi. The tissue expression profile revealed constitutive expression in the midgut and ovaries and induction of transcription in the hemolymph upon feeding. In salivary glands, upregulation was observed following ingestion of Gram-positive bacteria. In chapter 4, the tissue expression profile of O. savignyi defensin was investigated. It was found that transcription is induced following the ingestion of Gram-positive bacteria, while in the hemolymph upregulation was observed upon feeding. Furthermore, chapter 4 saw the attempts made at the RNAi mediated silencing of the lysozyme and defensin transcripts. Silencing, analysed by real time PCR, was not efficient as no statistically significant silencing was observed. Observation of the phenotype revealed mortality. However, statistical analysis of silencing revealed that the mortality observed was not due to silencing, but non-specific and possibly the result of injury during injection. Overall, the abovementioned experiments revealed the tissue specificity of expression of ixodid microplusin and that a more strategic approach is required for the elucidation of the argasid homolog. The partial O. savignyi lysozyme sequence was elucidated together with the tissue expression profile of this molecule and O. savignyi defensin. The RNAi experiments require optimization for future studies. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-20T17:14:55Z No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-27T17:52:07Z (GMT) No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) / Made available in DSpace on 2018-04-27T17:52:07Z (GMT). No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) Previous issue date: 2015-04-20 / CAPES / FAPEMAT / Arbovírus são transmitidos por artrópodes hematófagos, representando um problema de saúde pública em áreas tropicais. O objetivo deste estudo foi investigar a diversidade de espécies de culicídeos e sua frequência de infecção por Alphavirus e Flavivirus em Cuiabá, MT. Foram realizadas capturas com aspirador de Nasci e puçá entre janeiro e abril de 2013 em três locais de 200 setores censitários, definidos aleatoriamente. Os culicídeos foram identificados com chave dicotômica de Forattini, alocados em pools (1-20 mosquitos) segundo sexo, espécie, data, local de coleta e armazenados a -80˚C. Pools de fêmeas foram submetidos à extração de RNA e DNA total, à multiplex semi-nested-RT-PCR para cinco alphavírus e 11 flavivírus e Nested- PCR para identificação de Culex (Cx.) quinquefasciatus. Amostras positivas para SLEV, DENV-1, -4 e MAYV foram submetidas a single RT-PCR e sequenciamento nucleotídico. Pools positivos para o MAYV foram inoculados em células Vero e submetidos a RT-PCR para o gene de envelope E1 dos alphavirus. Pools positivos para flavivirus foram inoculados em células C6/36 (Flavivirus). Calculou-se a taxa de infecção mínima (MIR). Foram capturados 11.090 mosquitos, 4.556 fêmeas de 14 espécies, perfazendo 610 pools. Foram analisados 171 pools de Aedes (Ae.) aegypti; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp; 6 Limatus sp; 2 Mansonia wilsoni; 5 Psorophora (Ps.) sp; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Encontrou-se 1/171 (MIR=0,92) pool de Ae. aegypti positivo para DENV-1; 1/403 (MIR= 0,2) Cx. quinquefasciatus para SLEV genótipo V-A, 12/403 (MIR=3,5) de Cx. quinquefasciatus, 4/171 (MIR=3,67) de Ae. aegypti para MAYV; destes, cinco pools apresentaram co-infecção com DENV-4. Um produto de 1,3 kb obtido com o protocolo para o gene de envelope de três pools positivos para o MAYV resultou em sequências nucleotidicas inespecíficas. O MAYV foi isolado de dois pools contendo duas fêmeas não ingurgitadas de Ae. aegypti (#958) e duas de Cx. quinquefasciatus (#489). Positividade para DENV-4 foi identificada em 58/171 (MIR=53,35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp, 2/11 (MIR=142,85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor e 1/1 (MIR=1000) Aedes sp. O DENV-4 foi isolado de dois pools contendo três (#329) e 16 (#806) fêmeas de Cx. quinquefasciatus não ingurgitadas. O SLEV, MAYV e os sorotipos do DENV foram identificados em pacientes com suspeita de dengue na cidade de Cuiabá em estudos prévios do Laboratório de Virologia. Experimentalmente, Culex e Aedes spp. são vetores competentes do MAYV. A identificação do vírus em fêmeas não ingurgitadas sugere que estas espécies podem estar envolvidas no ciclo urbano do MAYV em Cuiabá. Dentre os sorotipos do DENV, somente o DENV-1 e o DENV-4 foram identificados em culicídeos. O DENV-4 tem produzido importantes epidemias em MT desde 2012. A positividade para DENV-4 em diferentes espécies de culicídeos pode ser decorrente de infecção natural ou da hematofagia em humanos, pois muitos destes pools apresentavam fêmeas ingurgitadas. Cuiabá possui ecossistema favorável para a ocorrência de arboviroses e proliferação de vetores. Estudos envolvendo vigilância entomológica e virológica são importantes para estimar a situação epidemiológica dos arbovírus no estado. / Arbovirus are transmitted by hematophagous arthropods, posing a public health issue in tropical areas. The aim of this study was to investigate the diversity of culicidae and their frequency of infection by arboviruses from Alphavirus and Flavivirus genus in Cuiabá, MT. To achieve that, culicids were captured with Nasci aspirators and hand net between January and April 2013 in three locations of 200 censitary sectors randomly defined. The specimens were identified using the Forattini dichotomy key, allocated in pools (1-20 mosquitoes), according to sex, species, day and place of capture, and stored at -80˚C. Female pools were subjected to total RNA and DNA extraction and to multiplex semi-nested-RT-PCR for five alphaviruses and 11 flaviviruses and Nested-PCR for Culex (Cx.) quinquefasciatus identification. The pools positive for SLEV, DENV-1, -4 and MAYV were subjected to single RT-PCR and nucleotide sequencing. MAYVpositive pools were inoculated in Vero cells and subjected to RT-PCR for the E1 envelope gene of alphaviruses. Pools positive for flaviviruses were inoculated in C6/36 cells. The minimum infection rate (MIR) was calculated. 11,090 mosquitoes were captured, 4,556 females belonging to 14 species, comprising 610 pools, 171 pools of Aedes (Ae.) aegypti specimens; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp.; 6 Limatus sp.; 2 Mansonia wilsoni; 5 Psorophora sp.; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Among them, 1/171 (MIR=0.92) Ae. aegypti pool was positive for DENV-1 and 1/403 (MIR=0.3) Cx. quinquefasciatus for SLEV genotype V-A. For MAYV, 12/403 (MIR=3.5) Cx. quinquefasciatus, 4/171 (MIR=3.67) Ae. aegypti were positive, five of them were also infected by DENV-4. A DNA product with 1.3 kb obtained from three pools positive for MAYV with the protocol for the envelope gene resulted in unspecific nucleotide sequences. MAYV was isolated from two pools, both containing two non-engorged females of Ae. aegypti (#958) and Cx. quinquefasciatus (#489). DENV-4 was detected in 58/171 (MIR=53.35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp., 2/11 (MIR=142.85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor and 1/1 (MIR=1000) Aedes sp. DENV-4 was isolated from two pools containing three (#329) and 16 (#806) non-engorged females of Cx. quinquefasciatus. The SLEV, MAYV and the four DENV serotypes were identified in patients suspected of harboring dengue infection in Cuiabá in previous studies of the Virology Laboratory. Experimentally, Culex and Aedes spp. are competent vectors for MAYV. The identification of the virus in nonengorged females suggests these species may be involved in the urban cycle of MAYV in Cuiabá. Among the DENV serotypes, only DENV-1 and DENV-4 were identified in culicids captured in the city. DENV-4 has been responsible for major outbreaks in MT since 2012. The identification of DENV-4 in several mosquito species might be resultant either from natural infection or hematophagy in humans, since several of these pools presented engorged females. Cuiabá presents a favorable ecosystem for the occurrence of arboviruses and vector proliferation. Studies involving entomological and virological surveillance are important to estimate the epidemiological situation of arboviruses in the state.

Avec la mise en place des politiques de conservation des espèces sauvages, l'extension de l'urbanisation et l'accroissement des populations humaines, le contact homme-faune a considérablement augmenté au cours de ces dernières décennies. Par conséquent, le nombre de maladies d'origines zoonotiques a explosé avec six apparitions d'agents infectieux par an, dont 75% sont susceptibles d’être transmises par un vecteur. La plupart de ces maladies n'ayant pas encore de vaccins, les principales méthodes d'évitement sont basées sur les stratégies de lutte anti-vectorielle adaptées à l'écologie et au comportement alimentaire des vecteurs. Au Gabon, particulièrement dans les parcs nationaux, nous avons identifié six espèces de glossines (Glossina palpalis palpalis, G. fuscipes fuscipes, G. fusca congolense, G. pallicera newsteadi, G. caliginea et G tabaniformis) vivant principalement en milieux forestiers, six espèces de stomoxes (Stomoxys calcitrans, S. inornatus, S. niger niger, S. niger bilineatus, S. omega omega et S. transvittatus) inféodées aux milieux ouverts types forêt secondaire, savane et villages. Nous avons également identifié six espèces de tabanides (Ancala sp., Atylotus sp., Chrysops sp., Haematopota sp., Tabanus par et T. taeniola), mais leur distribution n'était pas claire dans les milieux prospectés. Par ailleurs, nous constatons que ces mouches hématophages ont un régime alimentaire très diversifié, comprenant les mammifères terrestres et aquatiques, les reptiles et les oiseaux. Elles se nourrissent à 86% sur la faune, contre seulement 14% sur l'homme. Cependant, dans les milieux anthropisés les repas sanguins d'origine humaine sont très importants, notamment dans les villages (100%) et autour des camps de recherche implantés dans les parcs (24%). Ainsi en l'absence de faune dans le milieu, ces mouches hématophages se nourrissent sur l'homme. Comme 75% des maladies émergentes chez l'homme proviennent de la faune sauvage et que près de ¾ d'entre elles circulent via le sang, elles sont donc susceptibles d’être détectées dans les repas sanguins de mouches hématophages. Cette technique d'échantillonnage non-invasif de la faune sauvage semble être un bon moyen d'identifier les agents infectieux à ADN (plasmodiums et trypanosomes), mais reste encore imprécise pour les agents infectieux à ARN (arbovirus). / The contact between human and wild fauna has considerably increased during these last decades due to the increase of human population size but also to conservation policies. As a consequence, the number of zoonotic diseases soared with a mean of six new infectious diseases per year, 75% of whom being vectorially transmitted. The way to avoid the human contamination by these emergent diseases is based on the efficient vector control resulting from a deep knowledge of the ecology and the feeding behavior of the different vector species. During our work, we have identified and characterized the ecology of 6 tsetse species (Glossina palpalis palpalis, G. fuscipes fuscipes, G. fusca congolense, G. pallicera newsteadi, G. caliginea and G. tabaniformis) that live in forests and 6 stomoxe species (Stomoxys calcitrans, S. inornatus, S. niger niger, S. niger bilineatus, S. omega omega and S. transvittatus) that live in and around (anthropized places) conservation areas. We have also identified 6 tabanid species (Ancala sp., Atylotus sp., Chrysops sp., Haematopota sp., Tabanus par and T. taeniola). The feeding ecology of the tsetse species have been studied through the determination of host extracted from blood meals in the insect caught with molecular techniques. These hematophagous insects had a diversified diet that was constituted of diverse mammal species but also reptiles and birds. The food intake results mostly from wild fauna (86%) and more rarely from humans (14%). However, in anthropised habitats (villages and research’s camps within the parks), the blood intakes from human origin were important, in particular in the villages (100%), suggesting that without wild fauna the flies shift on human host. In the last part of our work, we tried to identify pathogens in the blood samples extracted from the tsetse species in order to test whether these species could be used as living sampling syringe of the wild fauna. This new proposed non-invasive sampling techniques allowed to detect the DNA of various infectious agents (plasmodiums and trypanosomes), but failed to detect the RNA of viruses (arbovirus) suggesting that this approach could be useful but need to be improved.