NEDD8 conjugation in Schistosoma mansoni : genome analysis and expression profiles.

NEDD8 is an ubiquitin-like molecule that covalently binds to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to bind to p53 and p73, as well as all Cullin family proteins, which are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes. Here, we focused on a genomic analysis of the genes involved in the NEDD8 conjugation pathway in Schistosoma mansoni. The results revealed seven genes related to NEDD8 conjugation that are conserved in Schistosoma japonicum, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We performed quantitative RT-PCR (qRT-PCR), which showed differential profiles for Smnedd8, Smapp1, Smuba3, Smube2f, Smdcn1, Smrbx and Smsenp8 throughout the life cycle of S. mansoni. Upregulation was observed in 3-day-old schistosomula and adult worms for all analysed genes. We also analysed the transcription levels of Cullin family members Smp63 and Smp73, and observed upregulation in early schistosomula, while cercariae and adult worms showed expression levels similar to one another. Taken together, these results suggest that the NEDDylation/ DeNEDDylation pathway controls important cellular regulators during worm development from cercariae to schistosomula and, finally, to adult

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects

The spatial and temporal scales of local dengue virus transmission in natural settings:a retrospective analysis

Background Dengue is a vector-borne disease caused by the dengue virus (DENV). Despite the crucial role of Aedes mosquitoes in DENV transmission, pure vector indices poorly correlate with human infections. Therefore there is great need for a better understanding of the spatial and temporal scales of DENV transmission between mosquitoes and humans. Here, we have systematically monitored the circulation of DENV in individual Aedes spp. mosquitoes and human patients from Caratinga, a dengue endemic city in the state of Minas Gerais, in Southeast Brazil. From these data, we have developed a novel stochastic point process pattern algorithm to identify the spatial and temporal association between DENV infected mosquitoes and human patients. Methods The algorithm comprises of: (i) parameterization of the variogram for the incidence of each DENV serotype in mosquitoes; (ii) identification of the spatial and temporal ranges and variances of DENV incidence in mosquitoes in the proximity of humans infected with dengue; and (iii) analysis of the association between a set of environmental variables and DENV incidence in mosquitoes in the proximity of humans infected with dengue using a spatio-temporal additive, geostatistical linear model. Results DENV serotypes 1 and 3 were the most common virus serotypes detected in both mosquitoes and humans. Using the data on each virus serotype separately, our spatio-temporal analyses indicated that infected humans were located in areas with the highest DENV incidence in mosquitoes, when incidence is calculated within 2.5–3 km and 50 days (credible interval 30–70 days) before onset of symptoms in humans. These measurements are in agreement with expected distances covered by mosquitoes and humans and the time for virus incubation. Finally, DENV incidence in mosquitoes found in the vicinity of infected humans correlated well with the low wind speed, higher air temperature and northerly winds that were more likely to favor vector survival and dispersal in Caratinga. Conclusions We have proposed a new way of modeling bivariate point pattern on the transmission of arthropod-borne pathogens between vector and host when the location of infection in the latter is known. This strategy avoids some of the strong and unrealistic assumptions made by other point-process models. Regarding virus transmission in Caratinga, our model showed a strong and significant association between high DENV incidence in mosquitoes and the onset of symptoms in humans at specific spatial and temporal windows. Together, our results indicate that vector surveillance must be a priority for dengue control. Nevertheless, localized vector control at distances lower than 2.5 km around premises with infected vectors in densely populated areas are not likely to be effective

Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host. [Corrigendum]

published erratum2016 Apr 202016 01 21importedErratum for : Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host. [Nucleic Acids Res. 2015

Identificação, classificação e anotação de enzimas desubiquitinadoras e ubiquitina-símile em Trypanosoma cruzi.

O sequenciamento do genoma de Leishmania major, Trypanosoma cruzi e T. brucei mostrou que cada genoma contém entre 8300-1200 genes que codificam proteínas, dos quais aproximadamente 6500 são comuns entre estas espécies. Neste estudo nós focamos a busca nos bancos de dados de tripanossomatídeos por proteínas envolvidas com o metabolismo de ubiquitina. Existem basicamente nove subfamílias distintas de enzimas envolvidas com esta via, denominadas DUBs: ubiquitina C-terminal hidrolases (UCHs), proteases específicas ao processamento de ubiquitina (USPs), proteases relacionadas à doença de Machado-Joseph (MJDs), proteases de tumor ovariano, também chamadas otubaínas (OTUs), proteases com motivo JAMM, sentrinas, autofaginas, peptidases semelhantes à papaína de vírus de RNA de fita dupla e eucariotas (PPPDEs), e metaloproteases Wss1p-símile (WLMs). Neste trabalho nós utilizamos ferramentas de bioinformática para identificar esse repertório de enzimas nos bancos de dados públicos de genomas das espécies L. infantum, L. major, L. braziliensis, T. brucei e T. cruzi. Nossa pesquisa in silico obteve 163 possíveis entradas que codificam para DUBs nestas espécies quando comparadas por BLASTx à proteínas ortólogas conhecidas no GenBank. Destas, 84 pertenciam à subfamília USP, 10 à JAMM, 10 à UCH, 15 à OTU, 5 à sentrina, 24 à PPPDE, 10 à autofagina, 5 à WLM, e não foram encontradas sequências correspondentes à MJD. No genoma do T. cruzi foi também observado a presença de genes duplicados, onde 63 entradas puderam ser resumidas, com no mínimo 95% de similaridade, à 34 entradas. No entanto, em cada genoma de Leishmania estudado foram encontrados em média 32 entradas para DUBs, e sua maioria possuindo ortólogos no mesmo gênero. Análises comparativas dos genes responsáveis pela remoção de ubiquitina mostraram diferenças significativas nos tamanhos das suas sequências de nucleotídeos bem como em suas composições de aminoácidos, especialmente nas regiões não cobertas por domínios conservados, sugerindo que há manutenção da função, mas diferente especificidade aos substratos. Além disso, a expressão relativa dos genes TcUSP7, -10, -14, -15 e TcUCH-L3 determinadas por RT-qPCR mostraram perfis similares de expressão em formas epimastigotas das cepas Berenice-62, Berenice-78, Colombiana e Y do T. cruzi. No entanto, os níveis de TcUSP15 foram menores quando comparados às outras cepas analisadas neste estudo, enquanto os níveis de TcUCH-L3 foram os maiores. Finalmente, nós determinamos a atividade DUB utilizando o substrato CBZ-Gly-Gly- Arg-7-amido-4-metilcumarina em extratos nucleares e citosólicos e a conjugação de proteínas com ubiquitina e SUMO nos mesmos extratos utilizando western blot e anticorpos específicos para ubiquitina e SUMO. Nossos resultados mostraram que a atividade é predominantemente citosólica. A técnica de western blotting mostrou que os conjugados ubiquitinados são preferencialmente encontrados no citoplasma e os sumorilados no núcleo. Esses resultados evidenciam a complexidade e diversidade das DUBs em tripanossomatídeos e abre a possibilidade de explorar a relevância das interações na regulação das vias mediadas por ubiquitina nesses parasitos.The genome sequences of Leishmania major, Trypanosoma brucei and T. cruzi revealed that each genome contains 8300-12000 protein-coding genes, of which approximately 6500 are common to their genomes. In this study we focused the mining the trypanosomatid databases looking for proteins involved in ubiquitin metabolism. There are basically nine distinct subfamilies of enzymes involved in this pathway, denominated DUBs: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific processing proteases (USPs), Machado-Joseph disease proteases (MJDs), ovarian tumour proteases (OTUs), JAMM motif proteases, sentrins, autophagins, Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes (PPPDEs), and Wss1p-like metalloproteases (WLMs). In this work we used bioinformatic approaches to identity these set of enzymes in L. infantum, L. major, L. braziliensis, T. brucei and T. cruzi public genome databases. Our in silico search retrieved 163 putative entries coding to DUBs in these species among databases when compared to known GenBank ortholog proteins through BLASTx. Of these, 84 belong to USP subfamily, 23 to JAMM, 10 to UCH, 15 to OTU, 5 to sentrin, 24 to PPPDE, 10 to autophagin, 5 to WLM, and where not found entries to MJD. In T. cruzi genome was also observed the presence of duplicated genes, where the 63 entries might be resumed, with at least 95% of similarity, in 34 sequences. However, in each Leishmania genome studied were found on average 32 entries for DUBs, and most of them have its orthologues into the same genus. Comparative analysis of genes responsible for ubiquitin removal showed significant difference in their respective nucleotide sequence lengths as well as amino acid composition, especially with regards to regions outer conserved domains, suggesting maintaining of function, but different substrate specificity. In addition, the relative gene expression of TcUSP7, -10, -14, -15 and TcUCH-L3 determined by RT- qPCR showed similar profile in epimastigotes forms between Berenice-62, Berenice-78, Colombian and Y strains of T. cruzi. However, the levels of TcUSP15 were lowest when compared to strains analyzed in this study, while the levels of TcUCH-L3 where highest. Finally, we determinate the DUB activity using the substrate CBZ-Gly-Gly- Arg-7-amido-4-methylcoumarin in nuclear and citosolic extracts and the ubiquitin and SUMO protein conjugation in the same extracts using western blot and specific antibody to ubiquitin and SUMO. Our results showed that the activity were predominantly citosolic. The western blotting showed that preferentially conjugate ubiquitin was found in the cytoplasm and sumoylation in the nucleus. These results evidence the complexity and diversity of DUBs in tripanosomatids and open the possibility to explore the relevance of their interactions in regulation of ubiquitin- mediated pathways in these parasites

The virome of vector mosquitoes

International audienceMosquitoes are the major vectors for arthropod-borne viruses (arboviruses) of medical importance. Aedes aegypti and A. albopictus are the most prolific and widespread mosquito vectors being responsible for global transmission of dengue, Zika and Chikungunya viruses. Characterizing the collection of viruses circulating in mosquitoes, the virome, has long been of special interest. In addition to arboviruses, mosquitoes carry insect-specific viruses (ISVs) that do not directly infect vertebrates. Mounting evidence indicates that ISVs interact with arboviruses and may affect mosquito vector competence. Here, we review our current knowledge about the virome of vector mosquitoes and discuss the challenges for the field that may lead to novel strategies to prevent outbreaks of arboviruses

Les insectes : un fantastique réservoir de virus et de gènes antiviraux

Les insectes forment le groupe d’animaux qui présente la plus grande diversité. Des travaux récents de métagénomique montrent qu’ils peuvent être infectés par une diversité extraordinaire de virus. Parmi eux, les arbovirus (arthropod-borne viruses) peuvent être transmis à l’Homme par les insectes hématophages, notamment les moustiques. Le séquençage à haut débit des petits ARN des insectes fournit des informations sur leur virome, un paramètre qui pourrait contribuer à expliquer la dynamique de la transmission des maladies infectieuses par des insectes vecteurs. D’autre part, la caractérisation des mécanismes qui restreignent les infections virales chez les insectes révèle des innovations génétiques qui pourraient à terme inspirer de nouvelles stratégies antivirales

Combining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control

Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01-0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors

Combining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control

International audienceChemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01-0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors