La mise en évidence de l'assemblage et de la recombinaison XerCD-dif à l'échelle de la molécule unique

Les recombinases à tyrosine sont bien connues pour catalyser des recombinaisons spécifiques de site de l'ADN des bactéries, des archées et des eucaryotes. Chez les bactéries, ces recombinases sont impliquées dans l'intégration programmée, l'excision ou l'inversion de segments d'ADN. Les recombinases XerC et XerD sont des recombinases à tyrosine très conservées et dévouées à la recombinaison du site dif localisé dans le domaine ter des chromosomes bactériens circulaires. Le système XerCD-dif résout les dimères de chromosomes en monomères avant la ségrégation et est donc requis pour une ségrégation correcte des chromosomes frères dans les cellules filles. Pour cela, son activité est soumise à un contrôle précis durant le cycle bactérien. Chez Escherichia coli, l'activité de XerCD-dif est contrôlée par la protéine de division cellulaire FtsK. En l'absence de FtsK, XerC effectue le premier échange de brins, mais la jonction de Holliday ainsi formée est résolue en reformant le substrat de départ. En la présence de FtsK, XerD interagit avec le sous-domaine gamma de FtsK et effectue le premier échange de brins. La jonction de Holliday ainsi formée est résolue par XerC. Cependant, la façon dont cette translocase à ADN contrôle l'activité de recombinaison de XerCD-dif n'est pas bien comprise mais implique un contrôle de l'assemblage de la synapse de recombinaison. Afin de mieux comprendre le mécanisme de la recombinaison XerCD-dif et son contrôle par FtsK, nous avons étudié l'assemblage et l'activité des complexes de recombinaison par une méthode molécule unique : le Tethered Particle Motion. Dans un premier temps, nous avons montré que FtsK n'est pas requise dans la formation de la synapse XerCD-dif. Cependant, elle augmente le taux de formation des synapses. Nous n'avons détecté aucune recombinaison en l'absence du sous-domaine gamma de FtsK mais ce dernier est suffisant pour activer la recombinaison dans les synapses formées. La fixation de XerD seule à l'ADN entraine un changement structural de l'ADN tandis que XerC seule n'a aucun effet décelable sur l'ADN. Ces résultats suggèrent que seule la synapse " XerD-ready " est formée et que XerD est un leader durant l'assemblage de la synapseTyrosine recombinases are well known to catalyse site-specific DNA recombination in bacteria, archeae and eukaryotes. In bacteria, these recombinases are extensively used for programmed integration, excision and inversion of DNA segments. XerC and XerD are highly conserved tyrosine recombinases devoted to recombine dif sites, located in the terminal domain of circular bacterial chromosomes. XerCD-dif recombination resolves chromosome dimers to monomers before segregation and is thus required for the faithful segregation of sister chromosomes in daughter cells. To do so, its activity is precisely tuned and controlled during the bacterial cell cycle. In Escherichia coli, XerCD-dif activity is controlled by FtsK. In the absence of FtsK, XerC mediates the first strand exchange but the Holliday junction formed is resolved back to substrate. In its presence, XerD interacts with the gamma subdomain of FtsK and mediates the first strand exchange. The Holliday junction is then resolved by XerC. However, the way this septal DNA translocase acts on XerCD-dif recombination is not completely understood but involves the control of the assembly of the synaptic complex where recombination takes place. To understand the XerCD-dif recombination and its FtsK-mediated control, we studied the assembly and the activity of the recombination complexes on single DNA molecules using the Tethered Particle Motion method. In a first part, we demonstrated that FtsK is no needed for the synapse assembly but increases its formation rate. No recombination was detected within these synapses in the absence of the gamma subdomain of FtsK. In a second part, we showed that the gamma subdomain of FtsK is sufficient to activate recombination within these synapses. We also showed that XerD but not XerC leads to a DNA structural modification. Taken together, our results suggest that only the XerD-ready synapse is formed and XerD is a leader during the synapse assembl

Vector competence of Aedes vexans (Meigen), Culex poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African lineages of Rift Valley fever virus

Background Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is a mosquito–borne, zoonotic pathogen. In Senegal, RVFV was first isolated in 1974 from Aedes dalzieli (Theobald) and thereafter from Ae. fowleri (de Charmoy), Ae. ochraceus Theobald, Ae. vexans (Meigen), Culex poicilipes (Theobald), Mansonia africana (Theobald) and Ma. uniformis (Theobald). However, the vector competence of these local species has never been demonstrated making hypothetical the transmission cycle proposed for West Africa based on serological data and mosquito isolates. Methods Aedes vexans and Cx. poicilipes, two common mosquito species most frequently associated with RVFV in Senegal, and Cx. quinquefasciatus, the most common domestic species, were assessed after oral feeding with three RVFV strains of the West and East/central African lineages. Fully engorged mosquitoes (420 Ae. vexans, 563 Cx. quinquefasciatus and 380 Cx. poicilipes) were maintained at 27 ± 1 °C and 70–80 % relative humidity. The saliva, legs/wings and bodies were tested individually for the RVFV genome using real-time RT-PCR at 5, 10, 15 and 20 days post exposure (dpe) to estimate the infection, dissemination, and transmission rates. Genotypic characterisation of the 3 strains used were performed to identify factors underlying the different patterns of transmission. Results The infection rates varied between 30.0–85.0 % for Ae. vexans, 3.3–27 % for Cx. quinquefasciatus and 8.3–46.7 % for Cx. poicilipes, and the dissemination rates varied between 10.5–37 % for Ae. vexans, 9.5–28.6 % for Cx. quinquefasciatus and 3.0–40.9 % for Cx. poicilipes. However only the East African lineage was transmitted, with transmission rates varying between 13.3–33.3 % in Ae. vexans, 50 % in Cx. quinquefasciatus and 11.1 % in Cx. poicilipes. Culex mosquitoes were less susceptible to infection than Ae. vexans. Compared to other strains, amino acid variation in the NSs M segment proteins of the East African RVFV lineage human-derived strain SH172805, might explain the differences in transmission potential. Conclusion Our findings revealed that all the species tested were competent for RVFV with a significant more important role of Ae. vexans compared to Culex species and a highest potential of the East African lineage to be transmitted

Malaria Risk Factors in Dielmo, A Senegalese Malaria-Endemic Village, Between October and November of 2013: A Case-Control Study

International audienceThe incidence of malaria has decreased recently in parts of Africa, coinciding with the widespread use of long-lasting insecticide-treated nets (LLINs) and artemisinin-based combination therapy (ACT). This reduction was also observed in Dielmo, our study area, and it was associated with the use of ACT as the first-line treatment against malaria beginning in 2006 and the implementation of LLINs in 2008. However, an unexplained slight increase in malaria incidence was observed in October and November of 2013. The aim of this study was to identify individual and environmental risk factors for malaria using a case-control study approach. Thirty cases and sixty controls were investigated. The use of LLINs was protective against malaria (adjusted odds ratio [AOR] = 0.10; 95% confidence interval [95% CI] = 0.02–0.45; P = 0.003). The risk of malaria transmission was high among villagers who watched television outside the house or the bedroom during the night (AOR = 8.83; 95% CI = 1.39–56.22; P = 0.021). The use of LLINs should be reinforced by the use of individual protection measures to avoid malaria transmission outside of the home. BACKGROUN

Mobile laboratory reveals the circulation of Dengue virus serotype I of Asian origin in Medina Gounass (Guediawaye), Senegal

With the growing success of controlling malaria in Sub-Saharan Africa, the incidence of fever due to malaria is in decline, whereas the proportion of patients with non-malaria febrile illness (NMFI) is increasing. Clinical diagnosis of NMFI is hampered by unspecific symptoms but early diagnosis is a key factor for better patient care and for disease control. The aim of this study was to determine the arboviral aetiologies of NMFI in a low resources settings using a mobile laboratory based on Recombinase Polymerase Amplification (RPA) assays. The panel of tests for this study was expanded to five arboviruses including (Dengue virus (DENV), Zika virus (ZIKV), Yellow fever virus (YFV), Chikungunya virus (CHIKV), and Rift valley fever virus (RVFV). One hundred and four children aged between one month and 115 months were enrolled and screened. Three of 104 blood samples of children < 10 years presenting at an outpatient clinic tested positive for DENV. The results were confirmed by real-time RT-PCR, partial sequencing, and non structural protein 1 (NS1) antigen capture ELISA (Biorad, France). Phylogenetic analysis of the derived DENV-1 sequences clustered them with sequences of DENV-1 isolates from Guangzhou in China in 2014. In conclusion, this mobile setup proved reliable for the rapid identification of the causative agent of NMFI, with results consistent with those obtained in the reference laboratory`s settings

Sperm characters of the digenean Nephrotrema truncatum (Troglotrematidae): a kidney parasite of Crocidura russula (Soricidae) and their phylogenetic significance

Spermatological characteristics of the troglotrematid digenean Nephrotrema truncatum, a parasite of the shrew Crocidura russula, have been investigated by means of transmission electron microscopy. The ultrastructural study reveals that the mature spermatozoon of N. truncatum exhibits many ultrastructural characters previously described in most gorgoderoideans. These are two axonemes of the 9+'1' trepaxonematan pattern, four attachment zones, a lateral expansion, an external ornamentation of the plasma membrane associated with spine-like bodies and cortical microtubules, and in the posterior part of the anterior spermatozoon region, two bundles of parallel cortical microtubules with the maximum number located in the anterior part of the spermatozoon, a nucleus, two mitochondria, and granules of glycogen. The obtained results are compared with those of other digeneans, particularly the Gorgoderoidea. The sperm cells gorgoderoideans are of type IV, characterised by a 9+'1' pattern of axonemes, the presence of an external ornamentation associated with cortical microtubules and located in the posterior area of the anterior extremity, the presence of two bundles of cortical microtubules, the maximum number of cortical microtubules located in the anterior region of the spermatozoon, and the presence of generally two mitochondria. However, dicrocoeliids and troglotrematids have spermatozoa with ornamentation of the plasma membrane and lateral expansions

Comparative study of the physicochemical quality of water of wells and drilling consumed in the commune of Sinthiou Maléme in the area of Tambacounda (Senegal)

In Senegal, the majority of the regions are not served by the drinking water supply networks. The phenomenon is more pronounced in rural areas, particularly in Sinthiou Maléme commune. For example, communities living in these areas often use well water and borehole. This work has been undertaken to evaluate the physicochemical quality of the water resources consumed by these populations. A total of 24 water samples were taken from the single borehole and 02 publics wells, let be 8 samples per source of water. To assess the quality of these different sources, the physical parameters (electrical conductivity, pH, total dissolved solids and hardness) and chemical parameters (F-, SO42-, PO43-, Fe and NO2-) were analyzed by the photometric method. The results obtained show that, from the physical point of view, drilling water is highly mineralized, slightly hard and has a basic tendency, unlike wells. Chemical analysis shows that well waters are heavily loaded with phosphate ions and nitrites. Based on the parameters analyzed, the quality of the drilling water is chemically acceptable. Studies on the elements of metallic traces will be envisaged to better assess the quality of this drinking water. Au Sénégal, la majeure partie des régions n’est pas desservie par les réseaux d'adduction d’eau potable. Le phénomène est plus accentué en milieu rural notamment dans la commune de Sinthiou Maléme. Ainsi, les communautés qui vivent dans ces zones ont souvent recours à l'eau des puits et des forages. Ce présent travail a été entrepris en vue d’évaluer la qualité physico-chimique des ressources en eaux consommées par ces populations. Au total 24 échantillons d’eaux ont été prélevés de l’unique forage et de 02 puits publics, soit 8 prélèvements par sources d’eaux. Pour apprécier la qualité de ces différentes sources, les paramètres physiques (conductivité électrique, pH, totale des solides dissous et dureté) et chimiques (F-, SO42-, PO43-, Fe and NO2-)&nbsp;ont été analysés par la méthode photométrique. Les résultats obtenus montrent que, du point de vue physique, les eaux de forage sont fortement minéralisées, légèrement dures et présentent une tendance basique contrairement à celles des puits. L’analyse chimique montre que les eaux de puits sont fortement chargées en ions phosphates et nitrites. Sur la base des paramètres analysés, la qualité des eaux de forage est chimiquement acceptable. Des études sur les éléments des traces métalliques seront envisagées pour mieux apprécier la qualité de ces eaux de consommation

Molecular Diagnostics of Ebola Patient Samples by Institut Pasteur de Dakar Mobile Laboratory in Guinea 2014–2016

As part of the laboratory response to the Ebola virus outbreak in Guinea, the Institut Pasteur de Dakar mobile laboratory (IPD-ML) was set up in Donka hospital from 2014 to 2016. EBOV suspected samples collected at Ebola Treatment Centers (ETC) and from community deaths were sent daily to IPD-ML. Analysis was performed using dried oligonucleotide mixes for real-time RT-PCR designed for field diagnostic. From March 2014 to May 2015, a total of 6055 patient samples suspected for EBOV collected from seven regions of Guinea were tested by real-time RT-PCR. These patients’ clinical included serum samples (n = 2537 samples) and swabs (n = 3518 samples) with positivity rates of 36.74 and 6.88% respectively. Females were significantly more affected than males with positivity rates of 22.39 and 17.22% respectively (p-value = 5.721e-7). All age groups were exposed to the virus with significant difference (p-value <= 2.2e-16). The IPD-ML contributed significantly to the surveillance and patient management during the EBOV outbreak in Guinea. Furthermore, dried reagents adapted for field diagnostic of EVD suspect cases could be useful for future outbreak preparedness and response

Epidemiology of West Nile virus in Africa: an underestimated threat

12openInternationalInternational coauthor/editorBackground West Nile virus is a mosquito-borne flavivirus which has been posing continuous challenges to public health worldwide due to the identification of new lineages and clades and its ability to invade and establish in an increasing number of countries. Its current distribution, genetic variability, ecology, and epidemiological pattern in the African continent are only partially known despite the general consensus on the urgency to obtain such information for quantifying the actual disease burden in Africa other than to predict future threats at global scale. Methodology and principal findings References were searched in PubMed and Google Scholar electronic databases on January 21, 2020, using selected keywords, without language and date restriction. Additional manual searches of reference list were carried out. Further references have been later added accordingly to experts’ opinion. We included 153 scientific papers published between 1940 and 2021. This review highlights: (i) the co-circulation of WNV-lineages 1, 2, and 8 in the African continent; (ii) the presence of diverse WNV competent vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more competence studies to be addressed on ticks; (iv) evidence of circulation of WNV among humans, animals and vectors in at least 28 Countries; (v) the lack of knowledge on the epidemiological situation of WNV for 19 Countries and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa. Conclusions This study provides the state of art on WNV investigation carried out in Africa, highlighting several knowledge gaps regarding i) the current WNV distribution and genetic diversity, ii) its ecology and transmission chains including the role of different arthropods and vertebrate species as competent reservoirs, and iii) the real disease burden for humans and animals. This review highlights the needs for further research and coordinated surveillance efforts on WNV in Africa.openMencattelli, G.; Dior Ndione M.H.; Rosa', R.; Marini, G.; Diagne, C.T.; Diagne, M.M.; Fall, G.; Faye, O.; Diallo, M.; Faye, O.; Savini, G.; Rizzoli, A.Mencattelli, G.; Dior Ndione, M.H.; Rosa', R.; Marini, G.; Diagne, C.T.; Diagne, M.M.; Fall, G.; Faye, O.; Diallo, M.; Faye, O.; Savini, G.; Rizzoli, A

Development, validation and clinical evaluation of a broad-range pan-filovirus RT-qPCR

Background: During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. Objectives: The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. Study design: A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. Results: The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Tai Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOVspecific assay. Discussion: Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.Peer reviewe