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Background The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. Results Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (C t values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at C t values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with C t values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. Conclusion We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches.

With 40% of the world population at risk, infections with dengue virus (DENV) constitute a serious threat to public health. While there is no antiviral therapy available against this potentially lethal disease, the efficacy of the only approved vaccine is not optimal and its safety has been recently questioned. In order to develop better vaccines based on attenuated and/or chimeric viruses, one must consider how the human immune system is engaged during DENV infection. The activation of the innate immunity through the detection of viruses by cellular sensors is the first line of defence against those pathogens. This triggers a cascade of events which establishes an antiviral state at the cell level and leads to a global immunological response. However, DENV has evolved to interfere with the innate immune signalling at multiple levels, hence dampening antiviral responses and favouring viral replication and dissemination. This review elaborates on the interplay between DENV and the innate immune system. A special focus is given on the viral countermeasure mechanisms reported over the last decade which should be taken into consideration during vaccine development.

Toxoplasma gondii is a common parasite of humans and animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The robust oocyst and sporocyst walls protect the infective sporozoites from deleterious external attacks including disinfectants. Upon oocyst acquisition, these walls lose their integrity to let the sporozoites excyst and invade host cells following a process that remains poorly understood. Given the resistance of the oocyst wall to digestive enzymes and the ability of oocysts to cause parenteral infections, the present study investigated the possible contribution of macrophages in supporting sporozoite excystation following oocyst internalisation. By using single cell micromanipulations, real-time and time-point imaging techniques, we demonstrated that RAW macrophages could interact rapidly with oocysts and engulfed them by remodelling of their actin cytoskeleton. Internalised oocysts were associated to macrophage acidic compartments and showed evidences of wall disruption. Sporozoites were observed in macrophages containing oocyst remnants or in new macrophages, giving rise to dividing tachyzoites. All together, these results highlight an unexpected role of phagocytic cells in processing T. gondii oocysts, in line with non-classical routes of infection, and open new perspectives to identify chemical factors that lead to oocyst wall disruption under physiological conditions.

En l'absence de traitements ou de vaccins, les infections par les virus de la dengue (VDEN) et Zika (VZIK), constituent un enjeu majeur de santé publique dans le monde. Lors de l’infection, les VDEN et VZIK remodèlent le réticulum endoplasmique (ER) pour former des usines de réplication virale (URv) qui incluent les convolutions de membrane (CM). De plus, ils induisent l'élongation des mitochondries (par NS4B pour VDEN) en faveur de la réplication virale suggérant une modulation des fonctions mitochondriales. Des observations antérieures ont identifié des contacts physiques entre les mitochondries et les CM en même temps qu'une diminution des contacts de l’interface réticulo-mitochondriale (IRM). Par conséquent, nous avons étendu nos recherches au niveau moléculaire, par comment les VDEN et VZIK exploitent l’IRM pour leur réplication, et si cela modifie le métabolisme respiratoire mitochondriale et l'apoptose.Des analyses en microscopie électronique à transmission démontrent que les VDEN et VZIK altèrent l’IRM. Celle-ci repose sur plusieurs interactions entre des protéines situées à la surface des deux organites, telles que PTPIP51-VAPB, SYNJ2BP-RRBP1 ou VDAC1-IP3R1. L'infection par les VDEN et VZIK induit une altération globale de ces interactions. Simultanément, l'infection virale altère le profil d'expression de RRBP1 et SYNJ2BP, suggérant qu'ils ciblent spécifiquement cette interaction protéine-protéine. Par ailleurs, l'inhibition de l'expression des protéines de l’IRM augmente la réplication virale, ce qui soutient que l’IRM est altérée au bénéfice de la réplication virale. De plus, l'infection par VDEN/VZIK et l'expression de NS4B modulent de manière significative la phosphorylation oxydative mitochondriale. Nos analyses métabolomiques et mitoprotéomiques révèle que cela est corrélé à une diminution de l'abondance de plusieurs métabolites du cycle de Krebs et à des changements dans la stoechiométrie des protéines de la chaîne de transport d'électrons. Par ailleurs, la déstabilisation de l’IRM par diminution de l'expression de protéines lors de l'infection virale entraîne une diminution drastique de l'apoptose, démontrant l'importance de l'altération de l’IRM par VDEN et VZIK pour leur réplication.Dans l'ensemble, nos données suggèrent fortement que les perturbations morphologiques des mitochondries par les VDEN et VZIK modulent leur activité métabolique via NS4B pour répondre aux besoins énergétiques du cycle de réplication virale. Cette étude soutient également un modèle selon lequel ces virus perturbent l’IRM afin de s'approprier des facteurs spécifiques de l’hôte nécessaires à la morphogenèse des CM et de diminuer ou retarder des mécanismes cellulaires importants défavorables au cycle de vie des Flavivirus.

The fight against the COVID-19 pandemic has created an urgent need to detect and isolate infected people. The challenge for clinical laboratories has been finding a high throughput, cheap, and efficient testing method in the context of extraction reagent shortages on a planetary scale. To answer this need, we studied SARS-CoV-2 detection in nasopharyngeal swabs stored in UTM (Universal Transport Media) or RNAse-free water by rRT-PCR with the Seegene Allplex TM 2019-nCoV assay without RNA extraction. Optimal results were obtained with 1/2 dilution for swabs in RNAse free water (30/30 detected) and 1/5 dilution for swabs in UTM (29/30 detected) followed by thermal lysis. In addition, a proteinase K (PK) treatment allows a significant reduction of invalid results and increases sensitivity for detection of low viral load specimens. In a panel of 90 known positives with all 3 viral genes present and N gene Ct values from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a panel of 60 low positives with only the N gene detectable at Ct values > 30, the detection rate was 76.7% with PK vs 53.3% without it and the invalid rate fell off from 18.3% to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, however false negatives become frequent above this range. Finally, we show that swabs should be stored at -70 o C rather than 4 o C when testing cannot be performed within 72 hours of collection when laboratories are overwhelmed.

Mitochondria are crucial metabolic organelles that are regulated by both intracellular and extracellular cues. The extracellular matrix (ECM) is a key component of the cellular environment that controls cellular behavior and metabolic activity. Here, we determined how ECM signalling regulates mitochondrial structure and activity. To distinguish mitochondrial regulation from the general survival cues generated by the ECM, we used breast cancer-derived spheres (mammospheres) because of their ability to grow in suspension culture in the absence of ECM. Using this system, we demonstrate that the association of mammospheres with the ECM results in dramatic mitochondrial elongation, along with enhanced mitochondrial respiration and ATP production. This remodeling occurs independently of DRP1 activity, but relies on integrin signaling and actin polymerization. Therefore, our findings demonstrate that ECM-driven actin polymerization plays a crucial role in remodeling mitochondrial networks to promote OXPHOS, which represents a vital step for migrating cells to enhance cellular adhesion and facilitate cell growth.Competing Interest StatementThe authors have declared no competing interest.

With no therapeutics available, there is an urgent need to better understand the pathogenesis of flaviviruses which constitute a threat to public health worldwide. During infection, dengue virus (DENV) and Zika virus (ZIKV), two flaviviruses induce alterations of mitochondria morphology to favor viral replication, suggesting a viral co-opting of mitochondria functions. Here, we performed an extensive transmission electron microscopy-based quantitative analysis to demonstrate that both DENV and ZIKV alter endoplasmic reticulum-mitochondria contacts (ERMC). This correlated at the molecular level with an impairment of ERMC tethering protein complexes located at the surface of both organelles. Furthermore, virus infection, as well as NS4B expression modulated the mitochondrial oxygen consumption rate. Consistently, metabolomic and mitoproteomic analyses revealed a decrease in the abundance of several metabolites of the Krebs cycle and changes in the stoichiometry of the electron transport chain. Most importantly, ERMC destabilization by protein knockdown increased virus replication while dampening ZIKV-induced apoptosis. Overall, our results support the notion that flaviviruses hijack ERMCs to generate a cytoplasmic environment beneficial for sustained and efficient replication.