The Flavivirus genus comprises many viruses (including dengue, Zika, West Nile and yellow fever viruses) which constitute important public health concerns worldwide. For several of these pathogens, neither antivirals nor vaccines are currently available. In addition to this unmet medical need, flaviviruses are of particular interest since they constitute an excellent model for the study of spatiotemporal regulation of RNA metabolism. Indeed, with no DNA intermediate or nuclear step, the flaviviral life cycle entirely relies on the cytoplasmic fate of a single RNA species, namely the genomic viral RNA (vRNA) which contains all the genetic information necessary for optimal viral replication. From a single open reading frame, the vRNA encodes a polyprotein which is processed to generate the mature viral proteins. In addition to coding for the viral polyprotein, the vRNA serves as a template for RNA synthesis and is also selectively packaged into newly assembled viral particles. Notably, vRNA translation, replication and encapsidation must be tightly coordinated in time and space via a fine-tuned equilibrium as these processes cannot occur simultaneously and hence, are mutually exclusive. As such, these dynamic processes involve several vRNA secondary and tertiary structures as well as RNA modifications. Finally, the vRNA can be detected as a foreign molecule by cytosolic sensors which trigger upon activation antiviral signaling pathways and the production of antiviral factors such as interferons and interferon-stimulated genes. However, to create an environment favorable to infection, flaviviruses have evolved mechanisms to dampen these antiviral processes, notably through the production of a specific vRNA degradation product termed subgenomic flavivirus RNA (sfRNA). In this review, we discuss the current understanding of the fates of flavivirus vRNA and how this is regulated at the molecular level to achieve an optimal replication within infected cells.
Toxoplasma gondii is a coccidian parasite with the cat as its definitive host but any warm-blooded animal, including humans, may act as intermediate hosts. It has a worldwide distribution where it may cause acute and chronic toxoplasmosis. Infection can result from ingestion either of tissue cysts in infected meat of intermediate hosts or oocysts found in cat faeces via contaminated water or food. In this review, we highlight how the oocyst and sporocyst walls sustain the persistence and transmission of infective T. gondii parasites from terrestrial and aquatic environments to the host. We further discuss why targeting the oocyst wall structure and molecules may reduce the burden of foodborne and waterborne T. gondii infections.
With no available therapies, infections with Zika virus (ZIKV) constitute a major public health concern as they can lead to congenital microcephaly. In order to generate an intracellular environment favourable to viral replication, ZIKV induces endomembrane remodelling and the morphogenesis of replication factories via enigmatic mechanisms. In this study, we identified the AAA+ type ATPase valosin‐containing protein (VCP) as a cellular interaction partner of ZIKV non‐structural protein 4B (NS4B). Importantly, its pharmacological inhibition as well as the expression of a VCP dominant‐negative mutant impaired ZIKV replication. In infected cells, VCP is relocalised to large ultrastructures containing both NS4B and NS3, which are reminiscent of dengue virus convoluted membranes. Moreover, short treatment with the VCP inhibitors NMS‐873 or CB‐5083 drastically decreased the abundance and size of ZIKV‐induced convoluted membranes. Furthermore, NMS‐873 treatment inhibited ZIKV‐induced mitochondria elongation previously reported to be physically and functionally linked to convoluted membranes in case of the closely related dengue virus. Finally, VCP inhibition resulted in enhanced apoptosis of ZIKV‐infected cells strongly suggesting that convoluted membranes limit virus‐induced cytopathic effects. Altogether, this study identifies VCP as a host factor required for ZIKV life cycle and more precisely, for the maintenance of viral replication factories. Our data further support a model in which convoluted membranes regulate ZIKV life cycle by impacting on mitochondrial functions and ZIKV‐induced death signals in order to create a cytoplasmic environment favourable to viral replication.
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 (Ct 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 Ct 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 Ct 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.
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