The Multiples Fates of the Flavivirus RNA Genome During Pathogenesis

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

Complications after partial nephrectomy: robotics overcomes open surgery and laparoscopy: the PMSI French national database

Abstract Purpose To evaluate three partial nephrectomies (PN) procedures: open (OPN), standard laparoscopy (LPN), and robot-assisted laparoscopy (RAPN), for the risk of initial complications and rehospitalization for two years after the surgery. Materials and methods From the French national hospital database (PMSI-MCO), every hospitalization in French hospitals for renal tumor PN in 2016–2017 were extracted. Complications were documented from the initial hospitalization and any rehospitalization over two years. Chi-square and ANOVA tests compared the frequency of complications and length of initial hospitalization between the three surgical procedures. Relative risks (RR) and 95% confidence intervals were computed. Results The 9119 initial hospitalizations included 4035 OPN, 1709 LPN, and 1900 RAPN; 1475 were excluded as the laparoscopic procedure performed was not determined. The average length of hospitalization was 8.1, 6.2, and 4.5 days for OPN, LPN, and RAPN, respectively. Compared to OPN, there were fewer complications at the time of initial hospitalization for the mini-invasive procedures: 29% for OPN vs. 20% for LPN (0.70 [0.63;0.78]) and 12% for RAPN (RR=0.43, 95%CI [0.38;0.49]). For RAPN compared to LPN, there were fewer haemorrhages (RR=0.55 [0.43;0.72]), anemia (0.69 {0.48;0.98]), and sepsis (0.51 [0.36;0.71]); during follow up, there were fewer urinary tract infections (0.64 [0.45;0.91]) but more infectious lung diseases (1.69 [1.03;2.76]). Over the two-year postoperative period, RAPN was associated with fewer acute renal failures (RR=0.73 [0.55;0.98]), renal abscesses (0.41 [0.23;0.74]), parietal complications (0.69 [0.52;0.92]) and urinary tract infections (0.54 [0.40;0.73]) than for OPN. Conclusions Conservative renal surgery is associated with postoperative morbidity related to the surgical procedure fashion. Mini-invasive procedures, especially robot-assisted surgery, had fewer complications and shorter hospital lengths of sta

Comparison of surgical procedures for benign prostatic hyperplasia of medium-volume prostates: evaluation of the causes of rehospitalization from the French National Hospital Database (PMSI-MCO)

International audiencePurpose: In France, transurethral resection of the prostate (TURP) is still the most commonly used surgical treatment for medium sized benign prostatic hyperplasia (BPH), but the Holmium Laser Enucleation of the Prostate (HoLEP) and laser vaporization procedures are becoming more common. For these three surgical procedures, we evaluate the initial complications, the short term (3 months) and the 4–12-month postoperative complications necessitating re-hospitalization. Methods: From the French national hospital data base (PMSI-MCO), all hospitalizations for BPH treatment in 2018 were extracted. We document the complications during the initial hospitalization and any subsequent rehospitalizations during the one-year postoperative period. Results: In 2018, 67,220 patients were treated for BPH: 46,242 TURP, 13,509 HoLEP and 7469 laser vaporization. Age and anticoagulation medications were similar for men treated by the three procedures, but TURP patients were more often hypertensive. Infections and hemorrhagic complications were the most common complications at the initial hospitalization: 17%, 10%, 13% for infections and 15%, 8.1%, 11% for hemorrhagic complications respectively, and TURP performed worse than the other two procedures at the initial hospitalization. During the first three months and then the subsequent nine months, there were fewer complications than initially, with little difference between the three procedures, all differences being less than 1%. Conclusion: Laser vaporization techniques led to fewer complications. However, the PMSI-MCO only registers complications during hospitalizations. This study should be extended to non-hospitalized, more minor complications

The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein

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

N-Phenylpyridine-3-Carboxamide and 6-Acetyl-1H-Indazole Inhibit the RNA Replication Step of the Dengue Virus Life Cycle.

Dengue virus (DENV) is a Flavivirus that causes the most prevalent arthropod-borne viral disease. Clinical manifestation of DENV infection ranges from asymptomatic to severe symptoms that can lead to death. Unfortunately, no antiviral treatments against DENV are currently available. In order to identify novel DENV inhibitors, we screened a library of 1,604 chemically diversified fragment-based compounds using DENV reporter viruses that allowed quantification of viral replication in infected cells. Following a validation screening, the two best inhibitor candidates were N-phenylpyridine-3-carboxamide (NPP3C) and 6-acetyl-1H-indazole (6A1HI). The half maximal effective concentration of NPP3C and 6A1H1 against DENV were 7.1 μM and 6.5 μM, respectively. 6A1H1 decreased infectious DENV particle production up to 1,000-fold without any cytotoxicity at the used concentrations. While 6A1HI was DENV-specific, NPP3C also inhibited the replication of other flaviviruses such as West Nile virus and Zika virus. Structure-activity relationship (SAR) studies with 151 analogues revealed key structural elements of NPP3C and 6A1HI required for their antiviral activity. Time-of-drug-addition experiments identified a postentry step as a target of these compounds. Consistently, using a DENV subgenomic replicon, we demonstrated that these compounds specifically impede the viral RNA replication step and exhibit a high genetic barrier-to-resistance. In contrast, viral RNA translation and the de novo biogenesis of DENV replication organelles were not affected. Overall, our data unveil NPP3C and 6A1H1 as novel DENV inhibitors. The information revealed by our SAR studies will help chemically optimize NPP3C and 6A1H1 in order to improve their anti-flaviviral potency and to challenge them in in vivo models