RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

Antiviral immunity in Drosophila requires systemic RNA interference spread.

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila.

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.

Multifaceted contributions of Dicer2 to arbovirus transmission by Aedes aegypti.

Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.

Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster.

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. Here, we sought to study this evolutionary arms-race and assessed the impact of the innate immune system on viral population diversity and evolution, using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). We isogenized eight fly genotypes generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen-sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV and the virus was serially passaged ten times via reinfection in naive flies. Viral population diversity was studied after each viral passage by high-throughput sequencing and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. We found that the absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, our results indicate that the innate immune system as a whole and not specific antiviral defence pathways in isolation, generally constrains viral diversity and evolution.

Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts.

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.

Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects.

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.

Regional variations of low-density lipoprotein oxidizability in hemodialysis patients may explain discrepancies in interventional therapy on oxidative profile.

BACKGROUND/AIMS: This study aimed at evaluating oxidative stress (OS) markers (i) in a cross-sectional study of hemodialysis (HD) patients to investigate potential regional effects of these markers and (ii) in a prospective crossover study to evaluate vitamin E-coated membrane (VE) effects. METHODS: At baseline, OS parameters including low-density lipoprotein (LDL) oxidizability were measured in HD patients from five dialysis facilities. Patients were then randomly assigned to two treatment groups: group I patients (n = 33) switching to VE, and group II patients (n = 29) still using reference polysulfone (PS) membrane. After 3 months, patients were switched from VE to PS and vice versa for 6 months. The same OS parameters were measured after each period. RESULTS: At baseline, the cross-sectional analysis of LDL oxidizability showed a regional effect. By contrast, the crossover study did not show beneficial effects of VE on this parameter. CONCLUSION: Regional variations of LDL oxidizability in HD patients exist and may explain discrepancies in interventional therapy on OS.

Immune priming and clearance of orally acquired RNA viruses in Drosophila.

Immune responses in insects are differentially triggered depending on the infection route used by the pathogen. In most studies involving Drosophila melanogaster and viruses, infection is done by injection, while oral infection, which is probably the most common route of viral entry in nature, remains unexplored. Here, we orally infected adults and larvae from wild-type and RNA interference (RNAi) mutant flies with different RNA viruses. We found that, in contrast with what is observed following virus injection, oral infections initiated at larval or adult stages are cleared in adult flies. Virus elimination occurred despite a larger infectious dose than for injected flies and evidence of viral replication. RNAi mutant flies suffered greater mortality relative to wild-type flies following oral infection, but they also eliminated the virus, implying that RNAi is not essential for viral clearance and that other immune mechanisms act during oral infections. We further showed that information of infection by RNA viruses acquired orally leaves a trace under a DNA form, which confers protection against future reinfection by the same virus. Together, this work presents evidence of clearance and immune priming for RNA viruses in insects and challenges the current view of antiviral immunity in insects.

Dicer-2-Dependent Generation of Viral DNA from Defective Genomes of RNA Viruses Modulates Antiviral Immunity in Insects.

The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.

Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut.

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

Virus-derived DNA drives mosquito vector tolerance to arboviral infection.

Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes.

Biochemical and spectrophotometric significance of advanced oxidized protein products.

We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The present study was carried out to further investigate how myeloperoxidase (MPO)-catalyzed reactions could contribute to AOPP generation in the plasma. First, patterns of plasma protein oxidation obtained after in vitro incubation of control plasma with hypochlorous acid (HOCl) were compared to those from HD patients and control plasma. The use of various analytical techniques enabled localising and identifying the main oxidized proteins with albumin (HSA) after protein separation by size-exclusion chromatography and SDS-PAGE electrophoresis. The characterization of the oxidation level of the individual plasma proteins in terms of carbonyl groups and 3-nitrotyrosine formations was performed by immunoblotting. Secondly, to highlight the significance of AOPP index monitored by spectrophotometry, spectra were established for plasma fractions from HD patients and compared to data for control plasma and HOCl-treated plasma. The corresponding absorbance difference spectra were matched with external standards such as dityrosine, nitrotyrosine and pentosidine and elaborated chromophoric probe models. Indeed, HSA was chlorinated by HOCl reagent or HOCl generated via the MPO/H(2)O(2)/Cl(-) system and was nitrated by tetranitromethane. Increased absorbances at the range of 340 nm were observed both with chlorinated and nitrated HSA. Finally, our results indicate that HOCl, and not NO(2)(*), generated via MPO activity, could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes.

Iron therapy, advanced oxidation protein products, and carotid artery intima-media thickness in end-stage renal disease.

BACKGROUND: Increased common carotid artery intima-media thickness (CCA-IMT) is a marker of early atherosclerosis. Low-grade inflammation is associated with the pathogenesis of atherosclerosis. Low-grade inflammation and increased CCA-IMT are observed in end-stage renal disease (ESRD). Oxidative stress is involved in uremia-related inflammation. Advanced oxidation protein products (AOPP) are markers of oxidant-mediated protein damage in ESRD. Intravenous iron given to patients on hemodialysis (HD) might induce oxidative stress. We investigated the relationships between AOPP, iron therapy, and CCA-IMT in stable HD patients. METHODS AND RESULTS: Plasma AOPP and blood chemistry, including iron status, were analyzed in a cohort of 79 ESRD patients on HD. Measurements of CCA-IMT and CCA diameter, as assessed by B-mode ultrasonography, were obtained in 60 patients. AOPP levels were elevated in ESRD patients, and in univariate (r=0.42, P<0.0001) and multivariate analyses (r=0.38, P<0.001), they correlated with serum ferritin and with the intravenous iron dose received during the 12 months preceding the study (ferritin, P<0001; AOPP, P<0.01). Univariate and multivariate analyses identified the AOPP concentration as being significantly associated with CCA-IMT (P=0.0197) and CCA wall-to-lumen ratio (r=0.560, P<0.0001). Independently of AOPP concentration, cumulative iron dose was positively related to CCA-IMT (P=0.015) in patients <60 years. CONCLUSION: In ESRD patients, CCA-IMT and CCA wall-to-lumen ratio were associated with plasma AOPP, serum ferritin, and the annual intravenous iron dose administered. These findings support the concept of a role of oxidative stress in the early atherosclerosis of ESRD patients, which may be increased by the usually recommended doses of intravenous iron.

Advanced oxidation protein products as risk factors for atherosclerotic cardiovascular events in nondiabetic predialysis patients.

BACKGROUND: Inflammation and oxidative stress are established risk factors for atherosclerosis, but whether they contribute to the accelerated atherogenesis associated with chronic kidney disease (CKD) remains to be assessed at the predialysis stage. METHODS: We prospectively examined the relationship between plasma levels of C-reactive protein (CRP), fibrinogen, and advanced oxidation protein products (AOPPs), as selected markers of inflammation and oxidative stress, and incident first occlusive atherosclerotic cardiovascular (CV) events (ASCVEs) in a single-center cohort of 80 uremic predialysis patients without diabetes with a creatinine clearance ranging from 20 to 40 mL/min/1.73 m2 . RESULTS: During follow-up (median, 7 years), 21 patients developed coronary, cerebral, or peripheral artery occlusive accidents, an incidence of 44/1,000 patient-years. Except for older age, their conventional risk factors did not differ compared with the 59 patients who remained free of such accidents. Conversely, plasma levels of CRP (4.3 +/- 2.7 versus 2.3 +/- 2 mg/L; P = 0.005), fibrinogen (5.6 +/- 1.4 versus 4.4 +/- 1.2 mg/L; P = 0.0009), and AOPPs (58 +/- 20 versus 42 +/- 14 micromol/L; P = 0.0002) were significantly greater at baseline, although serum creatinine levels did not differ between the 2 groups. By multivariate Cox regression analysis, age and CRP, fibrinogen, and AOPP levels were significant independent predictors of ASCVEs. Risk factor-adjusted hazard ratios were as follows: age, 1.13 (95% confidence interval, 1.04 to 1.22; P = 0.002); CRP level, 1.37 (95% confidence interval, 1.05 to 1.79; P = 0.02); fibrinogen level, 2.23 (95% confidence interval, 1.20 to 4.13; P = 0.011); and AOPP level, 1.68 (95% confidence interval, 1.12 to 2.51; P = 0.011). CONCLUSION: CRP, fibrinogen, and AOPP levels independently predict ASCVEs in patients with CKD in the predialysis phase and might directly contribute to the uremia-associated accelerated atherogenesis.

Are advanced oxidation protein products potential uremic toxins?

Oxidative stress, defined as a disruption of the equilibrium between the generation of oxidants and the activity of anti-oxidant systems, plays a significant role in the development of the inflammatory syndrome associated with chronic renal failure and hemodialysis. In our recent work, the aim of which was to better characterize oxidative stress in dialysis patients, we described the presence of oxidized protein products, which we have termed advanced oxidation protein products (AOPP), in the plasma of dialysis patients and we proposed AOPP as new markers of oxidative stress and potential inflammatory mediators. AOPP represent an exquisite marker of phagocyte-derived oxidative stress, and their role in the pathophysiology of chronic renal failure and dialysis-related complications might be of great importance. Regarding the mechanisms of generation of AOPP, we pointed out the importance of myeloperoxidase and the subsequent generation of chlorinated oxidants, previously considered solely as microbicidal agents, in the formation of AOPP. Indeed, AOPP appear to act as true inflammatory mediators since they are able to trigger the oxidative burst and the synthesis of inflammatory cytokines in neutrophils, as well as in monocytes. Thus, it could be hypothesized that the AOPP, which arise from the reaction between chlorinated oxidants and plasma proteins, constitute new uremic toxins with pro-inflammatory effects.

Viral small RNA cloning and sequencing.

At the current rate of technological progress, high-throughput sequencing of nucleic acids has become a commodity. These techniques are perfectly suitable for viral small RNAs sequencing and contribute to the understanding of many aspects of virus biology in the context of host-pathogen interaction. However, the generation of high quality data is still an issue and the preparation of small RNAs libraries that accurately reflect the viral siRNAs in the sample remains a challenge. In this chapter we describe how to clone and sequence libraries of viral small RNAs from infected insect samples (mosquito, drosophilidae, insect-derived cell lines).

Respective role of uraemic toxins and myeloperoxidase in the uraemic state.

BACKGROUND: In haemodialysis (HD) patients, advanced oxidation protein products (AOPP) were previously ascribed to oxidized plasma proteins, resulting mainly from increased myeloperoxidase (MPO) activity. The aim of the present study was to assess the mechanisms leading to the generation of AOPP during the course of chronic kidney disease including end-stage renal disease, with particular focus on AOPP and MPO characterization in the plasma at decreasing levels of kidney function. METHODS: Phagocyte activation was evaluated by whole blood NADPH oxidase and MPO activities. In plasma, MPO protein concentration was quantified by ELISA and catalytic activity assayed by the spectrophotometric detection of phenol and 4-aminoantipyrine (AAP) co-oxidation in the presence of hydrogen peroxide (H(2)O(2)). RESULTS: In HD patients, plasma AOPP concentration was linked to neutrophil oxidative activity. Such an association was not found in control subjects or predialysis patients, suggesting that in the latter, AOPP generation did not mainly result from MPO released by activated neutrophils. Similarly, plasma AOPP correlated with plasma MPO protein concentration in HD patients, but not in control subjects or predialysis patients, suggesting that in the latter AOPP did not predominantly result from MPO activity. This interpretation was supported by the observation of a greater degree of co-oxidation of phenol and AAP in the absence of H(2)O(2) in predialysis patients than in HD patients or control subjects. The contribution of MPO dramatically differed between predialysis and HD patients (2+/-5 vs 46+/-6%; P<0.001). CONCLUSION: Our observations suggest that AOPP generation in predialysis patients mainly results from MPO-independent oxidation mechanisms.

Myeloperoxidase promoter polymorphism -463G is associated with more severe clinical expression of cystic fibrosis pulmonary disease.

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.

Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils.

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.