Marcos P Thomé
email: marcos.thome@ufrgs.br

Chloé Borde

Annette K Larsen

Joao A P Henriques

Guido Lenz

Alexandre E Escargueil

Vincent Maréchal

Dipyridamole as a new drug to prevent Epstein-Barr virus reactivation

Keywords: Epstein-Barr virus, dipyridamole, drug repurposing, EBV reactivation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Epstein-Barr virus (EBV) is a human gamma-herpesvirus that persistently infects more than 90% of the adult population worldwide. Primary infection occurs mostly during childhood without apparent symptoms but it can cause infectious mononucleosis in young adults. Following primary infection, EBV establishes a lifelong persistent infection that combines lytic replication and latent phases, supported by its dual tropism for B lymphocytes and epithelial cells [START_REF] Chiu | Epstein-Barr Virus: The Path from Latent to Productive Infection[END_REF][START_REF] Hutt-Fletcher | Epstein-Barr virus replicating in epithelial cells[END_REF]. Whereas EBV infection is usually tightly controlled by the immune response, it has been associated with various malignancies including Burkitt's lymphoma, Hodgkin's disease, various B-cell lymphomas linked to immunosuppression, including Post-Transplant Lymphoproliferative Diseases (PTLD), some forms of T-cell lymphomas, undifferentiated nasopharyngeal carcinoma and gastric cancers [START_REF] Jha | Epstein-Barr Virus: Diseases Linked to Infection and Transformation[END_REF].

Sporadic reactivations leading to the production of viral progeny may occur from latently infected B-lymphocytes in response to physiological factors that are still largely unknown in vivo. Reactivation can be induced ex vivo by phorbol esters [START_REF] Zur Hausen | Persisting oncogenic herpesvirus induced by the tumour promotor TPA[END_REF], calcium ionophores [START_REF] Faggioni | Calcium modulation activates Epstein-Barr virus genome in latently infected cells[END_REF], transforming growth factor beta (TGF-β) [START_REF] Renzo | Endogenous TGF-β contributes to the induction of the EBV lytic cycle in two burkitt lymphoma cell lines[END_REF], sodium butyrate [START_REF] Luka | Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate[END_REF], hypoxia [START_REF] Jiang | Hypoxia can contribute to the induction of the Epstein-Barr virus (EBV) lytic cycle[END_REF] or following activation of B-cell receptor with antibodies directed against surface immunoglobulins (anti-sIg) [START_REF] Takada | Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines[END_REF].

Viral reactivation is a highly coordinated process that is initiated by the expression of two immediate early viral genes, namely BZLF1 (encoding for Zta, Z, Zebra, EB1) and BRLF1 (encoding for Rta, R, EB2) that encode transactivators required for early gene expression. Zta also activates the lytic origin of replication OriLyt. Lytic replication of the viral genomes precedes the expression of late viral proteins that eventually leads to virion assembly and release [START_REF] Feederle | The Epstein-Barr virus lytic program is controlled by the co-operative functions of two transactivators[END_REF][START_REF] Murata | Regulation of Epstein-Barr virus reactivation from latency[END_REF]. Although B-cell transformation by EBV is usually associated with latently expressed viral oncogenes, there is a need for non-toxic antiviral drugs that can inhibit EBV lytic replication, notably in immunosuppressed patients. Indeed, immunosuppression favors EBV reactivation, thereby increasing viral load and promoting B-cell reinfection and lymphomagenesis. In addition, it has been demonstrated that viral reactivation directly and indirectly contributes to lymphomagenesis both in mouse models and in humans [START_REF] Cohen | Epstein-Barr virus infection[END_REF]Hong et al., 2005;[START_REF] Ma | A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas[END_REF][START_REF] Manners | Contribution of the KSHV and EBV lytic cycles to tumourigenesis[END_REF]. EBV lytic replication may also contribute to carcinogenesis in epithelial tumors [START_REF] Wu | Perspective: Contribution of Epstein-Barr virus (EBV) Reactivation to the Carcinogenicity of Nasopharyngeal Cancer Cells[END_REF]. Whereas antiviral agents have a limited impact for the treatment of active infections such as infectious mononucleosis, their prophylactic administration resulted in a reduced incidence of PTLD [START_REF] Hay | Efficacy and safety of valganciclovir in liver-transplanted children infected with Epstein-Barr virus[END_REF][START_REF] Höcker | Val-)Ganciclovir prophylaxis reduces Epstein-Barr virus primary infection in pediatric renal transplantation[END_REF][START_REF] Malouf | Anti-viral prophylaxis reduces the incidence of lymphoproliferative disease in lung transplant recipients[END_REF].

So far, antiviral molecules against EBV mainly target viral DNA replication.

These include nucleoside analogues (acyclovir, ganciclovir) and their prodrugs (valaciclovir, valganciclovir, valomaciclovir), nucleotides (cidofovir) and pyrophosphate (foscarnet) analogs that are active against alpha-and/or betaherpesviruses. These drugs have been assayed in vitro or used experimentally against EBV, but none received approval by the Food and Drug Administration nor the European Medicines Agency for the treatment of EBV related diseases [START_REF] Andrei | Novel Therapeutics for Epstein-Barr Virus[END_REF][START_REF] Pagano | Antiviral Drugs for EBV[END_REF]. Therefore, identifying new and non-toxic antiviral drugs that inhibit EBV reactivation remains an important challenge.

Recent advances in drug screening technologies have helped identify new molecules. However, drug development is an uncertain process due to high attrition rates combined with the time and the cost necessary to bring new compounds to market. This makes new drug development a less desirable choice for pharmaceutical industries and investors. In that context, drug repurposing strategies propose new medical indications for either investigational or approved drugs that are outside the scope of the original targets [START_REF] Pushpakom | Drug repurposing: progress, challenges and recommendations[END_REF]. This approach reduces the risk of failure because of safety considerations and the timeframe for drug development, thus requiring less investment. To be efficient, however, a drug repurposing strategy first requires identifying a candidate molecule for the indication of interest. This systematic approach mostly relies on previously published experimental data describing both pharmacological features and mechanism of action. Dipyridamole (DIP) (2,6-bis [diethanolamino]-4,8-dipiperidino-pyrimido 5,4d pyrimidine) is a safe drug with favorable and broad pharmacological properties.

Initially, DIP was used as a coronary vasodilator and soon after it was recognized as an antithrombotic agent. This allowed its long use as a prophylactic compound to prevent strokes and other vascular diseases [START_REF] Chakrabarti | Dipyridamole, cerebrovascular disease, and the vasculature[END_REF]. DIP inhibits both vasodilatation and platelet aggregation through two complementary mechanisms: (1) the inhibition of phosphodiesterase (PDE), in particular PDE3 and PDE5 [START_REF] Gresele | Anti-platelet therapy: phosphodiesterase inhibitors[END_REF], and (2) the blockade of nucleoside transport from the extracellular compartment to the cytoplasm of the cells [START_REF] Harker | Mechanism of action of dipyridamole[END_REF][START_REF] Knabb | Effects of dipyridamole on myocardial adenosine and active hyperemia[END_REF]. The inhibition of nucleoside uptake into platelets, endothelial and red blood cells increases plasmatic adenosine levels, which in turn activates adenosine receptors, including the A2-receptor, thus stimulating adenylate cyclase and the production of cyclic-3′,5′-adenosine monophosphate (cAMP). This effect, concomitant with PDEs inhibition, promotes the elevation of cAMP and cGMP levels in cells, impairing platelet aggregation and promoting arteriolar smooth muscle relaxation [START_REF] Chakrabarti | Dipyridamole, cerebrovascular disease, and the vasculature[END_REF].

DIP antiviral activity was previously demonstrated against Herpes simplex virus in vitro [START_REF] Tenser | Inhibition of herpes simplex virus reactivation by dipyridamole[END_REF] and in a mouse model [START_REF] Hay | Efficacy and safety of valganciclovir in liver-transplanted children infected with Epstein-Barr virus[END_REF] and it was shown to inhibit Mengovirus RNA replication [START_REF] Fata-Hartley | Dipyridamole Reversibly Inhibits Mengovirus RNA Replication[END_REF]. Additionally, in a combined treatment, DIP potentiated the antiviral activity of nucleoside analogs against human immunodeficiency virus (HIV) [START_REF] Patel | Differential inhibition of 2'-deoxycytidine salvage as a possible mechanism for potentiation of the anti-human immunodeficiency virus activity of 2',3'-dideoxycytidine by dipyridamole[END_REF].

Cellular nucleotides are essential components required for rapid viral genome replication [START_REF] Sanchez | Viral activation of cellular metabolism[END_REF]. In vivo, nucleotides can be synthesized de novo through enzymatic reactions or recycled through the salvage pathway of extracellular nucleosides, refueling the intracellular nucleotide pools [START_REF] Nyhan | Nucleotide Synthesis via Salvage Pathway[END_REF]. Since viral transcription and replication rely on the sources of nucleotides from the host cells, the present work aimed at targeting nucleoside uptake with DIP and to characterize its ability to inhibit EBV reactivation.

Material and methods

2.1.Antibodies and reagents

Anti-EBV ZEBRA (Zta) (sc-53904), anti-EBV EA-D (sc-58121), anti-β-actin (sc-47778) were purchased from Santa Cruz and horseradish peroxidase (HRP) linked-secondary antibody (#7076) was from Cell Signaling Technology. Dipyridamole (D9766), adenosine (A9251), thymidine (T9250), S-(4-Nitrobenzyl)-6thioinosine (NBMPR) (N2255) and dimethyl sulfoxide (DMSO) (D2650) were purchased from Sigma-Aldrich and H89 was purchased from Biomol GmbH. DIP, NBMPR and H89 stock solutions were dissolved in DMSO; adenosine and thymidine were dissolved in water.

2.2.Cell lines and treatments

Akata, Mutu-I and B95-8 cells were cultured in RPMI 1640 medium (Gibco BRL) supplemented with 10 % heat-inactivated fetal bovine serum and 2 mM L-glutamine (Life Technologies) at 37°C in a humidified 5% CO2 incubator. All cell culture media and supplements were obtained from Gibco (Thermo Fisher Scientific). EBV reactivation in Mutu-I cells was induced by treating cells for eight hours with 2 ng/ ml TGF-β (Eurobio). EBV reactivation in Akata cells was triggered by the crosslinking of the surface immunoglobulins with 7.5 µg/mL polyclonal rabbit antihuman IgG (α-sIg) (A0423, Dako) for five, eight or twenty-four hours for viral RNA, proteins or genomic viral DNA (gDNA)/virion production analysis, respectively. The cells were treated with DIP and/or adenosine or thymidine for the indicated times and concentrations. The cells were treated with NBMPR at the indicated drug concentrations for four hours prior addition of α-sIg. H89 was used at 5 µM and added one hour prior the addition of DIP. The absence of DIP toxicity was assessed by Trypan blue exclusion counting in light microscopy and propidium iodide (Molecular Probes, Life Technology, Cergy Pontoise, France) staining followed by flow cytometry detection. Treatment with 0.01% Triton-X100 was used as a positive control to assess membrane permeabilization to propidium iodide.

2.3.RNA extraction and RT-qPCR analysis

Total RNA was extracted and purified from cell cultures using TRIzol reagent (Invitrogen; Thermo Fisher Scientific) following the manufacturer's instructions.

Seven micrograms of purified RNA were submitted to DNAse treatment using TURBO DNA-free kit (Life technologies). Then, 1.5 micrograms of DNAse-treated RNA were reverse-transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Invitrogen; Thermo Fisher Scientific). Real-time PCR was performed using SensiFAST SYBR No-Rox Kit (Bioline). Primers are listed in Supplementary table 1.

Cellular β-globin RNA levels were used as an internal control to normalize viral mRNA and fold-changes were calculated by using the ΔΔCT method.

2.4.Analysis of virion production and intracellular viral DNA

Total intracellular DNA was isolated from cell cultures using DNeasy Blood and Tissue Kit (Qiagen) as described previously [START_REF] Masud | Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production[END_REF]. Intracellular viral DNA was quantified by real-time PCR using primers designed for detecting BHRF1. β-globin coding gene amplification was used as an internal control. Primers are listed in Supplementary table 1. Fold-changes were calculated by using the ΔΔCT method. For virion production analysis, cell cultures supernatants were collected and treated with DNase I (New England BioLabs) to eliminate naked free DNA. To estimate EBV virion production, real-time PCR was performed as described for intracellular DNA and the absence of amplification for β-globin was used as a control of external DNA elimination.

2.5.Western blot analysis

Cells were washed twice in PBS and lysed for 30 minutes at 4°C in denaturing lysis buffer [50 mM Tris HCl (pH 6.8), 2 % SDS, 2 % 2-mercaptoethanol].

Proteins were resolved on 12 % SDS-PAGE and blotted on to PVDF membranes (Amersham; GE Healthcare Life Sciences). Membranes were saturated by non-fat dried milk and antigens revealed by immunoblotting overnight at 4°C using the following primary antibodies: mouse monoclonal IgG anti-Zta (1:1000), mouse monoclonal IgG anti-EA-D (1:1000) and mouse monoclonal IgG anti-β-actin (1:5000).

Primary antibodies were detected by an anti-mouse IgG, HRP-linked antibody (1:10000) and antigens revealed using an enhanced chemiluminescence kit (Amersham; GE Healthcare Life Sciences) and X-ray film exposure (Carestream Kodak BioMax light film). Digital images were collected, and band intensities measured using ImageJ software and normalized to β-actin signal. Western blots are representative of 3 independent experiments.

2.6.Statistical analysis

Statistical analysis was performed using Prism 6 (GraphPad Software, La Jolla).

The level of significance was determined with a one-way analysis of variance test (ANOVA) followed by Bonferroni's multiple comparisons test. P-value < 0.05 was considered as statistically significant.

Results

3.1.DIP inhibits spontaneous and induced EBV reactivation

Akata is a well-characterized EBV-positive Burkitt's lymphoma-derived cell line in which EBV reactivation can be induced with antibodies directed against surface immunoglobulins (anti-sIg/α-sIg), a process that mimics physiological reactivation following B cell receptor-mediated stimulation [START_REF] Takada | Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines[END_REF]. Following this treatment, around 15% of the cells enter reactivation, as observed by flow cytometry immunodetection of the proteins Zta and EA-D (data not shown).

To investigate DIP antiviral activity, Akata cells were incubated with increasing concentration of DIP prior to reactivation. Preliminary experiments indicated that four hours pre-treatment was optimal (Supp. Fig. 1A andB). The production of virus particles was monitored by quantifying virion DNA in the cell culture supernatant after 24 hours reactivation. As illustrated in figure 1A, virion production was inhibited in a dose-dependent manner. Importantly, cell viability was not affected when Akata cells were grown in the presence of up to 20 µM DIP for 48 hours (Supp. Fig. 1C and1D). Moreover, DIP concentrations used herein are in the range attained after oral administration in humans [START_REF] Grem | Enhancement of 5-fluorouracil's anticancer activity by dipyridamole[END_REF]. B95-8 is an EBV positive marmoset B-cell line in which a minority of cells spontaneously enters into reactivation. Treatment of B95-8 cells with DIP also resulted in a significant decrease in virus production albeit to a lesser extent (Fig. 1B). This lower antiviral activity was expected since only 3 to 5% of cells expressed lytic antigens before treatment (data not shown).

3.2.DIP inhibits viral lytic replication as well as early and immediate early gene expression

To further characterize DIP antiviral activity, we evaluated its effect on viral DNA replication. For this purpose, intracellular viral DNA was extracted from Akata cells and quantified following reactivation in the presence or absence of DIP.

Whereas DIP had no significant effect on latent genomes (i.e. non-reactivated cells), the treatment significantly repressed viral replication following reactivation (Fig. 1C). Accordingly, DIP also repressed viral replication in B95-8 cells (Fig. 1D).

During the lytic phase of the cycle, the replication of EBV genomes is placed under the control of immediate early and early viral proteins, including viral DNA polymerase, auxiliary factors and the viral transacting factor Zta. The expression of early (EA-D) and immediate early (Zta) proteins was analyzed by immunoblotting following 8 hours reactivation in the presence of increasing concentrations of DIP.

As shown in figure 2A, a significant dose-dependent inhibition of both early and immediate early proteins was observed. Inhibition reached 80 and 90%, respectively, for the highest dose of DIP in Akata cells whereas a 50 and 40% decrease in EA-D and Zta protein expression, respectively, was observed in B95-8 cells at 48 hours for the highest concentration of DIP (Fig. 2B). Importantly, this effect of DIP was not limited to B cells in which EBV reactivation is induced by antibodies directed against surface immunoglobulins or observed spontaneously.

DIP indeed showed a similarly strong inhibitory effect on the Zta protein expression levels that is induced by TGF-β in Mutu-I cells, an EBV-positive Burkitt's lymphomaderived cell line expressing both TGF-β receptors I and II (Supp. Fig. 1E). These results suggest that DIP acts on EBV reactivation independently of the activation pathway.

We next wondered whether early and immediate early protein accumulation was inhibited at the transcriptional or translational level. For this purpose, a quantitative reverse transcription PCR-based assay was designed to measure the expression of viral (immediate early BZLF1, which encodes for Zta transactivator; early genes BMRF1 and BHRF1, which encode for EA-D and EA-R, respectively) and cellular genes (β-globin, β-actin and cyclophilin). As shown in figure 2C, DIP significantly repressed viral gene transcription following reactivation. This effect was specific since expression of control cellular genes was unaffected in the same conditions. It is notable that repression of early transcription was expected since early gene expression is under the control of immediate early proteins Zta and Rta, whose expression was strongly inhibited by DIP.

3.3.DIP antiviral activity is associated with its ability to inhibit nucleoside uptake

Our data suggested that DIP could act upstream of viral gene transcription.

DIP is indeed a known inhibitor of phosphodiesterases (PDE) [START_REF] Gresele | Anti-platelet therapy: phosphodiesterase inhibitors[END_REF], leading to an increase of intracellular concentration of cAMP with possible effect on the cAMP/PKA signaling pathway. However, an activation of the cAMP/PKAdependent pathway by DIP was unlikely to explain DIP antiviral activity since, in our model, its effect on EBV reactivation was not affected by the PKA inhibitor H89 which acts downstream of DIP in this specific pathway (Supp. Fig. 1F).

Since DIP was reported to interfere with nucleotide metabolism by potently and directly inhibiting equilibrative nucleoside transporters (especially ENT1 and ENT2), we next wondered whether DIP inhibition of nucleoside uptake may explain its antiviral activity. To test this hypothesis, we first evaluated the impact of S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of ENT1 [START_REF] Griffiths | Cloning of a human nucleoside transporter implicated in the Cellular uptake of adenosine and chemotherapeutic drugs[END_REF], on immediate early and early protein expression. As shown on figure 3A, NBMPR significantly repressed Zta and EA-D accumulation. The effect may be lower than DIP-mediated inhibition because NBMPR promotes the inhibition of ENT1 but not ENT2, which are both sensitive to DIP (Boswell-Casteel and Hays, 2017; [START_REF] Wang | Dipyridamole analogues as pharmacological inhibitors of equilibrative nucleoside transporters. Identification of novel potent and selective inhibitors of the adenosine transporter function of human equilibrative nucleoside transporter 4 (hENT4)[END_REF]. If DIP antiviral activity was indeed associated with its ability to inhibit nucleoside transport, we assumed that this process could be reversed if the cells were reactivated in the presence of a large excess of either adenosine or thymidine, therefore promoting a competition for the binding to the transporters.

To test this assumption, Akata cells were treated with DIP in the presence of 100 µM adenosine or thymidine for 4 hours prior to treatment with anti-sIg. Then, the expression of viral proteins was analyzed by immunoblotting following 8 hours reactivation. As shown on figure 3B, both adenosine and thymidine reversed DIP inhibition of Zta and EA-D expression, although this effect seemed to be less effective on Zta than on EA-D. Importantly, when given alone, neither adenosine nor thymidine induced EBV reactivation or interfered with it (Fig. 3C). Altogether, these data confirmed that DIP antiviral activity on EBV reactivation was related to its ability to prevent nucleoside import.

Discussion and conclusions

Although several drugs have been successfully evaluated for their ability to inhibit EBV replication ex vivo, their clinical use has been limited so far. It was initially suggested to use antivirals against EBV in clinical situations where active EBV replication was supposedly associated with the clinical symptoms, such as infectious mononucleosis (IM). However, antiviral therapy proved to have only a limited interest in this setting. Indeed, although acyclovir, a nucleoside analog, efficiently reduced EBV shedding in the saliva during IM, it was not associated with a clear clinical benefit [START_REF] Pagano | Antiviral Drugs for EBV[END_REF].

Although EBV-associated tumors are mainly linked to latently expressed oncogenes, a growing set of basic and clinical evidence suggested that EBV lytic replication might also directly and indirectly contribute to EBV-associated lymphomagenesis. This is notably supported by experimental data demonstrating that EBV mutants unable to undergo lytic viral replication were defective in promoting EBV-mediated lymphoproliferative disease in severe combined immunodeficient mice (Hong et al., 2005). This observation was, at least in part, explained by the increased production of B-cell growth factors, such as cellular IL-10, viral IL-10 and cellular IL-6 in response to EBV infection by wild-type viruses compared to Zta-knock out mutants. The contribution of lytic expression to the development of lymphomas was further confirmed in a humanized mouse model, in which both human fetal CD34 hematopoietic stem cells and thymus/liver tissue were transplanted [START_REF] Ma | A new model of Epstein-Barr virus infection reveals an important role for early lytic viral protein expression in the development of lymphomas[END_REF].

The benefit of antivirals to prevent primary EBV infection in high-risk patients and/or PTLD patients is supported by several clinical investigations.

Antiviral prophylaxis with ganciclovir or valganciclovir resulted in a significant decrease in EBV primary infections in a group of EBV-negative pediatric renal transplant patients receiving a graft from EBV-positive donors [START_REF] Höcker | Val-)Ganciclovir prophylaxis reduces Epstein-Barr virus primary infection in pediatric renal transplantation[END_REF].

Malouf and colleagues reported a reduction in the incidence of PTLD in high-risk EBV-seronegative lung transplant recipients who received a continuous, specific anti-viral prophylaxis with acyclovir, valaciclovir or ganciclovir [START_REF] Malouf | Anti-viral prophylaxis reduces the incidence of lymphoproliferative disease in lung transplant recipients[END_REF]. Another study reported that antiviral prophylaxis could prevent late onset PTLD [START_REF] Ville | Impact of antiviral prophylaxis in adults Epstein-Barr Virus-seronegative kidney recipients on early and late post-transplantation lymphoproliferative disorder onset: a retrospective cohort study[END_REF]. Antiviral therapy inhibiting lytic EBV replication may also be advantageous for treating epithelial tumors, such as EBV-positive nasopharyngeal carcinoma, where lytic proteins have been proved to directly contribute to carcinogenesis [START_REF] Wu | Perspective: Contribution of Epstein-Barr virus (EBV) Reactivation to the Carcinogenicity of Nasopharyngeal Cancer Cells[END_REF][START_REF] Yoshizaki | Treatment of locally recurrent Epstein-Barr virus-associated nasopharyngeal carcinoma using the anti-viral agent cidofovir[END_REF]. Importantly, antiviral therapies might be more effective if they target the expression of immediate early and early proteins. Indeed, Zta and Rta have been shown to promote IL-6 secretion, a cytokine that has been involved in a variety of hematological and epithelial cancers [START_REF] Jones | Roles of lytic viral infection and IL-6 in early versus late passage lymphoblastoid cell lines and EBV-associated lymphoproliferative disease[END_REF]. Moreover, lytically infected B-cells secrete cytokines, including cellular IL-10, viral IL-10 and VEGF, that promote B-cell proliferation and angiogenesis [START_REF] Beatty | Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines[END_REF]Hong et al., 2005).

Most antiviral therapies target the virus directly, for example impairing the activity of virus-encoded enzymes and decreasing virus production. However, emerging adaptive mutations of the viral genome lead to drug resistance, remaining one of the major causes of reduced effectiveness [START_REF] De Clercq | Approved Antiviral Drugs over the Past 50 Years[END_REF]. Considering that viruses interplay with cellular signaling pathways [START_REF] Altmann | Epstein-Barr Virus Provides a New Paradigm: A Requirement for the Immediate Inhibition of Apoptosis[END_REF][START_REF] Boulant | Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis[END_REF] and rely on the cellular supply of metabolites to ensure productive infection [START_REF] Sanchez | Viral activation of cellular metabolism[END_REF], cellular mechanisms have gained increasing interest as targets for novel antiviral therapies [START_REF] Planz | Development of cellular signaling pathway inhibitors as new antivirals against influenza[END_REF][START_REF] Scheuch | Targeting intracellular signaling as an antiviral strategy: aerosolized LASAG for the treatment of influenza in hospitalized patients[END_REF]. Hereof, EBV reactivation was shown to be repressed by the inhibition of Cyclin-Dependent Kinase in B cells [START_REF] H804 Kudoh | Inhibition of S-Phase Cyclin-Dependent Kinase Activity Blocks Expression of Epstein-Barr Virus Immediate-Early and Early Genes, Preventing Viral Lytic Replication[END_REF] and by targeting p38 Mitogen-Activated Protein Kinase pathway [START_REF] Gao | Inhibition of Epstein-Barr Virus (EBV) Reactivation by Short Interfering RNAs Targeting p38 Mitogen-Activated Protein Kinase or c-myc in EBV-Positive Epithelial Cells[END_REF][START_REF] Matusali | Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds[END_REF]. Thereby, antiviral compounds targeting cellular pathways present the potential of broad-spectrum activity and are still welcome.

In that sense, we here report that DIP, a safe and widely used drug, could prevent EBV reactivation from B-cell lines and inhibit immediate early protein Zta expression. Based on the central role played by this protein in EBV reactivation, its transcriptional (and therefore translational) inhibition is indeed expected to have a major impact on the lytic cycle. However, the regulation of BZLF1 transcription is still puzzling, and several hypotheses can be proposed. First, DIP might act on the signaling pathways that activate BZLF1. Nonetheless, this hypothesis is unlikely since DIP antiviral activity was observed in three distinct situations: (1) spontaneous reactivation in B95-8 cells, (2) activation of B-cell receptor in Akata cells and (3) induction by TGF-β dependent pathway in Mutu-I cells [START_REF] Kenney | Regulation of the latent-lytic switch in Epstein-Barr virus[END_REF]. Second, DIP may inhibit the regulation of BZLF1 promoter. It is indeed regulated through a complex interplay between cellular trans-activating factors (TF) and CpG methylation occurring on specific binding sites within the BZLF1 promoter [START_REF] Li | Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis[END_REF]. In this model, DIP would inhibit BZLF1 promoter activation by modulating one or several TF binding, although there is no experimental evidence so far for such hypothesis. Third, DIP antiviral activity would be mostly due to its ability to block nucleoside import. This assumption is strongly supported by our data demonstrating that (1) DIP-mediated EBV inhibition could be reversed by an excess of extracellular adenosine or thymidine and (2) EBV reactivation could be reduced by NBMPR, an independent class of equilibrative nucleoside transporter inhibitor. DIP would so interfere with the high nucleotide demand that is required for viral transcription and replication as it was previously described for Herpes simplex virus [START_REF] Sanchez | Viral activation of cellular metabolism[END_REF]. Interestingly, DIP may directly act on viral RNA synthesis. Indeed, a previous report showed that DIP can reversibly inhibit RNA synthesis in a viral in vitro transcription system [START_REF] Fata-Hartley | Dipyridamole Reversibly Inhibits Mengovirus RNA Replication[END_REF]. Taken together, these reports and our data suggest that DIP antiviral activity is likely a multifactorial process, with nucleoside uptake a key determinant of its anti-EBV activity. The relative importance of salvage versus de novo nucleotide synthesis for viral and host nucleic acids metabolism is still unclear, and the precise characterization of the molecular mechanisms underlying DIP antiviral activity deserves a more detailed examination.

Compared to the several studies focused on the antioxidant [START_REF] Ciacciarelli | Dipyridamole: a drug with unrecognized antioxidant activity[END_REF] and anticancer [START_REF] Liu | A Multifunctional Drug Combination Shows Highly Potent Therapeutic Efficacy against Human Cancer Xenografts in Athymic Mice[END_REF][START_REF] Spano | Dipyridamole prevents triple-negative breast-cancer progression[END_REF][START_REF] Thomé | Dipyridamole impairs autophagic flux and exerts antiproliferative activity on prostate cancer cells[END_REF] properties of DIP, its antiviral activity has been poorly explored. Of pharmacological importance is the fact that antiviral nucleoside analogs use different transporters to enter cells than those targeted by DIP that block nucleoside uptake [START_REF] Pastor-Anglada | Cell entry and export of nucleoside analogues[END_REF]. Therefore, DIP is unlikely to interfere with the import of this important class of antiviral compounds. On the contrary, combining nucleoside analogs with DIP might potentiate their antiviral activity by increasing the incorporation of their phosphorylated metabolites into the viral DNA. In that sense, it was described that DIP potentiates the antiviral activity of dideoxynucleoside drugs against HIV, probably by inhibiting the salvage of competing physiological nucleosides [START_REF] Patel | Differential inhibition of 2'-deoxycytidine salvage as a possible mechanism for potentiation of the anti-human immunodeficiency virus activity of 2',3'-dideoxycytidine by dipyridamole[END_REF]. Importantly, DIP concentrations used in the present study were close to the range of the plasma concentration attained after oral administration in humans [START_REF] Grem | Enhancement of 5-fluorouracil's anticancer activity by dipyridamole[END_REF]. Remarkably, overdose case reports showed that higher DIP doses might be tolerated in humans, suggesting that even higher dosing could be used in new clinical settings [START_REF] Lagas | Toxicokinetics of a dipyridamole (Persantin) intoxication: case report[END_REF]. As a drug repurposing strategy, another benefit of DIP is that it could be readily evaluated in clinical trials, alone or in combination with other classes of antiviral drugs. Finally, it is worthwhile to proceed with additional studies to determine whether DIP has a broad inhibitory effect on other virus families.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. EBV immediate-early and early gene expression is impaired in a dosedepend manner. (A) Akata cells were exposed for 4 hours to the indicated DIP concentrations and then EBV reactivation induced or not by anti-sIg (α-sIg) treatment for 8 hours. Total protein extracts were prepared and analyzed by immunolabelling with antibodies directed against EA-D, Zta and β-actin. β-actin was used as a loading control. Column "C" corresponds to the control mock-treated cells. Column "C+" corresponds to the control mock-treated cells in which EBV reactivation was induced by the crosslinking of the surface immunoglobulins. (B) Spontaneously reactivating B95-8 cells were exposed for the indicated times to 10 or 20 µM DIP. Total protein extracts were prepared and analyzed by

Figure 3 .

 3 Figure 3. Dipyridamole antiviral activity is related to its ability to block nucleoside uptake. (A) Akata cells were exposed for 4 hours to the indicated NBMPR concentrations and then EBV reactivation induced or not by anti-sIg (α-sIg) treatment for 8 hours. Total protein extracts were prepared and analyzed by immunolabelling with antibodies directed against EA-D, Zta and β-actin. β-actin was used as a loading control. Column "C" corresponds to the control mock-treated cells. Column "C+" corresponds to the control mock-treated cells in which EBV reactivation was induced by the crosslinking of the surface immunoglobulins. (B)

  Figure 1

Funding/acknowledgments

This work was supported by CAPES/COFECUB (French-Brazilian collaborative research grant No. Me917/18), ICGEB/CNPq 405231/2015-6 and by ANRS. Marcos Thomé was supported by a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brasil. Chloé Borde was supported by Sorbonne Université as an ATER.

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary table 1 Real-time PCR primer sequences

Supplementary Figure legend

Supplementary figure 1.


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The Challenges and Opportunities for the Power Transmission Grid of Vietnam

Keywords: Energy grid, energy mix, HVDC, Vietnam, renewable energy

a) 3-core HVAC 110 kV cable; outer diameter 193 mm, (b) +/-160 kV HVDC cable; outer diameter 111 mm Figure 1. Examples of structures of HV submarine cables rated for 200 MW. Weight reduced by over 60% [8]. water-blocking conductor conductor screen insulation insulation screen semi-conductive water-blocking tape lead sheath semi-conductive PE sheath optical fiber formed filler banding tape PP layer zinc-coated wire PP layer water-blocking conductor conductor screen insulation insulation screen semi-conductive water-blocking tape lead sheath pitch + PE sheath PE filler optical fiber banding tape PP layer zinc-coated wire pitch + PP layer

INTRODUCTION

HVDC technologies will certainly represent a growing part of energy transmission systems owing to the new sources of energy introduced, especially renewable energy, and to the need for strengthening energy networks with the decarbonation of the energy. To cite an example of such an evolution, in prospects proposed by Cigre working group C1.35 on global energy network, 99% of the lines would be HVDC, either submarine between continents or overhead on the land [START_REF] Sanchis | Global electricity network feasibility study: Results of the CIGRE C1.35 working group[END_REF]. China is also developing extra high voltage corridors to transmit energy from the west of the country where most of the hydropower is concentrated to the consumption area of megapoles of the east of the country with some innovative challenges of hybrid LCC and VSC technologies in the same 'branched' network [START_REF] Fu | Technologies for the global energy grid[END_REF].

This massive introduction of DC links is a reality today for example in Europe with the France-Spain 320kV line made with extruded insulation cables in operation since 2015 [START_REF] Labra Francos | INELFE -Europe's first integrated onshore HVDC interconnection[END_REF], the construction of the Savoy-Piemont 320kV line [START_REF] Benato | HVDC cables along with highway infrastructures: the "Piedmont-Savoy" Italy-France intertie[END_REF], the building of offshore windfarms connected to the shore with DC links [START_REF] Elliott | A comparison of AC and HVDC options for the connection of offshore wind generation in Great Britain[END_REF][START_REF] Srinil | Cabling to connect offshore wind turbines to onshore facilities[END_REF], and the decision to build HVDC corridors through Germany fully made with buried cables [START_REF] Thomas | Efficiency of superconducting transmission lines: An analysis with respect to the load factor and capacity rating[END_REF]. All these examples are with HVDC cables. The switch from HVAC to HVDC systems has this great advantage that there is virtually no limit on transmission length, even for cable technology owing to the fact that the compensation of the capacitive current is no longer a necessity. Besides, for off-shore cables, at equivalent capacity, DC cables technology constitutes a great simplification in manufacture, reducing the cable from Year a 3-core for HVAC to a single core (Figure 1), and also reducing the weight and facilitating transport and laying of the cables [START_REF] Zhang | Application status of XLPE insulated submarine cable used in offshore wind farm in China[END_REF]. In this example for a 200 MW transmission, the diameter is reduced by 40% and the weight by 60%. Today, it seems that the main limitation to the development of cable links is the cost itself vs. overhead solutions. So, in developing countries, overhead lines will certainly be preferred for a while but when technologies will be mature no doubt that cable technologies will spread.

However, still with DC cables, some issues remain. The progressive switch from oil-filled insulation to extruded insulation that has been operated over the last 40 years for HVAC cables is much more recent for HVDC. Insulations involving pressurized oil tend to be avoided for environmental and maintenance reasons. The behavior of extruded cables under DC stress, regarding space charge build-up and field redistribution, particularly at polarity reversal stages as used in converter technologies using IGBT, is not fully under control [START_REF] Mazzanti | A first step towards predicting the life of HVDC cables subjected to load cycles and voltage polarity reversal[END_REF]. For these reasons, materials improvement, characterization and modelling are still intensively researched, both for cables and accessories and in general for the power conversion equipment.

The paper is a summary report of a workshop [START_REF]Workshop on Green Energy and Networks[END_REF] organized in Hanoi in Sept 2018 on green energy and networks. The objective was to present opportunities and challenges with HVDC cable links and to put it in relation to the current situation of the energy network in Vietnam and to the evolution forecasted for the energy generation and management in the country and at its borders. It was also a milestone of a joint project in which a relevant case study of energy transmission link that could be turned to HVDC. We report herein mainly on the current evolution of the Vietnamese electricity network and we discuss the way DC energy transmission could be incorporated in such context.

FACTS AND FIGURES FOR THE ENERGY SOURCES AND DEMAND IN VIETNAM

Vietnam has a relatively late development of electricity system compared to other countries in the world due to the historical impact of the wars. The power system only really developed when the 500 kV lines are put into use, along with the strong opening of the economy. This late development is illustrated in Figure 2 that represents the evolution in terms of power consumption and in peak power over the last 17 years [START_REF]Annual Report 2017[END_REF][START_REF]Annual Report 2018[END_REF]. The peak power is a particularly critical quantity for sizing the electrical network planning and investment [START_REF] Al-Shobaki | Modeling and forecasting of electrical power demands for capacity planning[END_REF][START_REF] Gajowniczek | Two-stage electricity demand modeling using machine learning algorithms[END_REF]. The growth rate is of the order 10-15%/year over the last 15 years.

Fast growing demand

According to the last figures available, for 2018, the total electricity production and importation of Vietnam was 212.9 TWh, increasing by 10% compared to 2017 [START_REF]Annual Report 2018[END_REF], making Vietnam ranked at 2nd among ASEAN countries and 23rd in the world. The peak demand of the system shares the same growing rate, being of 35 GW in 2018 and increasing by 14% compared to 2017 (Figure 2b). According to the forecast (Figure 2a), the growth rate should remain at ~ 11% per year up to 2020, decreasing to 8.5 ~ 9% per year for the period of 2021 -2030. The peak demand would be nearly 100 GW in 2030, meaning that the system will require over 3 GW/year of new generators to satisfy the demand.

The fast increase in demand is due to the current trend of using a wide range of technological equipment, especially electrical appliances and devices such as air conditioners in all households. In addition, there is a strong increase of industry investment and a gradual shift from heat engine vehicles to electrical ones. Therefore, the peak load growth rate may continue to stay high, even increase in the future. The fast increase in the demand poses challenges for adapting the electrical network and a lot of difficulties to secure the supply, especially with the reduction of domestic fuel sources supply like coal and natural gas.

The split in energy sources

According to the recent EVN annual report [START_REF]Annual Report 2018[END_REF], EVNstate owner corporation, contributed by the end of 2018 up to 58% of the national power generation system, including large-scale hydropower, coal-fired, gas-fired power plants with a total installed capacity of 28.2 GW out of a total of 48.6 GW for the country. The largest capacity was from hydropower (40%) before coal-fired thermal power (38%) in 2018. In recent years there has been significant development in coal fired power plants (from about 23% in 2013 to 38% now of total installed capacity, mostly located in the North and central regions of Vietnam) as well as gas-fired plants in the South (which account for approximately 18% of total installed capacity). Hydropower and coal-fired thermal power had equivalent contributions at 40% in 2018 (Figure 3). The production of electricity has increased by 75% between 2012 and 2018 (Figure 3a). In recent years, the increase in production was covered principally by the increase in capacity of coal fired power plants.

Figure 4 compares the forecasted distribution of power sources according to the revised Power Development Master Plan VII (PDP7) for the period 2020 to 2030 [START_REF]Vietnam Power Development Plan for the period 2011 -2020: Highlights of the PDP 7[END_REF]. For an understanding of the roles played by the different stakeholders in the definition of the plan, please consider a recent paper addressing current institutional framework and energy market structure of the country [START_REF] Nguyen-Trinh | Market conditions and change for low-carbon electricity transition in Vietnam[END_REF]. From 2020 to 2030, the total installed capacity for Vietnam would be increased from 60 GW up to 129.5 GW and the production from 265 TWh up to 572 TWh. Coal-fired thermal power is projected to be the main source by far for electricity production. The capacity of hydropower does not evolve substantially during the period. Other renewable energies are showing an upward trend. The specificities for these 3 different energy sources are detailed below.

Contribution from hydroelectricity

With sloping areas of relief and many rivers, a tropical climate with high annual rainfall, a long-lasting rainy season (from June to December), Vietnam has a huge hydropower potential. With a total current capacity of 19.7 GW, hydro power accounts nearly for a half (42%) of the total installed capacity of the country. Hydro-power plants represent a major and indispensable part of the system. They are spread throughout the country, but are still concentrated in the Northern and the Central Highlands. Some big hydro-power plants like Son La (2400 MW), Hoa Binh (1920 MW), Lai Chau (1200 MW), Ialy (720 MW) play as strategic and multipurpose hydro power plants: They act at supplying the water and reducing floods, as river transportation means, and for the power grid ensure frequency regulation and voltage stabilization of the entire system and contribute to a large amount of electricity production.

Hydropower plants have a great advantage in operation compared to other generation technologies, such as low operating costs, simple operation with low probability of failure, the ability to increase or decrease capacity in a short time, the ability to produce or receive reactive power. Hydroelectricity is thus utilized in the system for frequency control, provision for spinning and fast start reserve, hence for grid voltage regulation.

However, its operation depends a lot on natural conditions, especially on hydrological conditions and is always a very complex task. Optimizing the operation of the reservoirs to avoid spilling in flood season and depletion of water, ensuring capacity in dry season is always a problem for system operator as well as power plants. Moreover, as the characteristics of agriculture rely heavily on irrigation which is supplied from rivers, hydropower plants in Vietnam, in addition to electricity generation, also take a responsibility for providing water for agriculture. For this purpose, Vietnam government has issued many regulations to tightly monitor the operation of hydropower plants, stipulating the water level of the reservoir from time to time during the year, leading to more complicated and more constrained operation.

By encouraging the involvement of private investors in the electricity sector, reducing the pressure on state investment and leveraging on energy from the rivers, the government has adopted "an avoidable cost" policy. Many small hydropower plants (with power under 30 MW) have been built and operated, with a total capacity of around 4 GW. In the past few years, with concerns about the effects of hydropower on the environment as well as the dependence of the electricity sector on natural conditions, the planning and construction of new hydropower plants has been undertaken by the government, tightening the construction standards, introducing strictly operating regulations. Thus, from nearly 50% of the capacity of the system in 2012, hydropower now is only about 40%.

Dependence on coal

With the recent controlling and tightening hydropower development policies, hydropower development will not be as high as it is now. In addition, with the decline in supply of natural gas as well as delayed in the construction of gas turbine power plants, the power supply of the system will mainly rely on coal alone. This is all the more the case as the demand growth rate stays high and other energy sources are limited: the potential for hydropower has been fully exploited, the availability of natural gas supply declines, nuclear power projects have been abandoned and renewable energy demands high initial investment and offer low stability.

Currently, coal-fired power plants constitute the main development strategy with lower investment cost and shorter construction time compared to the same capacity for hydropower. From 15% of the system in 2010, there are now 18.5 GW of installed capacity, accounting for nearly 40% of the total capacity of the whole system, using 36 Mtons of coal The environmental impact is also a major problem: many power plants have been built since the 1980s, using old technology, releasing large quantities of COx and NOx. Within PDP7, the government has clarified measures to minimize the impact of environmental pollution, which requires the use of ash wastes for the production of building materials and other industries to reduce the area of waste disposal sites, ensuring compliance with regulations. At the same time, to encourage the use of modern technological lines to reduce environmental pollution, care has to be taken to fully carry out the work of monitoring, measurement and management of environmental impact.

Renewable other than hydropower

Compared with other countries, renewable energy in Vietnam is developing relatively late. European countries have begun to shift from fossil fuels to renewable energy in the early years of the 21st century, with as target to reach 20% in gross final consumption of energy from renewable sources by 2020 and at least 27% by 2030. In Europe wind production became the first renewable source in 2017 [START_REF] Nissen | Renewable energy in Europe -2018: Recent growth and knock-on effects[END_REF], before hydropower, and far ahead solar. For 2018, 14% of the energy was produced by wind [START_REF] Komusanac | Wind energy in Europe in 2018: Trends and statistics[END_REF], and nearly 50% of new capacity installed was for wind, with a growing part from offshore production. In Asia, China is a very successful country in the development of renewable energy. With the advantage of low production cost for solar panels, large area of land, renewable energy now accounts for a quarter of the total annual electricity production.

Located in the tropical area, close to the equator with hot climate and long sunshine duration (~3000 h/year), a 3400 km long coastal line with an average wind speed of 6 m/s, Vietnam has enormous potential for renewable energy, especially with solar and wind. But for now, solar power and wind power are still relatively modest in Vietnam: with only about 300 MW of wind and 150MW of solar, it accounts for less than 1% of the all electrical power production. To promote the development of renewable energy while declining traditional energy sources, the government decided a new policy in September 2018 with guaranteed purchase prices to attract investors. The goal is that by 2020, renewable energy will account for 10% of the total electricity production, 16 TWh of wind and 36 TWh of solar energy in 2030.

Vietnam's Wind energy potential is considerably high in comparison to Thailand, Laos or Cambodia [START_REF] Rosly | Wind energy potential in asean countries -special attention to Malaysia[END_REF]. According to a report by the GIZ Vietnam [START_REF] Nguyen | Green finance in Viet Nam: barriers and solutions[END_REF], the potential of wind power is about 27 GW. Presently, the capacity in operation is about 300 MW of wind power plants [START_REF] Tran | Smart grid development road map in Viet Nam -Achievements and future plans[END_REF]. According to PDP7, the total capacity of installed wind power is expected to reach 800 MW in 2020, 2 GW in 2025 and 6 GW in 2030, which account for 0.8% of the share of produced electricity in 2020, 1% in 2025 and 2.1% in 2030.

Vietnam has also a high potential in solar energy, particularly in its central and southern regions [START_REF] Nguyen | Performance comparison between tracking and fixed photovoltaic system: A case study of Hoa Lac Hi-tech Park, Hanoi[END_REF]. The solar energy density is lower in the North due to the annual winterspring cloudy and drizzle sky. A theoretical potential was estimated to about 2 -5 GW for residential and commercial rooftops, and 20 GW for solar photovoltaic (PV) plants. However, the deployment of solar power is still modest, as well as studies related to grid-connected PV systems are still limited to the demonstration phases. An ambitious development of solar is proposed in PDP 7: the capacity of solar Photovoltaic shall increase from around 84 MW at end 2018 [START_REF] Tran | Smart grid development road map in Viet Nam -Achievements and future plans[END_REF] to 850 MW by 2020 and to 12 GW by 2030. With the support policies, the number of renewable energy projects has increased sharply in the past two years. At present, 110 renewable energy projects with as total capacity 7.5 GW of solar and 2 GW of wind power were approved. However, besides many advantages that are zero operating costs, low emissions and support from the government, renewable energy also has many disadvantages. As wind and solar energy production are indeterminate and difficult to predict, the system needs to have a corresponding power supply in case of renewable energy production affected by the weather. In addition, the increase of renewable energy in the system will have impacts on grid operational characteristics such as grid congestion, power quality, redundancy, system stability and system reliability. Already, some researches also address the impact of transmitted energy fluctuation due to introduction of renewable energy on the lifetime of insulated systems [START_REF] Müller | Influences of changed grid utilization by renewable energies on the ageing behaviour of medium voltage cables[END_REF]. Therefore, to manage the increase in global energy demand and exploit safely renewable sources, the electrical network has to evolve. We present below the current network structure and the needs for strengthening it.

CURRENT ENERGY GRID AND EVOLUTIONS

The structure of the network

Vietnam electricity grid system, affected by history and natural topography, is divided into separate regions: North, Central and South, which had initially their own standards and technologies. Over time, with the unification of the country and the economic development, especially in the Southern region, it was necessary to unify the entire grid into a unified standard, to connect separate regional electrical systems, so as to transmit a large amount of capacity from the North to the South. The first 500 kV line with 1500 km was put into operation in 1994 after more than 2 years of construction. It integrates the 3 regional systems (previously operated independently), thereby enhancing the coordination, increasing the stability and the general reliability of the whole system. Since then, after 25 years of operation and expansion, the system has over 7500 km of 500 kV and nearly 40.000 km of 110 and 220 kV lines (Figure 5). It actually contributes for an essential part in the economic development of Vietnam.

Being not only an internal grid, the system also connects with neighboring networks to import and export electricity, see Figure 6 [24] and Table 1. Since 2005, to minimize the risk of power shortage in peak periods (usually in the dry season), Vietnam has begun to connect and import electricity from China Southern Grid. The annual purchase volume is about 1.5 TWh through two 220 kV lines with 800 MW peak. The connection to Laos is to take advantage of abundant hydropower resources. In the Southern Vietnam, a 220 kV transmission line with 200 MW peak connects to Cambodia's grid since 2000, for exporting about 1 TWh/year to Cambodia.

With the rapid development of the demand, the transmission system is heavily solicited for balancing the capacity of the system. The demand is not homogeneously distributed over the country. The North (with region of Hanoi) and the South (with Ho Chi Minh City) absorb about 45% of the power each while the Central part acts mainly as a transmission corridor and production site. The total transmitted energy between regions in 2017 is 33 TWh. In particular, transmission lines between North and Central part of Vietnam are always in high load status, especially in flood season. In the near future, with the slowly construction progress of new power plants in the South and limitations of primary energy sources, the shortage of power supply in the Southern region will continue and transmission system will still assume a large capacity transmission from the North and Central to the South. So, the transmission congestion will still be a problem to be solved for Vietnam's system in the near future. The reinforcement of the 220 kV/500 kV AC grid is very significant with 25 projects of sub-stations extension and lines build-up by 2025. This includes notably the third 500 kV line which is under construction. The congestion is also seen on the regional grid due to a large concentration of residents in cities. The local transmission network connecting from the substations, to the power plants and to the cities is often heavily loaded, especially in the two biggest cities that are Hanoi and Ho Chi Minh City. In addition, the natural topography divides the Northern region in two areas: east side where most of the largest hydropower plants are located, and west where a number of coal-fired thermal power plants are installed. With that configuration, the transmission lines linking these two areas often carry high loads. Oil, Gas

The impact of renewable energy

Along with above issues, the strong increase of renewable energy in the system has also brought many challenges. Technically, wind or large solar power plants are often installed in areas far away from the load so the transmission line is heavy and expensive. This power source is unstable and fluctuates in very large capacity in the short term and needs backup resources such as hydropower or electrical storage devices. Besides, Vietnam has little operational experience integrating the wind and solar power. For renewable energy integration, Vietnam should address important issues that are network infrastructure development, incorporation of energy storage systems and precise and agile management of the system through grid automation.

The grid system, that presently receives 300 MW from solar power plants, could face an increase of the installed capacity to over 4 GW in 2019 from plants concentrated mainly in the Southern and Central regions, where the transmission grid system (110 and 220 kV) has not been developed to meet such a large capacity. According to EVN report [START_REF]Annual Report 2017[END_REF], the current grid can only absorb for 2 GW of renewable energy. Therefore a large part of the capacity would be curtailed and the situation could be worse in the coming years.

In addition to investing in the upgrade and building of new transmission lines to release all the power from renewable power plants which licensed for construction, EVN expects to build and put into operation new energy storage systems, apply a smart grid to control and optimize the system.

The massive incorporation of renewable energy has to go with a strong transmission and distribution grid with strong interconnections to neighboring power markets. Denmark for example has the majority of its power from wind, but this goes with interconnectors to Norway and Sweden making it possible to balance wind power and hydro power [START_REF]Vietnam Energy output report[END_REF]. To the North of Vietnam, the energy exchange with China could be strengthened by building a 500 kV line (Honghe -Soc Son project with 400 km transmission line) [START_REF] Nguyen-Mau | HVDC application for enhancing power system stability[END_REF].

Besides the development of production and transmission infrastructures, energy storage constitutes one strategy for smoothing the needs in maximum power, optimizing the inputs from renewable and smoothing the transmission congestion, as depicted in Figure 7 [START_REF]Commercial energy storage 101[END_REF]. Because of its strong infrastructure in hydropower, hydraulic energy storage could be a solution for Vietnam. The other solution to storage could be with using batteries: from the fleet of electrical vehicles on the one hand, and with dedicated ESS -Electrical storage systems. Although there is a huge potential for development and the market seems to be booming, yet ESS are still in its early stages, so the investment to own and operate the system is still relatively high. This will require improvements in performances as well as a reduction in initial investment costs so that ESS can be easily accessible to the majority of users. Besides, up to now the presence of a large and easily regulated source through hydropower and the possibility of developing it from Laos did not justify the investment in hydroelectric storage infrastructures [START_REF] Gerner | Is Pumped storage hydroelectric power right for Vietnam? Live Wire[END_REF].

Finally, to meet challenges in strengthening the national transmission network, the disruptions of wind and solar energy can be addressed by upgrading the power transmission system with integrated power technology and by modern digital technology.

Master plan and uncertainties

As for many other countries, with today's rapid evolution in this field, the future of electricity network will substantially depend on evolutions in the demand, on the national and internal policies and on the technical and economical progresses in power engineering.

The incorporation of electrical vehicles will have a strong impact on the network but the speed of market penetration is Figure 7. Daily peak-smoothing in power by associating renewable energy and storage [START_REF]Commercial energy storage 101[END_REF]. difficult to anticipate. The impact is not only on the consumed power; it can also contribute to the general equilibrium of the network with providing a storage capacity. More generally the energy used in transport is complex in Vietnam [START_REF]Vietnam Energy output report[END_REF] even though there is objectively more penetration of electrical propulsion to come. Besides cars, the marked of motorbikes (representing presently one of the main transport means in Vietnam) is developing fast [START_REF] Nguyen | Electric vehicles use in Vietnam[END_REF]. High speed train projects have been postponed several times. Despite competition from budget airlines and the unpopularity of rail travel, developing a modern high speed railway system between Hanoi and Ho Chi Minh City may just be more a matter of pride for Vietnam.

The choice of compensating the demand by massive incorporation of coal exposes to two uncertainties of international nature. First, although more efficient technologies are coming, it is not in favor of the compliance to the national committed target at COP21 about CO 2 emission reduction and it can be affected by carbon tax policies. Second, it brings some dependence to other countries for primary energy as the coal will be imported e.g. from Australia or Indonesia. By 2030 and beyond, with the increase of price of fossil energy and progress for cheaper renewable, it can be anticipated that new orientation in favor of renewable energies will emerge.

Potentialities for HVDC and cable energy transmission

As stated previously, the massive introduction of renewable energy requires strong inter-regional cooperation. In this perspective, power lines at the boarder should be viewed no more as a one-way transmission of energy from source to charge but as an exchange with energy flux that can be reversed on a daily basis.

The fast increase in demand of electrical energy poses a challenge to the supply capacity. Interconnection links with foreign countries to import energy are considered as a solution. The 220 kV HVAC Malutang (China) -Ha Giang -Thai Nguyen transmission line was thus built and put into operation in 2007. The capacity of this line could be increased in the future with either switching to DC or realizing higher voltage line. However, for the massive exchange in electricity in the future, HVDC links tend to be preferred. Examples of increase in the power flow of existing overhead 380 kV AC lines are available notably in Germany [START_REF] Lundberg | Convert from AC to HVDC for higher power transmission[END_REF]. In a previous study in 2011, Nguyen-Mau et al. [START_REF] Nguyen-Mau | HVDC application for enhancing power system stability[END_REF] analyzed some of the weaknesses of the power grid such as the interconnection with Chinese and Laos power systems, the power swing protection (especially at Hatinh -Danang 500 kV transmission line in the central part of Vietnam), or over-voltages occurring when tripping heavily loaded lines. HVDC technologies could be a solution to those features. It was shown by simulation that, due to the capability of fast and independent active and reactive power control, VSC HVDC can greatly improve the stability of a power network. According to Lerch et al [START_REF] Lerch | Enhancing the stability and reliability of the Vietnamese power system -Part 2: Dynamic power transmission system studies[END_REF], strengthening the power transmission system by a separate 500 kV transmission system or HVDC connection between North and South can avoid the system separation at single faults. An alternative is to distribute more power generation across the country. In a report from World Bank technically realized by CESI it was considered that developing a long 500 kV HVDC line (700-800 km) would constitute a viable solution and a pillar for the Smart Grid initiative in the power network of Vietnam [START_REF]Smart grid to enhance power transmission in Vietnam[END_REF].

DC energy transmission has application also in the Medium Voltage domain (MVDC). Beyond renewable energy transmission, several applications are targeted ranging as DC microgrids, electric ships, data centers powering, etc. [START_REF] Abb Corp | Medium voltage direct current applications[END_REF]. The expansion of wind power and solar energy systems has huge consequences for the transmission and distribution of electricity making it more complicated and decentralized, with risks for power quality and grid stability. Solutions are provided in the 30-150 kV range [START_REF]MVDC PLUS direct current transmission solution[END_REF][START_REF] Rentschler | Addressing the microgrid stability challenge with MVDC[END_REF] that are suited for instance, to connect wind turbines installed on offshore islands or medium-sized solar plants to the AC grid and sensitive, weak distribution networks can be stabilized. Offshore windfarms is an obvious case where cable connections are necessary and it is considered that DC solutions may become competitive beyond 80 km of transmission from the farm to the shore. The world's first HVDC link to connect an offshore wind farm with an AC grid is the BorWin1 project that connects the Bard Offshore 1 wind farm in the North Sea to Germany's mainland AC grid. The 80 wind turbines feed their power into a 36 kV AC cable system. This voltage is transformed to 155 kV AC and then converted into ±150 kV DC to feed two 125 km sea cables, which continue into two 75 km land cables, transmitting 400 MW power to the land. However, the trade-off between HVAC and HVDC solutions is still complex to determine, as besides losses, questions of reliability and architecture of the offshore grid come into the balance [START_REF] Warnock | Failure rates of offshore wind transmission systems[END_REF][START_REF] Maciver | A reliability evaluation of offshore HVDC grid configuration options[END_REF]. While there is a project for backing HVDC for the Norfolk Vanguard windfarm (UK, 1.8 GW), the longest cable connection in the world for an offshore windfarm has just been installed: the 200 km-long cable is powered under 220 kV HVAC [START_REF] Zouraraki | Hornsea projects 1 and 2 -Design and optimisation of the cables for the world largest offshore wind farms[END_REF].

Back to the case of Vietnam, building offshore windfarms seems out of scope at the moment. There are however several PV plants projects of medium size capacity, such as Hong Phong 1A-B at Binh Thuan (250 MWp), BIM 2 at Ninh Thuan (250 MW) provinces, etc. that are planned to be built connected to the 110 kV transmission lines.

The above analysis shows that DC energy transmission is a possibility for improving the quality of the grid in Vietnam, especially at a time where a strong strengthening and stabilization of the network is necessary. As to HVDC cables the investment it represents is certainly still an issue for today. The first step will be to integrate more power electronics and upgrading existing overhead HVAC lines for HVDC. The MVDC links are still in the infancy, and have not yet really penetrated the market. Perhaps in the far future, offshore windfarms with effective resources of the country along the very long coast could benefit from these technologies.

CONCLUSION

As a rapidly developing country and with the changes in the primary source of energy, the electrical grid of Vietnam will necessary evolve and strengthen in the next decades with a demand in energy estimated to twice as today in a twelveyear time.

The short term energy provision is thought with development of fired coal-based electricity with advantages of agility, localization of the production on demand and low investment cost. In parallel, an ambitious development of renewable energy is planned, and it can be forecasted an amplification effect once technologies are well implanted, provided the grid can be conveniently managed. HVDC energy links will certainly be integrated on a middle term basis, as a way to stabilize the network. The last pillar of the grid will be the implantation of ESS, in which hydraulic or battery solutions will probably contribute. Though identification of best suited application of HVDC cable links is probably a bit early, a case as the 220 kV HVAC Malutang (China) -Ha Giang -Thai Nguyen transmission line will be considered as case study for equipment modeling.

  a) Annual electricity consumption b) Annual peak demand.

Figure 2 .

 2 Figure 2. Evolution of the annual electricity needs over the period 2001-2018 (in blue) and forecasted figures (in green) according to PDP7.

Figure 3 .

 3 Figure 3. Power generation output portfolio.

  (a) Total installed capacity (b) Total energy production

Figure 4 .

 4 Figure 4. The forecasted structure of targeted power sources for 2020-2030 [10, 15].

  per year. According to PDP7, Vietnam will have 26 GW of coal-fired power in 2020 and 48 GW in 2025.Years ago, because of the proximity of coal mines, coalfired power plants were only located in the North-East and their capacity was still small (50 -100 MW/unit). Nowadays, most of new constructed coal-fired power plants are located in Southern area, where the demand in electricity is high. They have larger unit capacity (~ 600 MW/unit), like in the thermal power complexes of Vinh Tan or Duyen Hai. Unlike old coal-fired power plants which use domestic coal in the north, existing and future plants in Southern Vietnam will use imported coal, often from Australia or Indonesia. By 2030, Vietnam will need to import about 85 Mtons of coal a year to supply for thermal power plants.

Figure 5 .

 5 Figure 5. Evolution of grid transmission line length from 1998 to 2018.

Figure 6 .

 6 Figure 6. The broad distribution of energy sources (left) and network structure (right). Adapted from [24].

Table 1 .

 1 Capability and annual volume of import and export transmission lines.

	Nation	Line	Capacity (MW)	Annual volume (MWh)
	China	Malutang -Ha Giang Xinqiao -Lao Cai	450 350	750 ~ 800 700 ~ 750
	Laos	Xekaman 1 -Thanh My Xekaman 3 -Thanh My	290 250	900 ~ 1100 900 ~ 1200
	Cambodia	Takeo -Chau Doc	200	700 ~ 800

ACKNOWLEDGMENT

We would like to acknowledge the C.N.R.S. for financial support to the ModHVDC project n° PICS07965.


Sombel Diaham

Petru Notingher

Gilbert Teyssèdre

Report on the 1 st Thematic School on Dielectrics 2018

SCOPE OF THE SCHOOL

The ageing, breakdown and life estimation of insulators are ancient problems though of utmost importance in many domains of electrical engineering and electronics. The evolution of power electronics technologies and new high voltage direct currents (HVDC) application introduce new threats for insulation, e.g. high frequency stresses, direct current issues, challenges of thermal endurance, etc. At the same time, material compounding and processing is becoming more and more flexible, and new design possibilities are offered through material functionalization, use of nanocomposites, etc.

This first edition of the Thematic School on Dielectrics 2018 proposed to form a bridge between the old theories The lectures addressed, on the part dedicated to the physics and withstanding of insulations, practical aspects of environmental stresses, new demands for materials and various examples of failure mode. On the materials side, new opportunities for designing materials, with flexibility in properties and opportunities for greener materials were presented. Beyond the courses, the opportunity for participants from different horizons to interact was certainly an invaluable aspect of the school.

Aside from these main courses, which were organized mainly as morning sessions, two activities took place. First, a round table with questions raised by younger researchers was organized in the last session of the school. The most original and demanding aspect of the school in terms of organization and logistics was the training of all participants to various measurement techniques on lab test benches. This latter unique activity was greatly appreciated by the participants.

ORGANIZATION

The Thematic School on Dielectrics was supported by the French National Centre of Scientific Research (CNRS) and the IEEE Dielectrics and Electrical Insulation Society (DEIS). Moreover, the organizers were supported by the French Working Group on Dielectric Materials belonging to the SEEDS-CNRS Electrical Engineering Research Network (http://seeds.cnrs.fr/).

The Thematic School was held at the IGESA Center of Porquerolles Island, belonging to the French army as a recreation and meeting site. Porquerolles Island, on the French Riviera, is a protected area classified as 'Heart of National Park' since 2012. Due to accommodation restriction, the island is nearly free from tourists in the evenings making it a quiet and natural place.

The dates were chosen to allow the overseas participants to attend the IEEE International Conference on Dielectrics (ICD), which was organized in Budapest during the following week.

Continuing the discussions during lunchtime and coffee breaks in front of a beautiful sea view.

PARTICIPANTS

For this Thematic School, 64 attendees registered and participated. Among them, 34 were from academia, 13 coming from industry and 17 were students. They came from many countries: UK, Italy, Sweden, Switzerland, Netherlands, Czech Republic, Algeria, South Korea and France. In total, 80 persons joined the event, making it a very successful event in respect to the ambition of the organizers as the full accommodation capacity was used.

LECTURES, PRACTICALS AND INSTRUCTORS

The most specific feature of this Thematic School on Dielectrics was to confront the attendees with direct practical situations by performing specific experimental tasks by themselves.

Thierry Lebey (LAPLACE, left) and Lionel Flandin (Univ. Savoie-Mont Blanc, right) during their lectures with all the attendees.

For that purpose, six experimental setups were brought to the island from LAPLACE and IES Institutes. Characterization techniques covered the different theoretical concepts taught during the lectures. The attendees were trained on dielectric spectroscopy, transient and conduction current measurements, space charge characterization by pulsed electro-acoustic and thermal step methods and dielectric breakdown and statistical analysis. Each attendee followed a total of 8 hours of practical training.

LESSONS LEARNT AND FUTURE OF THE SCHOOL

A questionnaire was distributed among the participants asking for comments regarding school technical content, site facilities and environment as well as suggestions for improvement. The possibility to attend the practical laboratory training was definitively the very well acknowledged feature of the school, rarely encountered at similar events or forums. As expected, the location was also admired, the latter being strengthened by the sunny weather through the week. Among points for improvement, some comments pointed to the spartan accommodation. As a site intended mainly for leisure of army staff and their families, the comfort is not always a main concern.

As this Thematic School on Dielectric had well responded to the need of our interdisciplinary research community, its second edition is planned to be organized either in 2020 or 2021. The venue will probably remain the same, Porquerolles Island, as it offers the perfect tradeoff between a convenient to reach location and, at the same time, an 'isolated' study environment. The attendees will have a great opportunity to expand their professional network inside and outside the fields of their specialties. We also hope that IEEE DEIS memberships will again be offered to the registered students.

  and the new technological opportunities, for researchers, engineers and PhD students in the field of electrical engineering and, most importantly also, opening our field and challenges to materials scientists, chemists and physicists. The School was in the spirit of the Electrical Ageing Workshop, Autrans, France, organized in 2005 and 2008 with IOP support. The organizers of the Thematic School on Dielectrics. From left to right: Gilbert Teyssèdre, Petru Notingher and Sombel Diaham. A total of 19 hours of plenary lectures were given on the following topics, involving lecturers from both the electrical engineering domain and from materials science: • Issues and challenges related to insulation materials • Theoretical basis on dielectric properties of materials • Characterization techniques for insulations • Classical models of ageing and failure modes of dielectrics • Physico-chemical phenomena in ageing of insulators • Formulation and elaboration processes of ceramics • Process and properties of dielectric nanocomposites • Bio-sourced insulating polymers

  Zarel Valdez-Nava (right) ruling the practical work on dielectric breakdown to Ondrej Sefl (PhD student, TU Prague, Czech Rep.), Marie-Laure Locatelli (CNRS Researcher, France) and Silvia De Giovanni (Engineer, Elantas, Italy). PhD students and young engineers working in-group on Weibull data analysis after breakdown tests. They are Simone Suraci (Univ. Bologna), Hasti Haghighi (Univ. Manchester), Maria Mitronika (Univ. Nantes), Kim Yu-Min (Engineer, Hyundai Electric and Energy Systems, South Korea) and Jung-Hun Kwon (Engineer, Power Technology Research, South Korea). Nicolas Guibert from LAPLACE, one of the instructors for the space charge measurements. Jean-Charles Laurentie from IES, one of the instructors for the dielectric spectroscopy measurements. The whole program ended with a 2 hour long general open discussion. It was led by Alun Vaughan and Christian Laurent, who earlier asked the PhD students and postdocs to prepare interesting questions. Alun Vaughan (Univ. Southampton, left) and Christian Laurent (LAPLACE, right) leading the final general discussion with the attendees, particularly the PhD students.

ACKNOWLEDGMENT

The organizers would like to thank the French Scientific National Research Council (CNRS), the SEEDS French Electrical Engineering Network, the IEEE Dielectric and Electrical Insulation Society (DEIS), the LAPLACE and IES Institutes for their financial supports.

The organizers are very grateful to Odile Bastide, André Bastide, Marc Von Dort, Anne Pontier, Karine Fleurot, Paul Leblanc for their technical contribution to the event organization.

The organizers also thank all the lecturers and instructors for their contributions to setting up the course program and the practical works on the 'insulated island'. Finally, the organizers warmly thank all the participants for coming to share a better insight on Dielectrics.

School on Dielectrics was organized under the sponsorship of CNRS -French National Research Center, and supported by DEIS, on Porquerolles Island in France, from 24 th to