BUSCADOR RECOLECTA

The COVID pandemic has evidenced how
vulnerable we are to emerging infectious diseases and how short
our current armamentarium is. Flavivirus, single stranded RNA
viruses transmitted by arthropods, are considered a global health
challenge. No drugs to treat these infections have been approved.
In this Viewpoint, we analyze the advantages and disadvantages of
two different, but probably also complementary, therapeutic
approaches: virus-targeting antivirals and host-targeting drugs, Our own research in the search of antivirals against West Nile
virus and other flaviviruses is financed by grants PID2019-
105117RR-C22 and PID2019-105117RR-C21 funded by
Ministerio de Ciencia e Innovación, Agencia Estatal de
Investigación MCIN/AEI/10.13039/501100011033, Peer reviewed

Centro de Biotecnología y Genómica de Plantas (CBGP), Zika virus (ZIKV) is a mosquito-borne pathogen with public health importance due to the high risk of its mosquito vector dissemination and the severe neurological and teratogenic sequelae associated with infection. Vaccines with broad immune specificity and control against this re-emerging virus are needed. Here, we described that mice immunized with a priming dose of a DNA plasmid mammalian expression vector encoding ZIKV prM-E antigens (DNA-ZIKV) followed by a booster dose of a modified vaccinia virus Ankara (MVA) vector expressing the same prM-E ZIKV antigens (MVA-ZIKV) induced broad, polyfunctional and long-lasting ZIKV-specific CD4+ and CD8+ T-cell immune responses, with high levels of CD4+ T follicular helper cells, together with the induction of neutralizing antibodies. All those immune parameters were significantly stronger in the heterologous DNA-ZIKV/MVA-ZIKV immunization group compared to the homologous prime/boost immunizations regimens. Collectively, these results provided an optimized immunization protocol able to induce high levels of ZIKV-specific T-cell responses, as well as neutralizing antibodies and reinforce the combined use of DNA-based vectors and MVA-ZIKV as promising prophylactic vaccination schedule against ZIKV., This research was supported by MINECO Spanish grants SAF-2013-45232-R and SAF-2017-88089-R (to M.E.) and Grant PID2019-105117RR-C21/AEI/10.13039/501100011033 from Agencia Estatal de Investigación to M.A.M.-A. P.P was supported by a fellowship from the Ministry of Science and Innovation of Spain and this work is part of her Thesis defence., Peer reviewed, 16 Pág.

20 Pág., Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile, Japanese encephalitis, Zika, and dengue viruses. USUV emerged in 1996 in Europe, where quickly spread across the continent causing a considerable number of bird deaths and varied neurological disorders in humans, including encephalitis, meningoencephalitis, or facial paralysis, thus warning about USUV as a potential health threat. USUV replication takes place on the endoplasmic reticulum (ER) of infected cells, inducing ER stress and resulting in the activation of stress-related cellular pathways collectively known as the integrated stress response (ISR). The alpha subunit of the eukaryotic initiation factor eIF2 (eIF2α), the core factor in this pathway, is phosphorylated by stress activated kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated inhibitor kinase (HRI), and general control non-repressed 2 kinase (GCN2). Its phosphorylation results, among others, in the downstream inhibition of translation with accumulation of discrete foci in the cytoplasm termed stress granules (SGs). Our results indicated that USUV infection evades cellular stress response impairing eIF2α phosphorylation and SGs assembly induced by treatment with the HRI activator ArsNa. This protective effect was related with oxidative stress responses in USUV-infected cells. Overall, these results provide new insights into the complex connections between the stress response and flavivirus infection in order to maintain an adequate cellular environment for viral replication., This work was supported by grants RTA2015-009 (J-C. S.) and E-RTA20017-003-C02 (J-C. S.) from INIA, PID2019-105117RR-C21(HOSTMEVIR) (J-C. S.) from the Spanish Ministerio de Ciencia e Innovación, (J-C. S.) from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA-2) (J-C. S.) from the Comunidad Autónoma de Madrid., Peer reviewed

Flavivirus comprises globally emerging and re-emerging pathogens such as Zika virus
(ZIKV), Dengue virus (DENV), and West Nile virus (WNV), among others. Although some vaccines
are available, there is an unmet medical need as no effective antiviral treatment has been approved for
flaviviral infections. The development of host-directed antivirals (HDAs) targeting host factors that
are essential for viral replication cycle offers the opportunity for the development of broad-spectrum
antivirals. In the case of flaviviruses, recent studies have revealed that neutral sphingomyelinase 2,
(nSMase2), involved in lipid metabolism, plays a key role in WNV and ZIKV infection. As a proof of
concept, we have determined the antiviral activity of the non-competitive nSMase2 inhibitor DPTIP
against WNV and ZIKV virus. DPTIP showed potent antiviral activity with EC50 values of 0.26 μM
and 1.56 μM for WNV and ZIKV, respectively. In order to unravel the allosteric binding site of DPTIP
in nSMase2 and the details of the interaction, computational studies have been carried out. These
studies have revealed that DPTIP could block the DK switch in nSMase2. Moreover, the analysis
of the residues contributing to the binding identified His463 as a crucial residue. Interestingly, the
inhibitory activity of DPTIP on the H463A mutant protein supported our hypothesis. Thus, an
allosteric cavity in nSMase2 has been identified that can be exploited for the development of new
inhibitors with anti-flaviviral activity., This work was supported by AECSIC, grant number PIE-201980E100 (to M.-J.P.-P.), by the
Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 grants PID2019-105117RR-
C21 (to M.A.M-A) and PID2019-105117RR-C22 (to M.-J.P.-P) and by the European
Commission—NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Plat-
form, PTI+ Salud Global, grants SGL2103053 (to M.A.M.-A.) and SGL2103051 to (M.-J.P.-P.). P.M.-C.
was supported by an FPI fellowship (PRE2020-093374) from AEI/10.13039/501100011033. H.Á.-F.
was supported by an AEI contract (PEJ2018-002006-A)., Peer reviewed

3 pág., EP is supported by the National Institutes of Health (NIGMS 1T34GM145503). JS is supported by Spanish Ministry of Science and Innovation (AEI/10.13039/501100011033 under grant PID2019-105117RR-C2). JN is supported by the Genesis Biotechnology Group., Peer reviewed

8 pág., West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 μM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1-4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 μM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors., This work was supported by Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under grant PID2020-115432RB-I00 (to R.A) grant PID2019-105117RR-C21 (to MAM-A); PID2019-105117RR-C22 (to M-JP-P); and from AECSIC under grant PIE-201980E100 (to M-JP-P). This research work was also funded by the European Commission-NextGeneration EU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global)., Peer reviewed

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of West Nile disease (WND) can curse with meningitis, encephalitis or acute flaccid paralysis. A better understanding of the physiopathology associated with disease progression is mandatory to find biomarkers and effective therapies. In this scenario, blood derivatives (plasma and serum) constitute the more commonly used biofluids due to its ease of collection and high value for diagnostic purposes. Therefore, the potential impact of this virus in the circulating lipidome was addressed combining the analysis of samples from experimentally infected mice and naturally WND patients. Our results unveil dynamic alterations in the lipidome that define specific metabolic fingerprints of different infection stages. Concomitant with neuroinvasion in mice, the lipid landscape was dominated by a metabolic reprograming that resulted in significant elevations of circulating sphingolipids (ceramides, dihydroceramides, and dihydrosphingomyelins), phosphatidylethanolamines and triacylglycerols. Remarkably, patients suffering from WND also displayed an elevation of ceramides, dihydroceramides, lactosylceramides, and monoacylglycerols in their sera. The dysregulation of sphingolipid metabolism by WNV may provide new therapeutic opportunities and supports the potential of certain lipids as novel peripheral biomarkers of WND progression., This study was supported by Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under grant PID2019-105117RR-C21 (to MAMA) and by the European Commission – NextGenerationEU through CSIC's Global Health Platform (PTI Salud Global). PMC was supported by a FPI fellowship from AEI/10.13039/501100011033 under grant PRE2020-093374. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication., Peer reviewed

3 Pág., Mosquito-borne flaviviruses include medically important pathogens that are responsible for a variety of human diseases, such as dengue, Zika congenital syndrome, and West Nile fever [...]., This research was funded by The Spanish Ministry of Science and Innovation, grant number PID2019-105117RR-C21 (to M.A.M.-A.), the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, (INIA), grants RTA-2015-00009-00-00 and E-RTA-2017-00003-C02-01, and Comunidad Autónoma de Madrid grant S2018/BAA-4370, PLATESA2-CM (to J.-C.S.)., Peer reviewed

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of the disease include meningitis, encephalitis, acute flaccid paralysis, or even death, and survivors develop long-lasting sequelae. The damage induced by WNV does not only stem from viral multiplication but also arises from immune-related pathology linked to neuroinflammation. Certain sphingolipids are key players in WNV infection, neurodegenerative diseases and inflammatory disorders. Neutral sphingomyelinase 2 (nSMase2), catalyzes the conversion of sphingomyelin into ceramide and is essential for flavivirus multiplication. Thus, nSMase2 constitutes a promising therapeutic target for the development of dual-acting antiviral and anti-inflammatory therapies against WNV. The effect of an orally bioavailable and brain-penetrating prodrug of the potent nSMase2 inhibitor DPTIP (DPTIP-P1) was studied in WNV-infected mice. While no reduction in the viral load in the brain of infected animals was observed, WNV-induced expression of inflammatory markers was modulated, resulting in a reduced IL-1β expression in brain. Transcriptomic analyses revealed that treatment with the DPTIP prodrug also modulated the expression of various genes related to immune cell function without altering antiviral innate response. Among others, DPTIP-P1 reduced the expression of Lyz2, an inducible genetic marker associated with macrophage infiltration, and modified the expression of genes related to T cell activation such as Trbc1 and Ptnp22. These results identify nSMase2 inhibitors as a new type of immune modulators of WNV infection. The neuroprotective effects exerted by DPTIP-P1 could contribute to mitigate WNV-induced neuroinflammation and sequelae., This work was supported by the Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under Grants PID2019- 105117RR-C21 (to MAMA), PID2019-105117RR-C22 (to MJPP) and PID2020-119195RJ-I00 (to NJO); Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 and FEDER, EU under grants PID2022-137372OR-C21 (to MAMA), PID2022-137372OR-C22 (MJPP and EMP), by Comunidad de Madrid under grant TEC-2024/BIO-66/ SALAINDEC-CM (to MAMA) and by the European Commission—NextGenerationEU through CSIC’s Global Health Platform (PTI Salud Global). PMC was supported by an FPI fellowship from AEI/ 10.13039/501100011033 under Grant PRE2020-093374. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication., Peer reviewed