scholarly journals Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination

2021 ◽  
Vol 15 (6) ◽  
pp. e0009442
Author(s):  
Alejandro Marin-Lopez ◽  
Junjun Jiang ◽  
Yuchen Wang ◽  
Yongguo Cao ◽  
Tyler MacNeil ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Aedes Aegypti ◽  
Dengue Virus ◽  
Denv Infection ◽  
Infected Mosquito ◽  
Mammalian Host ◽  
Susceptible Host ◽  
Calcium Transporter

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.

scholarly journals Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination

2020 ◽  
Author(s):  
Alejandro Marin-Lopez ◽  
Junjun Jiang ◽  
Yuchen Wang ◽  
Yongguo Cao ◽  
Tyler MacNeil ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Aedes Aegypti ◽  
Dengue Virus ◽  
Denv Infection ◽  
Infected Mosquito ◽  
Viral Dissemination ◽  
Protein Components ◽  
Calcium Transporter

AbstractDengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, being transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.ImportanceAedes aegypti mosquitoes are the major vector of different flaviviruses that cause human diseases, including dengue virus. There is a great need for better therapeutics and preventive vaccines against flaviviruses. Flaviviruses create complex virus-host and virus-vector interactions. The interactions between viral particles and protein components in the vector is not completely understood. In this work we characterize how two mosquito proteins, “AeSNAP” and “ATPase”, influence DENV viral dissemination within A. aegypti, using both in vitro and in vivo models. These results elucidate anti-vector measures that may be potentially be used to control dengue virus spread in the mosquito vector.

scholarly journals Differential replication of dengue virus serotypes 2 and 3 in coinfections of C6/36 cells and Aedes aegypti mosquitoes

2014 ◽  
Vol 8 (07) ◽  
pp. 876-884 ◽  
Author(s):  
Diana Carolina Quintero-Gil ◽  
Marta Ospina ◽  
Jorge Emilio Osorio-Benitez ◽  
Marlen Martinez-Gutierrez
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Viability ◽  
Denv Infection ◽  
Replication Capacity ◽  
Denv Serotypes ◽  
Viral Genomes ◽  
Diphenyltetrazolium Bromide

Introduction: Different dengue virus (DENV) serotypes have been associated with greater epidemic potential. In turn, the increased frequency in cases of severe forms of dengue has been associated with the cocirculation of several serotypes. Because Colombia is a country with an endemic presence of all four DENV serotypes, the aim of this study was to evaluate the in vivo and in vitro replication of the DENV-2 and DENV-3 strains under individual infection and coinfection conditions. Methodology: C6/36HT cells were infected with the two strains individually or simultaneously (coinfection). Replication capacity was evaluated by RT-qPCR, and the effects on cell viability were assessed with an MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Additionally, Aedes aegypti mosquitoes were artificially fed the two strains of each serotype individually or simultaneously. The viral genomes were quantified by RT-qPCR and the survival of the infected mosquitoes was compared to that of uninfected controls. Results: In single infections, three strains significantly affected C6/36HT cell viability, but no significant differences were found in the replication capacities of the strains of the same serotype. In the in vivo infections, mosquito survival was not affected, and no significant differences in replication between strains of the same serotype were found. Finally, in coinfections, serotype 2 replicated with a thousandfold greater efficiency than serotype 3 did both in vitro and in vivo. Conclusions: Due to the cocirculation of serotypes in endemic regions, further studies of coinfections in a natural environment would further an understanding of the transmission dynamics that affect DENV infection epidemiology.

scholarly journals Activation of the autophagy pathway affects Dengue virus infection in Aedes aegypti

2021 ◽  
Author(s):  
Tse-Yu Chen ◽  
Chelsea T. Smartt
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Small Molecules ◽  
Virus Titer ◽  
Denv Infection ◽  
Control Group ◽  
Mosquito Vector ◽  
Autophagy Pathway

AbstractMosquito-borne Dengue virus (DENV) has caused major disease worldwide, impacting 50 to 100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology and developing new control strategies becomes an important issue to eliminate DENV. We focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential anti-viral target in the mosquito. To understand the role played by autophagy in Ae. aegypti, we examined the expression of the pathway in vitro (Aag-2 cell) and in vivo (Ae. aegypti). The results indicated that DENV infection in Aag-2 cells caused the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) protein levels to increase which indicated the activation of the autophagy pathway. Rapamycin and 3-Methyladenine were used to activate or suppress the autophagy pathway, respectively. Rapamycin treatment decreased the virus titer in the Aag-2 cells, but the 3-Methyladenine treatment did not affect DENV titer. In Ae. aegypti, microinjected rapamycin increased the DENV titer after one-day infection and was significantly different compared to the control group titer. Two ATG genes, ATG4 and ATG12, were expressed differentially under the rapamycin treatments. Although the results differed between in vitro and in vivo studies, findings from both support the interaction between autophagy and DENV. Our studies revealed the activation of the autophagy pathway through rapamycin could be related to DENV infection in the mosquito. The possibility of autophagy being associated with different antiviral mechanisms at different extrinsic incubation times and tissues in Ae. aegypti is discussed.Author SummaryDengue virus (DENV) has been a great threat to public health and has not developed an efficient method to stop the transmission. To understand the complex interaction between virus and mosquito, we investigate the autophagy pathway and its role during the infection process. We noticed the induction of autophagy pathways from DENV infection in Aag-2 cells and blood meal from Ae. aegypti. Moreover, activation of the autophagy pathway from rapamycin could alter the DENV titer. Our results indicated the autophagy pathway is associated with DENV and could be crucial during the DENV infection. Furthermore, we proved the practicality of small molecules in altering the autophagy pathway in mosquitoes, and thus the usage of small molecules as possible mosquito pathogen vaccines should be evaluated.

scholarly journals Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo

2021 ◽  
Vol 102 (10) ◽  
Author(s):  
Wisam-Hamzah Al Shujairi ◽  
Luke P. Kris ◽  
Kylie van der Hoek ◽  
Evangeline Cowell ◽  
Gustavo Bracho-Granado ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Denv Infection ◽  
Brain Morphology ◽  
Moderate Increase ◽  
Tnf Α ◽  
Significant Difference

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip−/−) showed enhanced DENV infection, accompanied by increased IFN-β and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip−/− mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip−/− compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-β, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip−/− mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip−/− DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-β, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip−/− mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

scholarly journals Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
Artem Baidaliuk ◽  
Elliott F. Miot ◽  
Sebastian Lequime ◽  
Isabelle Moltini-Conclois ◽  
Fanny Delaigue ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Content Type ◽  
Cells In Culture ◽  
Mosquito Cells ◽  
Wide Range ◽  
Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

scholarly journals Epitope Determinants of a Chimpanzee Dengue Virus Type 4 (DENV-4)-Neutralizing Antibody and Protection against DENV-4 Challenge in Mice and Rhesus Monkeys by Passively Transferred Humanized Antibody

2007 ◽  
Vol 81 (23) ◽  
pp. 12766-12774 ◽  
Author(s):  
Ching-Juh Lai ◽  
Ana P. Goncalvez ◽  
Ruhe Men ◽  
Claire Wernly ◽  
Olivia Donau ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Type ◽  
High Titer ◽  
Neutralizing Antibody ◽  
Denv Infection ◽  
Passive Transfer ◽  
Escape Mutant ◽  
Protective Capacity

ABSTRACT The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID50) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys174-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 106 MID50 of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.

scholarly journals Discovery of Dengue Virus NS4B Inhibitors

2015 ◽  
Vol 89 (16) ◽  
pp. 8233-8244 ◽  
Author(s):  
Qing-Yin Wang ◽  
Hongping Dong ◽  
Bin Zou ◽  
Ratna Karuna ◽  
Kah Fei Wan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Transmembrane Protein ◽  
Selective Inhibition ◽  
Denv Infection ◽  
Viral Pathogens ◽  
Replication Complex ◽  
First Time

ABSTRACTThe four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV bothin vitroandin vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has goodin vivopharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.IMPORTANCEDengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) bothin vitroandin vivo. Our results validate, for the first time, that NS4B inhibitors could potentially be developed for antiviral therapy for treatment of DENV infection in humans.

scholarly journals Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takeshi Kurosu ◽  
Keiko Hanabara ◽  
Azusa Asai ◽  
Sabar Pambudi ◽  
Supranee Phanthanawiboon ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Evaluation Process ◽  
Denv Infection ◽  
Interferon Α ◽  
Double Knockout ◽  
Virus Envelope

AbstractIn a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/β–γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

scholarly journals Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

2014 ◽  
Vol 95 (8) ◽  
pp. 1712-1722 ◽  
Author(s):  
Amber M. Paul ◽  
Yongliang Shi ◽  
Dhiraj Acharya ◽  
Jessica R. Douglas ◽  
Amanda Cooley ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Dengue Virus ◽  
Sirna Delivery ◽  
Vero Cells ◽  
Denv Infection ◽  
Small Interfering Rnas ◽  
Virion Release ◽  
Life Threatening

Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.

scholarly journals SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication

2016 ◽  
Vol 90 (9) ◽  
pp. 4308-4319 ◽  
Author(s):  
Chan-I Su ◽  
Chung-Hsin Tseng ◽  
Chia-Yi Yu ◽  
Michael M. C. Lai
Keyword(s):  
Dengue Virus ◽  
Protein Stability ◽  
Posttranslational Modification ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Denv Infection ◽  
Cellular Protein ◽  
Viral Rna

ABSTRACTSmall ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular geneUbc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. Byin vivoandin vitroSUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication.IMPORTANCESUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.

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