Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex.

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

The E glycoprotein plays an essential role in the high pathogenicity of European-Mediterranean IS98 strain of West Nile virus.

West Nile virus (WNV) is the most widespread arbovirus in the world. Several recent outbreaks and epizootics have been reported in Europe and the Mediterranean basin with increased virulence. In contrast to the well-characterized American and Australian strains, little is known about the virulence determinants of the WNV European-Mediterranean strains. To investigate the viral factors involved in the virulence of these strains, we generated chimeras between the highly neuropathogenic Israel 1998 (IS-98-ST1, IS98) strain and the non-pathogenic Malaysian Kunjin virus (KJMP-502). In vivo analyses in a mouse model of WNV pathogenesis shows that chimeric virus where KJMP-502 E glycoprotein was replaced by that of IS98 is neuropathogenic, demonstrating that this protein is a major virulence determinant. Presence of the N-glycosylation site had limited impact on virus virulence and the 5'UTR does not seem to influence pathogenesis. Finally, mice inoculated with KJMP-502 virus were protected against lethal IS98 infection.

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts.

UNLABELLED: Japanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuation in vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virus in vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts. IMPORTANCE: JEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND: Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS: A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE: Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

A Japanese encephalitis virus genotype 5 molecular clone is highly neuropathogenic in a mouse model: impact of the structural protein region on virulence.

UNLABELLED: Japanese encephalitis virus (JEV) strains can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles were studied in various cell lines in vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance of viruses that express JEV g5 structural proteins in BALB/c mice blood, which correlated with viral invasion of the central nervous system and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity for entering the central nervous system, compared to JEV g3. Overall, in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia in the development of JEV encephalitis. IMPORTANCE: Genotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis.

Measles vaccine expressing the secreted form of West Nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of West Nile virus infection.

West Nile virus (WNV) is a mosquito-borne flavivirus that emerged in North America and caused numerous cases of human encephalitis, thus urging the development of a vaccine. We previously demonstrated the efficacy of a recombinant measles vaccine (MV) expressing the secreted form of the envelope glycoprotein from WNV to prevent WNV encephalitis in mice. In the present study, we investigated the capacity of this vaccine candidate to control WNV infection in a primate model. We first established experimental WNV infection of squirrel monkeys (Saimiri sciureus). A high titer of virus was detected in plasma on day 2 after infection, and viremia persisted for 5 days. A single immunization of recombinant MV-WNV vaccine elicited anti-WNV neutralizing antibodies that strongly reduced WNV viremia at challenge. This study demonstrates for the first time the capacity of a recombinant live attenuated measles vector to protect nonhuman primates from a heterologous infectious challenge.

Transgenic expression of full-length 2',5'-oligoadenylate synthetase 1b confers to BALB/c mice resistance against West Nile virus-induced encephalitis.

Susceptibility of inbred strains to infection with West Nile virus (WNV) has been genetically associated with an arginine-to-a nonsense codon substitution at position 253 (R253X) in the predicted sequence of the murine 2',5'-oligoadenylate synthetase 1B (OAS1B) protein. We introduced by transgenesis the Oas1b cDNA from MBT/Pas mice carrying the R253 codon (Oas1b(MBT)) into BALB/c mice homozygous for the X253 allele (Oas1b(BALB/c)). Overexpression of Oas1b(MBT) mRNA in the brain of transgenic mice prior and in the time course of infection provided protection against the neuroinvasive WNV strain IS-98-ST1. A 200-fold induction of Oas1b(MBT) mRNA in the brain of congenic BALB/c mice homozygous for a MBT/Pas segment encompassing the Oas1b gene was also efficient in reducing both viral growth and mortality, whereas a 200-fold induction of Oas1b(BALB/c) mRNA was unable to prevent virally-induced encephalitis, confirming the critical role of the R253X mutation on Oas1b activity in live mice.

Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses.

Dengue disease is an increasing global health problem that threatens one-third of the world's population. To control this emerging arbovirus, an efficient preventive vaccine is still needed. Because four serotypes of dengue virus (DV) coexist and antibody-dependent enhanced infection may occur, most strategies developed so far rely on the administration of tetravalent formulations of four live attenuated or chimeric viruses. Here, we evaluated a new strategy based on the expression of a single minimal tetravalent DV antigen by a single replicating viral vector derived from pediatric live-attenuated measles vaccine (MV). We generated a recombinant MV vector expressing a DV construct composed of the four envelope domain III (EDIII) from the four DV serotypes fused with the ectodomain of the membrane protein (ectoM). After two injections in mice susceptible to MV infection, the recombinant vector induced neutralizing antibodies against the four serotypes of dengue virus. When immunized mice were further inoculated with live DV from each serotype, a strong memory neutralizing response was raised against all four serotypes. A combined measles-dengue vaccine might be attractive to immunize infants against both diseases where they co-exist.

Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2',5'-oligoadenylate synthetase 1b.

The interferon-inducible 2',5'-oligoadenylate synthetase 1b (Oas1b) protein inhibits West Nile virus (WNV) infection by preventing viral RNA (vRNA) accumulation in infected cells. Serial passage of WNV in Oas1b-expressing mouse cells selected a virus variant with improved growth capacity. Two major amino acid substitutions were identified in this Oas1b-resistant WNV variant: NS3-S365G in the ATPase/helicase domain of NS3 and 2K-V9M in the C-terminal segment of NS4A. To assess their effect on antiviral activity of Oas1b, the NS3 and 2K mutations were engineered into an infectious WNV cDNA clone. The NS3 mutation alters requirement of ATP for ATPase activity and attenuates Oas1b-mediated suppression of vRNA accumulation. However, growth of NS3-mutant virus remains impaired in Oas1b-expressing cells. Only the 2K-V9M mutation efficiently rescued viral growth by promoting vRNA replication. Thus, WNV resistance to Oas1b antiviral action could be attributed to the 2K-V9M substitution with a potential role of NS3-S365G through rescue of vRNA accumulation.

DNA vaccines expressing retrovirus-like particles are efficient immunogens to induce neutralizing antibodies.

We aimed at improving DNA vaccination efficiency for inducing neutralizing antibodies. We used plasmids encoding Gag of MLV and envelope proteins of VSV or WNV. Upon in vivo injection, they generate retrovirus-derived VLPs pseudotyped with these envelopes expressed in their wild-type conformation. We show that these plasmo-retroVLPs induce potent humoral responses, the efficacy of which could be improved by co-administration of DNA encoding adjuvant cytokines. Antibodies against VSV or WNV were detected earlier than with plasmids not generating VLPs, and had higher neutralizing activities. These results highlight the potential of this approach for vaccination strategies aiming at neutralizing antibody induction.

The large form of human 2',5'-Oligoadenylate Synthetase (OAS3) exerts antiviral effect against Chikungunya virus.

Chikungunya virus (CHIKV) becomes one of the most important mosquito-borne alphavirus in the medical field. CHIKV is highly sensitive to antiviral activity of Type-I interferons (IFN-alpha/beta). Here, we investigated the role of IFN-induced 2',5'-Oligoadenylate Synthetase (OAS) family in innate immunity to CHIKV. We established inducible human epithelial HeLa cell lines expressing either the large form of human OAS, OAS3, or the genetic variant OAS3-R844X which is predicted to lack about 20% of the OAS3 protein from the carboxy terminus. HeLa cells respond to ectopic OAS3 expression by efficiently inhibiting CHIKV growth. The characteristic of the antiviral effect was a blockade in early stages of virus replication. Thus, OAS3 pathway may represent a novel antialphaviral mechanism by which IFN-alpha/beta controls CHIKV growth. HeLa cells expressing the truncated form of OAS3 were less resistant to CHIKV infection, raising the question on the involvement of OAS3 genetic polymorphism in human susceptibility to alphavirus infection.

Protective antiviral immunity conferred by a nonintegrative lentiviral vector-based vaccine.

Lentiviral vectors are under intense scrutiny as unique candidate viral vector vaccines against tumor and aggressive pathogens because of their ability to initiate potent and durable specific immune responses. Strategies that alleviate safety concerns will facilitate the clinical developments involving lentiviral vectors. In this respect, the development of integration deficient lentiviral vectors circumvents the safety concerns relative to insertional mutagenesis and might pave the way for clinical applications in which gene transfer is targeted to non-dividing cells. We thus evaluated the potential use of nonintegrative lentiviral vectors as vaccination tools since the main targeted cell in vaccination procedures is the non-dividing dendritic cell (DC). In this study, we demonstrated that a single administration of nonintegrative vectors encoding a secreted form of the envelope of a virulent strain of West Nile Virus (WNV) induces a robust B cell response. Remarkably, nonintegrative lentiviral vectors fully protected mice from a challenge with a lethal dose of WNV and a single immunization was sufficient to induce early and long-lasting protective immunity. Thus, nonintegrative lentiviral vectors might represent a safe and efficacious vaccination platform for the development of prophylactic vaccines against infectious agents.

Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population.

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19(+)B220(-) BST-2(+), which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.

Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein.

Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA.

Pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus.

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.

Characterization of reemerging chikungunya virus.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

Human muscle satellite cells as targets of Chikungunya virus infection.

BACKGROUND: Chikungunya (CHIK) virus is a mosquito-transmitted alphavirus that causes in humans an acute infection characterised by fever, polyarthralgia, head-ache, and myalgia. Since 2005, the emergence of CHIK virus was associated with an unprecedented magnitude outbreak of CHIK disease in the Indian Ocean. Clinically, this outbreak was characterized by invalidating poly-arthralgia, with myalgia being reported in 97.7% of cases. Since the cellular targets of CHIK virus in humans are unknown, we studied the pathogenic events and targets of CHIK infection in skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistology on muscle biopsies from two CHIK virus-infected patients with myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells (designed as satelllite cells), and not in muscle fibers. To evaluate the ability of CHIK virus to replicate in human satellite cells, we assessed virus infection on primary human muscle cells; viral growth was observed in CHIK virus-infected satellite cells with a cytopathic effect, whereas myotubes were essentially refractory to infection. CONCLUSIONS/SIGNIFICANCE: This report provides new insights into CHIK virus pathogenesis, since it is the first to identify a cellular target of CHIK virus in humans and to report a selective infection of muscle satellite cells by a viral agent in humans.

Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.

BACKGROUND: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis. METHODS AND FINDINGS: We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector. CONCLUSIONS: The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.

A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus.

BACKGROUND: Lentiviral vectors, due to their capacity to transduce non-dividing cells, have become precious and worldwide used gene transfer systems. Their ability to efficiently and stably transduce dendritic cells (DCs) has led to their successful use as vaccination vectors for eliciting strong, specific and protective cellular immune responses mostly in anti-tumoral but also in anti-viral applications. However, the ability of lentiviral vectors to elicit an antibody-based protective immunity has, to date, not been evaluated. In the present study, we evaluated the potential of a lentiviral vector-based vaccine to elicit humoral immunity against West Nile virus (WNV). WNV is a mosquito-borne flavivirus that emerged in North America and causes encephalitis in humans, birds and horses. Neutralizing anti-WNV antibodies have been shown to be crucial for protection against WNV encephalitis. METHODS: The ability of lentiviral vector TRIP/sE(WNV), expressing the secreted soluble form of the envelope E-glycoprotein (sE(WNV)) from the highly virulent IS-98-ST1 strain of WNV, to induce a specific humoral response and protection against WNV infection was assessed in a mouse model of WNV encephalitis. RESULTS: Remarkably, a single immunization with a minute dose of TRIP/sE(WNV) was efficient at eliciting a long-lasting, protective and sterilizing humoral immunity, only 1 week after priming. CONCLUSIONS: This study broadens the applicability of lentiviral vectors as efficient non-replicating vaccines against pathogens for which a neutralizing humoral response is one active arm of the protective immunity. The TRIP/sE(WNV) lentiviral vector appears to be a promising tool for veterinary vaccination against zoonotic WNV.