[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferonstimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus.
Journal of Virology 06/2015; 89(17). DOI:10.1128/JVI.00354-15 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although plasmacytoid dendritic cells (pDCs) represent a rare immune cell type, they are the most important source of type I interferons (IFNs) upon viral infection. Phagocytosed RNA viruses and RNA virus–infected cells are detected by pDCs with the endosomal pattern recognition receptor (PRR) toll-like receptor 7 (TLR7). We showed that replication of the yellow fever live vaccine YF-17D in human pDCs and pDC-like cell lines stimulated type I IFN production through RIG-I (retinoic acid–inducible gene I), a member of the RIG-I–like receptor (RLR) family of cytosolic PRRs. Thus, human pDCs sense replicative viral RNA. In contrast, direct contact between pDCs and YF-17D–infected cells stimulated a TLR7-dependent, viral replication–independent production of type I IFN. We also showed that the RLR pathway was dampened by the activities of interleukin-1 receptor–associated kinases 1 and 4 (IRAK1 and IRAK4), which are downstream effectors of the TLR7 pathway, suggesting that both kinases play opposing roles downstream of specific PRRs. Together, these data suggest that a virus can stimulate either TLR or RLR signaling in the same cell, depending on how its nucleic acid content is delivered.
[Show abstract][Hide abstract] ABSTRACT: Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.
[Show abstract][Hide abstract] ABSTRACT: West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.
[Show abstract][Hide abstract] ABSTRACT: The oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins that mediate antiviral activity through the synthesis of 2'-5'-linked oligoadenylates and subsequent activation of the endoribonuclease RNase L. However, we have recently demonstrated that exogenous recombinant OAS1 is taken up by cells and reduces viral replication both in cell culture and in vivo, independent of RNase L. These results demonstrate a novel paracrine antiviral activity of OAS working in parallel with the classical RNase L pathway. In this study, we investigate the uptake kinetics of recombinant porcine OAS1 and show that it is rapidly and efficiently internalized in a manner that can be blocked by heparin. Heparin, furthermore, abolishes the antiviral activity of OAS1, demonstrating the requirement of the intracellular localization of OAS1 to inhibit the virus. In addition, we demonstrate that exogenous OAS1 affects an early step of the viral replication cycle.
Journal of Interferon & Cytokine Research 12/2014; 35(5). DOI:10.1089/jir.2014.0140 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS.
We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2'-5'-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.
Journal of Virology 10/2014; 88(24). DOI:10.1128/JVI.01763-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to climate change and the propagation of competent arthropods worldwide, arboviruses have become pathogens of major medical importance. Early transmission to vertebrates is initiated by skin puncture and deposition of virus together with arthropod saliva in the epidermis and dermis. Saliva components have the capacity to modulate skin cell responses by enhancing and/or counteracting initial replication and establishment of systemic viral infection. Here, we review the nature of the cells targeted by arboviruses at the skin level and discuss the type of cellular responses elicited by these pathogens in light of the immunomodulatory properties of arthropod vector-derived salivary factors injected at the inoculation site. Understanding cutaneous arbovirus–host interactions may provide new clues for the design of future therapeutics.
Virology 09/2014; s 464–465(1):26–32. DOI:10.1016/j.virol.2014.06.023 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.
Journal of Visualized Experiments 05/2014; DOI:10.3791/51222 · 1.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Our therapeutic arsenal to treat viral diseases is extremely limited, and there is a critical need for molecules that could be used against multiple viruses. Among possible strategies, there is a growing interest for molecules stimulating cellular defense mechanisms. We recently developed a functional assay to identify stimulators of antiviral genes, and selected compound DD264 from a chemical library using this approach. While searching for its mode of action, we identified this molecule as an inhibitor of pyrimidine biosynthesis, a metabolic pathway that fuels the cell with pyrimidine nucleobases for both DNA and RNA synthesis. Interestingly, it was recently shown that inhibitors of this metabolic pathway prevent the replication of RNA viruses. Here, we established a functional link between pyrimidine biosynthesis pathway and the induction of antiviral genes, and demonstrated that pyrimidine biosynthesis inhibitors like DD264 or brequinar critically rely on cellular immune response to inhibit virus growth. Thus, pyrimidine deprivation is not directly responsible for the antiviral activity of pyrimidine biosynthesis inhibitors, which rather involves the induction of a metabolic stress and subsequent triggering of cellular defense mechanisms.