Uukuniemi Virus as a Tick-Borne Virus Model.

International audienceIn the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged on different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome close to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection was persistent. The glycoprotein GC was mainly highly mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H). Treatment with β-mercaptoethanol did not impact the apparent molecular weight of GN On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F, and the electrophoretic mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per focus forming unit differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells in investigations of the cell biology of arbovirus transmission and entry into mammalian hosts. Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research on not only tick-borne phleboviruses but also all viruses and other pathogens transmitted by ticks

A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses

Renforcement mécanique du verre (nouvelles compositions chimiques et dépôt de films minces élaborés par voie sol-gel)

Ce travail, articulé en deux parties, s inscrit dans une démarche exploratoire visant à améliorer la résistance mécanique du verre. Tout d abord, nous avons abordé la recherche de nouvelles compositions vitreuses à hautes propriétés mécaniques. Les compositions ont été synthétisées à l aide d un dispositif original développé au laboratoire opérant sous atmosphère contrôlée. Les verres ont été caractérisés en termes de propriétés mécaniques et de structure pour d identifier les paramètres gouvernant les propriétés mécaniques macroscopiques. Nous nous sommes intéressés dans la deuxième partie au dépôt sur la surface de films minces inorganiques élaborés par voie sol-gel (silice, alumine et zircone amorphes). Les échantillons ont été caractérisés par micro-indentation et micro-rayage. Des observations ont également été réalisées par microscopie à force atomique, dans le but de déterminer l influence du traitement thermique et de la composition sur ces propriétés.This work aims at improving mechanical resistance of glass. Two aspects of reinforcement have been studied. First, we focused on the research of new glassy compositions exhibiting high mechanical properties. The compositions were synthesized using an original home-made device. The mechanical characteristics were then determined, and an exploratory study of the structure was conducted in order to establish the role of the reticulation of the network. The second part deals with the mechanical reinforcement of the surface. Indeed, small defects existing on the surface often affect the strength of the whole piece of glass. To minimize this effect, we deposited inorganic thin films obtained via sol-gel process by a dip-coating method. This way, ordinary soda-lime silicate glass substrates were coated and then characterized both mechanically and morphologically, to determine the influence of thermal treatment, as well as composition, on mechanical properties.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Complete protection against influenza virus H1N1 strain A/PR/8/34 challenge in mice immunized with non-adjuvanted Novirhabdovirus vaccines

Novirhabdoviruses like Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) are fish-infecting Rhabdoviruses belonging to the Mononegavirales order. By reverse genetics, we previously showed that a recombinant VHSV expressing the West Nile Virus (WNV) E glycoprotein could serve as a vaccine platform against WNV. In the current study, we aimed to evaluate the potential of the Novirhabdovirus platform as a vaccine against influenza virus. Recombinant Novirhabdoviruses, rVHSV-HA and rIHNV-HA, expressing at the viral surface the hemagglutinin HA ectodomain were generated and used to immunized mice. We showed that mice immunized with either, rVHSV-HA or rIHNV-HA, elicited a strong neutralizing antibody response against influenza virus. A complete protection was conferred to the immunized mice when challenged with a lethal dose of influenza H1N1 A/PR/8/34 virus. Furthermore we showed that although acting as inert antigen in mice, since naturally inactivated over 20 degrees C, mice immunized with rVHSV-HA or rIHNV-HA in the absence of adjuvant were also completely protected from a lethal challenge. Novirhabdoviruses platform are of particular interest as vaccines for mammals since they are cost effective to produce, relatively easy to generate and very effective to protect immunized animals

Cultures séquentielles bactéries-levures pour l'épuration et la valorisation des matières azotées et carbonées des effluents

National audienc

Efficient Co-Replication of Defective Novirhabdovirus

We have generated defective Viral Hemorrhagic Septicemia Viruses (VHSV) which express either the green fluorescent protein (GFP) or a far-red fluorescent protein (mKate) by replacing the genes encoding the nucleoprotein N or the polymerase-associated P protein. To recover viable defective viruses, rVHSV-ΔN-Red and rVHSV-ΔP-Green, fish cells were co-transfected with both deleted cDNA VHSV genomes, together with plasmids expressing N, P and L of the RNA-dependent RNA polymerase. After one passage of the transfected cell supernatant, red and green cell foci were observed. Viral titer reached 107 PFU/mL after three passages. Infected cells were always red and green with the very rare event of single red or green cell foci appearing. To clarify our understanding of how such defective viruses could be so efficiently propagated, we investigated whether (i) a recombination event between both defective genomes had occurred, (ii) whether both genomes were co-encapsidated in a single viral particle, and (iii) whether both defective viruses were always replicated together through a complementation phenomenon or even as conglomerate. To address these hypotheses, genome and viral particles have been fully characterized and, thus, allowing us to conclude that rVHSV-ΔN-Red and rVHSV-ΔP-Green are independent viral particles which could propagate only by simultaneously infecting the same cells

Complete Protection against Influenza Virus H1N1 Strain A/PR/8/34 Challenge in Mice Immunized with Non-Adjuvanted Novirhabdovirus Vaccines.

Novirhabdoviruses like Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) are fish-infecting Rhabdoviruses belonging to the Mononegavirales order. By reverse genetics, we previously showed that a recombinant VHSV expressing the West Nile Virus (WNV) E glycoprotein could serve as a vaccine platform against WNV. In the current study, we aimed to evaluate the potential of the Novirhabdovirus platform as a vaccine against influenza virus. Recombinant Novirhabdoviruses, rVHSV-HA and rIHNV-HA, expressing at the viral surface the hemagglutinin HA ectodomain were generated and used to immunized mice. We showed that mice immunized with either, rVHSV-HA or rIHNV-HA, elicited a strong neutralizing antibody response against influenza virus. A complete protection was conferred to the immunized mice when challenged with a lethal dose of influenza H1N1 A/PR/8/34 virus. Furthermore we showed that although acting as inert antigen in mice, since naturally inactivated over 20°C, mice immunized with rVHSV-HA or rIHNV-HA in the absence of adjuvant were also completely protected from a lethal challenge. Novirhabdoviruses platform are of particular interest as vaccines for mammals since they are cost effective to produce, relatively easy to generate and very effective to protect immunized animals

Attenuated infectious hematopoietic necrosis virus with rearranged gene order as potential vaccine

The genome of infectious hematopoietic necrosis virus (IHNV), a salmonid novirhabdovirus, has been engineered to modify the gene order and to evaluate the impact on a possible attenuation of the virus in vitro and in vivo. By reverse genetics, eight recombinant IHNVs (rIHNVs), termed NxGy according to the respective positions of the nucleoprotein (N) and glycoprotein (G) genes along the genome, have been recovered. All rIHNVs have been fully characterized in vitro for their cytopathic effects, kinetics of replication, and profiles of viral gene transcription. These rIHNVs are stable through up to 10 passages in cell culture. Following bath immersion administration of the various rIHNVs to juvenile trout, some of the rIHNVs were clearly attenuated (N2G3, N2G4, N3G4, and N4G1). The position of the N gene seems to be one of the most critical features correlated to the level of viral attenuation. The induced immune response potential in fish was evaluated by enzyme-linked immunosorbent spot assay (ELISPOT) and seroneutralization assays. The recombinant virus N2G3 induced a strong antibody response in immunized fish and conferred 86% of protection against wild-type IHNV challenge in trout, thus representing a promising starting point for the development of a live attenuated vaccine candidate. IMPORTANCE In Europe, no vaccines are available against infectious hematopoietic necrosis virus (IHNV), one of the major economic threats in fish aquaculture. Live attenuated vaccines are conditioned by a sensible balance between attenuation and pathogenicity. Moreover, nonsegmented negative-strain RNA viruses (NNSV) are subject to a transcription gradient dictated by the order of the genes in their genomes. With the perspective of developing a vaccine against IHNV, we engineered various recombinant IHNVs with reordered genomes in order to artificially attenuate the virus. Our results validate the gene rearrangement approach as a potent and stable attenuation strategy for fish novirhabdovirus and open a new perspective for design of vaccines against other NNSV

Schematic representation of the engineered rIHNV-HA and rVHSV-HA genomes.

<p>GS: gene start; GE: gene end; SP: signal peptide; TM: transmembrane.</p