Usutu virus: an arbovirus on the rise

This article belongs to the Special Issue Transmission Dynamics of Insect VirusesThe Usutu virus (USUV) is a flavivirus that is drawing increasing attention because of its potential for emergence. First isolated in Africa, it was introduced into Europe where it caused significant outbreaks in birds, such as in Austria in 2001. Since then, its geographical distribution has rapidly expanded, with increased circulation, especially in the last few years. Similar to West Nile virus (WNV), the USUV enzootic transmission cycle involves Culex mosquitoes as vectors, and birds as amplifying reservoir hosts, with humans and other mammals likely being dead-end hosts. A similarity in the ecology of these two viruses, which co-circulate in several European countries, highlights USUV’s potential to become an important human pathogen. While USUV has had a severe impact on the blackbird population, the number of human cases remains low, with most infections being asymptomatic. However, some rare cases of neurological disease have been described, both in healthy and immuno-compromised patients. Here, we will discuss the transmission dynamics and the current state of USUV circulation in Europe

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

International audienceChikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition

Design of a genetically stable high fidelity coxsackievirus B3 polymerase that attenuates virus growth in vivo

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stoppedflow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg2+ ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe364 and Pro357, which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe364 to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis

RNA Replicons - A New Approach for Influenza Virus Immunoprophylaxis

RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7

Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase

The kinetic, thermodynamic, and structural basis for fidelity of nucleic acid polymerases remains controversial. An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness. Here we show that a conformational change step preceding phosphoryl transfer is a key fidelity checkpoint for the poliovirus RdRp (3D pol). We provide evidence that this conformational change step is orientation of the triphosphate into a conformation suitable for catalysis, suggesting a kinetic and structural model for RdRp fidelity that can be extrapolated to other classes of nucleic acid polymerases. Finally, we show that a site remote from the catalytic center can control this checkpoint, which occurs at the active site. Importantly, similar connections between a remote site and the active site exist in a wide variety of viral RdRps. The capacity for sites remote from the catalytic center to alter fidelity suggests new possibilities for targeting the viral RdRp for antiviral drug development. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Chikungunya virus vaccine candidates with decreased mutational robustness are attenuated in vivo and have compromised transmissibility

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates

Complete genome sequence of a novel recombinant Citrus tristeza virus, a resistance-breaking isolate from Uruguay

We report here the complete genome sequence of a Citrus tristeza virus (CTV) from Uruguay, sequenced by using Illumina and Sanger sequencing technology. This CTV DSST-17 genome clustered within genotype resistance breaking (RB) and presents two recombination events

Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis