Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

Development of iminosugar-based glycosidase inhibitors as drug candidates for SARS-CoV-2 virus via molecular modelling and in vitro studies.

We developed new iminosugar-based glycosidase inhibitors against SARS-CoV-2. Known drugs (miglustat, migalastat, miglitol, and swainsonine) were chosen as lead compounds to develop three classes of glycosidase inhibitors (α-glucosidase, α-galactosidase, and mannosidase). Molecular modelling of the lead compounds, synthesis of the compounds with the highest docking scores, enzyme inhibition tests, and in vitro antiviral assays afforded rationally designed inhibitors. Two highly active α-glucosidase inhibitors were discovered, where one of them is the most potent iminosugar-based anti-SARS-CoV-2 agent to date (EC90 = 1.94 µM in A549-ACE2 cells against Omicron BA.1 strain). However, galactosidase inhibitors did not exhibit antiviral activity, whereas mannosidase inhibitors were both active and cytotoxic. As our iminosugar-based drug candidates act by a host-directed mechanism, they should be more resilient to drug resistance. Moreover, this strategy could be extended to identify potential drug candidates for other viral infections.

IRF8 regulates efficacy of therapeutic anti-CD20 monoclonal antibodies.

Anti-CD20 monoclonal antibodies such as Rituximab, Ofatumumab, and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc-dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome-wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti-CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement-dependent-cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B-cell lines. IRF8 KO also decreased the efficacy of antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti-CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting capacity of anti-CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20-targeting immunotherapies.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

APOBEC3C S188I Polymorphism Enhances Context-Specific Editing of Hepatitis B Virus Genome.

Single-nucleotide polymorphism in APOBEC3C (resulting in a serine to isoleucine in position 188) is present in approximately 10% of African populations and greatly enhances restriction against human immunodeficiency virus-1 and simian immunodeficiency virus by improving dimerization and DNA processivity of the enzyme. In this study, we demonstrated in culture and in infected patients that hepatitis B virus (HBV) could be edited by APOBEC3CS188I. Using next-generation sequencing, we demonstrated that APOBEC3CS188I led to enhanced editing activity in 5'TpCpA→5'TpTpA context. This constitutes a new hallmark of this enzyme, which could be used to determine its impact on HBV or nuclear DNA.

RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes.

Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods, that is, next-generation sequencing (NGS) technologies and related bioinformatics algorithms. Although many algorithms were developed, their sensitivity and reproducibility were mostly assessed on simulated data. Here, we introduce DG-seq, a time-efficient pipeline for DVG detection and quantification, and a set of biological controls to assess the performance of not only our bioinformatics algorithm but also the upstream NGS steps. Using these tools, we provide the first rigorous comparison of the two commonly used sample processing methods for RNA-seq, with or without a PCR preamplification step. Our data show that preamplification confers a limited advantage in terms of sensitivity and introduces size- but also sequence-dependent biases in DVG quantification, thereby providing a strong rationale to favor preamplification-free methods. We further examine the features of DVGs produced by wild-type and transcription-defective (PA-K635A or PA-R638A) influenza viruses, and show an increased diversity and frequency of DVGs produced by the PA mutants compared to the wild-type virus. Finally, we demonstrate a significant enrichment in DVGs showing direct, A/T-rich sequence repeats at the deletion breakpoint sites. Our findings provide novel insights into the mechanisms of influenza virus DVG production.

Intra- and Inter-cellular Modeling of Dynamic Interaction between Zika Virus and Its Naturally Occurring Defective Viral Genomes.

Here, we examine in silico the infection dynamics and interactions of two Zika virus (ZIKV) genomes: one is the full-length ZIKV genome (wild type [WT]), and the other is one of the naturally occurring defective viral genomes (DVGs), which can replicate in the presence of the WT genome, appears under high-MOI (multiplicity of infection) passaging conditions, and carries a deletion encompassing part of the structural and NS1 protein-coding region. Ordinary differential equations (ODEs) were used to simulate the infection of cells by virus particles and the intracellular replication of the WT and DVG genomes that produce these particles. For each virus passage in Vero and C6/36 cell cultures, the rates of the simulated processes were fitted to two types of observations: virus titer data and the assembled haplotypes of the replicate passage samples. We studied the consistency of the model with the experimental data across all passages of infection in each cell type separately as well as the sensitivity of the model's parameters. We also determined which simulated processes of virus evolution are the most important for the adaptation of the WT and DVG interplay in these two disparate cell culture environments. Our results demonstrate that in the majority of passages, the rates of DVG production are higher inC6/36 cells than in Vero cells, which might result in tolerance and therefore drive the persistence of the mosquito vector in the context of ZIKV infection. Additionally, the model simulations showed a slower accumulation of infected cells under higher activation of the DVG-associated processes, which indicates a potential role of DVGs in virus attenuation. IMPORTANCE One of the ideas for lessening Zika pathogenicity is the addition of its natural or engineered defective virus genomes (DVGs) (have no pathogenicity) to the infection pool: a DVG is redirecting the wild-type (WT)-associated virus development resources toward its own maturation. The mathematical model presented here, attuned to the data from interplays between WT Zika viruses and their natural DVGs in mammalian and mosquito cells, provides evidence that the loss of uninfected cells is attenuated by the DVG development processes. This model enabled us to estimate the rates of virus development processes in the WT/DVG interplay, determine the key processes, and show that the key processes are faster in mosquito cells than in mammalian ones. In general, the presented model and its detailed study suggest in what important virus development processes the therapeutically efficient DVG might compete with the WT; this may help in assembling engineered DVGs for ZIKV and other flaviviruses.

Interactions between vector competence to chikungunya virus and resistance to deltamethrin in Aedes aegypti laboratory lines?

The urban mosquito species Aedes aegypti is the main vector of arboviruses worldwide. Mosquito control with insecticides is the most prevalent method for preventing transmission in the absence of effective vaccines and available treatments; however, the extensive use of insecticides has led to the development of resistance in mosquito populations throughout the world, and the number of epidemics caused by arboviruses has increased. Three mosquito lines with different resistance profiles to deltamethrin were isolated in French Guiana, including one with the I1016 knock-down resistant allele. Significant differences were observed in the cumulative proportion of mosquitoes with a disseminated chikungunya virus infection over time across these lines. In addition, some genes related to resistance (CYP6BB2, CYP6N12, GST2, trypsin) were variably overexpressed in the midgut at 7 days after an infectious bloodmeal in these three lines. Our work shows that vector competence for chikungunya virus varied between Ae. aegypti laboratory lines with different deltamethrin resistance profiles. More accurate verification of the functional association between insecticide resistance and vector competence remains to be demonstrated.

Vaccine-Derived Polioviruses, Central African Republic, 2019.

Since May 2019, the Central African Republic has experienced a poliomyelitis outbreak caused by type 2 vaccine-derived polioviruses (VDPV-2s). The outbreak affected Bangui, the capital city, and 10 districts across the country. The outbreak resulted from several independent emergence events of VDPV-2s featuring recombinant genomes with complex mosaic genomes. The low number of mutations (<20) in the viral capsid protein 1-encoding region compared with the vaccine strain suggests that VDPV-2 had been circulating for a relatively short time (probably <3 years) before being isolated. Environmental surveillance, which relies on a limited number of sampling sites in the Central African Republic and does not cover the whole country, failed to detect the circulation of VDPV-2s before some had induced poliomyelitis in children.

High-Throughput Fluorescence-Based Screen Identifies the Neuronal MicroRNA miR-124 as a Positive Regulator of Alphavirus Infection.

MicroRNAs (miRNAs) are small regulatory RNAs which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs, and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified 16 miRNAs showing a positive effect on Sindbis virus (SINV) expressing green fluorescent protein (GFP), among which were a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 and silent mutations to disrupt this binding site in the viral RNA abolished positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved in other alphaviruses, as its inhibition reduces chikungunya virus (CHIKV) production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.IMPORTANCE Arthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arbovirus infection, neurological diseases such as meningitis and encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphavirus infections.

Host nutritional status affects alphavirus virulence, transmission, and evolution.

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.

Mathematical modelling of SARS-CoV-2 infection of human and animal host cells reveals differences in the infection rates and delays in viral particle production by infected cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2), a causative agent of COVID-19 disease, poses a significant threat to public health. Since its outbreak in December 2019, Wuhan, China, extensive collection of diverse data from cell culture and animal infections as well as population level data from an ongoing pandemic, has been vital in assessing strategies to battle its spread. Mathematical modelling plays a key role in quantifying determinants that drive virus infection dynamics, especially those relevant for epidemiological investigations and predictions as well as for proposing efficient mitigation strategies. We utilized a simple mathematical model to describe and explain experimental results on viral replication cycle kinetics during SARS-CoV-2 infection of animal and human derived cell lines, green monkey kidney cells, Vero-E6, and human lung epithelium cells, A549-ACE2, respectively. We conducted cell infections using two distinct initial viral concentrations and quantified viral loads over time. We then fitted the model to our experimental data and quantified the viral parameters. We showed that such cellular tropism generates significant differences in the infection rates and incubation times of SARS-CoV-2, that is, the times to the first release of newly synthesised viral progeny by SARS-CoV-2-infected cells. Specifically, the rate at which A549-ACE2 cells were infected by SARS-CoV-2 was 15 times lower than that in the case of Vero-E6 cell infection and the duration of latent phase of A549-ACE2 cells was 1.6 times longer than that of Vero-E6 cells. On the other hand, we found no statistically significant differences in other viral parameters, such as viral production rate or infected cell death rate. Since in vitro infection assays represent the first stage in the development of antiviral treatments against SARS-CoV-2, discrepancies in the viral parameter values across different cell hosts have to be identified and quantified to better target vaccine and antiviral research.

Quantitative evaluation of PpSP15-LmSTI1 fusion gene expression following transfection with an alphavirus-derived self-amplifying mRNA and conventional DNA vaccine platforms.

New vaccine platforms are crucial to address complex parasitic infections such as cutaneous leishmaniasis. Self-amplifying mRNA (SAM) based vaccines represent the next generation nucleic acid-based platform. In the present study, we compared the expression levels of PpSP15-LmSTI1 fusion gene in BHK-21 cells following transfection with Semliki Forest virus (SFV)-derived SAM, SFV-derived plasmid DNA (pSFV-PD) and conventional plasmid DNA (pcDNA3.1+). PpSP15-LmSTI1 fusion gene expression levels were evaluated at different time points, using quantitative Real-time PCR. All data were validated and normalized by two internal control genes. According to the results, mean values of relative expression were significantly higher for SFV-PD SAM/fusion than pcDNA/fusion and pSFV-PD/fusion at all concentrations and time points. Our results showed that higher levels of PpSp15-LmSTI1 antigen expression could be achieved using a SAM vector than pcDNA and pSFV-PD, making it a valuable and efficient alternative to conventional plasmid DNA-based vaccines against leishmaniasis.

Homologous recombination is an intrinsic defense against antiviral RNA interference.

RNA interference (RNAi) is the major antiviral defense mechanism of plants and invertebrates, rendering the capacity to evade it a defining factor in shaping the viral landscape. Here we sought to determine whether different virus replication strategies provided any inherent capacity to evade RNAi in the absence of an antagonist. Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacity of positive- and negative-stranded RNA viruses to cope with this selective pressure. Applying this defense against four distinct viral families revealed that the capacity to undergo homologous recombination was the defining attribute that enabled evasion of this defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidly excised genomic material, while negative-stranded viruses were effectively targeted and cleared upon RNAi-based selection. These data suggest a dynamic relationship between host antiviral defenses and the biology of virus replication in shaping pathogen prevalence.

[A cold case: non-replicative recombination in positive-strand RNA viruses]. / Une affaire non résolue : la recombinaison non réplicative chez les virus à ARN de polarité positive.

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.

A cold case: non-replicative recombination in positive-strand RNA viruses.

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and the frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.

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.IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.