Altered Spatial and Temporal Gait Parameters in Mice Infected with Ross River Virus

Mouse models that accurately replicate the immunopathogenesis and clinical disease of alphavirus infection are vital to the preclinical development of therapeutic strategies that target alphavirus infection and disease. Current models rely on subjective scoring made through experienced observation of infected mice.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Current Understanding of the Role of Cholesterol in the Life Cycle of Alphaviruses

Enveloped viruses rely on different lipid classes present in cell membranes to accomplish several steps of their life cycle in the host. Particularly for alphaviruses, a medically important group of arboviruses, which are part of the Togaviridae family, cholesterol seems to be a critical lipid exploited during infection, although its relevance may vary depending on which stage of the virus life cycle is under consideration and whether infection takes place in vertebrate or invertebrate hosts. In this review, the role of cholesterol in both early and late events of alphavirus infection and how viral replication may affect cholesterol metabolism are summarized, taking into account studies on Old World and New World alphaviruses in different cell lines. Moreover, the importance of cholesterol for the structural stability of alphavirus particles is also discussed, shedding light on the role played by this lipid when they leave the host cell.

DDX56 Binds to Chikungunya Virus RNA To Control Infection

ABSTRACT DEAD box RNA helicases regulate diverse facets of RNA biology. Proteins of this family carry out essential cellular functions, and emerging literature is revealing additional roles in immune defense. Using RNA interference screening, we identified an evolutionarily conserved antiviral role for the helicase DDX56 against the alphavirus Sindbis virus (SINV), a mosquito-transmitted pathogen that infects humans. Depletion of DDX56 enhanced infection in Drosophila and human cells. Furthermore, we found that DDX56 also controls the emerging alphavirus chikungunya virus (CHIKV) through an interferon-independent mechanism. Using cross-linking immunoprecipitation (CLIP-Seq), we identified a predicted stem-loop on the viral genomic RNA bound by DDX56. Mechanistically, we found that DDX56 levels increase in the cytoplasm during CHIKV infection. In the cytoplasm, DDX56 impacts the earliest step in the viral replication cycle by binding and destabilizing the incoming viral genomic RNA, thereby attenuating infection. Thus, DDX56 is a conserved antiviral RNA binding protein that controls alphavirus infection. IMPORTANCE Arthropod-borne viruses are diverse pathogens and include the emerging virus chikungunya virus, which is associated with human disease. Through genetic screening, we found that the conserved RNA binding protein DDX56 is antiviral against chikungunya virus in insects and humans. DDX56 relocalizes from the nucleus to the cytoplasm, where it binds to a stem-loop in the viral genome and destabilizes incoming genomes. Thus, DDX56 is an evolutionarily conserved antiviral factor that controls alphavirus infection.

Clinical and Serological Findings of Madariaga and Venezuelan Equine Encephalitis Viral Infections: A Follow-up Study 5 Years After an Outbreak in Panama

Background Human cases of Madariaga virus (MADV) infection were first detected during an outbreak in 2010 in eastern Panama, where Venezuelan equine encephalitis virus (VEEV) also circulates. Little is known about the long-term consequences of either alphavirus infection. Methods A follow-up study of the 2010 outbreak was undertaken in 2015. An additional survey was carried out 2 weeks after a separate 2017 alphavirus outbreak in a neighboring population in eastern Panama. Serological studies and statistical analyses were undertaken in both populations. Results Among the originally alphavirus-seronegative participants (n = 35 of 65), seroconversion was observed at a rate of 14.3% (95% CI, 4.8%–30.3%) for MADV and 8.6% (95% CI, 1.8%–23.1%) for VEEV over 5 years. Among the originally MADV-seropositive participants (n = 14 of 65), VEEV seroconversion occurred in 35.7% (95% CI, 12.8%–64.9%). In the VEEV-seropositive participants (n = 16 of 65), MADV seroconversion occurred in 6.3% (95% CI, 0.2%–30.2%). MADV seroreversion was observed in 14.3% (95% CI, 1.8%–42.8%) of those who were originally seropositive in 2010. VEEV seroconversion in the baseline MADV-seropositive participants was significantly higher than in alphavirus-negative participants. In the population sampled in 2017, MADV and VEEV seroprevalence was 13.2% and 16.8%, respectively. Memory loss, insomnia, irritability, and seizures were reported significantly more frequently in alphavirus-seropositive participants than in seronegative participants. Conclusions High rates of seroconversion to MADV and VEEV over 5 years suggest frequent circulation of both viruses in Panama. Enhanced susceptibility to VEEV infection may be conferred by MADV infection. We provide evidence of persistent neurologic symptoms up to 5 years following MADV and VEEV exposure.

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

ABSTRACT 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.

Clinical and serological findings of Madariaga and Venezuelan equine encephalitis viral infections: A follow-up study five years after an outbreak in Panama

BackgroundHuman cases of Madariaga virus (MADV) infection were first detected during an outbreak in 2010 in eastern Panama, where Venezuelan equine encephalitis virus (VEEV) also circulates. Little is known about the long-term consequences of either alphavirus infection.MethodsA follow-up study of the 2010 outbreak was undertaken in 2015. An additional survey was carried out two weeks after a separate 2017 alphavirus outbreak in a neighboring population in eastern Panama. Serological studies and statistical analysis were undertaken in both populations.ResultsAmongst the originally alphavirus-seronegative subjects (n=35 of 65), seroconversion was observed at a rate of 14.3% (95% CI: 4.8%-30.3%) for MADV and 8.6% (95% CI: 1.8%-23.1%) for VEEV over 5 years. Amongst the originally MADV seropositive subjects (n=14 of 65), VEEV seroconversion occurred in 35.7% (95% CI: 12.8%-64.9%). In the VEEV seropositive subjects (n=16 of 65), MADV seroconversion occurred in 6.3% (95% CI: 0.2%-30.2%). MADV seroreversion was observed in 14.3% (95% CI: 1.8%-42.8%) of those originally seropositive in 2010. VEEV seroconversion in the baseline MADV-seropositive subjects was significantly higher than in alphavirus-negative subjects. In the population sampled in 2017, MADV and VEEV seroprevalence was 13.2% and 16.8%, respectively. Memory loss, insomnia, irritability and seizures were reported significantly more frequently in alphavirus-seropositive subjects than in seronegative.ConclusionsHigh rates of 5-year seroconversions to MADV and VEEV suggest continuous circulation of both viruses in Panama. Enhanced susceptibility may be conferred by MADV towards VEEV. We provide evidence of persistent neurologic symptoms up to 5 years following MADV and VEEV exposure.summaryWe estimate seroconversion rates over a 5-year period to Madariaga (MADV) and Venezuelan equine encephalitis (VEEV) alphaviruses in Panama. Individuals with MADV antibodies seroconverted to VEEV at a rate greater than individuals who were alphavirus-negative at baseline. This was not observed in individuals with VEEV antibodies, suggesting asymmetric cross-immunity. Neurological sequelae were reported more frequently by MADV and/or VEEV seropositive-versus seronegative subjects.