Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. Injury to the joint is believed to occur because of viral and host immune-mediated effects. However, the exact involvement of the different immune mediators in CHIKV-induced pathogenesis is unknown. In this study, we assessed the roles of T cells in primary CHIKV infection, virus replication and dissemination, and virus persistence, as well as in the mediation of disease severity in adult RAG2−/−, CD4−/−, CD8−/−, and wild-type CHIKV C57BL/6J mice and in wild-type mice depleted of CD4+ or CD8+ T cells after Ab treatment. CHIKV-specific T cells in the spleen and footpad were investigated using IFN-γ ELISPOT. Interestingly, our results indicated that CHIKV-specific CD4+, but not CD8+, T cells are essential for the development of joint swelling without any effect on virus replication and dissemination. Infection in IFN-γ−/− mice demonstrated that pathogenic CD4+ T cells do not mediate inflammation via an IFN-γ–mediated pathway. Taken together, these observations strongly indicate that mechanisms of joint pathology induced by CHIKV in mice resemble those in humans and differ from infections caused by other arthritogenic viruses, such as Ross River virus.

The Togaviridae is a family of small, enveloped viruses with single-stranded, positive-sense RNA genomes of 10–12 kb. Within the family, the genus Alphavirus includes a large number of diverse species, while the genus Rubivirus includes the single species Rubella virus. Most alphaviruses are mosquito-borne and are pathogenic in their vertebrate hosts. Many are important human and veterinary pathogens (e.g. chikungunya virus and eastern equine encephalitis virus). Rubella virus is transmitted by respiratory routes among humans. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Togaviridae, which is available at www.ictv.global/report/togaviridae.

Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.

Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that has caused severe epidemics in Africa and Asia and occasionally in Europe. As of today, there is no licensed vaccine available to prevent CHIKV infection. Here we describe the development and evaluation of novel CHIKV vaccine candidates that were attenuated by deleting a large part of the gene encoding nsP3 or the entire gene encoding 6K and were administered as viral particles or infectious genomes launched by DNA. The resulting attenuated mutants were genetically stable and elicited high magnitudes of binding and neutralizing antibodies as well as strong T cell responses after a single immunization in C57BL/6 mice. Subsequent challenge with a high dose of CHIKV demonstrated that the induced antibody responses protected the animals from viremia and joint swelling. The protective antibody response was long-lived, and a second homologous immunization further enhanced immune responses. In summary, this report demonstrates a straightforward means of constructing stable and efficient attenuated CHIKV vaccine candidates that can be administered either as viral particles or as infectious genomes launched by DNA.IMPORTANCESimilar to other infectious diseases, the best means of preventing CHIKV infection would be by vaccination using an attenuated vaccine platform which preferably raises protective immunity after a single immunization. However, the attenuated CHIKV vaccine candidates developed to date rely on a small number of attenuating point mutations and are at risk of being unstable or even sensitive to reversion. We report here the construction and preclinical evaluation of novel CHIKV vaccine candidates that have been attenuated by introducing large deletions. The resulting mutants proved to be genetically stable, attenuated, highly immunogenic, and able to confer durable immunity after a single immunization. Moreover, these mutants can be administered either as viral particles or as DNA-launched infectious genomes, enabling evaluation of the most feasible vaccine modality for a certain setting. These CHIKV mutants could represent stable and efficient vaccine candidates against CHIKV.