Plants extracts from Cameroon pharmacopeia strongly inhibit the Chikungunya virus infection by targeting entry and replication steps.

ETHNOPHARMACOLOGICAL RELEVANCE: Cameroon is one of the sub-Saharan African countries affected by Chikungunya virus (CHIKV). With the absence of approved treatment, this disease represents globally a major public health concern. Several plants are traditionally used in Cameroon for the treatment of virus induced fever and arthralgia. But to date there is no study that validate the efficacy of these plants for the treatment of Chikungunya virus infection. AIM OF THE STUDY: This study aims to explore the inhition effect, mechanism of action of plant extracts against Chikungunya virus. MATERIAL AND METHODS: An ethnobotanical survey conducted in some regions of Cameroon, led to the identification of nine medicinal plants used in traditional medicine for the healing of fever-related diseases and arthritis. Crude hydro-ethanolic extracts of each plant were prepared by maceration and their effects against CHIKV infection were investigated. CHIKV S27 strain was used to infection in Vero cell line. The antiviral activities were determined by plaque assay and/or RT-PCR targeting E1 envelope gene of CHIKV. Dose-response studies of the active plants were also determined by flow cytometry and Western blot. RESULTS: Four extracts, Entada africana Guill et Pers. (E4), Entandrophragma cylindricum Sprague (EI), Khaya grandifoliola C. D.C. Sapindales (E2) and Macaranga hurifolia Beille (E6) showed antiviral activity with the half-maximal inhibitory concentration of 8.29; 8.14; 12.81 and 26.89 µg/mL respectively. All extracts were nontoxic up to the concentration of 100 µg/µL. Entandrophragma cylindricum Sprague (EI), Khaya grandifoliola C. D.C. Sapindales (E2), and Entada africana Guill et Pers. (E4) showed strong inhibition on the entry step of viral infection. At the same time, only Entandrophragma cylindricum Sprague (EI) inhibited the viral titer significantly in replication and intercellular assembly steps. Four plant extracts namely Entandrophragma cylindricum Sprague (EI), Macaranga hurifolia Beille (E6), Phragmentera capitata (Sprengel) Balle (E12), and Detarium microcarpum (E13) were effective against egression step. CONCLUSIONS: Together, the results of this study showed anti-chikungunya activities of Entandrophragma cylindricum Sprague (EI) and Macaranga hurifolia Beille (E6), with therapeutics perspectives and can be promising sources of the development of anti-CHIKV molecule in future.

Evaluation of novobiocin and telmisartan for anti-CHIKV activity.

Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved drugs with a good safety profile used in other diseases for their new property of anti-chikungunya activity. It provides a base for a fast and efficient approach to bring a novel therapy from bench to bedside by the process of drug-repositioning. We utilized an in-silico drug screening with FDA approved molecule library to identify inhibitors of the chikungunya nsP2 protease, a multifunctional and essential non-structural protein required for virus replication. Telmisartan, an anti-hypertension drug, and the antibiotic novobiocin emerged among top hits on the screen. Further, SPR experiments revealed strong in-vitro binding of telmisartan and novobiocin to nsP2 protein. Additionally, small angle x-ray scattering suggested binding of molecules to nsP2 and post-binding compaction and retention of monomeric state in the protein-inhibitor complex. Protease activity measurement revealed that both compounds inhibited nsP2 protease activity with IC50 values in the low micromolar range. More importantly, plaque formation assays could show the effectiveness of these drugs in suppressing virus propagation in host cells. We propose novobiocin and telmisartan as potential inhibitors of chikungunya replication. Further research is required to establish the molecules as antivirals of clinical relevance against chikungunya.

Green synthesis of nanometal impregnated biomass - antiviral potential.

Due to the epidemic nature, Chikungunya virus (CHIKV), arthropod-borne alphaviruses, is considered as a potential public health threat worldwide. Currently, no antiviral drug or vaccine is available against alphaviruses. Nanotechnology with green synthesis of nanoparticles is a novel and emerging interdisciplinary field of science that involves the production and usage of nanomaterials. Nano-biomaterials are clean, safe, non-toxic, cost effective and environment friendly which have been largely exploited to develop novel antiviral drugs in recent years. In the current work, different nanometals (Ag, Fe and Zn) were impregnated into raw Citrus limetta peels. The characterization of synthesized biomaterials was done using XRD and XPS which confirmed the presence of metallic forms of Fe and Ag whereas ZnO was formed during the impregnation process. Based on TEM analysis, the average size of nanoparticles was found to be 5, 32 and 12 nm for Ag, Fe and ZnO respectively. For evaluation of antiviral activity, nano-biomaterials AgNps, FeNps and ZnONps were processed for cell culture based experiments. All the three nano-biomaterials significantly reduced CHIKV viral titer and viral RNA level as determined by plaque assay, real time PCR and immunofluorescence assay. The results revealed that silver and iron nano-biomaterials have higher antiviral potential in comparison to zinc against CHIKV. In conclusion, green synthesized nano-biomaterials can act as good antiviral agents. These nano-biomaterials can further be processed for formulation of effective and promising nanomedicines against CHIKV and other similar viruses which are a threat to human health.

Antiviral activity of selected flavonoids against Chikungunya virus.

This study focuses on the antiviral activity of selected flavonoids against the Chikungunya virus (CHIKV), a mosquito-transmitted virus that can cause incapacitating arthritis in infected individuals. Based on the results of screening on Vero cells, the tested compounds were evaluated further with various assays, including cytotoxicity assay, virus yield assay by quantitative reverse transcription polymerase chain reaction (qRT-PCR), virus RNA replication assay with a CHIKV replicon cell line, Western blotting, and quantitative immunofluorescence assay. Baicalein, fisetin, and quercetagetin displayed potent inhibition of CHIKV infection, with 50% inhibitory concentrations [IC50] of 1.891 µg/ml (6.997 µM), 8.444 µg/ml (29.5 µM), and 13.85 µg/ml (43.52 µM), respectively, and with minimal cytotoxicity. The time-of-addition studies and various antiviral assays demonstrated that baicalein and quercetagetin mainly inhibited CHIKV binding to the Vero cells and displayed potent activity against extracellular CHIKV particles. The qRT-PCR, immunofluorescence assay, and Western blot analyses indicated that each of these flavonoids affects CHIKV RNA production and viral protein expression. These data provide the first evidence of the intracellular anti-CHIKV activity of baicalein, fisetin, and quercetagetin.

Utilization and Assessment of Throat Swab and Urine Specimens for Diagnosis of Chikungunya Virus Infection.

Chikungunya is a mosquito-borne infection with clinical presentation of fever, arthralgia, and rash. The etiological agent Chikungunya virus (CHIKV) is generally transmitted from primates to humans through the bites of infected Aedes aegypti and Aedes albopictus mosquitoes. Outbreaks of Chikungunya occur commonly with varied morbidity, mortality, and sequele according to the epidemiological, ecological, seasonal, and geographical impact. Investigations are required to be conducted as a part of the public health service to understand and report the suspected cases as confirmed by laboratory diagnosis. Holistic sampling at a time of different types would be useful for laboratory testing, result conclusion, and reporting in a valid way. The use of serum samples for virus detection, virus isolation, and serology is routinely practiced, but sometimes serum samples from pediatric and other cases may not be easily available. In such a situation, easily available throat swabs and urine samples could be useful. It is already well reported for measles, rubella, and mumps diseases to have the virus diagnosis from throat swabs and urine. Here, we present the protocols for diagnosis of CHIKV using throat swab and urine specimens.

Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.

Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis.

We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

High-throughput screening for broad-spectrum chemical inhibitors of RNA viruses.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

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.