Screening of viral inhibitors through induction of cytopathic effects (CPE) by conventional method has been applied for various viruses including Chikungunya virus (CHIKV), a significant arbovirus. However, it does not provide the information about cytopathic effect from the beginning and throughout the course of virus replication. Conventionally, most of the approaches are constructed on laborious end-point assays which are not capable for detecting minute and rapid changes in cellular morphology. Therefore, we developed a label-free and dynamical method for monitoring the cellular features that comprises cell attachment, proliferation, and viral cytopathogenicity, known as the xCELLigence real-time cell analysis (RTCA). In this chapter, we provide a RTCA protocol for quantitative analysis of CHIKV replication using an infected Vero cell line treated with ribavirin as an in vitro model.
Chikungunya virus (CHIKV) represents a reemerging global threat to human health. Recent outbreaks across Asia, Europe, Africa, and the Caribbean have prompted renewed scientific interest in this mosquito-borne alphavirus. There are currently no vaccines against CHIKV, and treatment has been limited to nonspecific antiviral agents, with suboptimal outcomes. Herein, we have identified β-d-N4-hydroxycytidine (NHC) as a novel inhibitor of CHIKV. NHC behaves as a pyrimidine ribonucleoside and selectively inhibits CHIKV replication in cell culture.
Chikungunya virus is a reemerging arbovirus transmitted mainly by Aedes mosquitoes. As there are no specific treatments available, Chikungunya virus infection is a significant public health problem. This study investigated 120 extracts from selected medicinal plants for anti-Chikungunya virus activity. The plant materials were subjected to sequential solvent extraction to obtain six different extracts for each plant. The cytotoxicity and antiviral activity of each extract were examined using African monkey kidney epithelial (Vero) cells. The ethanol, methanol and chloroform extracts of Tradescantia spathacea (Commelinaceae) leaves showed the strongest cytopathic effect inhibition on Vero cells, resulting in cell viabilities of 92.6% ± 1.0% (512 μg/ml), 91.5% ± 1.7% (512 μg/ml) and 88.8% ± 2.4% (80 μg/ml) respectively. However, quantitative RT-PCR analysis revealed that the chloroform extract of Rhapis excelsa (Arecaceae) leaves resulted in the highest percentage of reduction of viral load (98.1%), followed by the ethyl acetate extract of Vernonia amygdalina (Compositae) leaves (95.5%). The corresponding 50% effective concentrations (EC50) and selectivity indices for these two extracts were 29.9 ± 0.9 and 32.4 ± 1.3 μg/ml, and 5.4 and 5.1 respectively. Rhapis excelsa and Vernonia amygdalina could be sources of anti-Chikungunya virus agents. [Int Microbiol 19(3):175-182 (2016)].
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.
The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection.
Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC50 of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.
The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 μM, ≈ 350 μM and ≈110 μM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 μM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of -9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 μM followed by the inhibition of synthesis of positive-strand genomic (EC50 ≈ 13 μM) and subgenomic RNAs (EC50 ≈ 14 μM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 μM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.