A combination of doxycycline and ribavirin alleviated chikungunya infection

Abstract

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.

Fig 1. The inhibitory effect of antimicrobial agents against CHIKV replication in Vero cells.

(A) Viral load was significantly reduced after the treatment with TETRA derivatives and the antiviral drugs compared with the untreated CHIKV-infected cells. DOXY showed highest inhibitory effect against CHIKV replication in Vero cells compared to the control, ROLI and TETRA. RIBA showed higher inhibitory effect (p<0.01) against CHIKV compared to ACIC and other TETRA derivatives except DOXY. (B) The chemical structure of the medical compounds (One way ANOVA, P<0.01).

Fig 2. Docking studies of DOXY to CHIKV cysteine protease and E2 glycoprotein.

The input files for AutoDock Vina were prepared using AutoDock Tools 1.5.6 where a grid box of 30x30x30 ų was used by the default settings of 10 docked conformations for each run with the Iterated Local Search global optimizer algorithm. (A) DOXY and viral protease binding is due to non-polar and van der Waal interactions. The predicted binding of DOXY to protease was not at the binding site indicating to possible non-competitive inhibition of DOXY against CHIKV protease. (B) Binding of DOXY to viral E2 glycoprotein involved the amino acids L16, P240 and L241 in addition to known E2 binding site to cell receptors (circled).

Fig 3. Inhibition of CHIKV after the treatment with DOXY, RIBA and DOXY+RIBA.

(A) ELISA-like cell-based assay was used to evaluate the antiviral activity of the drugs. Viral antigen was detected by CHIKV antibody and anti-mouse IgG conjugated with alkaline phosphatase. The 50% effective concentration (EC₅₀) of the DOXY was calculated to be 10.95±2.12 μM and the RIBA drug was 15.51±1.62 μM, while DOXY+RIBA (1:1) showed an EC₅₀ of 4.52±1.42 μM. (B) Levels of viral proteins after the treatment with drugs were measured by ELISA-like cell-based assay of triplicates at 4, 8, 24, 48 and 72 h post infection. The OD values were measured by an ELISA reader at 490–650 nm. The result showed considerable improvement on performance when the test compound were administered together compared to individual treatment (Two way ANOVA, *P<0.05).

Fig 4. Mode of inhibition of DOXY and its combination with RIBA against CHIKV.

(A) Viral inactivation assay showed significant viral inhibition after the treatment with DOXY compared with RIBA; while DOXY+RIBA showed the highest inhibition percentage. (B) Viral attachment assay showed that DOXY significantly inhibited virus attachment to target cells compared to RIBA and the combination of DOXY+RIBA showed the highest inhibitory effect. (C) Post-infection treatment showed that DOXY inhibitory effect against CHIKV was lower than the inhibitory effect of RIBA. The combination treatment significantly inhibited virus replication compared with the individual treatments. (D) Cell viability of CHIKV-infected cells after treatment with DOXY, RIBA and a DOXY+RIBA compared with untreated cells (One Way ANOVA, *P<0.05, **P<0.01).

Fig 5. CHIKV-infected mice showed liver and spleen hypertrophy, but reduction in size was evident after treatment.

(A) Treatment with DOXY+RIBA led to significant reduction in liver weight (p<0.05) compared with infected group. (B) Treatment with DOXY+RIBA showed considerable reduction in spleen weight (p<0.01), as evidenced by Kruskal-Wallis test followed by Dunn’s post-test. (C) Viral titer in blood of infected mice was quantified by plaque formation assay (limit of detection was 10 pfu) after 7 days post infection. Although RIBA significantly (One Way ANOVA, P<0.05) reduced viral titer, DOXY+RIBA showed a more prominent reduction compared to control (One Way ANOVA, P<0.01).

Fig 6. Histopathology of the liver and spleen of CHIKV-infected mice after the treatment with DOXY-RIBA.

The liver of infected mice showed accumulated infiltrated fluid due to inflammation (black arrows), many swollen hepatocyte (white arrows) and few shrinking apoptotic cells (small arrows). DOXY- or RIBA- treated mice showed reduction in abnormal cells and inflammation. DOXY+RIBA showed lesser shrinking apoptotic cells (small arrows) while other infection features disappeared. The existence of binucleate hepatocytes indicated accelerated liver cell renewal (arrowheads). The spleen of infected mice showed shrinking apoptotic cells and accumulated infiltrated fluid due to inflammation (black arrows), and this symptoms were reduced due to treatment with DOXY+RIBA (scale bars 50 μM).