Chikungunya virus (CHIKV) infection is one of the most challenging human Arboviral infections with global significance and without any specific antiviral. In this investigation, 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) was synthesised as a molecular hybrid of 2-methyl benzimidazole and isatin-β-thiosemicarbazone and its anti-CHIKV property was evaluated. The release of infectious virus particles was calculated by plaque assay, expression profile of viral RNA was estimated by RT-PCR and viral protein profiles were assessed by Western blot and FACS analyses. The safety index of MBZM-N-IBT was found to be >21. The CHIKV infectious viral particle formation was abrogated around 76.02% by MBZM-N-IBT during infection in mammalian system and the viral RNA synthesis was reduced by 65.53% and 23.71% for nsP2 and E1 respectively. Surprisingly, the viral protein levels were reduced by 97% for both nsP2 and E2. In the time-of-addition experiment it abrogated viral infection at early as well as late phase of viral life cycle, which indicates about multiple mechanisms for its anti-CHIKV action. In silico analysis justified development of MBZM-N-IBT with good affinities for potential target proteins of CHIKV and related virus. With predictions of good drug-likeness property, it shows potential of a drug candidate which needs further experimental validation.

BackgroundThe high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV) emphasize the need to understand the biology of the virus for developing effective antiviral therapies.Methods and FindingsIn this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90) mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27) and Indian outbreak strain of 2006 (DRDE-06). Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA) can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production.ConclusionHsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06, which will be informative for developing effective anti-CHIKV therapies in future.

BackgroundThe recent re-emergence of Chikungunya virus (CHIKV) in India after 32 years and its worldwide epidemics with unprecedented magnitude raised a great public health concern.Methods and FindingsIn this study, a biological comparison was carried out between a novel 2006 Indian CHIKV outbreak strain, DRDE-06 and the prototype strain S-27 in mammalian cells in order to understand their differential infection pattern. Results showed that S-27 produced maximum number of progenies (2.43E+06 PFU/ml) at 20 to 24 hours post infection whereas DRDE-06 produced more than double number of progenies around 8 hours post infection in mammalian cells. Moreover, the observation of cytopathic effect, detection of viral proteins and viral proliferation assay confirmed the remarkably faster and significantly higher replication efficiency of DRDE-06. Moreover, our mutational analysis of whole genome of DRDE-06 revealed the presence of nineteen mutations as compared to S-27, whereas the analysis of 273 global isolates showed the consistent presence of fifteen out of nineteen mutations in almost all outbreak isolates. Further analysis revealed that ∼46% of recent outbreak strains including DRDE-06 do not contain the E1-A226V mutation which was earlier shown to be associated with the adaptation of CHIKV in a new vector species, Aedes albopictus.ConclusionsA novel 2006 Indian CHIKV outbreak strain, DRDE-06 exhibits different pattern of infection as compared to prototype strain, S-27. This might be associated to some specific mutations observed in genome wide mutational analysis in DRDE-06 which emphasizes the need of future experimental investigation.

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