Differential regulation of TLR mediated innate immune response of mouse neuronal cells following infection with novel ECSA genotype of Chikungunya virus with and without E1:A226V mutation.

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world including Africa, Indian Ocean Islands, India and South-East Asia. The appearance of many unusual severe manifestations including neurological disorders was reported in post resurgence epidemics with implication of novel East Central South African (ECSA) genotype with E1:A226V mutation. The molecular mechanism of CHIKV neuropathogenesis is not yet understood and very little is known about the host-pathogen interactions. In the present study replication kinetics and innate immune response of ECSA genotype of CHIKV with and without A226V mutation were determined in mouse neuroblastoma cell line (N2a). The 226V mutant strain was more replication competent in N2a cells with a peak titer of 10(8)PFU/ml compared to 10(6)PFU/ml for A226 virus. Besides, the 226V mutant virus showed relatively less induction of antiviral genes i.e. IFN-ß, OAS-3, MX-2, ISG-15 and Toll like receptors 3 and 7 as compared to non mutant strain (A226). Further pretreatment of N2a cells either with Poly I: C, IFN-ß or TNF-α resulted in inhibition of CHIKV replication hence confirming the role of TLR mediated innate immune response in CHIKV pathogenesis. Differential regulation of TLRs and associated down stream antiviral genes might have attributed for increased pathogenesis of the 226V mutant novel ECSA genotype of CHIKV during the recent epidemics.

Characterization of Chikungunya virus infection in human neuroblastoma SH-SY5Y cells: role of apoptosis in neuronal cell death.

Chikungunya infection is characterized by fever, rash and arthritis. The disease pathogenesis is still poorly understood. Hence, unveiling the molecular mechanisms that govern the survival and death of neuronal cells infected by Chikungunya virus (CHIKV) was the particular interest of this study. Human neuroblastoma SH-SY5Y cells infected with CHIKV showed characteristic features of apoptosis with activation of caspase-3, cleavage of PARP and translocation of Cyt-c. Cells also showed a loss in the intracellular level of GSH and an increase in the lipid peroxidation of the infected cells with the increasing time of infection, which indicated the involvement of oxidative stress in Chikungunya infection. There was observed a gradual decrease in the fold change of antioxidant enzymes and an increase in the fold change of pro-inflammatory cytokines. This study suggested the implication of virus induced apoptosis in disease pathogenesis which may give a fresh insight for CHIKV induced neuronal cell damage and antiviral therapeutics.

Cellular IMPDH enzyme activity is a potential target for the inhibition of Chikungunya virus replication and virus induced apoptosis in cultured mammalian cells.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotide, namely, the conversion of IMP to XMP. The depletion of the intracellular GTP and dGTP pools is the major event occurring in the cells exposed to the inhibitors such as mycophenolic acid. The present study was undertaken with an objective to assess the antiviral potential of mycophenolic acid (MPA) against Chikungunya virus via inhibition of IMPDH enzyme in Vero cells. The inhibitory potential of MPA on CHIKV replication was assessed by virus inhibition assay (cytopathic effect, immunofluorescence), virus yield reduction assay and cell viability assay. Inhibition of virus induced apoptosis was analyzed by Hoechst staining, DNA fragmentation, immunoblotting of Caspase-3, PARP and Bcl-2. Percentage apoptotic cell population was determined by flow cytometry. Total genome infectivity was determined by analyzing the ratio of total infectious viral particles to the genome copy number. Non-toxic concentration of MPA (10 µM) reduced ≥ 99.9% CHIKV titre in Vero cells. MPA via depletion of substrate for polymerase (GTP), inhibited CHIKV induced apoptosis. By limiting the rate of de novo synthesis of guanosine nucleotide, MPA could apparently block the formation of the CHIKV progeny. The antiviral activity of MPA against Chikungunya virus is mediated through depletion of GTP pool via inhibition of IMPDH as demonstrated by Immunoblotting and different microscopic analysis.