Evaluation of RNAi (RNA interference) technology in curing Chandipura virus infection in vitro and in vivo

Abstract

In parts of India, Chandipura Virus (CHPV) has emerged as an encephalitis causing pathogen in both epidemic and sporadic forms. This pediatric disease follows rapid course leading to 55-75% mortality. In the absence of specific treatment, effectiveness of RNA interference (RNAi) was evaluated. As a primary requisite, a highly sensitive method for quantifying CHPV RNA by real time one step reverse transcriptase PCR (real time one step RT-PCR) using TaqMan technology was developed and its performance was evaluated in clinical settings. Primers and probes corresponding to G and P genes were designed and used to standardize real time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which was considered as a gold standard.Real-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for both G and P genes (r2=0.99) from 50-1010 RNA copies/ reaction for G gene and 102-1010 for P gene. The newly developed real time one step RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml). Vero and PS cell-lines (1.2 ×103 PFU/ml) were least sensitive.Whereas specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.