doi:10.1128/jvi.74.13.6156-6161.2000. FIG?S2, TIF file, 1.5 MB. Copyright ? 2021 Bhaskar et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3. Relative type-1 interferon expression in RIG-I-depleted NSC34 cells upon JEV and CHPV contamination. Total RNA was extracted from mock-infected control, virus-infected NSC34, mock-infected RIG-I, or eGFP-transfected NSC34 and virus-infected RIG-I or eGFP-transfected NSC34 cells for studying type-1 IFN expression using qRT-PCR, with fold change being calculated with GAPDH as a loading control. Data are represented as mean SD from a minimum of 3 impartial experiments, where statistical significance (*, Bonferroni correction. Download FIG?S3, TIF file, 1.3 MB. Copyright ? 2021 Bhaskar et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Calpain Inhibitor II, ALLM International license. FIG?S5. Percent live/lifeless populace in RIG-I-transfected NSC34 versus negative-control (NC) transfected NSC34 upon computer virus infection. Cells were either transfected with RIG-I specific siRNA (Ddx58) or negative-control (NC) siRNA (eGFP) and were infected with JEV Calpain Inhibitor II, ALLM at an MOI of 1 1 and CHPV at an MOI of 0.1 for various time points postinfection. Green bars represent live NSC34 populace, and red bars represent lifeless NSC34 populace. Data plotted as percent live populace after JEV and CHPV contamination at various time points and are a representation of a minimum of 3 impartial experiments (mean SD) performed, where statistical significance (*, Bonferroni correction. Download FIG?S5, TIF file, 1.1 MB. Copyright ? 2021 Bhaskar et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S4. Studying relative expression of PAMP MDA5, TLR3, TLR4, and TLR7 upon contamination in RIG-I-depleted NASC34 cells. Protein lysates prepared from positive-control, mock-infected control, virus-infected NSC34, mock-infected RIG-I, or eGFP-transfected cells and virus-infected RIG-I or eGFP-transfected NSC34 cells were subjected to immunoblot analysis for studying expression of viral protein and various PAMPs, including MDA5, TLR3, TLR4, and TLR7, with -actin as a loading control. To rule HKE5 out the possibility of an experimental error in probing blots, positive control samples were included for each PAMP in respective gels. Lysate prepared from the AGS cell line was used as a positive control for MDA5. Similarly, JEV-infected N2A cells at 36 hpi were used as a positive control for TLR3 and TLR7, whereas lysate prepared from JEV-infected N9 cells (36 hpi) served as a positive control for TLR4. Data here are a representation of 3 impartial experiments. Download FIG?S4, TIF file, 0.7 MB. Copyright ? 2021 Bhaskar et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT Poliomyelitis-like illness is usually a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis computer virus (JEV) and Chandipura computer Calpain Inhibitor II, ALLM virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we exhibited that upon contamination, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic computer virus infections. values were decided (*, Bonferroni test. (C) Altered hindlimb clasping phenotype with common posture of hindlimbs retracted toward the midline or away from midline was scored as HLC (hindlimb clasping) score where data are represented as means SD.