Single-Round Infectious Particle Antiviral Screening Assays for the Japanese Encephalitis Virus

Abstract

Japanese Encephalitis virus (JEV) is a mosquito-borne flavivirus with a positive-sense single-stranded RNA genome that contains a big open reading frame (ORF) flanked by 5′- and 3′- untranslated regions (UTRs). Nearly 30,000 JE cases with 10,000 deaths are still annually reported in East Asia. Although the JEV genotype III vaccine has been licensed, it elicits a lower protection against other genotypes. Moreover, no effective treatment for a JE case is developed. This study constructed a pBR322-based and cytomegaloviruses (CMV) promoter-driven JEV replicon for the production of JEV single-round infectious particles (SRIPs) in a packaging cell line expressing viral structural proteins. Genetic instability of JEV genome cDNA in the pBR322 plasmid was associated with the prokaryotic promoter at 5′ end of the JEV genome that triggers the expression of the structural proteins in E. coli. JEV structural proteins were toxic E. coli, thus the encoding region for structural proteins was replaced by a reporter gene (enhanced green fluorescent protein, EGFP) that was in-frame fused with the first eight amino acids of the C protein at N-terminus and the foot-and-mouth disease virus (FMDV) 2A peptide at C-terminus in a pBR322-based JEV-EGFP replicon. JEV-EGFP SRIPs generated from JEV-EGFP replicon-transfected packaging cells displayed the infectivity with cytopathic effect induction, self-replication of viral genomes, and the expression of EGFP and viral proteins. Moreover, the combination of JEV-EGFP SRIP plus flow cytometry was used to determine the half maximal inhibitory concentration (IC50) values of antiviral agents according to fluorescent intensity and positivity of SRIP-infected packaging cells post treatment. MJ-47, a quinazolinone derivative, significantly inhibited JEV-induced cytopathic effect, reducing the replication and expression of JEV-EGFP replicon in vitro. The IC50 value of 6.28 µM for MJ-47 against JEV was determined by the assay of JEV-EGFP SRIP infection in packaging cells plus flow cytometry that was more sensitive, effective, and efficient compared to the traditional plaque assay. Therefore, the system of JEV-EGFP SRIPs plus flow cytometry was a rapid and reliable platform for screening antiviral agents and evaluating antiviral potency.

Tubacin, an HDAC6 Selective Inhibitor, Reduces the Replication of the Japanese Encephalitis Virus via the Decrease of Viral RNA Synthesis

“…TE671-CprME stable cell line generated in our laboratory is a packing cell line expressing JEV structure proteins (C, prM, and E) which was described in our previous work [34]. TE671-CprME cells were culture in MEM, 5% FBS, and 500 µg/mL G418 (Sigma, Saint Louis, MO, USA).…”

Structure analysis and antiviral activity of CW-33 analogues against Japanese encephalitis virus

“…7 ). Moreover, a CMV promoter-launched JEV-EGFP replicon reported in our prior study 10 was used to ascertain whether CW-33A influences on the viral protein expression and genome synthesis (Figs 8 and 9 ). Fluorescent microscopy revealed that CW-33A reduced the green fluorescence in the replicon-transfected cells and inhibited replicon-induced CPE in TE-671 cells (Fig.…”

“…After 15-min incubation, 10,000 cells from each sample are directly evaluated using flow cytometry with a 488 nm/620 nm. In the infectivity inhibition assay with immunofluorescence staining, JEV-infected cells with an MOI of 0.05 were fixed, permeabilized, and blocked after a 24-h incubation with the indicated concentrations of CW-33 and CW-33A, as described in our report 10 . Cells were stained with rabbit anti-JEV-NS3 (GeneTex, Inc) and secondary AF546 conjugated antibodies (ThermoFisher).…”

“…A JEV-EGFP replicon, previously constructed in our laboratory 10 , was used to explore whether CW-33A influenced viral protein translation and RNA genome replication stages of JEV life cycle. TE-671 cells were transfected with JEV-EGFP replicon and simultaneously treated with 10 μM of CW-33A or CW-33.…”

Selective Reactivity of Anti-Japanese Encephalitis Virus NS4B Antibody Towards Different Flaviviruses

“…The suggested optimal dilutions were 1:100-1:2000 for ICC/IFA, and 1:500-1:3000 for WB. This antibody has been utilized to study JEV [20], but not other members of the genus Flavivirus such as West Nile virus (WNV), dengue (DENV), and Zika virus (ZIKV). For the detection of NS4B protein in the present study, the anti-JEV NS4B antibody was diluted 1:150 (5.1 µg/mL) or 1:1500 (0.5 µg/mL) for IFA and WB assays, respectively.…”