Development of an improved RT-qPCR Assay for detection of Japanese encephalitis virus (JEV) RNA including a systematic review and comprehensive comparison with published methods

Abstract

BackgroundJapanese encephalitis virus (JEV) is a major cause of encephalitis in Asia, and the commonest cause of mosquito-borne encephalitis worldwide. Detection of JEV RNA remains challenging due to the characteristic brief and low viraemia, with 0–25% of patients positive, and the mainstay of diagnosis remains detection of anti-JEV IgM antibody.MethodsWe performed a systematic review of published RT-PCR protocols, and evaluated them in silico and in vitro alongside new primers and probes designed using a multiple genome alignment of all JEV strains >9,000nt from GenBank, downloaded from the NCBI website (November 2016). The new assays included pan-genotype and genotype specific assays targeting genotypes 1 and 3.ResultsTen RT-qPCR assays were compared, a pre-existing in-house assay, three published assays and six newly designed assays, using serial RNA dilutions. We selected three assays, one published and two novel assays, with the lowest limit of detection (LOD) for further optimisation and validation. One of the novel assays, detecting NS2A, showed the best results, with LOD approximately 4 copies/ reaction, and no cross-reaction on testing closely related viruses in the JEV serocomplex, West Nile Virus and St. Louis Virus. The optimised assays were validated in consecutive patients with central nervous system infections admitted to hospitals in Laos, testing paired CSF and serum samples.ConclusionsWe succeeded in developing a JEV specific RT-qPCR assay with at least 1 log10 improved sensitivity as compared to existing assays. Further evaluation is required, field-testing the assay in a larger group of patients.

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests

“…The specimens were extracted using the Biomerieux NucliSENS Easy Mag automated extraction system. NML performed real‐time reverse‐transcription (rRT‐PCR) assays for WNV and JEV (Taqman Fast Virus 1‐step Mastermix; Thermo‐Fisher) using the QuantStudio‐3 (Thermo‐Fisher) . The NML performed two different assays for each virus (JEV:NS2A and NS3 targets; WNV: ENV and 3′ non‐coding region); a specimen was considered positive if both assay targets were detected.…”

Investigation of Japanese encephalitis virus as a cause of acute encephalitis in southern Pakistan, April 2015–January 2018

“…Given the high incidence of dengue, ongoing evidence of WNV transmission, and limited but past evidence of JEV transmission in Pakistan, patients with acute encephalitis of unknown etiology should be tested for WNV, JEV, and DENV IgM in both CSF and serum samples, with convalescent serum testing at 10 days. For WNV and JEV, low viral titers in blood and CSF and typically high neutralizing antibodies at the time of presentation mean molecular or virus detection methods are usually unhelpful, so IgM detection followed by PRNT to confirm the infecting virus remains the gold standard for confirmatory diagnosis [27,28]. We therefore employed serologic tests to determine the possible presence of JEV infection in our study, despite the known challenges of cross-reactivity in serologic testing.…”

“…We therefore employed serologic tests to determine the possible presence of JEV infection in our study, despite the known challenges of cross-reactivity in serologic testing. Molecular diagnostic approaches would provide greater specificity, and recent developments in tests with greater sensitivity have shown promise, but these tests are still insufficiently sensitive for routine diagnostic purposes [27,28,29]. Systematic and reliable laboratory testing will be essential to identify transmission and emergence of JEV in Pakistan.…”

Low-Cost Battery-Powered and User-Friendly Real-Time Quantitative PCR System for the Detection of Multigene

“…Polymerase chain reaction (PCR) is a molecular reaction used to amplify a specific segment of DNA [1][2][3]. PCR plays an important role in many fields, including pathogen diagnosis, bioprospecting, and environmental protection [4][5][6][7][8][9][10][11][12]. In traditional PCR, the detection of amplified products largely relies on electrophoretic gel analysis [3], which is time-consuming and makes it difficult to quantify the target molecules [13].…”