AUTHOR=Tomar Priyanka Singh , Kumar Jyoti S. , Patel Sapan , Sharma Shashi 

TITLE=Polymerase Spiral Reaction Assay for Rapid and Real Time Detection of West Nile Virus From Clinical Samples

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 10 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.00426

DOI=10.3389/fcimb.2020.00426

ISSN=2235-2988

ABSTRACT=West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 630C for 60 minutes using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of RT-PSR assay was 1 copy which is 100 fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. In addition, a panel of positive serum samples of Chikungunya (n=12) along with samples from healthy volunteers (n=20) were also included as the negative control in this study. The specificities of the selected primer sets were established by cross-reactivity studies with other closely related members of the Flaviviruses Alphaviruses and Morbilliviruses group. No cross- reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. PSR assay has potential that can be used for rapid screening of large number of clinical samples in endemic areas during an outbreak.