Abstract
West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 101.5 TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.
Highlights
West Nile virus (WNV) infection leads to an acute febrile zoonosis, which can cause disease in birds, humans and horses1 (Gubler, 2001)
We developed a RT-Loop-mediated isothermal amplification (LAMP) assay coupled with a vertical flow (VF) visualization strip for rapid, simple, and accurate visual detection of WNV
To determine the optimal conditions for the RT-LAMP-VF assay, synthesized RNA transcripts were used as a template to optimize both assay temperature and assay time
Summary
West Nile virus (WNV) infection leads to an acute febrile zoonosis, which can cause disease in birds, humans and horses (Gubler, 2001). In America, as of January 12, 2016, a total of 48 states have reported WNV infection in humans, birds, or mosquitoes. WNV is an arthropod-borne, neurotropic, enveloped Flavivirus with a single-stranded, positive-sense RNA genome and is a member of the Japanese encephalitis virus (JEV) serogroup, which includes JEV, Murray Valley encephalitis virus (MVEV) and St. Louis encephalitis virus (Mukhopadhyay et al, 2003; Solomon et al, 2003; Lim et al, 2011). Phylogenetic analysis of the WNV E protein sequence has indicated that WNV can be separated into two main lineages (lineages 1 and 2) as well as several additional minor lineages (lineage 3, lineage 4, lineage 5 and the putative lineage 6; Lanciotti et al, 2002; Vazquez et al, 2010; Del Amo et al, 2013; Faggioni et al, 2014)
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