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. 2019 Sep 11;13(9):e0007700.
doi: 10.1371/journal.pntd.0007700. eCollection 2019 Sep.

Development and validation of a pen side test for Rift Valley fever

Affiliations

Development and validation of a pen side test for Rift Valley fever

Catherine Cêtre-Sossah et al. PLoS Negl Trop Dis. .

Abstract

Background: Rift Valley fever (RVF) is one of the main vector borne zoonotic diseases that affects a wide range of ruminants and human beings in Africa and the Arabian Peninsula. A rapid and specific test for RVF diagnosis at the site of a suspected outbreak is crucial for the implementation of control measures.

Methodology/principal findings: A first-line lateral flow immunochromatographic strip test (LFT) was developed for the detection of the nucleoprotein (N) of the RVF virus (RVFV). Its diagnostic performance characteristics were evaluated using reference stocks isolates recovered from different hosts and in geographic regions mimicking clinical specimens and from known RVF negative serum samples. A high level of diagnostic accuracy (DSe (35/35), DSp (167/169)) was observed, including the absence of cross-reactivity with viruses belonging to different genera.

Conclusion/significance: The fact no specialized reagents and laboratory equipment are needed, make this assay a valuable, first-line diagnostic tool in resource-poor diagnostic territories for on-site RVFV detection, however the staff require training.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Coproduction of RVFV N and GN/GC proteins in insect cells.
Controls (uninfected and wild-type AcMNPV-infected cells) gave no signal. A. SDS-PAGE/Western blot, anti-N detection. Lane 1: Sf9 crude cell lysates coproducing N and GN/GC proteins. Lane 2: Sf9 crude supernatant coproducing N and GN/GC proteins. Lane 3: purified N and GN/GC proteins. B. SDS-PAGE/Western blot, anti-N/GN/GC detection. Lane a: Sf9 crude supernatant coproducing N and GN/GC proteins. Lane b: copurified N/GN/GC proteins. A double band at/above 45 kDa might correspond to products of GN/GC cleavage and maturation, and possibly to partial and/or nonspecific processed forms.
Fig 2
Fig 2. Specificity of the two Mabs 8E10-4A4 and 10H3-4E4-3D5 used in combination in the LFT for RVFV.
Specific binding of the Mabs to RVFV N protein was examined by (A) ELISA with RVFV copurified N/GN/GC proteins (ELISA N/GN/GC), ELISA with coating of Bac-N nucleoprotein expressed by Sf9 insect cells (ELISA N). Readings are OD (Optical Density) values measured at 450 nm. Mabs were tested diluted 1:10 in culture medium and run in duplicates (mean +/- 3 SD). The positive cut-off value of the test is an OD value > 0.300, the positive control (mouse immunized with RVF N/GN/GC sampled at day 41, dilution 1:100) must have an OD value > 1.200 and the negative control (non immunized OF1 mouse control) must have an OD value < 0.300. A tested sample is detected positive when the OD value > 0.300. (B) Immunofluorescence Assay (IFA) x 10, green fluorescence: positive sample, Mab directed against peste des petits ruminants virus (PPRV), negative control, no fluorescence.
Fig 3
Fig 3. RVF LFT strip test for the detection of RVF infection using the two selected Mabs.
(A) Diagram of the rapid RVF LFT for the detection of the RVF N protein, (B) Results of LFT strip. The serum sample or the viral suspension (150 μl) was mixed with 150 μl of sample buffer and applied to the S hole of the strips for migration. Results were recorded after 15 minutes. The test is valid when a red band is visible at the same level as the label C. S stands for Sample, C for Control and T for Test.

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