Development of a high-sensitivity vertical flow immunoassay for the detection of Rift Valley fever virus

Rift Valley fever (RVF) is a vector-borne, zoonotic infectious disease with a proven history of morbidity and mortality in both humans and animals. Rift Valley fever virus (RVFV) is categorized as a high-priority biothreat agent by the Centers for Disease Control and Prevention and poses a serious national threat due to its ease of dissemination and potential for social disruption. RVF often presents as a febrile disease without specific symptoms, making early-stage detection particularly challenging. As such, it is critical that rapid, sensitive, and specific diagnostics are available for the detection of RVFV. While lateral flow immunoassays (LFIs) have been developed and validated for point-of-care (POC) diagnostics, vertical flow immunoassays (VFIs) provide enhanced analytical sensitivity and are equally suitable for POC use. In this study, we developed a VFI system for the detection of RVFV, achieving a limit of detection of 0.78 ng/mL, which is a 2.5-fold increase in analytical sensitivity compared to an LFI prototype. Furthermore, minimal cross-reactivity was demonstrated when performing the assay with target analytes of other high-priority biothreats and one other common viral nucleoprotein. This high-sensitivity VFI has the potential to prove useful for the detection of RVFV and other high-priority biothreat agents at the POC. IMPORTANCE In this study, we have developed a rapid, sensitive vertical flow immunoassay (VFI) for the detection of Rift Valley fever virus (RVFV) in spiked human serum. The prototype diagnostic described in this research was shown to be more sensitive than traditional methods, such as lateral flow dipstick tests. Moreover, the VFI is readily deployable at the point of care in resource-limited settings. The ability of the described diagnostic to accurately and rapidly detect RVFV in samples could expedite the delivery of life-saving care and thus improve patient outcomes.