The Use of Nanotrap Particles in the Enhanced Detection of Rift Valley Fever Virus Nucleoprotein

Abstract

BackgroundRift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.ResultsAfter screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC).ConclusionThis study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest.

Enhanced detection of respiratory pathogens with nanotrap particles

“…Furthermore, the particles are capable of concentrating analytes of interest from large volumes of complex biological fluids (such as nasal aspirates) into a significantly smaller volume (as low as 25 mL). 9,10 At the heart of Nanotrap technology is a base particle consisting of a network of crosslinked environmentally-responsive polymer chains that have been functionalized with affinity baits to facilitate analyte capture and retention. 11 The versatility of the Nanotrap particles stems from the variety of affinity baits that can be immobilized onto the polymer matrix.…”

“…[12][13][14] This novel sample preparation methodology has been utilized for the concentration and enhanced detection of infectious disease proteins, including a low-abundant bacterial antigen Outer surface protein A (OspA) used to diagnose Lyme disease 10 and the nucleoprotein of Rift Valley fever virus (RVFV). 9 While the Nanotrap particles were originally designed to specifically harvest proteins and other small molecules, recent findings by Shafagati et al have shown that the Nanotrap particles can also be used in the capture and detection of virions. 15 The ability of the Nanotrap particles to capture virions allows samples to be analyzed through numerous downstream analytical techniques (both protein and nucleic acid based) including standard sandwich ELISAs, lateral flow immunoassays, mass spectroscopy (MS) techniques, or q-RT-PCR assays.…”

Magnetic Nanotrap Particles Preserve the Stability of Venezuelan Equine Encephalitis Virus in Blood for Laboratory Detection

“…Data are plotted as the geometric mean ± SEM from results of two independent experiments (n = 2). red 120 affinity bait allows NT particles to capture a wide range of RNA viruses (19,29). Using the newly developed magnetic NT particles could simplify workflows allowing the isolation of NT particles in 3-5 s instead of long centrifugation steps.…”

“…It has been shown in our previous studies that NT particles capture and enrich various viruses, such as influenza virus and RVFV (19,21,29), but it has never been shown if NT particles can directly bind to viruses. Using transmission electronic microscopy (TEM) we visualized the direct interaction between NT particles and virions.…”

“…Previous studies have shown that NT particles are efficient in the preservation of RVFV and nucleoproteins in biologically relevant matrices (19,29). However, the effect of NT in the stabilization of virions in a complex matrix such as whole human blood has never been tested before.…”