Use of Nanotrap particles for the capture and enrichment of Zika, chikungunya and dengue viruses in urine

Abstract

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.

Fig 1. Screening of NT particles for febrile illness pathogen enrichment.

(A) CHIKV, (B) ZIKV and (C) DENV were spiked into urine and incubated with CN1030, CN2030, CN2010 or CN4000. Viral RNA was extracted and quantitate by RT-qPCR. Samples without NT particles (-NT) are included as controls. Numbers above the +NT bars indicate the fold-change enrichment based on the following calculating formula: genomic copies of NT positive were divided by those in Nanotrap negative (no-NT control) groups. (D) The diagrams of chemical structures of three affinity baits. Data was presented as mean ± SEM from three or four biological replicates. P value is ≤ 0.05; ** P ≤ 0.01.

Fig 2. CN1030 captures and enriches DENV, CHIKV, and ZIKV in co-infection model systems.

The viral preference for enrichment of CN1030 (+NT) was investigated in co-infection models. 10⁶ PFU/mL of (A) DENV and ZIKV, (B) CHIKV and ZIKV, and (C) CHIKV and DENV were spiked in urine and incubated with (+NT) or without CN1030 (-NT) followed by quantification of viral RNA by RT-qPCR. Numbers above the +NT bars indicate the fold-change enrichment generated by dividing with genomic copies in no-NT controls. Data was shown as mean ± SEM from four biological replicates where significance was indicated as *, P <0.05.

Fig 3. NT particles preserves ZIKV infectivity.

10⁶ PFU/mL of (A) ZIKV and (B) CHIKV were spiked in human saliva with or without CN1030 incubation in 1:10 ratio (CN1030: saliva-spiked virus) for 30 min at ambient temperature. Viral titers were subsequently determined by plaque assays at 0, 24, 48, and 72 hours post-incubation at 37°C. Inserted images in panel B show the plaque morphology and sizes of CHIKV. ND, not detected. Data were presented as mean ± SEM of at least three biological replicates and analyzed with two-way ANOVA (***, P<0.001).

Fig 4. NT particles enhance ZIKV and CHIKV detection at both low and high titers.

Detection of ZIKV (panel A) or CHIKV (panel B) by RT-qPCR with or without CN1030 was evaluated through the use of decreasing concentrations of viruses (PFU/mL) spiked in urine. Numbers above the +NT bars indicate the fold-change enrichment. Data are shown as mean ± SD for three biological replicates. The statistical significance was performed with two-way ANOVA and marked with an asterisk (*) symbol where *** indicates P value is ≤ 0.001.

Fig 5. Increasing volumes of urine enhanced ZIKV RNA in urine by CN1030.

ZIKV was spiked into various volume of urine (10 PFU/mL) and incubated with 100 μL of CN1030 for 30 min at ambient temperature followed by quantification of viral genomic copy via RT-qPCR. Data are presented as mean ± SEM for five biological replicates, except for the 1mL samples where three biological replicates were performed. The statistical significance was demonstrated with Kruskal-Willis test. *, P < 0.05. **, P < 0.01.

Fig 6. NT magnetic particles are capable of capturing and enriching ZIKV and CHIKV in urine.

Different volumes of NT magnetic particle, CN3080, were incubated with ZIKV and CHIKV spiked in urine and viral capture quantified by RT-qPCR. The viral enrichment of (A) ZIKV and (B) CHIKV were determined by q-RT-PCR. The binding efficacies of CN3080 were calculated based on the unbound (C) ZIKV and (D) CHIKV RNA in the supernatant after CN3080 incubation. Data represents results from three biological replicates as mean ± SD. The statistical significance was determined by comparing each +NT group with its -NT group with Kruskal-Wallis Test where * indicates p value is ≤ 0.05.