Optimization of cationic (Q)-paper for detection of arboviruses in infected mosquitoes

Abstract

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper's binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated. Sixteen variations of Q-paper were prepared with modifications of the derivatizing reagents and derivatization temperature. The binding capacities of these variations were determined first with 1,3,5-benzenetricarboxylic (BTCA), then viral RNA (purified or in infected mosquito samples) was used for validation. For this, samples with Zika (ZIKV) and chikungunya (CHIKV) RNA or virus-infected Aedes aegypti mosquito bodies were applied to sixteen Q-paper variants. Washing the paper samples with water versus elution with aqueous salt (1 M) gave samples that were analyzed for viral RNA by a PCR-based direct Luminex hybridization assay. The comparison ranked the Q-paper binding capacities from the lowest to the highest. The Q-paper with the highest RNA binding capability was further validated with ZIKV- and CHIKV-infected mosquito saliva.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes

“…All arboviruses were obtained from human clinical samples of patients residing in or having traveled to geographic regions during outbreaks (Table 1). We created virus stocks by propagating isolates in cultured African green monkey (Vero) cells as described in [35]. Viral titer was determined by plaque assay using procedures similar to established techniques [36–38].…”

“…Seven days after ingestion of DENV-2 and CHIKV-infected blood, females were individually transferred to 37-mL plastic tubes and saliva was collected as described in [35]. The chosen time to collect saliva was based on maximizing the amount of expectorated viruses in saliva [41−43].…”

“…The honey was dyed with blue food coloring to provide a visual marker indicating that mosquitoes ingested honey and expectorated saliva onto the Q-paper [41, 44]. Mosquitoes were examined as described in [35] for blue in their crop after 24 h. Mosquitoes and Q-paper were stored at − 80 °C, and Q-paper was tested for the presence of DENV2 or CHIKV RNA for mosquitoes that fed on blue honey.…”

“…Bodies and legs of individual mosquitoes were homogenized separately and viral RNA was isolated as described in [34, 35]. Quantitative RT-PCR for the presence of arboviral RNA was determined as in [35], using the Superscript III One-Step qRT-PCR with Platinum® Taq kit (Invitrogen, Carlsbad, CA) with the CFX96 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).…”

“…DENV2–4 RNA detection was evaluated on the background of nucleic acids from pooled mosquitoes. Infected mosquitoes and saliva were also positively assessed on the surface of a cationic (Q)-paper [34, 35] that bound mosquito and viral nucleic acids via electrostatic interactions.

Fig. 1Adapted from [32, 34].

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