One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: a protocol to study infection and gene expression during ZIKV infection

Ricardo Vieira Araujo,Tonya M Colpitts,Berlin Londono-Renteria,Alexander S Gold,Fabiana Feitosa-Suntheimer

doi:10.1186/s13071-020-4002-x

Ricardo Vieira Araujo, Tonya M Colpitts + Show 3 more

Open Access

https://doi.org/10.1186/s13071-020-4002-x

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Abstract

BackgroundZika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is quantitative reverse transcription polymerase chain reaction (qRT-PCR), yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito ribonucleic acid (RNA). In this paper, we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection.MethodsOur new oligo design to detect ZIKV RNA included in silico analysis of both viral and mosquito (Ae. aegypti and Ae. albopictus) genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedes genomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes. Standard curves constructed with in vitro transcribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.ResultsFinally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response to virus infection.ConclusionsThe experimental approach to detect ZIKV RNA in Aedes aegypti presented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

Highlights

Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito
Identification of conserved ZIKV region divergent from Aedes Aiming to design oligos optimized to minimize nonspecific amplification in one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that allows the analysis of mosquito gene expression during ZIKV infection in the same samples, the ZIKV Nonstructural protein 5 (NS5) gene sequence of the PRVABC59 strain was submitted for sequence alignment analysis with Ae. aegypti and Ae. albopictus sequences from “EST”, “Assembled transcriptome” and “Transcripts” datasets from VectorBase, using the BLAST tool
Confirmation of ZIKV detection in mammalian cells We performed qRT-PCR analysis using the designed NS5-2362F and NS5-2457R primers with ribonucleic acid (RNA) template isolated from Vero cells infected with ZIKV MR766 and PRVABC59 strains

Summary

Introduction

Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. We present a one-step qRT-PCR protocol that allows for the detec‐ tion of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing confident detection. Zika virus (ZIKV) was first isolated in Uganda, from a sentinel rhesus macaque in 1947 [1]. In 2016–2017, Zika fever autochthonous cases were reported in USA, in the states of Texas and Florida [8, 9]

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