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

Abstract

Background: Zika 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.

Methods: Our 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.

Results: Finally, 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.

Conclusions: The 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.

Keywords: Aedes aegypti; Mosquito gene expression; One-step qRT-PCR; Zika virus.

Fig. 1

Alignment of the target amplicon of ZIKV NS5 gene. The nucleotides in red differ from the consensus and the asterisks indicate identity with the consensus sequence on the top. Nucleotides highlighted in yellow correspond to primer (NS5-2362F and NS5-2457R) annealing sites. Clustal Omega tool (EMBL-EBI - https://www.ebi.ac.uk/Tools/msa/clustalo/)

Fig. 2

ZIKV strains detected from infected Vero cells. Total RNA from Vero cells infected with ZIKV MR766 and Puerto Rico strains was used as template for one-step RT-qPCR reaction, using NS5-2362F and NS5-2457R primers. Each sample was tested in duplicate

Fig. 3

Alignment of the target amplicon amplified by ZIKV 1086 and ZIK 1162c primers with Aedes aegypti mRNA sequences. The nucleotides in bold on Ae. aegypti sequences indicate identities with consensus sequence on the top (Zika virus - PRVABC59 strain: polyprotein). Nucleotides highlighted in yellow correspond to primer (ZIKV 1086 and ZIK 1162c) annealing sites. Sequence highlighted in green correspond to 1107-FAM probe binding site

Fig. 4

Limit of detection and efficiency of the one-step RT-qPCR assay for ZIKV RNA detection. RNAs transcribed in vitro, containing sequences of NS5 and Env genes from MR766 and PRVABC59 strains, were used as templates for RT-qPCR reactions using hydrolysis probe (1107-FAM) and SYBR Green (NS5-2362F and NS5-2457R primers) as fluorescent dyes. Cq values (mean ± standard deviation) were obtained from two technical replicates, performed in triplicate each. Coefficient of determination (R²) was calculated using GraphPad Prism 8.3.0 software

Fig. 5

Reproducibility intra and inter assay of the one-step RT-qPCR assay for ZIKV RNA detection. RNAs transcribed in vitro (10⁸ copies/reaction), containing sequences of NS5 and Env genes from MR766 and PRVABC59 strains, were used as templates for RT-qPCR reactions using hydrolysis probe (1107-FAM) and SYBR Green (NS5-2362F and NS5-2457R primers) as fluorescent dyes. Coefficients of variance were calculated from three technical replicates, performed in triplicate each. Coefficient of variance = standard deviation/mean × 100

Fig. 6

Detection of ZIKV from infected blood-fed (IBF) mosquitoes. Total RNA from ZIKV-infected (PRVABC59 strain) whole mosquitoes were extracted 7 days post-infection, and used as template for RT-qPCR reactions, using NS5-2362F/NS5-2457R primers and 1107-FAM probe. The absolute quantification of ZIKV RNA in mosquito samples was obtained using a standard curve constructed from in vitro transcribed RNA

Fig. 7

Defensin A gene expression in infected blood-fed (IBF) mosquitoes. Samples positive for ZIKV were used for RT-qPCR to evaluate Defensin A transcription levels. Actin was utilized as reference gene. Each sample was tested in duplicate