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.

Development and evaluation of RT-qPCR assays for two neglected orthobunyaviruses: Oya virus and Ebinur Lake virus

“…Moreover, the reproducibility and repeatability of our assays was excellent, with general %CV <3 in both intra- and -inter assay experiments. Compared to other studies, the%CV in our study was considered ideal ( Taylor et al., 2019 ; Araujo et al., 2020 ; Feng et al., 2018 ; Jaroenram et al., 2021 ), indicating that the results can be reliably reproduced, even when experiments were to be conducted in different days.…”

Dengue Virus-2 Infection Affects Fecundity and Elicits Specific Transcriptional Changes in the Ovaries of Aedes aegypti Mosquitoes

“…Aedes aegypti colony (Rockefeller strain) was obtained from Tulane University. Mosquitoes were maintained in rearing chambers in a secure insectary (arthropod containment level 3) ACL-3 in the NEIDL, following rearing conditions as described by Araujo et al (2020) .…”

“…The in vitro transcription of standard DENV-2 RNA (ssRNA) was obtained following the methods described by Araujo et al (2020) , using 5 μg of total RNA from DENV-2 NGC infected Vero-E6 cells. The first strand was generated using SuperScript III (Invitrogen, 18080051) with random hexamers, and the cDNA generated was used in PCR reaction with the T7 promoter sequence added to the forward primer DENV2_T7-5′-TAATACGACTCACTATAGGGAGAGCAGATCTCTGATGAA TAACCAACG-3′ and the reverse primer DENV2_Env_RC: 5′-CATTCCAAGTGAGAATCTCTTTGTCA-3′.…”