Zika virus (ZIKV) was first isolated in Uganda, from a sentinel rhesus macaque in 1947 . It is an arthropod-borne virus (arbovirus) belonging to the genus Flavivirus of the Flaviviridae family. ZIKV infection in humans usually results in mild disease or asymptomatic infections, however it can develop into to severe symptoms that can be lethal. The symptomatology can include fever, rash, arthritis and/or arthralgia and/or myalgia, conjunctivitis, and fatigue. Neurological complications caused by ZIKV infection were reported in adults (Guillain-Barré syndrome) and neonates (congenital malformations including microcephaly) .
Before a major outbreak of Zika cases in 2007 at the Pacific Island of Yap in the Federate States of Micronesia , ZIKV infections occurred in Africa and Asia without much attention. In 2015, the first Zika cases were reported in the Americas (Brazil) and quickly spread to more than 20 countries throughout the Caribbean, and South, Central, and North Americas [4, 5, 6, 7]. In 2016–2017, Zika fever autochthonous cases were reported in USA, in the states of Texas and Florida [8, 9].
ZIKV is transmitted to humans by the bite of an infected mosquito. The main vectors associated with transmission in the urban cycle are Aedes aegypti and Aedes albopictus. In the current state of globalization and climate change, the frequency of human disease outbreaks related to arboviruses, including Zika, has increased in urban centers with competent vectors [10, 11, 12, 13], revealing a need for continuous improvement of ZIKV detection in vector populations. In addition, there has been increased research involving ZIKV infection in the mosquito as an improved understanding of both pathogenesis and interactions in the vector will be crucial information. As such, successful detection of ZIKV infection in mosquito cells and samples is an important component of laboratory work involving the virus and vector.
Molecular detection of ZIKV RNA in mosquitoes can be challenging due to the limited number of primers and probes published [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27], as well as the presence of viral genetic material integrated in mosquito genomes [28, 29] which can reduce the specificity for RNA detections by RT-qPCR . Most of these ZIKV oligos were optimized in mammalian cells and samples and show often-unresolvable background when used to detect infection in mosquito tissues.
In recent studies utilizing RT-qPCR to detect ZIKV RNA in mosquitoes, which mostly analyzed vector competence, it is possible to observe a methodological trend favoring the utilization of hydrolysis probes as a fluorescent label (83% of the papers in literature) and the use of viral RNA extraction kits to obtain the RNA templates (56% of the studies). However the number of studies could actually be higher since some of them do not specify the extraction RNA method used for the experiments [9, 14, 16, 18, 19, 21, 22, 23, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51]. Although this approach has demonstrated to be relatively effective in detecting ZIKV RNA, since the isolation of viral RNA is prioritized, it does not permit the study of the gene expression in mosquito genes during viral infection using the same samples. The study of mosquito gene expression during ZIKV infection could elucidate phenomena not fully understand regarding ZIKV and mosquito interactions, as vector competence varies in mosquito populations infected with ZIKV isolates from different geographical regions [52, 53, 54, 55, 56]. In addition, these are very expensive approaches, a potential barrier to vector surveillance in developing countries.
In this study, we present a one-step qRT-PCR protocol that both detects ZIKV RNA and can be used to evaluate gene expression from the same sample of infected A. aegypti. In order to avoid nonspecific ZIKV RNA detection due to possible viral integration in the mosquito genome, in silico analysis of the A. aegypti, A. albopictus and ZIKV genomes were conducted to find sequences conserved between Asian and African ZIKV phylogenetic lineages [57, 58] but divergent from Aedes spp genomes. Primers were designed to detect ZIKV RNA using these determined target regions. Primers were tested on in vitro transcribed ZIKV RNA as well as RNA samples from mosquitoes infected with ZIKV, and the positive mosquito samples were used for transcriptional level analysis of Defensin A, an antimicrobial peptide (AMP) involved in Aedes aegypti immune response [59, 60].