Abstract
Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.
Highlights
Arthropod transmitted flaviviruses are highly adapted to arthropod vectors at the species level, causing several degrees of disease outcomes
The goal of this study was to assess the quantification of Zika virus (ZIKV) genomic copies in mosquito pools containing 10, 3 and 0 mosquitoes to estimate the influence of host genetic material on target quantification, assay sensitivity and variation of precision rates according to treatment
The experiment was designed to estimate the influence of different ZIKV concentrations in mosquito pool samples, ZIKV MR766 was serially diluted in mosquito pools with diluent [2]
Summary
Arthropod transmitted flaviviruses are highly adapted to arthropod vectors at the species level, causing several degrees of disease outcomes. Mosquito borne flaviviruses derived from Aedes sp. Flaviviruses, as most arboviruses, are established in sylvatic cycles involving animals and zoophilic arthropods as vectors. Spillover events involving forest dwelling mosquitoes infecting people can give rise to epidemics, with variable degrees of pathogenicity depending on the virus and the population affected [1]. Several strategies have been employed to investigate and recognize flaviviruses circulating in mosquitoes in sylvatic environments, and vector competence studies rely on viral detection in mosquito pools [2]. Zika Virus (ZIKV, Flavivirus: Flaviviridae) has become an important pathogen worldwide since the recent outbreaks that occurred in French Polynesia, Yap island and Northeastern Brazil, causing
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