Abstract
BackgroundThe monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures.ResultsWe developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes.ConclusionsBeing a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.
Highlights
The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions
In order to support Zika virus (ZIKV) surveillance in mosquito populations, we developed a SYBR Green-based RT-polymerase chain reaction (PCR) assay optimized for the detection of ZIKV in mosquitoes and compared its performance with one of the probebased assays [9] in field-caught mosquitoes
We further evaluated the sensitivity and specificity of four different primer pairs (ZIKV-F + R3, F + R4, F2 + R3, and F3 + R4) that amplified a range of amplicon sizes (76–257 bp)
Summary
The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. Zika virus (ZIKV) is an arbovirus that was first isolated in 1947 from a rhesus monkey in Uganda and was subsequently identified in Aedes africanus mosquitoes in 1948 [1]. The first authentic ZIKV infection in humans was described in early 1960s [2], ZIKV remained dormant until 2007 when it re-emerged in Micronesia [3]. As Aedes mosquitoes are widely distributed, there is an imminent threat of recurrent ZIKV epidemics, especially in the tropics and subtropics
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