Both adult and immature stages of Aedes mosquitoes were sampled in three neighborhoods with suspected CHIKV cases (Soyo Safari, Kinkanda, and Camp Molayi) (see Figure 1B). Adult mosquitoes were trapped using six battery-powered BG-sentinel traps and two Prokopack aspirators. The traps were placed in vegetation surrounding houses (preferably in the shade), activated in the morning and emptied in the evening. Aspirator collections were made from vegetations only and were performed in the late afternoon. Water-holding containers in the neighborhoods were inspected for larvae (both indoors and outdoors), reported on entomological survey forms, and sampled if positive. Collections were made during one day for each site. Larvae were reared to adults in the laboratory for morphological identification following Walter Reed’s identification keys: we looked at the characteristic patterns on the scutum (white longitudinal stripe for Ae. albopictus and lyre-shaped white markings for Ae. aegypti) and the white/black patterns of the tarsi on the legs [25]. To ensure the correct identification of Aedes albopictus in the area (as this species was never detected before in Matadi), we selected two individual mosquitoes for DNA barcoding (a technique based on the amplification of the partial mitochondrial cytochrome c oxidase subunit I (COI) gene), which we excluded from the pools and preserved in separate tubes [26]. Sanger sequencing of the 658-base pair barcode and phylogenetic analyses of these specimens followed laboratory protocols and methodologies described in [26] and by Mariën et al. (amplicons obtained from the Matadi specimens were included in the phylogenetic tree of the latter reference) [27]. In short, PCR amplicons (EPPO 2016, LCO1490, and HCO2198 universal primers) and negative controls were checked on an agarose gel, sequenced in both directions, and compared against BOLD Identification System with Species Level Barcode Records.
Larvae indices were calculated to estimate the level of infestation in the affected areas [28]:
Container index: number of containers positive for immature stages of Aedes spp. per 100 inspected containers.
House index: number of houses positive for at least one container with immature stages of Aedes spp. per 100 inspected houses.
Breteau index: number of containers positive for immature stages of Aedes spp. per 100 inspected houses.
All adult mosquitoes were killed by ethanol inhalation, identified, and separated by sex. The mosquitoes were pooled according to sampling site, sex, stage during capture (adult/larvae), and species in Eppendorf tubes with RNA shield for viral preservation (max. 50 individuals per tube). The mosquito pools were immediately homogenized in the field using Zymo Research bashing beads (vortex for 2 min). They were stored at 4 °C for one week during the field investigation and further kept at −20 °C at INRB in Kinshasa. All pooled samples were screened for the presence of CHIKV using the Zymo quick DNA/RNA pathogen extraction kit and RT-qPCR. All RT-qPCR reactions were performed in duplo and samples were considered to be positive if both reactions gave the same Ct-value (±1), as described by Selhorst et al. [19].
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