Identification of arboviruses circulating in mosquito populations in the Bloemfontein area, South Africa

Abstract

Globally there are more than 3 500 different species of mosquito. Many of these are known to be the primary insect vectors of many medically important diseases. Adequate surveillance programs should be put in place to develop effective control strategies and to prevent outbreaks of disease. For a surveillance programme to be effective, mosquito vectors need to be identified accurately. This is done through combining morphological, molecular and environmental data to get more accurate identification results. Currently the diversity of mosquito populations circulating in the Bloemfontein area is not well defined. Mosquitoes were captured from three different sites in the Bloemfontein area. A total of 318 mosquitoes were collected in four different genera. A total of ten different species were identified using morphological identification. Six specimens could only be identified to genus level, because of extensive damage to their external anatomy. Representative specimens were selected from selected species. These included Anopheles squamosus, Culex theileri, Aedes aegypti, Mansonia uniformis and two Aedes subgenus Ochlerotatus species. The Ochlerotatus species include Ochlerotatus harrisoni and Ochlerotatus juppi. DNA was extracted from these mosquitoes and sequenced bidirectionally making use of the barcoding primers, HCO2198 and LCO1490. Anopheles squamosus and Aedes aegypti were identified successfully using the barcoding primers. The primers were less useful for obtaining adequate sequence data for genetic identification of Ochlerotatus spp., Culex theileri and Mansonia uniformis and it is proposed that additional sequence data be obtained subsequent to cloning of fragments. The field caught mosquitoes were sorted and pooled, according to species, capture site and capture date. An RT-qPCR assay was developed to detect Sindbis virus (SINV) using a primer and probe set specifically targeting a region of the nsp2 gene. Another RT-qPCR assay was developed to detect West Nile virus (WNV) and Wesselsbron virus (WSLV) using a primer and probe set targeting a region of the NS5 gene. The assays were validated using cDNA reverse transcribed from RNA extracted from wild caught mosquitoes to ensure that there were no inhibitors in the mosquitoes that would interfere with downstream reactions. These assays proved to work efficiently. RNA controls were constructed for WNV and WSLV to be used as the positive controls to validate the RT-qPCR assays. RNA was extracted from mosquito pools and was screened using the RT-qPCR assays. All mosquito pools tested negative for the arboviruses that were screened for. Due to the small sample size and a low infectivity rate of mosquitoes, it is not surprising that no viral RNA was detected in this study. The number of mosquitoes caught in this study is too low to be used for surveillance but was rather used as a proof of concept for developing appropriate assays. Large scale surveillance programmes will be needed to determine the full extent of arbovirus circulation in the Bloemfontein area, Free State province, South Africa and the assays developed in this study can be used to execute such programmes.