A bstract: Background: Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. Results: The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. Conclusion: We have developed a rapid, sensitive and specific rRT – PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection.

Zika virus (ZIKV; genus Flavivirus, family Flaviviridae) is an emerging virus of medical importance maintained in a zoonotic cycle between arboreal Aedes spp. mosquitoes and nonhuman primates in African and Asian forests. Serological evidence and virus isolations have demonstrated widespread distribution of the virus in Senegal. Several mosquito species have been found naturally infected by ZIKV but little is known about their vector competence. We assessed the vector competence of Ae. aegypti from Kedougou and Dakar, Ae. unilineatus, Ae. vittatus and Ae. luteocephalus from Kedougou in Senegal for 6 ZIKV strains using experimental oral infection. Fully engorged female mosquitoes were maintained in an environmental chamber set at 27 ± 1 °C and 80 ± 5 % Relative humidity. At day 5, 10 and 15 days post infection (dpi), individual mosquito saliva, legs/wings and bodies were tested for the presence of ZIKV genome using real time RT-PCR to estimate the infection, dissemination, and transmission rates. All the species tested were infected by all viral strains but only Ae. vittatus and Ae. luteocephalus were potentially capable of transmitting ZIKV after 15 dpi with 20 and 50 % of mosquitoes, respectively, delivering epidemic (HD 78788) and prototype (MR 766) ZIKV strains in saliva. All the species tested here were susceptible to oral infection of ZIKV but only a low proportion of Ae. vittatus and Ae. luteocephalus had the viral genome in their saliva and thus the potential to transmit the virus. Further investigations are needed on the vector competence of other species associated with ZIKV for better understanding of the ecology and epidemiology of this virus in Senegal.

After 8 years of silence, dengue virus serotype 2 (DENV-2) reemerged in southeastern Senegal in 1999. Sixty-four DENV-2 strains were isolated in 1999 and 9 strains in 2000 from mosquitoes captured in the forest gallery and surrounding villages. Isolates were obtained from previously described vectors, Aedes furcifer, Ae. taylori, Ae. luteocephalus, and—for the first time in Senegal—from Ae. aegypti and Ae. vittatus. A retrospective analysis of sylvatic DENV-2 outbreaks in Senegal during the last 28 years of entomologic investigations shows that amplifications are periodic, with intervening, silent intervals of 5–8 years. No correlation was found between sylvatic DENV-2 emergence and rainfall amount. For sylvatic DENV-2 vectors, rainfall seems to particularly affect virus amplification that occurs at the end of the rainy season, from October to November. Data obtained from investigation of preimaginal (i.e., nonadult) mosquitoes suggest a secondary transmission cycle involving mosquitoes other than those identified previously as vectors.

Malaria is one of the leading causes of acute febrile illness (AFI) in Africa. With the advent of malaria rapid diagnostic tests, misdiagnosis and co-morbidity with other diseases has been highlighted by an increasing number of studies. Although arboviral infections and malaria are both vector-borne diseases and often have an overlapping geographic distribution in sub-Saharan Africa, information about their incidence rates and concurrent infections is scarce. From July 2009 to March 2013 patients from seven healthcare facilities of the Kedougou region presenting with AFI were enrolled and tested for malaria and arboviral infections, i.e., yellow fever (YFV), West Nile (WNV), dengue (DENV), chikungunya (CHIKV), Crimean Congo haemorrhagic fever (CCHFV), Zika (ZIKV), and Rift Valley fever viruses (RVFV). Malaria parasite infections were investigated using thick blood smear (TBS) and rapid diagnostics tests (RDT) while arbovirus infections were tested by IgM antibody detection (ELISA) and RT-PCR assays. Data analysis of single or concurrent malaria and arbovirus was performed using R software. A total of 13,845 patients, including 7387 with malaria and 41 with acute arbovirus infections (12 YFV, nine ZIKV, 16 CHIKV, three DENV, and one RVFV) were enrolled. Among the arbovirus-infected patients, 48.7 % (20/41) were co-infected with malaria parasites at the following frequencies: CHIKV 18.7 % (3/16), YFV 58.3 % (7/12), ZIKV 88.9 % (8/9), DENV 33.3 % (1/3), and RVF 100 % (1/1). Fever ≥40 °C was the only sign or symptom significantly associated with dual malaria parasite/arbovirus infection. Concurrent malaria parasite and arbovirus infections were detected in the Kedougou region from 2009 to 2013 and need to be further documented, including among asymptomatic individuals, to assess its epidemiological and clinical impact.

Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal. Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4). All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.