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.

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.

Usutu virus (USUV) has been isolated in several African and European countries mainly from mosquitoes and birds. However, previous benign and two recent severe cases of human infections point out the need of a tool for the identification of USUV in human samples. A published real-time reverse transcription (RT) PCR assay for the detection of USUV in human blood or cerebrospinal fluid does not take into account the genetic variability of USUV in different geographic regions. Therefore, this article presents a quantitative real-time RT-PCR assay based on sequences from Europe and Africa. Primers and probe were designed in conserved regions among USUV strains that differed from closely related flaviviruses. The specificity of the assay was investigated by testing 16 other flaviviruses circulating in Africa. The sensitivity was determined by testing serial dilutions of virus and RNA standard. Intra- and inter-assay coefficients of variation were evaluated by 10 reactions in a same and in different assays, respectively. The assay provides high analytical specificity for USUV and detection limits of 1.2pfu/reaction for virus dilutions in L-15 medium or human serum and 60 copies/reaction for the RNA standard. The assay needs to be evaluated in a clinical context and integrated in standard diagnosis of flaviviral diseases.

Four large outbreaks of Rift Valley Fever (RVF) occurred in Mauritania in 1998, 2003, 2010 and 2012 which caused lots of animal and several human deaths. We investigated rainfall and vegetation conditions that might have impacted on RVF transmission over the affected regions. Our results corroborate that RVF transmission generally occurs during the months of September and October in Mauritania, similarly to Senegal. The four outbreaks were preceded by a rainless period lasting at least a week followed by heavy precipitation that took place during the second half of the rainy season. First human infections were generally reported three to five weeks later. By bridging the gap between meteorological forecasting centers and veterinary services, an early warning system might be developed in Senegal and Mauritania to warn decision makers and health services about the upcoming RVF risk.

Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy.