Full genome analysis of circulating DENV-2 in Senegal reveals a regional diversification into separate clades.

To assess the genetic diversity of circulating dengue virus 2 (DENV-2) in Senegal, we analyzed nine newly generated complete genomes of strains isolated during the 2018 outbreaks and 06 sequences obtained in 2018 and 2019 from Thiès and Rosso, respectively. Phylogenetic analyses revealed that Senegalese strains belonged to the cosmopolitan genotype of DENV-2, but we observed intragenotype variability leading to a divergence in two clades associated with specific geographic distribution. We report two DENV-2 variants belonging to two distinct clades. Isolates from the "Northern clade" (n = 8) harbored three nonsynonymous mutations (V1183M, R1405K, P2266T) located respectively on NS2A, NS2B, and NS4A, while isolates from the "Western clade" (n = 7) had two nonsynonymous mutations (V1185E, V3214E) located respectively in the NS2A and NS5 genes. These findings call for phylogeographic analysis to investigate routes of introductions, dispersal patterns, and in-depth in vitro and functional study to elucidate the impact of observed mutations on viral fitness, spread, epidemiology, and pathology.

P. falciparum genetic markers associated with drug resistance from patients with treatment failure in the Southern part of Senegal in 2017.

Artemisinin Combination Therapies (ACT) stand as the most potent antimalarial treatments. In response to the emergence of ACT-resistant malaria parasites in Southeast Asia, the World Health Organization (WHO) has recommended continuous monitoring of the effectiveness of ACT and other antimalarials. To address this need, we collected dried blood spots from malaria patients during a 42-days drug efficacy trial evaluating the efficacy of Artesunate plus Amodiaquine (ASAQ), Artemether Plus Lumefantrine (AL) and Dihydroarthemisinine plus Piperaquine (DHAPQ) on simple P. falciparum malaria in 2017. Blood samples were collected on Day 0, prior to the patients' initial ACT dose, and on any days of recurrent parasitemia. Genetic markers such as Merozoite Surface Protein 1 (MSP1) and Merozoite Surface Protein 2 (MSP2) were genotyped to differentiate between recrudescence and re-infestation cases. Furthermore, PCR Single Specific Oligonucleotide Probes combined with-ELISA platform (PCR-SSOP-ELISA) and PCR-RFLP techniques were used to identify Pfcrt 72-76 mutant haplotype and Pfmdr1_86Y allele associated with chloroquine and amodiaquine resistance, respectively. Out of the 320 patients enrolled in the study, only 43 (13.43%) experienced relapses. Upon PCR correction, our analysis revealed that recrudescent infections affected 13 patients, with 8 in the ASAQ group, 5 in the AL group, and none in the DHAPQ group. Notably, no early treatment failures (within the first 3 days of treatment) were observed, and all recurrences occurred between Day 21 and Day 42. The prevalence of the Pfcrt wild-type haplotype CVMNK and Pfmdr N86 allele was 67.03% and 97.70%, respectively. In contrast, the mutant types CVIET and 86Y were found at 32.97% and 2.3%, respectively. The high prevalence of the CVMNK wild haplotype suggests that the parasites remain sensitive to chloroquine, while the low prevalence of the 86Y mutants indicates continued effectiveness of amodiaquine. Furthermore, the low prevalence of strains exhibiting the combination of CVIET and 86Y suggests that the use of multiple antimalarials is valuable for resistance control. Notably, none of the relapse cases carried the 86Y mutation or the combination of 86Y and CVIET.

Genomic Characterization of a Bataï Orthobunyavirus, Previously Classified as Ilesha Virus, from Field-Caught Mosquitoes in Senegal, Bandia 1969.

Bataï virus (BATV), belonging to the Orthobunyavirus genus, is an emerging mosquito-borne virus with documented cases in Asia, Europe, and Africa. It causes various symptoms in humans and ruminants. Another related virus is Ilesha virus (ILEV), which causes a range of diseases in humans and is mainly found in African countries. This study aimed to genetically identify and characterize a BATV strain previously misclassified as ILEV in Senegal. The strain was reactivated and subjected to whole genome sequencing using an Illumina-based approach. Genetic analyses and phylogeny were performed to assess the evolutionary relationships. Genomic analyses revealed a close similarity between the Senegal strain and the BATV strains UgMP-6830 from Uganda. The genetic distances indicated high homology. Phylogenetic analysis confirmed the Senegal strain's clustering with BATV. This study corrects the misclassification, confirming the presence of BATV in West Africa. This research represents the first evidence of BATV circulation in West Africa, underscoring the importance of genomic approaches in virus classification. Retrospective sequencing is crucial for reevaluating strains and identifying potential public health threats among neglected viruses.

Dengue 1 outbreak in Rosso, northern Senegal, October 2021: entomologic investigations.

Senegal has experienced periodic epidemics of dengue in urban areas with increased incidence in recent years. However, few data are available on the local ecology of the epidemic vectors. In October 2021, a dengue outbreak was reported in northern Senegal to the Institute Pasteur de Dakar. Entomologic investigations then were undertaken to identify the areas at risk of transmission and to identify the vector(s). Adult mosquitoes were collected indoors and outdoors at selected households, while containers with water were inspected for mosquito larvae. All the Aedes aegypti (L.) collected were tested for dengue virus NS1 protein using a rapid diagnostic test (RDT), and positive samples were confirmed by real-time RT-PCR. The qRT-PCR positive samples were subjected to whole genome sequencing using Nanopore technology. The majority of the larvae-positive containers (83.1%) were used for water storage. The Breteau and Container indices exceeded the WHO-recommended thresholds for the risk of dengue virus transmission except at 2 localities. Ae. aegypti, the only reputed dengue vector, was collected resting indoors as well as outdoors and biting during the day and night. The NS1 protein was detected in 22 mosquito pools, including one pool of females emerging from field-collected larvae. All NS1-positive results were confirmed by RT-PCR. Virus serotyping showed that the outbreak was caused by DENV-1. This study demonstrates the need for continuous control of adult and aquatic stages of Ae. aegypti to prevent future dengue epidemics in Senegal. RDTs appear to be a promising tool for dengue diagnostics and surveillance.

Molecular Detection and Genetic Characterization of Two Dugbe Orthonairovirus Isolates Detected from Ticks in Southern Senegal.

Dugbe virus (DUGV) is a tick-borne arbovirus first isolated in Nigeria in 1964. It has been detected in many African countries using such diverse methods as serological tests, virus isolation, and molecular detection. In Senegal, reports of DUGV isolates mainly occurred in the 1970s and 1980s. Here, we report a contemporary detection of three novel DUGV isolates upon screening of a total of 2877 individual ticks regrouped into 844 pools. The three positive pools were identified as Amblyomma variegatum, the main known vector of DUGV, collected in the southern part of the country (Kolda region). Interestingly, phylogenetic analysis indicates that the newly sequenced isolates are globally related to the previously characterized isolates in West Africa, thus highlighting potentially endemic, unnoticed viral transmission. This study was also an opportunity to develop a rapid and affordable protocol for full-genome sequencing of DUGV using nanopore technology. The results suggest a relatively low mutation rate and relatively conservative evolution of DUGV isolates.

Whole Genome Sequencing Analysis of African Orthobunyavirus Isolates Reveals Naturally Interspecies Segments Recombinations between Bunyamwera and Ngari Viruses.

Bunyamwera virus is the prototype of the Bunyamwera serogroup, which belongs to the order Bunyavirales of the Orthobunyavirus genus in the Peribunyaviridae family. Bunyamwera is a negative-sense RNA virus composed of three segments S, M, and L. Genetic recombination is possible between members of this order as it is already documented. Additionally, it can lead to pathogenic or host range improvement, if it occurs with viruses of public health and agricultural importance such as Rift Valley fever virus and Crimea-Congo hemorrhagic fever virus. Here, we characterize five African Orthobunyavirus viruses from different geographical regions. Our results suggest that the five newly characterized strains are identified as Bunyamwera virus strains. Furthermore, two of the five strains sequenced in this study are recombinant strains, as fragments of their segments are carried by Ngari and Bunyamwera strains. Further investigations are needed to understand the functional impact of these recombinations.

Detection of Crimean-Congo Haemorrhagic Fever Virus from Livestock Ticks in Northern, Central and Southern Senegal in 2021.

Crimean-Congo haemorrhagic fever virus (CCHFV) occurs sporadically in Senegal, with a few human cases each year. This active circulation of CCHFV motivated this study which investigated different localities of Senegal to determine the diversity of tick species, tick infestation rates in livestock and livestock infections with CCHFV. The samples were collected in July 2021 from cattle, sheep and goats in different locations in Senegal. Tick samples were identified and pooled by species and sex for CCHFV detection via RT-PCR. A total of 6135 ticks belonging to 11 species and 4 genera were collected. The genus Hyalomma was the most abundant (54%), followed by Amblyomma (36.54%), Rhipicephalus (8.67%) and Boophilus (0.75%). The prevalence of tick infestation was 92%, 55% and 13% in cattle, sheep and goats, respectively. Crimean-Congo haemorrhagic fever virus (CCHFV) was detected in 54/1956 of the tested pools. The infection rate was higher in ticks collected from sheep (0.42/1000 infected ticks) than those from cattle (0.13/1000), while all ticks collected from goats were negative. This study confirmed the active circulation of CCHFV in ticks in Senegal and highlights their role in the maintenance of CCHFV. It is imperative to take effective measures to control tick infestation in livestock to prevent future CCHFV infections in humans.

Quantitative real time PCR detection of Saboya virus: A flavivirus member of yellow fever genetic group.

The genus Flavivirus in the Flaviridae contains arthropod born viruses associated with high public health burdens like Zika, Dengue or Yellow fever. Saboya virus (SABV) is an understudied flavivirus grouping in the same genetic sub-group as Yellow Fever Virus (YFV) together with Sepik virus (SEPV) and Wesselbron virus (WSLV). Flavivirus infections are characterized by non-specific clinical presentations resulting in a high risk of misdiagnosis. SABV virus has been shown to circulate in the Sahelian zone and in central Africa. To study this virus we a qRT-PCR system based on TaqMan chemistry was developed to allow rapid and specific detection of SABV. The SABV assay was evaluated on available SABV isolates and others flaviviruses (DENV, ZIKV, YFV, WNV, KEDV). The system reliably detected all used SABV strains without cross amplification of other flaviviruses. In term of sensitivity the SABV assay detect up to 40.25 copies of SABV standard DNA molecule per ul. This system can be easily added to the available panel of arboviruses detection assays as a reliable tool to study virus prevalence in human, vertebrate and insect-vector samples.

Rapid On-Site Detection of Arboviruses by a Direct RT-qPCR Assay.

Arthropod-borne diseases currently constitute a source of major health concerns worldwide. They account for about 50% of global infectious diseases and cause nearly 700,000 deaths every year. Their rapid increase and spread constitute a huge challenge for public health, highlighting the need for early detection during epidemics, to curtail the virus spread, and to enhance outbreak management. Here, we compared a standard quantitative polymerase chain reaction (RT-qPCR) and a direct RT-qPCR assay for the detection of Zika (ZIKV), Chikungunya (CHIKV), and Rift Valley Fever (RVFV) viruses from experimentally infected-mosquitoes. The direct RT-qPCR could be completed within 1.5 h and required 1 µL of viral supernatant from homogenized mosquito body pools. Results showed that the direct RT-qPCR can detect 85.71%, 89%, and 100% of CHIKV, RVFV, and ZIKV samples by direct amplifications compared to the standard method. The use of 1:10 diluted supernatant is suggested for CHIKV and RVFV direct RT-qPCR. Despite a slight drop in sensitivity for direct PCR, our technique is more affordable, less time-consuming, and provides a better option for qualitative field diagnosis during outbreak management. It represents an alternative when extraction and purification steps are not possible because of insufficient sample volume or biosecurity issues.

Human and Livestock Surveillance Revealed the Circulation of Rift Valley Fever Virus in Agnam, Northern Senegal, 2021.

The mosquito-borne disease caused by the Rift Valley Fever Virus (RVFV) is a viral hemorrhagic fever that affects humans and animals. In 1987, RVFV emerged in Mauritania, which caused the first RVFV outbreak in West Africa. This outbreak was shortly followed by reported cases in humans and livestock in Senegal. Animal trade practices with neighboring Mauritania suggest northern regions of Senegal are at high risk for RVF. In this study, we aim to conduct a molecular and serological survey of RVFV in humans and livestock in Agnam (northeastern Senegal) by RT-PCR (reverse transcription real-time polymerase chain reaction) and ELISA (Enzyme-Linked Immunosorbent Assay), respectively. Of the two hundred fifty-five human sera, one (0.39%) tested RVFV IgM positive, while fifty-three (20.78%) tested positive for RVFV IgG. For animal monitoring, out of 30 sheep recorded and sampled over the study period, 20 (66.67%) showed seroconversion to RVFV IgG antibodies, notably during the rainy season. The presence of antibodies increased significantly with age in both groups (p < 0.05), as the force of RVF infection (FOI), increased by 16.05% per year for humans and by 80.4% per month for livestock sheep. This study supports the usefulness of setting up a One Health survey for RVF management.

Multiple genotypes of Crimean-Congo hemorrhagic fever virus detected in ticks during a one health survey in Agnam, Northeastern Senegal.

A Crimean-Congo Hemorrhagic Fever Virus (CCHFV) survey in Agnam (North Senegal) permits the detection of three isolates in ticks. These isolates belong genetically to multiple genotypes (I, II, III) and clustered with strains from Uganda, Sudan, Mauritania, and Senegal. The role of ticks in CCHF emergence and widespread is highlighted.

Crimean-Congo Hemorrhagic Fever Virus Survey in Humans, Ticks, and Livestock in Agnam (Northeastern Senegal) from February 2021 to March 2022.

Crimean-Congo hemorrhagic fever virus (CCHFV) is widespread in Asia, Europe, and Africa. In Senegal, sporadic cases of CCHFV have been reported since 1960. Bordering Mauritania in northeastern Senegal, Agnam is an arid area in the region of Matam where CCHFV is endemic, which harbors a pastoralist community. Given the drought conditions of Agnam, inhabitants are in constant movement with their animals in search of pasture, which brings them into contact with pathogens such as arboviruses. To identify CCHFV in this area, we established a One Health site in order to analyze animal livestock, ticks and human samples collected over a one-year period by qRT-PCR and ELISA. Our analysis showed one (1/364) patient carried anti-CCHFV IgM and thirty-seven carried anti-CCHFV IgG (37/364). In livestock, anti-CCHFV IgG was detected in 13 (38.24%) of 34 sentinel sheep. The risk of CCHFV infection increased significatively with age in humans (p-value = 0.00117) and sheep (p-value = 1.18 × 10-11). Additional risk factors for CCHFV infection in sheep were dry seasons (p-value = 0.004) and time of exposure (p-value = 0.007). Furthermore, we detected a total of three samples with CCHFV RNA within Rhipicephalus evertsi evertsi and Rhipicephalus guilhoni tick species. Our results highlighted the usefulness of a One Health survey of CCHFV in pastoral communities at risk of arboviruses.

Yellow Fever Outbreak in Eastern Senegal, 2020-2021.

Yellow fever virus remains a major threat in low resource countries in South America and Africa despite the existence of an effective vaccine. In Senegal and particularly in the eastern part of the country, periodic sylvatic circulation has been demonstrated with varying degrees of impact on populations in perpetual renewal. We report an outbreak that occurred from October 2020 to February 2021 in eastern Senegal, notified and managed through the synergistic effort yellow fever national surveillance implemented by the Senegalese Ministry of Health in collaboration with the World Health Organization, the countrywide 4S network set up by the Ministry of Health, the Institut Pasteur de Dakar, and the surveillance of arboviruses and hemorrhagic fever viruses in human and vector populations implemented since mid 2020 in eastern Senegal. Virological analyses highlighted the implication of sylvatic mosquito species in virus transmission. Genomic analysis showed a close relationship between the circulating strain in eastern Senegal, 2020, and another one from the West African lineage previously detected and sequenced two years ago from an unvaccinated Dutch traveler who visited the Gambia and Senegal before developing signs after returning to Europe. Moreover, genome analysis identified a 6-nucleotide deletion in the variable domain of the 3'UTR with potential impact on the biology of the viral strain that merits further investigations. Integrated surveillance of yellow fever virus but also of other arboviruses of public health interest is crucial in an ecosystem such as eastern Senegal.