Re-Emergence of Rift Valley Fever Virus Lineage H in Senegal in 2022: In Vitro Characterization and Impact on Its Global Emergence in West Africa.

Rift Valley fever (RVF) is a re-emerging vector-borne zoonosis with a high public health and veterinary impact. In West Africa, many lineages were previously detected, but since 2020, lineage H from South Africa has been the main cause of the outbreaks. In this study, clinical samples collected through national surveillance were screened for RVF virus (RVFV) acute infection by RT-PCR and IgM ELISA tests. Sequencing, genome mapping and in vitro phenotypic characterization in mammal cells were performed on RT-PCR positive samples in comparison with other epidemic lineages (G and C). Four RVFV human cases were detected in Senegal and the sequence analyses revealed that the strains belonged to lineage H. The in vitro kinetics and genome mapping showed different replication efficiency profiles for the tested RVFV lineages and non-conservative mutations, which were more common to lineage G or specific to lineage H. Our findings showed the re-emergence of lineage H in Senegal in 2022, its high viral replication efficiency in vitro and support the findings that genetic diversity affects viral replication. This study gives new insights into the biological properties of lineage H and calls for deeper studies to better assess its potential to cause a future threat in Senegal.

Detection and Diagnosis of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is a globally important mosquito-borne virus that can also be directly transmitted via aerosolization of body fluids from infected animals. RVFV outbreaks cause mass mortality of young livestock and abortions in animals. In most severe human cases, the disease can progress to hemorrhagic fever and encephalitis, leading to death. RVF has a significant economic impact due to the loss of livestock that is a great challenge for people who depend on animals for income and food. Several vaccines are available for animal use, but none are yet licensed for use in human populations. This situation emphasizes the need to have robust and efficient diagnostic methods that can be used for early case confirmation, assessment of seroprevalence, and virus surveillance as well as vaccine efficacy evaluation. Despite the existence of different diagnostic methods for RVFV, we still have untimely reporting or underreporting of cases, probably due to lack of appropriate surveillance systems or diagnostic tools in some endemic countries. Here, we describe different methods available for detection and diagnosis of RVFV.

An amplicon-based sequencing approach for Usutu virus characterization.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus.

Genomic characterization of a reemerging Chikungunya outbreak in Kedougou, Southeastern Senegal, 2023.

Chikungunya virus has caused millions of cases worldwide over the past 20 years, with recent outbreaks in Kedougou region in the southeastern Senegal, West Africa. Genomic characterization highlights that an ongoing epidemic in Kedougou in 2023 is not due to an introduction event but caused by the re-emergence of an endemic strain evolving linearly in a sylvatic context.

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.

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here we propose adding two sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present an assignment tool to show that the proposed lineages are useful for regional, national and sub-national discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

Crimean-Congo haemorrhagic fever outbreak in Northern Senegal in 2022: Prevalence of the virus in livestock and ticks, associated risk factors and epidemiological implications.

OBJECTIVE: Crimean-Congo haemorrhagic fever (CCHF) is a severe zoonotic arboviral disease that occurs widely in Eastern and Western Europe, Asia and Africa. The disease is becoming of growing public health importance in Senegal. However, analysis of tick infestation, CCHF virus (CCHFV) circulation extent and risk factors during ongoing outbreak are scarce. A thorough outbreak investigation was carried out during a CCHF outbreak in Podor (Northern Senegal) in August 2022. METHODS: Ticks and blood samples were collected from animals (cattle, goats and sheep) randomly selected from confirmed CCHF human cases houses, neighbourhoods and surrounding villages. Blood samples were tested for CCHFV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA) test. Tick samples were screened for CCHFV RNA by RT-PCR. RESULTS: Overall, tick infestation rate (TIR) and CCHFV seroprevalence of livestock were 52.12% (95% confidence interval (CI): 45.54%-58.64%) and 43.28% (95% CI: 36.33%-50.44%), respectively. The TIRs were 87.7% in cattle, 57.6% in sheep and 20.0% in goats. These rates were significantly associated with location, host species and tick control (p < 0.001) but not with animal age and sex (p > 0.7). CCHFV seroprevalence was 80.4% (95% CI: 67.57%-89.77%) in cattle, 35.4% (95% CI: 25.00%-47.01%) in sheep and 21.2% (95% CI: 12.11%-33.02%) in goats. Age, sex, location, animal host and presence of ticks were significantly associated to the presence of antibodies. The 950 ticks collected included among other species, Hyalomma impeltatum (48.84%) and H. rufipes (10.21%). Five pools of Hyalomma ssp. were found CCHFV RT-PCR positive. These infected ticks included 0.86% (4/464) of H. impeltatum collected on cattle and sheep and 1.03% (1/97) of H. rufipes collected on a sheep. CONCLUSIONS: To our knowledge, this is the first report on the extend of tick infestation and CCHFV infection in livestock during an outbreak in Senegal. The results highlight the risk of human infections and the importance of strengthening vector, animal and human surveillance as well as tick control measures in this area to prevent CCHF infections in humans.

Reemergence of Sylvatic Dengue Virus Serotype 2 in Kedougou, Senegal, 2020.

In 2020, a sylvatic dengue virus serotype 2 infection outbreak resulted in 59 confirmed dengue cases in Kedougou, Senegal, suggesting those strains might not require adaptation to reemerge into urban transmission cycles. Large-scale genomic surveillance and updated molecular diagnostic tools are needed to effectively prevent dengue virus infections in Senegal.

The Spatiotemporal Distribution and Molecular Characterization of Circulating Dengue Virus Serotypes/Genotypes in Senegal from 2019 to 2023.

Dengue virus is becoming a major public health threat worldwide, principally in Africa. From 2016 to 2020, 23 outbreaks were reported in Africa, principally in West Africa. In Senegal, dengue outbreaks have been reported yearly since 2017. Data about the circulating serotypes and their spatial and temporal distribution were limited to outbreaks that occurred between 2017 and 2018. Herein, we describe up-to-date molecular surveillance of circulating DENV serotypes in Senegal between 2019 to 2023 and their temporal and spatial distribution around the country. For this purpose, suspected DENV-positive samples were collected and subjected to dengue detection and serotyping using RT-qPCR methods. Positive samples were used for temporal and spatial mapping. A subset of DENV+ samples were then sequenced and subjected to phylogenetic analysis. Results show a co-circulation of three DENV serotypes with an overall predominance of DENV-3. In terms of abundance, DENV-3 is followed by DENV-1, with scarce cases of DENV-2 from February 2019 to February 2022. Interestingly, data show the extinction of both serotype 1 and serotype 2 and the only circulation of DENV-3 from March 2022 to February 2023. At the genotype level, the analysis shows that sequenced strains belong to same genotype as previously described: Senegalese DENV-1 strains belong to genotype V, DENV-2 strains to the cosmopolitan genotype, and DENV-3 strains to Genotype III. Interestingly, newly obtained DENV 1-3 sequences clustered in different clades within genotypes. This co-circulation of strains belonging to different clades could have an effect on virus epidemiology and transmission dynamics. Overall, our results highlight DENV serotype replacement by DENV-3, accompanied by a wider geographic distribution, in Senegal. These results highlight the importance of virus genomic surveillance and call for further viral fitness studies using both in vitro and in vivo models, as well as in-depth phylogeographic studies to uncover the virus dispersal patterns across the country.

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.

Emergence of Crimean-Congo Hemorrhagic Fever Virus in Eastern Senegal in 2022.

Crimean-Congo hemorrhagic fever (CCHF), the most widespread tick-borne viral human infection, poses a threat to global health. In this study, clinical samples collected through national surveillance systems were screened for acute CCHF virus (CCHFV) infection using RT-PCR and for exposure using ELISA. For any CCHF-positive sample, livestock and tick samples were also collected in the neighborhood of the confirmed case and tested using ELISA and RT-PCR, respectively. Genome sequencing and phylogenetic analyses were also performed on samples with positive RT-PCR results. In Eastern Senegal, two human cases and one Hyalomma tick positive for CCHF were identified and a seroprevalence in livestock ranging from 9.33% to 45.26% was detected. Phylogenetic analyses revealed that the human strain belonged to genotype I based on the available L segment. However, the tick strain showed a reassortant profile, with the L and M segments belonging to genotype I and the S segment belonging to genotype III. Our data also showed that our strains clustered with strains isolated in different countries, including Mauritania. Therefore, our findings confirmed the high genetic variability inside the CCHF genotypes and their introduction to Senegal from other countries. They also indicate an increasing CCHF threat in Senegal and emphasize the need to reinforce surveillance using a one-health approach.

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.

An amplicon-based Illumina and nanopore sequencing workflow for Chikungunya virus West Africa genotype.

The Chikungunya virus, a global arbovirus, is currently causing a major outbreak in the Western African region, with the highest cases reported in Senegal and Burkina Faso. Recent molecular evolution analyses reveal that the strain responsible for the epidemic belongs to the West African genotype, with new mutations potentially impacting viral replication, antigenicity, and host adaptation. Real-time genomic monitoring is needed to track the virus's spread in new regions. A scalable West African genotype amplicon-based Whole Genome Sequencing for multiple Next Generation Sequencing platforms has been developed to support genomic investigations and identify epidemiological links during the virus's ongoing spread. This technology will help identify potential threats and support real-time genomic investigations in the ongoing spread of the virus.

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.

Molecular Evolution of Dengue Virus 3 in Senegal between 2009 and 2022: Dispersal Patterns and Implications for Prevention and Therapeutic Countermeasures.

Dengue fever is the most prevalent arboviral disease worldwide. Dengue virus (DENV), the etiological agent, is known to have been circulating in Senegal since 1970, though for a long time, virus epidemiology was restricted to the circulation of sylvatic DENV-2 in south-eastern Senegal (the Kedougou region). In 2009 a major shift was noticed with the first urban epidemic, which occurred in the Dakar region and was caused by DENV-3. Following the notification by Senegal, many other West African countries reported DENV-3 epidemics. Despite these notifications, there are scarce studies and data about the genetic diversity and molecular evolution of DENV-3 in West Africa. Using nanopore sequencing, phylogenetic, and phylogeographic approaches on historic strains and 36 newly sequenced strains, we studied the molecular evolution of DENV-3 in Senegal between 2009 and 2022. We then assessed the impact of the observed genetic diversity on the efficacy of preventive countermeasures and vaccination by mapping amino acid changes against vaccine strains. The results showed that the DENV-3 strains circulating in Senegal belong to genotype III, similarly to strains from other West African countries, while belonging to different clades. Phylogeographic analysis based on nearly complete genomes revealed three independent introduction events from Asia and Burkina Faso. Comparison of the amino acids in the CprM-E regions of genomes from the Senegalese strains against the vaccine strains revealed the presence of 22 substitutions (7 within the PrM and 15 within the E gene) when compared to CYD-3, while 23 changes were observed when compared to TV003 (6 within the PrM and 17 within the E gene). Within the E gene, most of the changes compared to the vaccine strains were located in the ED-III domain, which is known to be crucial in neutralizing antibody production. Altogether, these data give up-to-date insight into DENV-3 genomic evolution in Senegal which needs to be taken into account in future vaccination strategies. Additionally, they highlight the importance of the genomic epidemiology of emerging pathogens in Africa and call for the implementation of a pan-African network for genomic surveillance of dengue virus.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

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.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.