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 2 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 assignment tools to show that the proposed lineages are useful for regional, national, and subnational 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.

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.

Molecular characterization of circulating Dengue virus 2 during an outbreak in Northern Senegal's Saint-Louis region in 2018.

Globally, 390 million people are at risk of dengue infection and over the past 50 years, the virus incidence increased thirty-fold. In Senegal, an unprecedented occurrence of outbreaks and sporadic cases have been noticed since 2017. In October 2018, an outbreak of Dengue virus 2 (DENV-2) was reported in the north of Senegal affecting multiple areas including Saint-Louis, Richard Toll, and Rosso which are located at the border with Mauritania. Of these 173 blood specimen samples collected from patients, 27 were positive for dengue by quantitative reverse transcription PCR (qRT-PCR), and eight were serologically confirmed to be positive for DENV immunoglobulin M (IgM). Serotyping using qRT-PCR reveals that isolates were positive for DENV-2. A subset of DENV-2 positive samples was selected and subjected to whole-genome sequencing followed by phylogenetic analysis. Analysis of six nearly complete genome sequences revealed that the isolates belong to the cosmopolitan genotype and are closely related to the Mauritanian strains detected between 2017 and 2018 and those detected in many West African countries such as Burkina Faso or Cote d'Ivoire. Our results suggest a transboundary circulation of the DENV-2 cosmopolitan genotype between Senegal and Mauritania and call for a need for coordinated surveillance of arboviruses between these two countries. Interestingly, a high level of homology between West African isolates highlights endemicity and calls for the set-up of subregional viral genomic surveillance which will lead to a better understanding of viral dynamics, transmission, and spread across Africa.

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.

Multifoci and multiserotypes circulation of dengue virus in Senegal between 2017 and 2018.

BACKGROUND: Dengue fever is a mosquito born disease associated with self-limited to life threatening illness. First detected in Senegal in the nineteenth century, and despite its growing incidence this last decade, significant knowledge gaps exist in our knowledge of genetic diversity of circulating strains. This study highlights the circulating serotypes and genotypes between January 2017 and December 2018 and their spatial and temporal distribution throughout all regions of Senegal. METHODS: We used 56 dengue virus (DENV) strains for the analysis collected from 11 sampling areas: 39 from all regions of Senegal, and 17 isolates from Thiès, a particular area of the country. Two real time RT-qPCR systems were used to confirm dengue infection and corresponding serotypes. For molecular characterization, CprM gene was sequenced and submitted to phylogenetic analysis for serotypes and genotypes assignment. RESULTS: Three dengue virus serotypes (DENV-1-3) were detected by all used methods. DENV-3 was detected in 50% (28/56) of the isolates, followed by DENV-1 and DENV-2, each representing 25% (14/56) of the isolates. DENV-3 belongs to genotype III, DENV-1 to genotype V and DENV-2 to Cosmopolitan genotype. Serotype 3 was detected in 7 sampling locations and a co-circulation of different serotypes was observed in Thiès, Fatick and Richard-toll. CONCLUSIONS: These results emphasize the need of continuous DENV surveillance in Senegal to detect DENV cases, to define circulating serotypes/genotypes and to prevent the spread and the occurrence of severe cases.

Seoul Orthohantavirus in Wild Black Rats, Senegal, 2012-2013.

Hantaviruses cause hemorrhagic fever in humans worldwide. However, few hantavirus surveillance campaigns occur in Africa. We detected Seoul orthohantavirus in black rats in Senegal, although we did not find serologic evidence of this disease in humans. These findings highlight the need for increased surveillance of hantaviruses in this region.

COVID-19 Outbreak, Senegal, 2020.

The spread of severe acute respiratory syndrome coronavirus 2 began later in Africa than in Asia and Europe. Senegal confirmed its first case of coronavirus disease on March 2, 2020. By March 4, a total of 4 cases had been confirmed, all in patients who traveled from Europe.

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.

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.

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.

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.

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.

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 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.

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.

Analysis of contact tracing data showed contribution of asymptomatic and non-severe infections to the maintenance of SARS-CoV-2 transmission in Senegal.

During the COVID-19 pandemic in Senegal, contact tracing was done to identify transmission clusters, their analysis allowed to understand their dynamics and evolution. In this study, we used information from the surveillance data and phone interviews to construct, represent and analyze COVID-19 transmission clusters from March 2, 2020, to May 31, 2021. In total, 114,040 samples were tested and 2153 transmission clusters identified. A maximum of 7 generations of secondary infections were noted. Clusters had an average of 29.58 members and 7.63 infected among them; their average duration was 27.95 days. Most of the clusters (77.3%) are concentrated in Dakar, capital city of Senegal. The 29 cases identified as super-spreaders, i.e., the indexes that had the most positive contacts, showed few symptoms or were asymptomatic. Deepest transmission clusters are those with the highest percentage of asymptomatic members. The correlation between proportion of asymptomatic and degree of transmission clusters showed that asymptomatic strongly contributed to the continuity of transmission within clusters. During this pandemic, all the efforts towards epidemiological investigations, active case-contact detection, allowed to identify in a short delay growing clusters and help response teams to mitigate the spread of the disease.