ABSTRACT
West Africa serves as a critical region for the co‐circulation of mosquito‐borne flaviviruses, which often precipitate sporadic outbreaks. This study investigated the seropositivity to dengue virus serotypes 1–4 (DENV‐1–4), Zika virus (ZIKV), yellow fever virus (YFV), and West Nile virus (WNV) in three regions of Senegal: Sindia, Thies, and Kedougou. We retrospectively analyzed 470 serum samples for flavivirus immunoglobulin G (IgG) using a DENV‐2 envelope (E) ELISA. Our findings revealed an overall flavivirus seroprevalence of 37.23%. Among the DENV‐2 E IgG positive samples, the proportion of subjects with IgG to DENV‐1–4, ZIKV, YFV, or WNV NS1 was 57.14%, 12.57%, 80.57%, and 17.14%, respectively, with 66.86% harboring neutralizing antibodies against two or more flaviviruses. We also identified that residents in Sindia (ZIKV, aOR, 9.428; 95% CI: 1.882–47.223 and WNV, aOR, 6.039; 95% CI: 1.855–19.658) and Kedougou (ZIKV, aOR, 7.487; 95% CI: 1.658–33.808 and WNV, aOR, 1.142; 95% CI: 0.412–3.164) were at significant risk for ZIKV and WNV exposure. This study underscores the complexity of flavivirus epidemiology in West Africa and the necessity for enhanced surveillance to inform public health strategies.
Keywords: dengue virus serotypes 1–4, seroprevalence, West Africa, West Nile virus, yellow fever virus, Zika virus
1. Introduction
Flavivirus, a genus within the Flaviviridae family, comprises over 70 arthropod‐borne viruses characterized by a single‐stranded, positive‐sense RNA genome [1]. Notable members of this group include dengue virus serotypes 1–4 (DENV‐1–4), Zika virus (ZIKV), yellow fever virus (YFV), and West Nile virus (WNV), all of which pose significant global health risks. Transmission occurs via distinct mosquito vectors, with Aedes species primarily responsible for spreading DENV‐1–4 [2], ZIKV [3] and YFV [4], while Culex mosquitoes are the main vectors of WNV [5]. The flavivirus genome encodes a single polyprotein that is cleaved into three structural (capsid, envelope [E], and pre‐membrane) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins [6]. Phylogenetic analysis suggests that all vector‐borne flaviviruses originated in Africa, with mosquito vectors likely playing a central role in their evolution and dissemination [7]. The abundant population of these mosquitoes is driven by warm, humid climates and human‐driven environmental changes, such as deforestation, which create ideal breeding conditions that can facilitate their spread into rapidly urbanizing areas [8]. In many resource‐constrained regions, limited healthcare infrastructure hampers the diagnosis and treatment of flavivirus infections [8]. Moreover, the clinical symptoms of flavivirus infections often overlap with those of other endemic diseases, like malaria or typhoid, leading to underreporting or misdiagnosis in the absence of specific, distinctive acute‐phase symptoms [9].
In Africa, where DENV‐1–4 is endemic in at least 34 countries [10], infections are frequently inapparent or manifest as mild disease [11]. However, outside of Africa, dengue is responsible for significant morbidity and mortality, with an estimated 400 million annual cases characterized by a spectrum of clinical manifestations, ranging from mild dengue fever to severe forms such as dengue hemorrhagic fever and dengue shock syndrome [12]. Despite its global prevalence, the true burden of dengue in Africa remains poorly understood, partly due to inadequate diagnostic and surveillance infrastructure [13].
In Senegal, the first documented dengue case was reported in 1970, underscoring its emerging public health importance [14]. The epidemiological transmission dynamics of DENV‐1–4 in Senegal remain unclear; however, all four serotypes have been isolated from patients in Senegal, with recent reports of sylvatic DENV‐2 causing human outbreaks in Kedougou [15]. Historically, DENV‐2 has predominated; however, the epidemiological landscape shifted in 2009, when DENV‐3 emerged, particularly in urban areas like Thies [16]. In recent years, the situation has escalated to hyperendemic levels, with annual outbreaks driven by multiple serotypes, such as the 2017–2018 outbreak of DENV‐1–3 [17, 18]. The availability of three tetravalent dengue vaccines signals progress in controlling dengue outbreaks, but only the Sanofi Pasteur dengue vaccine Dengvaxia has been approved for use in Africa [19]. However, Sanofi Pasteur's recent announcement to halt manufacturing of the dengue vaccine for children, emphasizes the urgent need for robust surveillance to mitigate future outbreaks across Africa [20].
YFV, believed to have originated in African rainforests, has plagued human populations for centuries and was first isolated during the 1927 epidemic in West Africa [21]. This strain served as the basis for the development of the YFV 17D vaccine, a live‐attenuated vaccine that induces long‐lasting immunity in 99% of vaccinated individuals [22]. In countries such as Senegal, estimates suggest that vaccination campaigns targeting early childhood have achieved coverage rates of about 80% in specific regions [23]. However, inconsistencies in vaccination coverage persist, leading to periodic outbreaks, such as those that occurred in eastern Senegal between 2020 and 2021 [24]. These gaps in coverage are attributed to limited healthcare infrastructure, suboptimal vaccine distribution, and under‐resourced immunization campaigns [25]. Consequently, YFV remains a major public health threat in West Africa, particularly in unvaccinated populations, with an estimated annual burden of 84 000 to 170 000 severe cases [25]. Surveillance of populations for neutralizing antibodies, whether from vaccination or natural infection, is crucial for assessing seroprevalence and understanding the role of pre‐existing immunity in modulating responses to subsequent flavivirus infections.
ZIKV, first identified in Uganda in 1947, has been reported intermittently in Senegal from 1962 through 2018 [26, 27]. As a neurotropic virus, ZIKV is linked to severe outcomes, such as microcephaly in neonates [28] and Guillain Barre syndrome in adults [29]. The 2015–2016 Latin American ZIKV outbreak highlighted the importance of monitoring emerging neglected tropical diseases. Initially, only Asian lineage ZIKV strains were thought to be associated with neurological complications in infants; however, recent studies indicate that African lineage ZIKV may exhibit greater transmissibility and pathogenicity [30]. A subsequent study found that ZIKV infections in pregnant women in Nigeria were associated with adverse neonatal outcomes, including microcephaly and other congenital anomalies [28]. In Senegal, ZIKV primarily circulates within a sylvatic transmission cycle involving nonhuman primates and arboreal Aedes mosquitoes, with human cases predominantly occurring in rural areas [31]. Despite this, there have been no large‐scale human outbreaks reported in Africa, and seroprevalence studies in Senegal have documented ZIKV seropositivity rates, ranging from 7.5%–13%, suggesting low‐level transmission [32].
Beginning in 1937, WNV was first discovered in Uganda and circulates primarily in an enzootic cycle between birds and mosquitoes [33]. With nine genetically distinct lineages identified, WNV exhibits considerable genetic diversity, and lineages 1, 2, 7, and 8 have been reported circulating in Africa [33]. Although most WNV infections are inapparent or mild, a subset of cases results in severe neurological manifestations, including encephalitis [33]. Notably, a large WNV outbreak affected thousands of individuals in South Africa in 1974, and sporadic outbreaks continue to occur across Africa [34]. While WNV has been documented in Senegal, no major outbreaks have been reported, despite serological evidence of its circulation. Hence, in‐depth seroprevalence studies are needed to assess the current burden of WNV in Senegal.
Given the increasing threat posed by flavivirus infections across Africa, there is a need to prioritize surveillance efforts and to better characterize the immune responses elicited by these infections. The objective of this retrospective study was to determine the seropositivity rates against DENV‐1–4, ZIKV, YFV, and WNV in non‐febrile serum collected from individuals residing in three regions in Senegal. Furthermore, we assessed the neutralizing capacity against these viruses. Our findings suggest a high proportion of individuals experiencing multiple and/or sequential flavivirus infections, potentially harboring cross‐neutralizing antibodies, which has significant implications for public health and vaccine development strategies.
2. Methods
2.1. Study Populations and Ethics Statement
The samples included in our study were originally collected as part of malaria and non‐malarial surveillance among residents of Sindia, Thies, and Kedougou, Senegal. Samples from Sindia and Thies were part of the surveillance of non‐malarial, afebrile individuals, while samples from Kedougou were part of genomic surveillance of malaria. Informed consent was obtained from all subjects and/or their legal guardians for the initial sample collection as well as for its future use. All methods were performed in accordance with the guidelines and regulations set forth by the Declaration of Helsinki.
The primary studies under which the samples and data were collected received ethical clearance from the CIGASS Institutional Review Board (IRB) (Protocol numbers: SEN15/46, 19300, and SEN14/49). All excess samples and corresponding data were banked and de‐identified before the analyses. This study received an exemption determination from the Rutgers Robert Wood Johnson Medical School IRB.
2.2. ELISAs
Human sera were subjected to DENV‐2 E Immunoglobulin G (IgG) enzyme‐linked immunosorbent assay (ELISA, as previously described [35]) to test for the presence of DENV‐2 E IgG antibodies to determine flavivirus serostatus. Nunc Maxisorp 96‐well plates (ThermoFisher) were coated overnight with 20 ng/well of native DENV‐2 E antigen (Native Antigen). Plates were then blocked for 1 h with 1× PBST supplemented with 1% BSA (ThermoFisher) and then washed three times with 1× PBST. Serum samples (1:400) were added and incubated for 2 h at room temperature, followed by three washes to remove unbound antibody. Plates were then incubated with HRP‐conjugated anti‐Human IgG (ThermoFisher), according to the manufacturer's instructions. The plates were washed three more times with 1× PBST, followed by the addition of the 1‐Step TMB substrate and incubated in the dark for 10 min. Absorbance was measured using an ELISA reader (Diasource) at 450 nm, with a reference wavelength of 650, as previously described [35].
Subsequently, NS1‐based IgG ELISAs, as previously described [35], were performed to determine the seropositivity rates against DENV1–4, ZIKV, YFV, and WNV among the DENV‐2 E IgG‐positive serum samples. Similar to DENV‐2 E‐based ELISA, 96‐well plates were individually coated with 20 ng/well of native DENV1–4, ZIKV, YFV, or WNV NS1 proteins (Native Antigen), blocked, washed, and incubated with serum samples (1:400). Bound serum IgGs were detected using an HRP‐conjugated anti‐Human IgG, followed by additional washes and treatment with 1‐Step TMB ELISA substrate (ThermoFisher) Absorbance was measured at 450 nm, with a reference wavelength of 650, as previously described [35].
2.3. Microneutralization Assays
Microneutralization assays were used to determine the proportion of samples harboring neutralizing antibodies to a specific flavivirus. Serum samples were diluted in a twofold series from 1:10 to 1:1280 in 1× PBS in a 96‐well plate. For each dilution, contact was performed with equal volume of 100 plaque forming units (PFU) of DENV‐1 (Hawaii strain), DENV‐2 (DakArA1247 strain), DENV‐3 (CH53489 strain), DENV‐4 (H241 strain), ZIKV (DAK AR 41524 strain), YFV (YFV17D strain), or WNV (Bird114 strain) for 90 min at 37°C in a 5% CO2 incubator. After neutralization, the complexes were then added to pre‐seeded Vero cell monolayers (CCL‐81, ATCC) in 96‐well plates and an additional 100 μl of cell culture medium was added without removal of the virus inoculum after adsorption. After 3–6 (ZIKV, YFV, WNV) to 7–10 (DENV‐1–4) days of incubation, the cytopathogenic effects were investigated. The cell culture medium was removed from the plates and the cells were stained with 0.2% crystal violet (SigmaAldrich) in 20% Ethanol (SigmaAldrich). After staining for 2 h, the plates were washed with copious amounts of water.
The 90% neutralization titer (NT90) of the test serum sample against each virus was defined as the reciprocal of the test serum dilution for which the virus infectivity was reduced by 90% relative to the challenge virus dose (without any antibodies). A titer lower than 1:20 was considered negative if cytopathogenic effects were not observed. For monotypic infections, samples were considered positive if the titer was ≥ 1:20. For samples that had neutralizing activity against two or more viruses, samples were considered positive if the titer was ≥ 1:40; a higher NT90 cutoff was used as a more stringent means for determination. Samples with titers ≥ 1:40 to two or more DENV serotypes were defined as secondary dengue (sDENV). Microneutralization of each virus was validated using immune serum from previously infected humans (BEI Resources) or mice. Additionally, at least 3 flavivirus‐naïve serum samples were incubated with DENV‐1, DENV‐2, DENV‐3, DENV‐4, ZIKV, YFV, or WNV before pipetting onto the Vero cells per plate. In parallel, 100 PFU of each virus was applied to Vero cells in triplicates without prior serum contact as a technical positive control.
2.4. Statistical Analysis
Categorical variables are represented as case counts and percentages, while continuous variables are represented as medians with ranges. To evaluate the performance of the NS1‐based ELISAs, we compared their results to those obtained from the microneutralization assay, which served as the gold standard for determining true positive and true negative rates. Sensitivity of the assay represents the proportion of true positives correctly identified, while specificity indicates proportion of true negatives. To provide precise estimates of the assay's sensitivity and specificity, 95% confidence intervals were calculated using the exact Clopper‐Pearson method.
To investigate the association between flavivirus (DENV, ZIKV, and WNV) seropositivity rates and demographic factors, such as age group, gender, geographic location, a univariate binary logistic regression analysis was employed. These models allowed for the estimation of the odds ratios (ORs) for each independent variable, quantifying the likelihood of flavivirus seropositivity. Adjusted odds ratios (aORs) were calculated to account for age and gender as confounding variables, ensuring that the observed associations were not biased by these variables. Statistical significance was assessed at a p‐value threshold of < 0.05, and 95% confidence intervals (CIs) were calculated to evaluate the precision of the odd ratios. All analyses were conducted using IBM SPSS Statistics software version 28. Heatmap and Venn diagram, representing neutralization titers against the flaviviruses, were generated by GraphPad Prism version 10.2.0 and Microsoft PowerPoint, respectively.
3. Results
A total of 470 serum samples were collected from three regions in Senegal: Sindia, Thies, and Kedougou. Of these, 94 samples each were obtained from Sindia and Thies in 2018, while 282 samples were collected from Kedougou between 2022 and 2023. Participants' median ages were 24 (range: 2–70 years) in Sindia, 20 (range: 6 months–69 years) in Thies, and 21 (range: 2–85 years) in Kedougou (Table 1). 47.66% (224/470) of subjects were aged 20 years or younger, 40% (188/470) were between 21 and 40 years, 11.7% (55/470) were older than 40 years, and 0.63% (3/470) did not report an age (Table 1). Samples from Sindia and Thies were from malaria‐negative individuals, while all samples from Kedougou were from malaria‐positive individuals (Table 1).
Table 1.
Baseline characteristics and prevalence of flavivirus exposure.
| Sindia | Thies | Kedougou | Total | |
|---|---|---|---|---|
| (n = 94) | (n = 94) | (n = 282) | (n = 470) | |
| Demographics | ||||
| Year(s) samples collected | 2018 | 2018 | 2022–2023 | |
| Median age (range), years | 24 (2–70) | 20 (0.5–69) | 21 (2–85) | 21 (0.5–85) |
| Age group, n (%) | ||||
| ≤ 20 years | 40 (42.55) | 48 (51.06) | 136 (48.23) | 224 (47.66) |
| 21–40 years | 42 (44.68) | 23 (24.47) | 123 (43.62) | 188 (40.00) |
| > 40 years | 11 (11.70) | 23 (24.47) | 21 (7.45) | 55 (11.70) |
| Unreported | 1 (1.06) | _ | 2 (0.71) | 3 (0.64) |
| Female gender, n (%) | 39 (41.49) | 51 (54.26) | 121 (42.91) | 211 (44.89) |
| History of malaria infection, n (%) | 0 (0.00) | 0 (0.00) | 282 (100.00) | 282 (60.00) |
| Serology | ||||
| DENV‐2 E IgG+, n (%) | 33 (35.11) | 27 (28.72) | 115 (40.78) | 175 (37.23) |
Out of the total samples included in the study, 175 samples (37.23%) tested positive for DENV‐2 E IgG by ELISA (Table 1). Among the 175 DENV‐2 E IgG positive samples, the proportion of subjects with IgG antibodies to DENV‐1–4, ZIKV, YFV, or WNV were assessed using NS1‐based IgG ELISAs. In Sindia (n = 33), the proportions were 75.76% for DENV‐1–4, 9.09% for ZIKV, 69.70 for YFV, and 39.39% for WNV (Table 2). In Thies (n = 27), the proportions were 62.96% for DENV‐1–4, 3.70% for ZIKV, 70.37% for YFV, and 14.81% for WNV (Table 2). In Kedougou (n = 115), the proportions were 50.43% for DENV‐1–4, 15.65% for ZIKV, 86.09% for YFV, and 11.30% for WNV (Table 2). Overall, 57.14% of the subjects had IgG antibodies to DENV‐1–4, 12.57% to ZIKV, 80.57% to YFV, and 17.14% to WNV (Table 2). Finally, the seropositivity rates were estimated at 21.28% for DENV‐1–4, 4.68% for ZIKV, 6.38% for WNV, and 30% for YFV (Supplementary Table 1).
Table 2.
Proportion of NS1‐specific IgG among DENV‐2 IgG‐positive individuals.
| Sindia | Thies | Kedougou | Total | |
|---|---|---|---|---|
| (n = 33) | (n = 27) | (n = 115) | (n = 175) | |
| DENV‐1–4 NS1, n (%) | 25 (75.76) | 17 (62.96) | 58 (50.43) | 100 (57.14) |
| ZIKV NS1, n (%) | 3 (9.09) | 1 (3.70) | 18 (15.65) | 22 (12.57) |
| WNV NS1, n (%) | 13 (39.39) | 4 (14.81) | 13 (11.30) | 30 (17.14) |
| YFV NS1, n (%) | 23 (69.70) | 19 (70.37) | 99 (86.09) | 141 (80.57) |
To assess the reliability of the NS1‐based ELISAs, we compared their performance against neutralization assays. Sensitivity and specificity rates for each virus were determined. For any DENV, the sensitivity and specificity were 89.72% and 94.12%, respectively (Supporting Information S1: Table 2). For ZIKV, the sensitivity was 87.50%, and the specificity was 99.34% (Supporting Information S1: Table 2). For YFV, the sensitivity was 91.39%, with a specificity of 87.50% (Supporting Information S1: Table 2). For WNV, the sensitivity was 88.46% and the specificity was 95.30% (Supporting Information S1: Table 2). These results underscore the high accuracy of NS1‐based ELISAs in distinguishing between flaviviruses.
Neutralization assays were performed to evaluate the proportion of subjects with neutralizing antibodies (nAbs) to all the DENV serotypes, ZIKV, YFV, and WNV, among the DENV E IgG positive subjects. The proportions were 18.86% for DENV‐1, 12.57% for DENV‐2, 32.57% for DENV‐3, and 5.71% for DENV‐4. (Table 3). The proportion of nAbs against any DENV serotype was 69.71% (Table 3). The proportions of nAbs against ZIKV, YFV, and WNV were 13.71%, 86.29%, and 14.85%, respectively (Table 3). In Sindia, the proportions of nAbs against DENV‐1, DENV‐2, DENV‐3, and DENV‐4 were 30.03%, 15.15%, 15.15%, and 12.12%, respectively, with an overall any DENV proportion of 72.73% (Table 3). ZIKV, YFV, and WNV proportions were 12.12%, 93.94%, and 36.36%, respectively (Table 3). In Thies, the proportions of nAbs against DENV‐1, DENV‐2, DENV‐3, and DENV‐4 were 18.52%, 22.22%, 37.04%, and 0%, respectively, with an overall any DENV proportion of 77.78% (Table 3). ZIKV, YFV, and WNV proportions were 3.70%, 66.67%, and 11.11%, respectively (Table 3). In Kedougou, the proportions of nAbs were 15.65% for DENV‐1, 9.57% for DENV‐2, 36.52% for DENV‐3, and 5.21% for DENV‐4, with an any DENV proportion of 66.96% (Table 3). ZIKV, YFV, and WNV proportions were 16.52%, 88.70%, and 9.56%, respectively (Table 3).
Table 3.
Proportion of DENV‐1–4, ZIKV, YFV, and WNV neutralizing antibodies among DENV‐2 IgG‐positive individuals.
| Sindia | Thies | Kedougou | Total | |
|---|---|---|---|---|
| (n = 33) | (n = 27) | (n = 115) | (n = 175) | |
| DENV‐1, n (%) | 10 (30.03) | 5 (18.52) | 18 (15.65) | 33 (18.86) |
| DENV‐2, n (%) | 5 (15.15) | 6 (22.22) | 11 (9.57) | 22 (12.57) |
| DENV‐3, n (%) | 5 (15.15) | 10 (37.04) | 42 (36.52) | 57 (32.57) |
| DENV‐4, n (%) | 4 (12.12) | 0 (0) | 6 (5.21) | 10 (5.71) |
| sDENV, n (%) | 2 (6.06) | 4 (14.81) | 9 (7.83) | 15 (8.57) |
| any DENV, n (%) | 24 (72.73) | 21 (77.78) | 77 (66.96) | 122 (69.71) |
| ZIKV, n (%) | 4 (12.12) | 1 (3.70) | 19 (16.52) | 24 (13.71) |
| WNV, n (%) | 12 (36.36) | 3 (11.11) | 11 (9.56) | 26 (14.86) |
| YFV, n (%) | 31 (93.94) | 18 (66.67) | 102 (88.70) | 151 (86.29) |
| Negative, n (%) | 2 (6.06) | 4 (14.81) | 7 (6.09) | 13 (7.43) |
Among the DENV E IgG‐positive subjects, 66.86% (117/175) had neutralizing activity against two or more flaviviruses, with 20.0% (35/175) of the subjects exhibiting neutralizing activity against three or more flaviviruses (Figure 1B; Supporting Information S1: Table 3). nAbs to DENV and YFV were observed in 32.0% of these subjects, while 6.86% of subjects exhibited neutralization patterns corresponding to secondary DENV (sDENV) plus YFV (Figure 1B; Supporting Information S1: Table 3). Additionally, 4.57% of DENV E IgG‐positive subjects had nAbs against ZIKV and YFV, while 7.43% demonstrated nAbs against both WNV and YFV. Finally, the prevalence of nAbs against DENV‐1, DENV‐2, DENV‐3, DENV‐4, any DENV, ZIKV, YFV, and WNV was estimated at 7.02%, 4.68%, 12.13%, 2.13%, 25.96%, 5.11%, 5.53%, and 32.13% (Supporting Information S1: Table 4).
Figure 1.
Neutralizing antibodies across the total population. (A) Heat map representing the neutralizing antibody titers against DENV‐1–4, ZIKV, YFV, and WNV for the samples tested from each Senegalese location. The representative sample size is the 175 subjects who were positive for DENV‐2 E ELISA. (B) Venn diagram depicting the flavivirus‐specific neutralization rates from positive PRNT90 results. Thirteen samples were negative for neutralization against all viruses tested. Abbreviations: DENV, dengue virus; sDENV, secondary dengue virus; WNV, West Nile virus; YFV, yellow fever virus; and ZIKV, Zika virus.
Univariate logistic regression analysis identified several factors associated with flavivirus infection based on the DENV‐2 E IgG‐positive subjects. Individuals aged 21–40 years (aOR, 2.912; 95% CI: 1.915–4.427) and those over 40 years old (aOR, 2.961; 95% CI: 1.604–5.465) were more likely to have flavivirus exposure, with the highest risk in individuals over 40 (Supporting Information S1: Table 5). Geographic location also played a marginal role; residents of Sindia (aOR, 1.323; 95% CI: 0.691–2.534) and Kedougou (aOR, 1.782; 95% CI: 1.034–3.072) had increased odds of flavivirus exposure even though the association was not statistically significant (Supporting Information S1: Table 5).
In contrast, univariate logistic regression based on DENV, ZIKV, or WNV NT90 confirmed subjects, revealed no significant association between DENV infection risk and any of the modeled variables (Table 4). However, residents in Sindia (ZIKV, aOR, 9.428; 95% CI: 1.882–47.223 and WNV, aOR, 6.039; 95% CI: 1.855–19.658) and Kedougou (ZIKV, aOR, 7.487; 95% CI: 1.658–33.808 and WNV, aOR, 1.142; 95% CI: 0.412–3.164) were associated with significantly increased odds of ZIKV and WNV exposure (Table 4).
Table 4.
Risk factors for DENV‐1–4, ZIKV and WNV exposure.
| Univariate | ||||
|---|---|---|---|---|
| Variable | OR (95% CI) | p value | aOR (95% CI) | p value |
| DENV NT90‐confirmed individuals | ||||
| Age group | ||||
| ≤ 20 years | Reference | 0.342 | Reference | 0.35 |
| 21–40 years | 1.104 (0.553–2.204) | 1.079 (0.539–2.162) | ||
| > 40 years | 2.162 (0.751–6.226) | 2.137 (0.736–6.205) | ||
| Gender | ||||
| Male | Reference | Reference | ||
| Female | 1.119 (0.597–2.097) | 0.727 | 1.058 (0.559–2.002) | 0.863 |
| City | ||||
| Thies | Reference | 0.638 | Reference | 0.582 |
| Sindia | 1.569 (0525–4.685) | 1.747 (0.568–5.369) | ||
| Kedougou | 1.062 (0.445–2.532) | 1.171 (0.479–2.86) | ||
| ZIKV NT90‐confirmed individuals | ||||
| Age group | ||||
| ≤ 20 years | Reference | 0.084 | Reference | 0.107 |
| 21–40 years | 2.274 (1.076–4.802) | 2.182 (1.028–4.629) | ||
| > 40 years | 1.36 (0.484–3.826) | 1.318 (0.464–3.74) | ||
| Gender | ||||
| Male | Reference | Reference | ||
| Female | 1.212 (0.644–2.280) | 0.552 | 1.165 (0.611–2.223) | 0.643 |
| City | ||||
| Thies | Reference | 0.016 | Reference | 0.021 |
| Sindia | 10.417 (2.114–51.335) | 9.428 (1.882–47.223) | ||
| Kedougou | 7.192 (1.622–31.893) | 7.487 (1.658–33.808) | ||
| WNV NT90‐confirmed individuals | ||||
| Age group | ||||
| ≤ 20 years | Reference | 0.062 | Reference | 0.05 |
| 21–40 years | 2.061 (0.988–4.298) | 2.106 (1.006–4.41) | ||
| > 40 years | 0.837 (0.281–2.492) | 0.842 (0.28–2.53) | ||
| Gender | ||||
| Male | Reference | Reference | ||
| Female | 0.984 (0.523–1.852) | 0.96 | 0.97 (0.507–1.857) | 0.928 |
| City | ||||
| Thies | Reference | < 0.001 | Reference | < 0.001 |
| Sindia | 7 (2.193–22.343) | 6.039 (1.855–19.658) | ||
| Kedougou | 1.235 (0.455–3.352) | 1.142 (0.412–3.164) | ||
4. Discussion
This study on the seropositivity to DENV‐1–4, ZIKV, YFV, and WNV in Senegal highlights the heterogenous distribution of these flaviviruses, driven by geographical and ecological factors that influence transmission dynamics. Among the 470 serum samples analyzed, 37.23% exhibited seropositivity to DENV‐2 E, suggestive of prior flavivirus exposure, with the highest seropositivity rate observed in Kedougou. This aligns with the region's longstanding status as an endemic zone, where extensive sylvatic surveillance since the 1960s has contributed valuable insights into the evolutionary ecology and circulation of arboviruses in these complex ecosystems [36]. Understanding these dynamics is crucial for the development of targeted public health interventions that reflect the transmission nuances of different regions.
The flavivirus envelope exhibits about 40% amino acid sequence homology across species, which poses a significant challenge for serological differentiation [37]. While E‐based ELISAs are commonly used due to their cost‐effectiveness and ability to detect broad flavivirus exposure, their specificity is limited by cross‐reactivity—a drawback exacerbated in regions where multiple flaviviruses co‐circulate [38, 39]. Nevertheless, our DENV‐2 E‐based ELISA provided an overall estimate of flavivirus seroprevalence of 37.23% within the study population, with prior flaviviral exposure rates of 35.11% in Sindia, 28.72% in Thies, and 40.78% in Kedougou (Table 1).
The implementation of NS1‐based IgG ELISAs, which offers less cross‐reactivity and higher specificity among closely related flaviviruses, has served as a valuable tool for distinguishing between virus‐specific infections [40, 41, 42]. In this study, NS1‐based ELISAs demonstrated high sensitivity and specificity (Supporting Information S1: Table 2), corroborated by neutralization assays that remain the gold standard for confirming prior exposure to a specific virus [43]. The results obtained underscore the necessity of employing complementary methods, such as neutralization assays, to confirm specific flavivirus infections and minimize diagnostic ambiguities.
Our findings revealed significant exposure to YFV, with a higher proportion of subjects exhibiting YFV‐specific IgG antibodies in Kedougou (86.09%), compared to 69.70% in Sindia and 70.37% Thies (Table 2). Additionally, the observed proportion of YFV nAbs at 86.29% further support our assertion of high YFV exposure, likely through YFV 17D vaccination, with natural infection contributing to a lesser extent (Table 3, Figure 1). This is consistent with prior reports documenting multiple distinct YFV lineages in Senegal, particularly concentrated in Kedougou, an epicenter of YFV due to its expansive forest habitats facilitating sylvatic transmission [44]. Additionally, differences in spatial distribution of Aedes species contributes to human rates of infections, where YFV‐bearing mosquitoes have been associated with peak human transmission when found in agricultural villages [45]. Also, the Senegalese government's vaccination campaign strategies, including the 2021 mass vaccination initiative, likely influenced these high seroposivity rates [24, 46]. Such efforts are essential for understanding regional immunity patterns and the persistence of YFV across different landscapes.
The DENV‐1–4 NS1 IgG positive subjects were notably high in Sindia (75.76%), followed by Thies (62.96%), and Kedougou (50.43%), reflecting the broad endemicity of DENV across Senegal. Urban centers, such as Thies, experience intensified transmission due to the proliferation of Aedes aegypti, whereas sylvatic transmission also contributes to persistent DENV‐2 circulation with outbreak potential in areas like Kedougou [15, 47]. The 2018 outbreak [48], which heavily impacted Sindia and Thies, likely contributed to these observations, a finding supported by the higher proportion of nAbs observed against DENV‐1 (30.03%) in Sindia, and DENV‐3 (37.04%) in Thies (Table 3, Figure 1). Overall, the proportion of subjects with any DENV nAb was 69.71%, with rates ranging from 5.71% for DENV‐4% to 32.57% for DENV‐3 (Table 3, Figure 1). There was also evidence of sDENV exposure across all three sites, with 6.06%, 14.81%, and 7.83% seropositivity rates in Sindia, Thies, and Kedougou, respectively. While we cannot ascertain the interval of time between the dengue infections in these subjects, it is possible that these infections occurred concomitantly, as one study has reported on the occurrence of co‐circulatory outbreaks of DENV‐1 and DENV‐2 in Louga city, Senegal in 2017 [49]. Reporting on patterns of cross‐reactive immunity is particularly insightful in the context of DENV infections, where antibody‐dependent enhancement remains a major concern.
In the present study, the proportion of subjects with ZIKV‐specific IgG was 12.57%, which is comparable to findings from similar studies in Senegal, which reported seropositivity rates of approximately 13% [32]. This is further supported by the presence of ZIKV nAbs in 13.71% of subjects with DENV E‐specific IgG (Table 3, Figure 1). Another study conducted in north central Nigeria found a seropositivity rate of 10% using both anti‐ZIKV IgM and IgG NS1‐based ELISAs [50]. The differences seen in prevalence rates across our three study sites (3.70%–15.65%), potentially reflect differences in geographical transmission dynamics as one study performed in six different sites in Cameroon reported a ZIKV seroprevalence range from 2%–10%, with an average rate of 5% compared to our study's 12.57% NS1 seropositivity rate [51].
Moreover, exposure to WNV was also observed, with a rate of 14.85%. This rate varied geographically, with 14.81% of subjects in Thies, 11.30% in Kedougou, and a markedly higher rate of 39.99% in Sindia. Furthermore, the 14.86% of subjects with nAbs against WNV further supports the circulation of WNV in Senegal (Table 3, Figure 1). Previous studies have demonstrated widespread WNV circulation among mosquito vectors and enzootic hosts, such as birds and horses, across Senegal [52]. Although WNV outbreaks have not been extensively reported, the evidence of nAbs indicates sporadic human exposure and the potential for future outbreaks [33]. Indeed one study found a 78.5% nAbs seroprevalence rate in wild horses in north central Senegal, an area with a high occurrence of transient water reservoirs. These water sources serve as breeding grounds for mosquitoes and migratory birds and horses exacerbating the spread of WNV [53]. Additionally, the proximity of animal sanctuaries in northcentral is an associated risk factor for increased WNV exposure to humans, one study found [33]. It is possible that the proximity to the Bandia reserve in Sindia serves as a favorable environment for Culex mosquitoes to proliferate and infect humans. Further studies need to be performed to assess other risk factors that may pre‐dispose some locations in Senegal to higher WNV exposure.
Moreover, our neutralization data also provided insights into sequential infections/exposures and/or cross‐neutralization. Among subjects with flavivirus exposure, a majority of them (66.86%) exhibited neutralizing activity against two or more flaviviruses, while 2.29% demonstrated potential cross‐neutralization against all tested flaviviruses. These findings underscore the complexity of immune responses in regions with persistent flavivirus co‐circulation. Differentiating between secondary DENV infections and potential DENV–ZIKV co‐infections remains challenging due to their substantial cross‐reactivity. Notably, 13 subjects who showed seropositivity in the DENV‐2 E ELISA, lacked nAbs, suggesting waning binding antibodies that no longer confer neutralization, as reported in a prior study [54]. Another possible explanation that cannot be excluded is false positivity of the DENV‐2 E‐based ELISA. Additionally, the waning potential of YFV nAbs post‐vaccination, particularily in infants and young children, has also been observed [55], warranting further investigation into the duration of immunity acquired through YFV natural infection or YFV 17D vaccination.
The role of pre‐existing immunity in modulating secondary flavivirus infections is of particular interest. Cross‐reactivity between DENV and YFV, and its potential protective or detrimental effects, has been documented in multiple studies [56, 57]. Our findings support this complex interplay, with individuals showing cross‐neutralization between DENV and YFV potentially reflecting an adaptive immune response that influences subsequent exposure outcomes. The lack of monotypic ZIKV and WNV infections further emphasizes the possibility that pre‐existing immunity to more prevalent flaviviruses, such as DENV, or through YFV 17D immunization, may help attenuate ZIKV or WNV outbreaks.
Our univariate logistic regression analysis did not reveal any significant association between DENV exposure and demographic factors, such as age, gender, or location (Table 4). However, our analysis revealed significant associations between geographic location and exposure risk, with Sindia and Kedougou residents showing higher odds of ZIKV and WNV exposure, respectively (Table 4). These findings highlight the favorable environmental and ecological conditions that support the propagation and survival of Aedes and Culex mosquito vectors in these regions. Additionally, the possible overlap of vector species in harboring these pathogens simultaneously may contribute to the observed exposure pattern [7].
Limitations of this study include its retrospective design and the absence of clinical data, preventing confirmation of recent or acute infections through IgM testing. Additionally, while we report the flavivirus seroprevalence rate (37.23%) based on the total samples included in the study, much of the analysis is centered on the subgroup of 175 samples that initially tested positive for DENV‐2 E by ELISA. This focus could introduce bias, as the reported seropositivity rates for DENV‐1–4, ZIKV, WNV, and YFV may not fully represent the broader population sampled. By extrapolating findings from this subgroup, there is potential for overestimating seropositivity rates among flavivirus‐naïve individuals or underestimating the burden in regions where DENV exposure is less prevalent. Nevertheless, we believe that focusing on the DENV‐2 E‐positive subgroup provides valuable insights into the epidemiology of flaviviruses among individuals with prior flavivirus exposure. Furthermore, distinguishing YFV 17D vaccination‐induced and natural infection‐induced antibodies was not feasible, which may affect interpretations of seroprevalence data. Despite these limitations, this study represents one of the most comprehensive evaluations of DENV‐1–4, ZIKV, YFV, and WNV seropositivity in Senegal to date, further corroborated by neutralization data. The findings emphasize the need for continued surveillance and deeper investigation into flavivirus molecular epidemiology, including flavivirus genome analyses in West Africa, particularly in areas with overlapping viral circulation and vaccination campaigns.
In conclusion, this study sheds light on the complex interactions between flaviviruses in Senegal, shaped by ecological, historical, and immunological factors. The high rates of flavivirus neutralization observed in our study underscore the need for ongoing research into the immune responses elicited by concurrent flavivirus exposures. Understanding these interactions will be critical for developing more effective vaccines and public health strategies to manage outbreaks in flavivirus‐endemic regions.
Conflicts of Interest
B.B.H. is a cofounder of Mir Biosciences Inc., a biotechnology company focused on T cell‐based diagnostics and vaccines for infectious diseases, cancer, and autoimmunity.
Supporting information
Supplementary Table 1. Seropositivity rates of DENV‐1–4, ZIKV, WNV, and YFV across all samples.
Supplementary Table 2. Performance of NS1‐based ELISAs.
Supplementary Table 3. Proportion of subjects with monotypic and/or multitypic viral exposure.
Supplementary Table 4. Prevalence of DENV‐1–4, ZIKV, WNV, and YFV neutralizing antibodies.
Supplementary Table 5. Risk factors for flavivirus exposure.
Acknowledgments
We would like to thank Rutgers Global Health Institute, Rutgers Robert Wood Johnson Medical School, and The Child Health Institute of New Jersey for their continued support.
Contributor Information
Daouda Ndiaye, Email: dndiaye@cigass.org.
Bobby Brooke Herrera, Email: bherrera@globalhealth.rutgers.edu.
Data Availability Statement
Data produced as part of the current study are available in the manuscript or upon request from the corresponding author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Table 1. Seropositivity rates of DENV‐1–4, ZIKV, WNV, and YFV across all samples.
Supplementary Table 2. Performance of NS1‐based ELISAs.
Supplementary Table 3. Proportion of subjects with monotypic and/or multitypic viral exposure.
Supplementary Table 4. Prevalence of DENV‐1–4, ZIKV, WNV, and YFV neutralizing antibodies.
Supplementary Table 5. Risk factors for flavivirus exposure.
Data Availability Statement
Data produced as part of the current study are available in the manuscript or upon request from the corresponding author.