1. IntroductionThe genus Flavivirus is composed of single-stranded RNA viruses, which include Dengue virus (DENV), Saint Louis encephalitis virus (SLEV), Zika virus (ZIKV), West Nile virus (WNV), and Yellow Fever virus (YFV) [1,2]. All four of which are arboviruses that cause mosquito borne diseases throughout the Americas and are responsible for thousands of deaths and hospitalizations every year [3,4]. Many factors are recognized as contributing to their wide dissemination and re-emergence, e.g., poorly planned urbanization, geographical expansion of vectors, changing environmental conditions, and deforestation [5,6,7].The transmission cycles of these viruses involve a wide variety of susceptible species such as humans, rodents, horses, birds, and nonhuman primates [8]. The clinical presentation of acute flavivirus infections in humans and vertebrates ranges from mild illness (e.g., asymptomatic infection) or self-limiting febrile episodes to severe and life-threatening diseases (hemorrhagic fever, shock syndrome, encephalitis, congenital defects) [9,10,11].WNV and SLEV belong to the Japanese encephalitis serocomplex [12]. Both are neurotropic flaviviruses that can cause encephalitis, seizure disorders, and paralysis in humans and equines [10,13,14,15]. In the United States, several mosquito species have been shown to have high or moderate vectorial capacity, including Culex tarsalis, Cx. pipiens, Cx restuans, Cx. quinquefasciatus, Cx. stigmatosoma, and Cx. nigripalpus [16,17,18]. Additionally, migratory birds can serve as dispersal vehicles when they move seasonally and stop at different sites during their journey, establishing possible dispersal events [19]. Most mammals, such as equines and humans, are dead-end hosts because of the low-level viremia produced after infection [16,20].Costa Rica is endemic for DENV [21,22]. Molecular epidemiological studies show broad circulation of DENV 1–3 in humans, and there is both molecular and serological evidence of DENV-4 circulation in wild animal samples [23,24]. Cocirculation of different flaviviruses adds complexity to clinical and laboratory diagnosis, because of significant cross-reactivity and similarities in the undifferentiated fever-like initial symptoms [25]. The National Health Service of Costa Rica does not currently include SLEV and WNV in their routine diagnostic panel for arbovirus detection; so, the epidemiology and/or local presence of these viruses in the human population is still poorly studied. In 2004–2005, serological evidence of contact with WNV was found in asymptomatic equines and sloths (Choloepus hoffmanni and Bradypus variegatus) in the regions of Guanacaste and Upala [26,27]. Additionally, sloths presented antibodies against SLEV [27]. In 2009, the first cases of clinical disease in equines associated with WNV were reported. After this first report, positive animals continue to be reported annually. Between 2009 and 2017, there were 32 cases of symptomatic equines positive for WNV [28]. Continuous monitoring in endemic areas, such as Costa Rica, and in other tropical areas for early detection and timely reporting are crucial to evaluate the risk of transmission to humans and animals [25,29]. In such areas, epidemiological surveillance based on regular sampling of equines, sentinel chickens, and wild birds has demonstrated good sensitivity [25,29].

Since the presence of zoonotic flaviviruses other than DENV and ZIKV has not been readily determined in humans in our country, the current study aimed to be a proof of concept of the cocirculation of WNV and SLEV in two rural areas that are hyperendemic for other human-infecting flaviviruses and aimed to detect putative reservoirs and vectors for these viruses. Humans, wild birds, equines, and mosquito samples were analyzed to better understand if the viral cycle was present in those areas. We sampled during the rainy and dry seasons of 2017–2018 and conducted plaque reduction neutralization tests (PRNT) for serology and reverse transcription polymerase chain reactions (RT-PCR) for virus detection. We reported, here, several seroconversion events in different peri-domestic and domestic species but found no evidence of active viral infection in any mosquito or bird samples. This seroconversion evidence supports recent circulation of SLEV and WNV in these two regions. This evidence must be taken into account for future prevention campaigns but, most importantly, in arboviruses differential diagnostics.

4. Discussion

In this study, we detected the circulation of WNV and SLEV in two regions of Costa Rica (Cuajiniquil and Talamanca). Although we did not detect WNV or SLEV RNA in wild bird organs or mosquito pools, active circulation of those viruses was evidenced by seroconversion events at both sampling sites and by the detection of neutralizing antibodies in wild bird samples. Interestingly, our results show simultaneous circulation of several flaviviruses in the sampled areas: WNV, SLEV, and DENV-1 in Cuajiniquil and DENV-1 and DENV-2 in Talamanca.

In these areas, equines positive against WNV by IgM ELISA detection have previously been reported [28,47]. In 2009, the first clinical case of WNV was reported in a horse also from Guanacaste, and since then, new equine cases are reported annually [28,48]. Likewise, human DENV and ZIKV cases are reported annually in those areas by the National Health authorities [30]. During the years sampled for this study (2017–2018), both sites reported cases of DENV and ZIKV, most of them diagnosed by symptoms in the clinic and passive surveillance but not as laboratory-confirmed cases, situation normal for endemic countries in which it is not possible to confirm with RT-PCR every single “dengue-like” illness [30]. Interestingly, Brazil and Argentina have reported sporadic cases of SLEV in people presenting mild febrile, “dengue-like” illness, thus resulting in a misdiagnosis of the causative agent [49,50,51]. It is, therefore, tempting to speculate that human WNV and SLEV infections are being mistaken for DENV and ZIKV in endemic areas, e.g., Costa Rica.

Serological analysis showed that neutralizing antibodies against WNV and SLEV are uniformly distributed in Cuajiniquil. There, in each household, at least one of the sampled species had neutralizing antibodies (wild birds, chickens, equines, and/or humans). On the other hand, in the Caribbean sampled area, no evidence of previous contact with WNV was recorded, but serological evidence against SLEV was documented in wild birds, horses, and humans. Likewise, each household had at least one species test positive. The strikingly high seropositivity to WNV in Cuajiniquil and to SLEV in both regions reveals that these viruses might be widely distributed within Costa Rica.

We detected four wild birds with neutralizing antibodies (three for SLEV and one for WNV) belonging to four different species. Three were resident wild birds (Campylorhynchus rufinucha, Myiozetetes similis, and Turdus grayi) suggesting local contact with the virus and raising the possibility that the mosquito–bird–mosquito virus cycle is well established locally. The fourth one, Empidonax virescens, is a migratory species that was captured in Talamanca. This area is one of the most important sites for wild bird migration in the world [46]. During the yearly migration period from October to November, thousands of wild birds fly over Talamanca on their way to South America [46,52]. This migratory behavior could lead to the introduction or local emergence of wild bird-hosted flaviviruses including new strains of SLEV and WNV [19,53]. In the Americas, the role of migratory birds in the spread of WNV is not clear; however, recent studies in other continents have shown the important role of migratory birds in the introduction of new variants of WNV to the territories to which they migrate each year [54,55]. Therefore, this same role could be present in the American continent. Nevertheless, Costa Rica has never reported massive avian deaths, suggesting that the local species might be less susceptible to WNV disease.WNV and SLEV share common mosquito vectors (Culex) and present comparable transmission cycles and clinical signs [15,17,56]. Costa Rica lacks information regarding which mosquitoes can be the possible vectors of WNV and SLEV (or other non-DENV or ZIKV arboviruses). However, some potential vectors are present in the country such as Cx. quinquefasciatus, Cx. thriambus, and Cx. nigripalpus [36]. At both sampling areas, Cx. quinquefasciatus was the most abundant mosquito species collected. Other species of Culex such as Cx. nigripalpus were also identified. The latter has been proposed as a vector for WNV and SLEV in America [20,57]. The blood meals in our study exemplify that Cx. quinquefasciatus, Cx. Coronator, and Cx. pseudostigmatosoma use humans and animals as a food source. This behavior favors virus transmission between different species, so their vectorial competence for WNV in Costa Rica should be investigated. Blood meals and species distribution further support the hypothesis that Culex species may be serving as bridging vectors capable of transmitting WNV between wild birds and final hosts, e.g., humans and equines.In Central America, there have been no reports of outbreaks caused by SLEV or WNV in humans. However, prior studies also found serological evidence of virus circulation [58]. In vitro studies suggest that prior infection with ZIKV or DENV modulates subsequent infection with a different flavivirus and might confer cross protection [59] and Central America is hyperendemic for these two viruses. Alternatively, SLEV (or other arboviruses) could out-compete with WNV for vector and virus amplifiers [60]. The lack of outbreak reports might also be explained by less virulent strains circulating in Central America, since migrating birds carrying outbreak-causing virus do succumb during long-distance migration [53,61].Costa Rica, like the rest of Central America, lacks information about the seroepidemiology of WNV and SLEV. In a literature review conducted by Ortiz et al., 2022, it was found that there is no recent literature or official information about the sanitary status of SLEV and WNV in Central America [58]. Our study demonstrates an ongoing circulation of WNV in the region of Cuajiniquil and SLEV in Talamanca. Our results also indicate the cocirculation of other flaviviruses such as DENV and ZIKV, and suggest that other flaviviruses could also be circulating [22]. Regions with multiple flaviviruses encounter a significant challenge in the clinical and serological diagnosis. Laboratory testing is crucial for accurate diagnosis because symptoms can overlap. Many ELISA kits are not completely devoid of cross-reactions (a necessity for accurate interpretation of results) so there is potential for misinterpretation [25]. Molecular diagnosis by RT-PCR of serum, plasma, and cerebrospinal fluid is of limited value for routine diagnosis, due to the low level and short-lived viremia generated by these viruses [25]. The PRNT ≥90% technique is the gold standard for identifying antibodies against different flaviviruses, but this technique is expensive, needs laboratory facilities, and requires careful interpretation. Thus, flavivirus serological diagnosis is indeed a real challenge [25].

Our study has some limitations. Specifically, the sample size, the limited number of areas that were sampled, and the lack of sentinel animal usage. Further studies must be focused on establishing nation-wide seroprevalence, identifying vectors and reservoirs, and identifying genotypes that might be circulating in the country. Costa Rica, as a tropical country, is susceptible to introduction and establishment of emerging and re-emerging flaviviruses that could result in an even more complex epidemiologic scenario.

Active surveillance for WNV and SLEV must be performed in flavivirus-endemic areas using mosquitoes, wild birds, and sentinel chickens to detect the viruses before re-emergence, the outburst of disease, outbreaks or even establishment of the virus in regions where all the components of the transmission cycle are present. Additionally, WNV and SLEV must be considered as a differential diagnosis in patients suspected for DENV and ZIKV infection. Here, we show that they are indeed circulating in these hyperendemic regions. Therefore, they should be considered by the health and epidemiology authorities for future prevention campaigns and arboviruses differential diagnostics.