Mapping Eastern (EEE) and Venezuelan Equine Encephalitides (VEE) among Equines Using Geographical Information Systems, Colombia, 2008–2019

Introduction: Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) viruses are zoonotic pathogens affecting humans, particularly equines. These neuroarboviruses compromise the central nervous system and can be fatal in different hosts. Both have significantly influenced Colombia; however, few studies analyse its behaviour, and none develop maps using geographic information systems to characterise it. Objective: To describe the temporal-spatial distribution of those viruses in Colombia between 2008 and 2019. Methods: Retrospective cross-sectional descriptive study, based on weekly reports by municipalities of the ICA, of the surveillance of both arboviruses in equines, in Colombia, from 2008 to 2019. The data were converted into databases in Microsoft Access 365®, and multiple epidemiological maps were generated with the Kosmo RC1®3.0 software coupled to shape files of all municipalities in the country. Results: In the study period, 96 cases of EEE and 70 of VEE were reported, with 58% of EEE cases occurring in 2016 and 20% of EEV cases in 2013. The most affected municipalities for EEE corresponded to the department of Casanare: Yopal (20), Aguazul (16), and Tauramena (10). In total, 40 municipalities in the country reported ≥1 case of EEE. Conclusions: The maps allow a quick appreciation of groups of neighbouring municipalities in different departments (1° political division) and regions of the country affected by those viruses, which helps consider the expansion of the disease associated with mobility and transport of equines between other municipalities, also including international borders, such as is the case with Venezuela. In that country, especially for EEV, municipalities in the department of Cesar are bordering and at risk for that arboviral infection. there is a high risk of equine encephalitis outbreaks, especially for VEE. This poses a risk also, for municipalities in the department of Cesar, bordering with Venezuela.


Introduction
Vector-borne diseases remain a significant public health problem in tropical and subtropical regions, especially those of viral aetiology. Beyond dengue, chikungunya, and Zika, other particularly zoonotic arboviruses, such as the case of equine encephalitides, Both viral pathologies (EEE and VEE) can cause severe compromise of the central nervous system and death, both in animals and in humans, with the ability to persist over time due to having multiple vertebrate hosts. Despite all of the above, there are few studies on VEE [14][15][16][17][18] and even more on EEE in Colombia [19,20]. The objective of the current study is to characterise the temporal-spatial distribution of EEE and VEE in Colombia between 2008 and 2019.

Type of Study
A retrospective cross-sectional descriptive observational study was conducted to assess the incidence of EEE and VEE in horses in Colombia. Their incidence per year for the Colombian departments was estimated, and epidemiological maps of the diseases were developed covering the years 2008 to 2019. Additionally, for 2016 to 2019, the annual number of equines per department was obtained. For that period, the incidence rate of EEE and VEE was estimated by calculating the number of cases per 100,000 equines per department per year, confirmed by RT-PCR, by the accredited ICA central reference laboratories.
This is a study of secondary sources where bulletins from the Columbian Agricultural Institute (ICA) were used to obtain local sociodemographic data and records of disease occurrences and their locations, including disease diagnosis. In addition, the total number of cases and their regional location were taken for the geographical characterisation of the incidence of VEE-EEE in the departments of Colombia.
For the respective geographical characterisation, the free access software Kosmo 3.1 was used, which comes with preloaded tools for the geographical analyses. For this investigation, West Bogotá (West Bogotá) (Magna SIRGAS) was used as a reference system (EPSG 21896) of the geographic coordinate system. The mapping was carried out (at a scale of 1: 1,365,207) for which two types of layers were used; the first layer was the departments, to which colours of greater and lesser intensity were added, defined by the epidemiological data layer, classified by ranges established by quartiles, which made it possible to differentiate the areas with a higher incidence of the disease from those with a lower incidence.

Population
Horses from Colombia between the periods 2008-2019 that were diagnosed positive for EEE and VEE, according to the ICA databases.

Sample
The sample was a census taken from the ICA databases, from which registered data will be taken from cases of diagnosis of EEE and VEE in horses in Colombia between 2008 and 2019, confirmed by molecular testing, RT-PCR, by the accredited ICA central reference laboratories.

Analyses
Descriptive statistics were performed on Stata IC ® version 14. The summary of cases per year and departments and the median number and their interquartile (IQR) cases per year for specific periods were calculated.
In 2016, there was a concentration of cases in the neighbouring municipalities in the Casanare department, amounting to 93% of the annual cases for that year ( Figure 5). In 2017, municipalities such as Cumaribo, La Primavera, and Arauca, which border Venezuela, reported cases of EEE ( Figure 6). In 2013, 2014, and 2016, a similar situation occurred for Tibú, Agustin Codazzi, Becerril, and Curumaní municipalities, which also border with that country and reported cases of VEE (Figures 9-11). In addition, one capital municipality reported cases of VEE in 2014 and 2016, Valledupar.    The most affected municipalities for EEE corresponded to the department of Casanare: Yopal (20), Aguazul (16) and Tauramena (10) (Figures 3-7). A total of 40 municipalities in the country reported ≥1 case of EEE in the period ( Figure 3). For VEE, the cases were less concentrated, with Montelíbano, Córdoba being the municipality with the most reported cases (4) (Figures 8-11). A total of 44 municipalities reported ≥1 case of VEE in the period ( Figure 8).                           For 2016-2019, the highest incidence rates of VEE (cases per 100,000 animals) were observed in 2016, 0.829 cases per 100,000 animals (8.29 cases per 1,000,000 animals) ( Table  1).
In 2016, the department with the highest incidence rate was Cesar (14.165 cases per During the first six years (2008-2013), a smaller number of cases and affected municipalities was observed for EEV ( Figure 3) compared with the following six years (2014-2019) (Figures 3-7). On the other hand, the highest concentration of cases and affected municipalities in VEE occurred from 2013 to 2016 (Figures 8-11).
In 2016, there was a concentration of cases in the neighbouring municipalities in the Casanare department, amounting to 93% of the annual cases for that year ( Figure 5). In 2017, municipalities such as Cumaribo, La Primavera, and Arauca, which border Venezuela, reported cases of EEE ( Figure 6). In 2013, 2014, and 2016, a similar situation occurred for Tibú, Agustin Codazzi, Becerril, and Curumaní municipalities, which also border with that country and reported cases of VEE (Figures 9-11). In addition, one capital municipality reported cases of VEE in 2014 and 2016, Valledupar.
For 2016-2019, the highest incidence rates of VEE (cases per 100,000 animals) were observed in 2016, 0.829 cases per 100,000 animals (8.29 cases per 1,000,000 animals) ( Table 1). In 2016, the department with the highest incidence rate was Cesar (14.165 cases per 100,000 animals). However, the national rates decreased in the following years to 0.346 cases per 100,000 animals (3.46 cases per 1,000,000 animals) in 2017, to 0.269 cases per 100,000 animals (2.69 cases per 1,000,000 animals) in 2018, and 0 cases per 100,000 animals in 2019 (Table 1). For 2017, the department with the highest incidence rate was Cordoba (1.842 cases per 100,000 animals). In 2018, at Chocó, the incidence rate was 18.150 cases per 100,000 animals. In 2019 no cases were reported (Table 1). For 2016-2019, the highest incidence rates of EEE (cases per 100,000 animals) were observed in 2016, 3.870 cases per 100,000 animals (38.7 cases per 1,000,000 animals) ( Table 2). In 2016, the department with the highest incidence rate was Casanare (62.999 cases per 100,000 animals). However, the national rates decreased in the following years to 1.176 cases per 100,000 animals (11.76 cases per 1,000,000 animals) in 2017, to 0.269 cases per 100,000 animals (2.69 cases per 1,000,000 animals) in 2018, and 0 cases per 100,000 animals in 2019 ( Table 2). For 2017, the department with the highest incidence rate was Vichada (56.417 cases per 100,000 animals). In 2018, at Guaviare, the incidence rate was 9.708 cases per 100,000 animals. In 2019 only Meta reported cases with an incidence rate of 3.159 per 100,000 animals ( Table 2). On the border with Venezuela, the Cesar department ( Figure 11) had 2016 high incidence rates of VEE (Table 3).

Discussion
Zoonotic arboviruses may be considered even more complex than those that are only anthroponotic or are predominantly anthroponotic [21]. Equine encephalitides are suitable examples of that. These arboviruses include many flavi and alphaviruses and have a wide range of animal hosts and humans. Birds and horses, specifically, are key animals in their enzootic cycles [21]. In the case of horses, these are animals that may serve as sentinels for these vector-borne diseases, as well as for others, mainly due to the ease of access to them, given their proximity to human beings. Regarding birds, the fact that many of them are migratory is a matter of concern for international spreading in countries related to migration routes.
Equine encephalitides such as EEE and VEE are still significant zoonotic arboviral threats in Colombia, Venezuela, and other countries of Latin America and probably the Caribbean islands [1,[22][23][24]. In the current study, a wide distribution during the study period was observed in both alphavirus infections. Nevertheless, with higher variations in EEE (0 to 56 cases in a year), reaching a peak of cases in 2016, without significant variations in the number of cases per year for VEE (0 to 14 cases per year) were observed.
As expected, circulation in municipalities bordering Venezuela was observed in both infections, which may create concern given the sanitary crisis in the country. Over the last few years, the sanitary crisis in Venezuela has implied the reemergence of multiple infectious diseases in humans, animals, zoonotic diseases, and a decrease in vaccination and control programmes, which have led to a complex situation. That may be related to vaccination problems and the critical situation and especially given the healthcare crisis in Venezuela [25], where there are no vaccination programmes for animals or humans (lack of immunisation for measles, mumps, yellow fever and multiple other vaccine-preventable diseases), and where numerous infectious diseases have re-emerged, including foot-andmouth disease that was reintroduced to Colombia from Venezuela [25][26][27][28][29][30][31]. Furthermore, the border crossing of human beings from Venezuela to Colombia has been documented among patients with imported infections, that in some cases, try to reach other borders, such as the one between Colombia and Ecuador in the South or between Colombia and Panama trying to reach the "American dream" after crossing the Darién gap [32][33][34][35]. Although humans are not important reservoir hosts for VEE or EEE, they may cross borders infected and may be diagnosed in other territories and countries.
Additionally, although they may not be a suitable source for transmission, they may impact the health system, care, and disease burden in destination areas. Domestic, farm and wild animals, including equines and birds, may cross the borders (in areas without border controls), sometimes even with their owners, with multiple implications, including EEE and VEE. Therefore, more controls and regulations regarding equines, including vaccination, should be considered, as they may add an additional burden to the healthcare systems of destination countries. In the case of VEE, live-attenuated vaccines have been used in the U.S. military and laboratory workers, and formalin-inactivated vaccines are available for use in horses [36]. One such live-attenuated vaccine is TC-83, developed initially by the U.S. army for vaccine use [37]. TC-83 was created by serially passaging the Trinidad Donkey VEEV strain in guinea pig heart cells [38]. In recent years, new vaccines for horses have been investigated [39], but even a phase 1, open-label, dose-escalation, randomised clinical trial, assessing the safety and immunogenicity of a trivalent virus-like particle vaccine against Western, Eastern, and Venezuelan Equine Encephalitis viruses in humans found a favourable safety profile and neutralising antibody responses including high response against VEE and EE. Along with pressing public health needs, evidence supports further evaluation of the vaccine in advanced-phase clinical trials [40]. In this context, especially vaccination against VEE plays a key role in control. However, the enhancement in the vaccination programme against eastern and Venezuelan equine encephalitis viruses should be performed not only in Colombia but across the border to Venezuela. Given the current sociopolitical situation, cooperation between both governments should be held in order to improve the disease control and vaccination programmes in both countries and specifically on the border. Such arboviral diseases may add an additional burden to the healthcare systems of destination countries.
In the case of EEE, this neuroarbovirus has been scarcely investigated in Colombia, with municipalities of the department of Casanare, such as Yopal, Aguazul, San Luis de Palenque, Trinidad, Orocué, Mani, and Tauramena, affected in 2016. Additionally, in 2017, many municipalities of the eastern plains or lowlands presented cases of EEE in the departments of Meta, Guaviare, Arauca, and Vichada. The maps developed show a clear circulation corridor that goes from the municipality of La Primavera (Vichada) on the border with Venezuela, passing through Cumaribo (Vichada), San José del Guaviare (Guaviare), Puerto Rico (Meta) and Puerto Lleras (Meta) ( Figure 6). Surveillance for eastern and Venezuelan equine encephalitis viruses needs to be increased in the country in humans and horses, as well as studies in birds are also crucial for understanding its transmission. In addition, entomological surveillance is critical in studying such cycles [41], including the study of the mosquito's host preference, which may be helpful in understanding vector feeding [16].
VEE is a persistent zoonosis in Colombia through long periods of apparent epidemiological silence in horses and humans [41][42][43]. In addition to equine studies, it is essential to know the behaviour of rodents as well as bats [24,44], which could have a potential role as dispersing hosts of this virus. Therefore, equine surveillance is helpful as a predictive or sentinel indicator to prevent human cases. Similarly, EEE, to date, has not been reported in Colombia in humans. Nevertheless, a member of the eastern equine encephalitis virus (EEEV) complex, Madariaga virus (MADV) is currently registered in multiple countries of Latin America [45], such as Brazil, Panama [46], Haiti [47], Venezuela [48], among others and in addition to Argentina [49], where it was described.

Conclusions
The developed GIS-based maps generated have allowed us to quickly appreciate groups of neighbouring municipalities in different departments and regions of the country affected by EEE and VEE, helps to appreciate the expansion of the disease associated with mobility and transportation of equines between different municipalities, also including international borders, as it is the case with Venezuela, especially for VEE, with municipalities of the department of Cesar among others. Furthermore, these results have implications for public health in planning and surveillance, especially in those municipalities close to those historically affected in the study period. Future assessments of equine encephalitides should extend to other potential threats, including MADV, Western Equine Encephalitis virus, West Nile Virus, Saint Louis encephalitis virus, reported in Brazil [18,[50][51][52], Rocio virus [53,54], among others, that may even be considered neglected arboviruses in Latin America [55].