
==== Front
One Health
One Health
One Health
2352-7714
Elsevier

S2352-7714(24)00202-7
10.1016/j.onehlt.2024.100876
100876
Research Paper
Prevalence of Flavivirus and Alphavirus in bats captured in the state of Yucatan, southeastern Mexico
Yeh-Gorocica Aaron a
Torres-Castro Marco a
Carrillo-Chan Claudia a
Suarez-Galaz Alejandro a
Suarez-Galaz Melissa a
Moguel-Chin Wilson a
Panti-May Alonso a
Lugo-Caballero Cesar b
Puerta-Guardo Henry cf
Chable-Santos Juan d
Manrique-Saide Pablo df
Ayora-Talavera Guadalupe c
Selem-Salas Celia d
Frias-Casas Mario eg
Rivero-Juarez Antonio antonio.rivero@imibic.org
eg⁎
a Laboratorio de Zoonosis y otras Enfermedades Transmitidas por Vector, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autonoma de Yucatan, Merida, Mexico
b Laboratorio de Enfermedades Emergentes y Reemergentes, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autonoma de Yucatan, Merida, Mexico
c Laboratorio de Virologia, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autonoma de Yucatan, Merida, Mexico
d Departamento de Zoologia, Facultad de Medicina Veterinaria y Zootecnia, Campus de Ciencias Biologicas y Agropecuarias, Universidad Autonoma de Yucatan, Merida, Mexico
e Virologia Clinica y Zoonosis, Instituto Maimonides de Investigacion Biomedica de Cordoba (IMIBIC), Hospital Universitario Reina Sofia de Cordoba, Universidad de Cordoba, Cordoba, Spain
f Unidad Colaborativa para Bioensayos Entomologicos, Facultad de Medicina Veterinaria y Zootecnia, Campus de Ciencias Biologicas y Agropecuarias, Universidad Autonoma de Yucatan, Merida, Mexico
g CIBERINFEC, ISCIII – CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain
⁎ *Corresponding author at: Virologia Clinica y Zoonosis, Instituto Maimonides de Investigacion Biomedica de Cordoba (IMIBIC), Avenida Menedez Pidal, s/n, 14004 Cordoba, Spain. antonio.rivero@imibic.org
15 8 2024
12 2024
15 8 2024
19 10087610 6 2024
12 8 2024
12 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Summary

Flavivirus (family Flaviviridae) and Alphavirus (family Togaviridae) are mosquito-borne viruses that poses a significant risk to public health worldwide. Examples of these viruses include Dengue virus (DENV) and Zika virus (ZIKV) in the Flavivirus genus, and Chikungunya virus (CHIKV) in the Alphavirus genus. The potential contribution of bats in the mosquito-to-human transmission cycle of these viral genera in the tropics has not been studied. Here, a total of 144 bats belonging to three families (Emballonuridae, Phyllostomidae, and Molossidae) and six species were captured for one year using mist nets in sites with different landscapes (forest and grassland) in the state of Yucatan, southeastern Mexico. Blood samples and rectal and oral swabs were collected to detect Flavivirus and Alphavirus RNA genomes through RT-PCR. Flavivirus RNA was detected in 53 individuals (36.8%; 95% CI: 29.4%–44.9%), and Alphavirus RNA was detected in 59 individuals (40.1%; 95% CI: 33.2%–49.2%). The sequences obtained were consistent with ZIKV and DENV, into the Flavivirus, and CHIKV into the Alphavirus positive samples. The prevalence of both Flavivirus and Alphavirus was higher during the dry season compared with the rainy season. This high positivity rate, highlighted in both Flavivirus and Alphavirus, suggests a potential contribution of bats in the circulation of these viral genera in sylvatic environments. Seasonal variation in viral genera prevalence, with higher prevalence during dry seasons than rainy seasons, may suggest specific viral activity patterns in response to climatic conditions.

Keywords

Orthoflavivirus
Alphavirus
Bats
Tropics
Yucatan
==== Body
pmc1 Introduction

Viruses belonging to the Flavivirus (currently renamed to Orthoflavivirus) and Alphavirus genera are mainly transmitted to vertebrate hosts, including humans, domestic animals, and pets, by the bite of an arthropod vector (vectorial route). These arthropod-borne viruses (arboviruses) have the widest distribution and potential for dissemination worldwide, sharing ecological characteristics, evolutionary patterns, and epidemiological elements such as vectors, susceptible vertebrate hosts, and pathways and mechanisms of transmission [1]. In recent years, they have caused significant outbreaks and epidemics in tropical and subtropical regions around the world, including the Americas, Africa, and Asia [2]. Additionally, autochthonous outbreaks are emerging in non-tropical regions such as the southeastern United States and parts of Europe due to climate change and changes in land use [[3], [4], [5]].

In the Americas, Orthoflavivirus such as Dengue virus (DENV) and Zika virus (ZIKV), and Alphavirus such as Chikungunya virus (CHIKV), have an emerging or re-emerging nature, leading to significant prevalence, incidence, and mortality rates in endemic areas of Latin America and the Caribbean [6,7]. In Mexico, particularly in tropical or subtropical areas including the state of Yucatan, there is a high prevalence of Orthoflavivirus infections, mainly occurring during the rainy seasons [8]. However, in recent years, Alphavirus infections have also affected residents of urban areas in the southern and southeastern states, posing a significant concern for public health authorities [8].

In the urban transmission cycle, mosquitoes and humans serve as amplifying hosts or reservoirs, while in the sylvatic transmission cycle, wild animals play a crucial role [9]. Bats are known hosts for numerous zoonotic pathogens and have been identified as potential reservoirs for several arboviruses [10]. While most epidemiological studies in America have focused on urban environments, there is growing evidence of the importance of sylvatic cycles involving wild animals, including bats [10,11]. Some studies have evaluated a range of wild animals, suggesting that bats may contribute to the maintenance and expansion of sylvatic transmission cycles (e.g., West Nile virus and Zika virus circulating in bats from suburban areas of Yucatan) [12]. Nevertheless, the exact role of these animals in the transmission cycles of Orthoflavivirus and Alphavirus remains unclear.

To address this knowledge gap, we designed a prospective and longitudinal study to evaluate the prevalence of Orthoflavivirus and Alphavirus in bats of Yucatan, a state enriched with bat species [13]. We aim to investigate potential factors linked to climate conditions and landscapes that may influence the prevalence of these viruses, with implications for public health interventions.

2 Materials and methods

2.1 Ethical considerations

This study was approved by the Bioethics Committee of the Faculty of Veterinary Medicine of the Autonomous University of Yucatan (official document: CB-CCBA-D-2022-004). The capture and extraction of bats were conducted with permission from the Ministry of Environment and Natural Resources (SEMARNAT) of Mexico (official documents: SPARN/DGVS/06447/22 and SPARN/DGVS/09663/23).

2.2 Study concept and design

This prospective longitudinal study aimed to evaluate the prevalence of Orthoflavivirus and Alphavirus in bats from Yucatan, Mexico. The capture of animals was conducted in two distinct areas: forest with secondary vegetation landscape and grassland landscape [14]. Sampling was carried out during the rainy and dry seasons to assess possible annual fluctuations.

2.3 Sampling areas

The study was conducted from August 2022 to July 2023 in two municipalities: Panaba (grassland landscape) and Tekax (forest with secondary vegetation landscape). In the grassland area, the study site was the Santa Maria cattle ranch, with coordinates 21.263405, −88.279978. In the forest area, two sampling sites were selected: the cavern “Las Sartenejas” (20.194345, −89.309258) and Kaalmankal Ecotourism Park (20.207045, −89.304474) (Fig. 1).Fig. 1 Geographic location of capture sites (diamonds) in grassland landscape (Panaba) and forest landscape (Tekax) of the state of Yucatan, Mexico. CONABIO.

Fig. 1

The climate in all study sites is warm and subhumid throughout the year, with two defined climatic seasons [15]. The rainy season, from June to October, has an average monthly temperature of 27.5 °C and an average precipitation of 1000 mm (with the highest peak at the beginning). The dry season, from November to May, has an average monthly temperature of 25.1 °C and an average rainfall of 300 mm.

2.4 Capture and data collection

Ten sampling sessions were planned in both study areas (five in each). In the grassland area, captures were conducted in September 2022, and January, March, May, and July 2023. In the forest area, sampling was conducted in August and October 2022, and February, April, and June 2023. Captures were made using three mist nets (6 m × 2 m) (Redes Ramirez®, Mexico). Bats were removed from the nets using protective gear and transferred to a mobile unit for handling and sample collection. Data on sex, age, trophic guild, study site, and date were recorded for every bat. Species were identified using a field identification key for bats of Mexico [16].

Feces and saliva samples were collected via anal and oral swabs, using 150 mm × 2 mm nylon flocked swabs (GlobalRoll®, China). Peripheral blood samples were collected by puncture of the marginal or brachial vein, following a proportion of 4 μL of blood for each gram of weight [17]. Samples were preserved in DMEM (GIBCO®, USA) and refrigerated until transfer to the Regional Research Center Dr. Hideyo Noguchi for storage at −80 °C until analysis.

To prevent or control cross-contamination during our study, each bat was sampled using sterile equipment, and gloves were changed when handling each specimen to avoid cross-contamination at the source. All personnel followed established for sample handling and processing, which are designed to minimize the risk of cross-contamination.

2.5 Molecular detection of Orthoflavivirus and Alphavirus RNA

All samples were processed in a dedicated laboratory area with strict protocols. Separate rooms were used for different stages of sample processing to minimize the risk of contamination. All laboratory equipment was thoroughly sterilized before and after use, and disposable materials were used whenever possible to reduce the risk of contamination further.

Total RNA was extracted using the RNAget® reagent (Biotecnologias Moleculares, Mexico). Subsequently, a pooling strategy was implemented, combining RNA extracted from feces, saliva, and blood samples from each bat for molecular detection. cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis Kit® (Thermo Scientific®, USA).

A heminested RT-PCR for Orthoflavivirus [18] and nested RT-PCR for Alphavirus [19] targeting a conserved fragment of the NS5 and NSP4 genes, respectively, were used for viral detection (supplementary Table 1). The first-round reagents in both reactions (heminested and nested) included: 1× Dream Taq Buffer, 200 μM dNTPs, 400 nM of each oligonucleotide, 1 U Dream Taq DNA polymerase® (Thermo Scientific®, USA), 5 μL of cDNA as template, and nuclease-free water (Thermo Scientific®, USA) for a final volume of 25 μL. Adaptations were made to the annealing, elongation, and final extension temperatures in the original protocols as follows: 94 °C for 3 min, followed by 25 cycles at 94 °C for 30 s, 52 °C for 40 s, and 72 °C for 30 s, with a final extension at 72 °C for 3 min. For the second round, the final concentration of reagents was the same as in the first round, using 3 μL of the first-round product as the template. The temperature conditions were the same as in the first round but with 30 cycles and an annealing temperature of 53 °C. The amplicon sizes were 220 bp for Orthoflavivirus and 195 bp for Alphavirus.

Each PCR reaction included a positive control for DENV (type 1: U88535, type 2: GU289914, type 3: DQ863638, and type 4: KU513442), ZIKV (KX830960), and CHIKV (MK028839) RNA supplied by the Virology Laboratory at the Regional Research Center Dr. Hideyo Noguchi (UADY) and included negative controls to monitor for any signs of cross-contamination. No contamination was detected in these controls, confirming the integrity of our procedures. After the second round, the amplified products were visualized on 2% agarose gels.

Positive samples with easily visible amplicons were selected for one-way Sanger sequencing for viral species identification, based on bat species, sampling site, and climatic season. Each second-round PCR reaction tube positive for viral genera was sent to Macrogen (Seoul, South Korea) for PCR purification and sequencing.

2.6 Sequencing and phylogenetic analysis

The received sequences were visualized, curated, and analyzed using the 4Peaks and UGENE v.47.0 programs. Viral genera and species were assigned through phylogenetic analysis and confirmed using nucleotide BLAST. The sequences generated in this study were submitted to GenBank. Phylogenetic analyses of NS5 (Orthoflavivirus) and NSP4 (Alphavirus) for viral species identification were conducted by comparing the sequences obtained in this study with reference virus sequences, positive control virus sequences, and sequences of viruses isolated from humans, mosquitoes, and other mammalian hosts available in GenBank. Each dataset was aligned using the MAFFT v.7 server (https://mafft.cbrc.jp/alignment/server/), and phylogenetic trees were constructed using the Neighbor-Joining method and Jukes-Cantor substitution model with 1000 bootstrap replicates. The resulting trees were visualized using MEGA 7.

2.7 Statistical analysis

Descriptive statistics were used to determine the relative frequency of bat species captured in each study area and during each climatic season. Additionally, the estimated prevalence of Orthoflavivirus and Alphavirus RNA in different bat species and the corresponding 95% confidence intervals (CI) were calculated. A chi-square test was performed to compare (P < 0.05) the prevalence of Orthoflavivirus and Alphavirus between bat families, bat species, trophic guilds, sex, age, sampling area (forest and grassland), and season (dry and rainy). All statistical analyses were performed using WINPEPI v.11.65.

3 Results

3.1 Study population

One hundred forty-four bats were captured, spanning three families (Emballonuridae, Phyllostomidae, and Molossidae), six species (Peropteryx macrotis, Glossophaga mutica, Sturnira parvidens, Artibeus jamaicensis, Dermanura phaeotis, and Molossus nigricans), and three trophic guilds (insectivores, nectarivores, and frugivores). Detailed characteristics of the studied specimens are presented in Table 1. Glossophaga mutica was the most frequently captured species (n = 61; 42.3%). Trophic guild distribution showed 72 frugivores (50%), 61 nectarivores (42.3%), and 11 insectivores (7.7%). The sex, area of capture, and seasonality distribution were similar, with 52.7% males and 47.3% females, 57.6% captured in forest area and 42.4% in grassland area, and 51.4% captured during the rainy season and 48.6% during the dry season. Most captured bats were adults (n = 115; 79.9%).Table 1 Main characteristics of sampled bats.

Table 1Family	Species	Trophic guild	Sex	Age	Area	Season	
Male	Female	Juvenil	Adult	Forest	Grassland	Rainy	Dry	
Phyllostomidae
(n = 133)	Artibeus jamaicensis
(n = 50)	Frugivore	27
(54%)	23
(46%)	11
(22%)	39
(78%)	21
(42%)	29
(58%)	32
(64%)	18
(36%)	
Dermanura phaeotis
(n = 6)	Frugivore	4
(66.7%)	2
(33.3%)	1
(16.7%)	5
(83.3%)	0
(0%)	6
(100%)	2
(33.3%)	4
(66.7%)	
Glossophaga mutica
(n = 61)	Nectarivore	32
(52.5%)	29
(47.5%)	10
(16.4%)	51
(83.6%)	54
(88.5%)	7
(11.5%)	27
(44.3%)	34
(55.7%)	
Sturnira parvidens
(n = 16)	Frugivore	11
(68.8%)	5
(31.3%)	5
(31.3%)	11
(68.8%)	0
(0%)	16
(100%)	6
(37.5%)	10
(62.5%)	
Emballonuridae
(n = 8)	Peropteryx macrotis
(n = 8)	Insectivore	1
(12.5%)	7
(87.5%)	1
(12.5%)	7
(87.5%)	8
(100%)	0
(0%)	7
(87.5%)	1
(112.5%)	
Molossidae
(n = 3)	Molossus nigricans
(n = 3)	Insectivore	1
(33.3%)	2
(66.7%)	1
(33.3%)	2
(66.7%)	0
(0%)	3
(100%)	0
(0%)	3
(100%)	
Total
(N = 144)			76
(52.7%)	68
(47.3%)	29
(20.1%)	115
(79.9%)	83
(57.6%)	61
(42.4%)	74
(51.4%)	70
(48.6%)	

3.2 Prevalence of Orthoflavivirus and Alphavirus

The results of the molecular screening for Orthoflavivirus and Alphavirus in bats from Yucatan are presented in Table 2. We detected viral RNA throughout the study year for both viral genera. Orthoflavivirus RNA was detected in 53 bats, indicating a prevalence of 36.8% (95% CI: 29.4%–44.9%). We observed a higher prevalence among juvenile individuals compared to adults (55.1% vs. 32.1%, p = 0.02) and among bats captured during the dry season compared to those captured during the rainy season (50% vs. 24.3%, p = 0.001).Table 2 Prevalence of Orthoflavivirus and Alphavirus of sampled bats according to baseline characteristics.

Table 2Variable	Category	Orthoflavivirus	P Value	Alphavirus	P Value	
Negative
(n = 91)	Positive
(n = 53)		Negative
(n = 85)	Positive
(n = 59)		
Family	Phyllostomidae	83
(62.4%)	50
(37.6%)	0.2	78
(58.6%)	55
(41.4%)	0.43	
Emballonuridae	7
(87.5%)	1
(12.5%)		6
(75%)	2
(25%)		
Molossidae	1
(33.3%)	2
66.7%)		1
(33.3%)	2
(66.7%)		
Specie	Glossophaga mutica	40
(65.6%)	21
(34.4%)	0.47	43
(70.5%)	18
(29.5%)	< 0.001*	
Artibeus jamaicensis	29
(58%)	21
(42%)		29
(58%)	21
(42%)		
Sturnira parvidens	11
(68.7%)	5
(31.3%)		6
(37.5%)	10
(62.5%)		
Dermanura phaeotis	3
(50%)	3
(50%)		0
(0%)	6
(100%)		
Peropteryx macrotis	7
(87.5%)	1
(12.5%)		6
(75%)	2
(25%)		
Molossus nigricans	1
(33.3%)	2
(66.7%)		1
(33.3%)	2
66.7%)		
Trophic guild	Nectarivore	40
(65.6%)	21
(34.4%)	0.62	43
(70.5%)	18
(29.5%)	0.03*	
Frugivore	43
(59.7%)	29
(40.3%)		35
(48.6%)	37
(51.4%)		
Insectivore	8
(72.7%)	3
(27.3%)		7
(63.6%)	4
(36.4%)		
Sex	Male	51
(66.2%)	26
(33.8%)	0.41	44
(57.1%)	33
(42.9%	0.62	
Female	40
(59.7%)	27
(40.3%)		41
(61.2%)	26
(38.8%)		
Age	Juvenile	13
(44.8%)	16
(55.2%)	0.02*	13
(44.8%)	16
(55.2%)	0.08	
Adult	78
(67.8%)	37
(32.2%)		72
(62.6%)	43
(37.4%)		
Area	Forest	55
(66.3%)	28
33.7%)	0.37	59
(69.4%)	24
(30.6%)	< 0.001*	
Grassland	36
(59%)	25
41%)		26
(42.6%)	35
(57.4%)		
Season	Dry	35
(50%)	35
(50%)	0.001*	33
(47.1%)	37
(52.9%)	0.004*	
Rainy	56
(75.7%)	18
(24.3%)		52
(70.3%)	22
(29.7%)		

Fifty-nine bats were positive for Alphavirus, resulting in a prevalence of 40.1% (95% CI: 33.2%–49.2%). We found a higher prevalence among individuals captured in the grassland area than in the forest area (57.4% vs. 30.6%, p = 0.001) and among those captured during the dry season compared to animals collected during the rainy season (52.9% vs. 29.7%, p = 0.004). Similarly, we observed a higher prevalence among frugivore individuals (51.4%) than insectivore and nectarivore bats (p = 0.03). However, differences in prevalence according to bat species cannot be interpreted in this context due to the low numbers of several species.

Twenty-five individuals tested positive for Orthoflavivirus and Alphavirus RNA, suggesting a co- prevalence of 17.3% (95% CI: 12%–24.4%). Among them, 12 (48%) were captured in the forest area, and 13 (52%) were captured in the grassland area. Most individuals testing positive for Orthoflavivirus and Alphavirus RNA were also captured during the dry season (n = 19; 76%).

3.3 Prevalence of viral genera in bats during main climatic season and landscape

The prevalence of the viral genera Orthoflavivirus and Alphavirus during the dry and rainy seasons in forest and grassland areas is presented in Table 3. In the dry season, the prevalence of Orthoflavivirus in bats from the grassland area was higher than that in bats from the forest area (64.7% vs. 36.1%, p = 0.019). Conversely, during the rainy season, we observed a higher prevalence of Orthoflavivirus in bats from the forest area than those from the grassland area (31.9% vs. 11.1%, p = 0.038). Additionally, the prevalence of Alphavirus in bats from the grassland area was higher when compared to bats from the forest area (55.6% vs. 14.9%, p < 0.001).Table 3 Prevalence of Orthoflavivirus and Alphavirus in bats captured in forest and grassland areas during the dry and rainy seasons.

Table 3Season	Area	Orthoflavivirus	P Value	Alphavirus	P Value	
Negative	Positive	Negative	Positive	
Dry
(n = 70)	Forest
(n = 36)	23
(63.9%)	13
36.1%	0.019*	19
(52.8%)	17
(47.2%)	0.291	
	Grassland
(n = 34)	12
(35.3%)	22
(64.7%)		14
(41.2%)	20
(58.8%)		
Rainy
(n = 74)	Forest
(n = 47)	32
(68.1%)	15
(31.9%)	0.038*	40
(85.1%)	7
(14.9%)	< 0.001*	
	Grassland
(n = 27)	24
(88.9%)	3
(11.1%)		12
(44.4%)	15
(55.6%)		

3.4 Phylogenetic analyses

In total forty-two positives samples were sequenced, comprising 20 individuals for Orthoflavivirus and 22 for Alphavirus. This sequenced selection represents 37.7% of the positive individuals for Orthoflavivirus (20 out of 53) and 26.8% of the positive bats for Alphavirus (22 out of 59). Regarding Orthoflavivirus, nine individuals were positive for ZIKV (45%), while 11 tested positive for DENV (55%). Among the DENV sequences (Fig. 2A), all sequences obtained in this study grouped in clades belonging to each DENV type. Four were DENV-1, one was DENV-2, and six were DENV-3, all sequences isolated in this study grouped in clades near human cases. Among ZIKV (Fig. 2B), nine individuals were grouped in clade belonging to ZIKV but not within clades with human and vectors isolated. For Alphavirus (Fig. 3), all 22 individuals were grouped with CHIKV, but not within clades with human, vectors or other host. Accession numbers for each sequence obtained in the present study are presented in Supplementary Table 2.Fig. 2 Phylogenetic analysis for Orthoflavivirus species identified in the study. (A) The DENV tree was constructed using 39 sequences, including reference sequences for each DENV (NC_001477, NC_001474, NC_001475, and NC_002640), sequences used as PCR positive controls (U88535, GU289914, DQ863638, and KU513442), 19 sequences from GenBank showing high homology with the 11 sequences isolated in this study, and the West Nile virus sequence (NC_009942) as the outgroup. Sequences were 125 nt in length. (B) The ZIKV tree comprised 38 sequences, including one reference sequence for ZIKV (NC_012532), one sequence used as a PCR positive control (KX830960), 26 sequences from GenBank, nine sequences isolated in this study, and the West Nile virus sequence (NC_009942) as the outgroup. Sequences were 121 nt in length. For both trees the evolutionary history was inferred by using the neighbor-joining method based on the Jukes-Cantor model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed.

Fig. 2

Fig. 3 Phylogenetic analysis for Alphavirus. The CHIKV tree included 51 sequences, consisting of a reference sequence (NC_004162), one sequence used as a PCR positive control (MK028839), 26 sequences from GenBank, 22 sequences isolated in this study, and a Mayaro virus sequence (NC_003417) as the outgroup. Sequences were 125 nt in length. The evolutionary history was inferred by using the neighbor-joining method based on the Jukes-Cantor model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed.

Fig. 3

4 Discussion

In this study, we unveil the presence of Orthoflavivirus, including DENV and ZIKV, in six bat species (P. macrotis, G. mutica, S. parvidens, A. jamaicensis, D. phaeotis, and M. nigricans) inhabiting different areas (forest and grassland) of Yucatan, Mexico, across rainy and dry seasons. While these viral species have been previously documented in bats through molecular studies in Mexico [12,[20], [21], [22]], our findings reveal a higher prevalence of Orthoflavivirus in bats from grassland area during the dry season compared to those from forest area, shedding light on the possible influence of environmental factors like landscape type (forest and grassland) and seasonal variations (dry and rainy season) on occurrence on these arboviruses in bat populations. Notably, our investigation also identifies factors associated with the prevalence of Alphavirus in bats, with a higher prevalence observed among individuals captured in grassland area during the dry season. This result, in the same way that Orthoflavivirus prevalence, underscores the complex interplay between environmental factors and the circulation of CHIKV in bat populations. For example, in case of DENV, in landscape with changes in land use due agriculture in areas that are enzootic for DENV could lead the interaction among vectors and hosts [23], similar observed in this study in grassland areas where found Orthoflavivirus and Alphavirus. Also, in China, in addition to the high risk of DENV transmission during rainy season to population, a risk of transmission has been observed during dry season in less urbanized areas [24]. On the other hand, contrary to previous findings our study suggests a paradigm shift for presence of DENV, ZIKV and CHIKV in bat population. While previous research across different regions of the Americas indicated low arbovirus prevalence in bat populations [[25], [26], [27], [28], [29], [30], [31]], our study unveils a high prevalence of Orthoflavivirus and Alphavirus in various bat species throughout the year. This defies the notion of bats serving only as incidental hosts and suggests a potentially significant role for circulation of these virus genera in sylvatic cycle.

Various ecological factors have been associated to the transmission dynamics of arboviruses in endemic areas, particularly those related to climate (such as rainfall, temperature, and humidity) and changes in land use (including urbanization and agricultural activities) [29]. These factors influence the composition of vector, susceptible hosts, and virus communities, thereby affecting the risk of vector-borne virus transmission [30]. In our study, we assessed the impact of climatic seasons (rainy and dry) and landscape types (forest and grassland) on the presence of Orthoflavivirus and Alphaviruses in bats inhabiting different perturbate areas. Despite the high prevalence of Orthoflavivirus and Alphaviruses observed throughout the year, the dry season significantly influences the simultaneous presence of these viral genera in the studied bats. Aedes aegypti and Ae. Albopictus, known vectors of DENV, ZIKV, and CHIKV [31], have been observed to present a lower abundance during dry season compared to the abundance in rainy season in rural and suburban areas [32,33]. However, in Yucatan, although the average temperature is 26 °C, reaching up to 36 °C during the dry season [34], higher temperatures have been associated with increased infection and transmission rates of DENV, ZIKV, and CHIKV in mosquitoes [35,36]. On the other hand, our study revealed that bats captured in the grassland area exhibited a higher prevalence of Alphavirus compared to those in forested areas. Grassland regions are linked to livestock and have a more significant presence of Ae. Albopictus [37], which is more suited to grassland landscapes than Ae. Aegypti, with a strong feeding preference for a wide range of mammals. In this regard, it has been observed that mosquito diversity in disturbed areas is higher than in forested areas [38,39]. A similar finding has been reported in Australia [40], where vector richness in grassland areas differs from other regions, influencing the transmission of pathogens to humans and wildlife. Further research is warranted to explore these findings ecological and epidemiological implications and to develop targeted strategies for disease surveillance in grassland landscapes.

Highly endemic countries for arbovirosis, such as Mexico, are not exempt from the increasing cases of arboviral diseases, primarily in the country's southeastern states, where almost 80% of the cases are concentrated [8]. The detection of DENV, ZIKV, and CHIKV in bat species suggests the active maintenance of these arboviruses in areas near urban zones (Fig. 1). This might imply a risk of infection to humans due to the constant presence and circulation of these viruses in bats. Disturbance of natural habitat of these species might favor the interaction between people, animals (pets, domestic, and wild), and vectors [41]. Particularly concerning DENV, the detection of DENV1, DENV2, and DENV3 offers a similar outlook to the circulation of DENV types in the human population during recent DENV outbreaks in the region. Perturbations in their natural habitats due to urbanization and tourism activities increase the risk of spillover events into human populations, particularly in urban areas locally and potentially contributing to the spread of these arboviruses.

Several limitations should be noted. Firstly, our study was conducted in specific landing use and vegetation areas (forest and grassland) and while, it offers significant findings, these may not be representative of bat populations across the region. Therefore, further epidemiological investigations in various ecological settings are warranted to comprehensively understand the prevalence and dynamics of arboviruses in bat populations in Mexico. Secondly, although our phylogenetic analyses of partial genomic regions provided valuable information about the circulating viral species, other genomic analyses encompassing wider genomic regions of the viral genomes could offer more detailed insights into viral ecological, diversity, and transmission dynamics. Lastly, the strategy of pooling samples of feces, saliva, and blood, may occasionally yield ambiguous results. For instance, the detection of arboviruses in saliva samples could, in some cases, be attributed to the inadvertent presence of virus particles in vector mosquitoes resulting from bat feeding behaviors, in the case of insectivore bats (M. nigricans and P. macrotis), but in case of frugivores (A. jamaicensis) and nectarivores (G. mutica) for complementary diet. Nevertheless, the consistent detection of arboviruses in several bat species suggests active viral circulation within the study area.

In conclusion, our study reveals the year-round circulation of DENV, ZIKV, and CHIKV among bats from Yucatan. Our data suggests a potential role of bats in maintaining these arboviruses in the sylvatic cycle. In addition, there is a potential risk of transmitting these arboviruses from the sylvatic cycle to the urban cycle in Yucatan. This risk might be exacerbated by the exponential growth of human activities such as tourism and urban development in the region, leading to the encroachment upon and disruption of wildlife habitats. Our findings underscore the importance of unceasing surveillance and research efforts to reach a better understanding and mitigate the risks posed by arboviruses in sylvatic transmission cycles, ultimately safeguarding public health.

Funding

This work was supported by the Andalusian General Secretariat for Research, Development, and Innovation in Health (PI-0287-2019 ), the Spanish Ministry of Health (RD12/0017/0012 ), co-financed by 10.13039/501100008530 European Regional Development Fund (ERDF). AYG was supported by CONAHCYT Grant no. 413387 . ARJ is supported by a contract from the Spanish Junta de Andalucia (Nicolas Monardes program: C1-0001-2023). The funders did not play any role in the design, conclusions, interpretation of the study, or decision to publish.

CRediT authorship contribution statement

Aaron Yeh-Gorocica: Writing – review & editing, Writing – original draft, Visualization, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Marco Torres-Castro: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Claudia Carrillo-Chan: Writing – review & editing, Visualization, Software, Investigation, Data curation. Alejandro Suarez-Galaz: Writing – review & editing, Visualization, Software, Methodology, Investigation, Data curation. Melissa Suarez-Galaz: Writing – review & editing, Visualization, Software, Investigation, Data curation. Wilson Moguel-Chin: Writing – review & editing, Visualization, Methodology, Data curation. Alonso Panti-May: Writing – review & editing, Visualization, Data curation. Cesar Lugo-Caballero: Writing – review & editing, Visualization, Funding acquisition, Data curation. Henry Puerta-Guardo: Writing – review & editing, Resources. Juan Chable-Santos: Writing – review & editing, Resources, Project administration. Pablo Manrique-Saide: Writing – review & editing, Resources, Project administration. Guadalupe Ayora-Talavera: Writing – review & editing, Validation, Supervision. Celia Selem-Salas: Writing – review & editing, Validation, Supervision. Mario Frias-Casas: Writing – review & editing, Validation, Supervision. Antonio Rivero-Juarez: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

Authors declare that we do not have use any generative AI or AI-assisted technologies in the design, analysis, interpretation of the results, or writing process of the present work.

Declaration of competing interest

The authors declare that they have no competing interests. Neither the authors nor their institutions have at any time received payment or services from a third party for any aspect of the submitted work (data monitoring board, study design, manuscript preparation, statistical analysis, or other aspects).

Appendix A Supplementary data

Supplementary tables

Image 1

Data availability

A total of 42 sequenced samples were submitted to the NCBI Genbank database, with accession numbers for DENV NS5 gene ranging from OR726071 to OR726081 (n = 11), ZIKV NS5 gene ranging from OR787460 to OR787468 (n = 9), and CHIKV NSP4 gene ranging from OR707486 to OR787507 (n = 22).

Acknowledgement

We gratefully acknowledge Rodolfo Chan-Chan, Raymundo Matu-Gongora, Bibiana Reyes-Hernandez, Angelica Andrade-Lopez, Rodrigo Ramos-Vazquez, Rosa Maria Galaz Avalos, K. Jaqueline Ciau Carrillo and Manuel Parra Cardeña for their technical support in sample collection, processing, and analysis. Also, we acknowledge the administrations of Rancho Santa Maria (Panaba, Yucatan), Grutas Las Sartenejas (Tekax, Yucatan), and Parque Ecoturistico Kaalmankal (Tekax, Yucatan) for their facilities for bat sampling.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.onehlt.2024.100876.
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