Abstract
Background:
In Mexico, two Mammarenavirus genotypes have been identified: Real de Catorce virus and Ocozocoautla de Espinosa virus (OCEV), which are found in rodents (Neotoma leucodon and Peromyscus mexicanus). Mammarenaviruses from clade B cause mild to severe infectious diseases in humans, particularly in South America. A 1967 outbreak in Chiapas, Mexico, was potentially linked to the OCEV.
Materials and Methods:
Between 2017 and 2020, we collected bats across southern/southeastern Mexico to identify Mammarenavirus reservoirs. We analyzed 81 liver samples from 14 bat species using nested PCR. For quality control, we synthesized and OCEV S-region fragment into an ampicillin-resistant pUC57 plasmid, cloned in One Shot Stbl3 chemically competent E. coli.
Results:
Here, we report the first detection of a Mammarenavirus (OCEV) in an Artibeus lituratus bat from Sabancuy, Campeche, southeastern Mexico. This finding represents the third Mammarenavirus recorded in this bat species in the Americas, suggesting it may harbor multiple Mammarenavirus species (OCEV and Mammarenavirus tacaribeense).
Conclusions:
While this represents the first documentation of OCEV in a bat, it remains unknown whether environmental stressors could facilitate potential transmission to humans, particularly given the close association between Artibeus bats and human populations.
Introduction
Bats are the second most diverse group of mammals worldwide, with approximately 1510 species (Mammal Diversity Database, 2023). They are known to be hosts of the following zoonotic RNA viruses: Coronaviridae, Circoviridae, Filoviridae, Poxviridae, Picornaviridae, Rhabdoviridae, and Arenaviridae, some of which have been associated with diseases and epidemics in humans (Williams et al., 2021). Arenaviridae was initially considered a family of viruses carried exclusively by rodents of the families Muridae (in the Old World) and Cricetidae (in the New World), with a single genus, Mammarenavirus (Radoshitzky et al., 2015). However, in the Americas, viruses of this genus have been detected in the fruit bats Artibeus jamaicensis and A. lituratus (M. tacaribeense) in Trinidad and Tobago (Downs et al., 1963) and, more recently, in Brazil, where a new virus was also described: M. tietense, carried by specimens of Carollia perspicillata (Bentim Góes et al., 2022).
Other mammarenaviruses have also been detected in shrews and pikas in Southeast Asia (Li et al., 2015; Luo et al., 2023) and in sea urchins in Europe (Reuter et al., 2023) totaling 53 species currently described and recognized by the International Committee on Taxonomy of Viruses, plus two proposed species: Real de Catorce virus and Ocozocoautla de Espinosa virus (OCEV) (Cajimat et al., 2012; Inizan et al., 2010). The family Arenaviridae currently includes four additional genera: Hartmanivirus, Reptarenavirus (both carried by snakes), Antennavirus (carried by fish), and Innmovirus (unknown reservoir or carrier) (Chen et al., 2022).
Bat-borne mammarenaviruses are grouped in the phylogenetic clade (clade B) that includes rodent-borne New World mammarenaviruses classified as pathogenic (Mammarenavirus guanaritoense, M. juninense, and M. machupoense), which have caused mild to severe hemorrhagic fevers in humans in the Americas. However, none of these diseases have been associated with bat-borne viruses (Radoshitzky et al., 2015). In Mexico, mammarenaviruses have been reported in rodents of the family Cricetidae, from which two genotypes have been described: RCTV carried by Neotoma leucodon and OCEV carried by Peromyscus mexicanus (Cajimat et al., 2012; Inizan et al., 2010), in the north and south of the country, respectively. To date, no other Mammarenavirus has been detected in wild rodents or bats in Mexico. In the state of Chiapas (southeastern Mexico), there was an epidemic hemorrhagic fever outbreak in humans hypothesized to have been caused by a Mammarenavirus carried by P. mexicanus (Cajimat et al., 2012; Goldsmith and Shields, 1971). This article reports the first evidence of the presence of an RNA arenavirus in a fruit bat collected in the state of Campeche, southeastern Mexico. In addition, we provide a novel technique for detecting this type of virus, which can set the basis for future zoonotic research.
Materials and Methods
Bat collection
Bats were collected during field trips in five states in south-southeastern Mexico: Campeche, Chiapas, Oaxaca, Tabasco, and Yucatan (Fig. 1) between 2017 and 2020 as part of a major project entitled Analysis and Assessment of Potential Ebola Virus Vectors and Reservoirs in Mexico, funded by the Sectoral Research Fund for Education SEP-CONACyT, and other minor projects that aimed at understanding the relationship between environmental disturbance and the presence of viruses in small mammals (Hernández-Aguilar et al., 2023). Bat specimens were collected in houses, parks, crops, or natural areas such as forests and scrubs 6 km from an urban location (Tapia-Ramírez et al., 2022a). The bats collected were processed in situ under the highest possible safety conditions. Each specimen was identified to species using specialized keys (Álvarez-Castañeda et al., 2017). They were sacrificed following the recommendations of the American Society of Mammalogists (Sikes and The Animal Care and Use Committee of the American Society of Mammalogists, 2016), and standard somatic metrics were recorded. The specimens were prepared as vouchers preserved in skin and skull or in 70% alcohol and deposited in the Mammalogical Collection of El Colegio de La Frontera Sur (ECOSUR; ECO-SC-M). A liver fragment was removed and preserved in RNA later in search of arenaviruses. The collection of specimens and protocol of study were authorized by the Ethics Committee for Research of El Colegio de La Frontera Sur (ECOSUR; CEI/1001/24). We applied an innovative arenavirus RNA extraction and detection method.
Location of the study area and the zone where Mammarenavirus was detected in Artibeus lituratus in the state of Campeche, southeastern Mexico.
RNA extraction
Total RNA was extracted from each liver tissue sample using the RNeasyt Mini Kit (250) (Cat.#74106, Qiagen, Hilden, Germany) following the manufacturer’s instructions. Briefly, the liver tissue was transferred to a new tube and homogenized with a polypropylene pistil until complete dissolution. Then, 600 µL of lysis buffer (RLT) containing beta-mercaptoethane (10 µL/mL of RLT buffer) was added. The lysate was transferred to a column included in the kit; the column was previously washed once with RW1 and twice with RPE diluted with ethanol. Finally, the total RNA was eluted with 40 µL of RNA storage buffer (Cat.#AM7001, Invitrogen, Waltham, Massachusetts). The samples were immediately subjected to PCR testing to detect Mammarenavirus.
Generation of a positive control of the mammarenavirus S segment
For quality control, a fragment of the S segment of OCEV was synthesized in an ampicillin-resistant pUC57 plasmid (BioBasic Inc., Ontario, Canada). The plasmid was cloned in One Shot Stbl3 chemically competent E. coli cells (Cat.#C7373-03, Invitrogen, Waltham, Massachusetts) following the manufacturer’s instructions. The plasmid identity was confirmed with the restriction enzymes HindIII-HF (Cat.#R3104V, NEB) and EcoRv-HF (Cat.#R3195S, New England BioLabs Inc.), adding 1 U of each enzyme to 1 µg of plasmid and incubating for 1 h at 37°C. The digestion product was examined on 1% agarose gel, where the two expected fragments could be observed: a 2700 bp fragment and a 499 bp fragment.
Detection of the S segment of mammarenavirus by nested PCR
For the amplification of the S segment of the Mammarenavirus, we used the first- and second-round primers previously published in (Castellar et al., 2017). The first round was performed using the Super Script III RT/Platinum Taq One-Step RT/PCR Mix enzyme (Cat.#11732-020), 10 µM of each primer (arena_1_pos and arena_2_neg), 1x buffer, and 5 µL of newly extracted RNA in a final volume of 25 µL to produce a 459 bp fragment. For the second round, 2 µL of the first round were used in a final 40 µL reaction with the following proportions: 1x of buffer without Mg, 10 µM of each second-round primer (arena_2_pos and arena_2_neg), 10 mM dNTPs, 50 mM MgSO4, and 1 U of Platinum Taq to produce a 200 bp fragment. The PCR product was run on a 2% agarose gel. Each PCR included one positive and one negative amplification control.
Next-generation sequencing of positive control and amplified samples
Individual libraries were prepared for each amplicon (M200-sample and ECOSUR-200-positive control) using the Nextera XT DNA Library Preparation Kit (FC-131-1096, Illumina) and the Nextera XT Index Kits (FC-131-2002, Illumina) to label each sample, respectively. Libraries were prepared according to the manufacturer’s protocols, with a modified labeling step. In this case, 40% of the working volumes were used for tagmentation reagents with 1 ng of DNA as input. The indexed amplicons were purified using AMPure XP beads (Cat.#A63880, Beckman Coulter Genomics); the library was analyzed in an Agilent 2100 bioanalyzer. The pooled libraries were denatured and diluted to a final concentration of 1.5 pM. The Illumina PhiX control was then added to the DNA libraries in a 1.5% ratio and sequenced on the Illumina MiniSeq platform using the 2 × 150 bp MiniSeq High Output Reagent Kit (Cat.#FC-420-1003, Illumina, San Diego, California, USA).
Bioinformatics analysis
The complete sequence of the S-segment of the Ocozocoautla de Espinosa virus was downloaded from the NCBI GenBank nucleotide database (JN897398.1). It was used as a reference for the alignment of the sequences obtained using the CLC genomics Workbench 23.0.5 sequence analysis program (QIAGEN). This program incorporates several assembly and quality control processes for Fastq sequence files retrieved from Illumina MiniSeq. These processes include preremoval of duplicates, filtering to discard misindexed reads, reading clipping based on quality thresholds, read mapping to the reference genome, output of 4000 reads per sample, read mapping visualizations, and consensus sequence generation considering the coverage depth and bases quality to make a consensus base call. A BLAST was used for genus assignment, obtaining 100% coverage and 100% identity with the Ocozocoautla de Espinosa virus. A phylogenetic tree was built with the MEGA 11 program using the general time-reversible nucleotide substitution model.
Results
Eighty-one liver samples corresponded to 14 species of bats were analyzed. Artibeus jamaicensis, A. lituratus, Carollia perspicillata, C. sowelli, Centurio senex, Chrotopterus auritus, Dermanura watsoni, Desmodus rotundus, Glossophaga mutica, Mimon cozumelae, Pteronotus fulvus, P. mesoamericanus, Saccopteryx bilineata, and Sturnira parvidens (Table 1). Arenavirus RNA was detected in one giant fruit bat, Artibeus lituratus (ECO-SC-M9236), an adult male with the following dimensions: total length, 82 mm; hind foot length, 16 mm; ear length, 19 mm; weight, 50 g; forearm length, 66 mm. The specimen looked healthy and was collected in a public park in Sabancuy, Campeche, a town of 7744 inhabitants surrounded by low deciduous forest in the Yucatan Peninsula (southeastern Mexico; Fig. 1). The viral detection rate was 1.23% (95% CI: 0.03 − 6.71%).
Species of Bats Analyzed in This Study, Sampled from the Area of Study in Southeastern Mexico
The phylogenetic analysis (Fig. 2) showed that the positive sequence detected in A. lituratus (GenBank accession number PV700624) was closely related to the OCEV in a well-supported clade. The statistical support of the branches was evaluated using 1000 start-up replicates, with the consensus obtained from the ECO-SC-M9236_Sabancuy sample and the positive control against 14 sequences with a 100% identity with the OCEV (Fig. 2). This is the first finding of this virus in bats and the first time that more than one Mammarenavirus has been detected in a carrier mammal. The sequence of the virus detected in Sabancuy is also closely related to sequences of M. tacaribeense (Fig. 2). This sequence from Sabancuy Campeche, belongs to the clade of pathogenic mammarenaviruses, together with M. juninense (clade B) (Fischer et al., 2024), which causes Argentine hemorrhagic fever; however, it is known that the virus is carried by rodents of the subfamily Sigmodontinae (Tapia-Ramírez et al., 2022b). The results presented here are similar to those reported by Bentim Góes et al. (2022), who also detected the presence of a mammarenavirus (M. tacaribeense) in A. lituratus specimens collected in Brazil (Fig. 3).
Maximum Likelihood tree based on sequences of mammarenaviruses from the Americas. Confidence support for branches (1000 replicates) is shown as a percentage next to the nodes. The units for the scale bar represent the number of substitutions per site. The Mammarenavirus sequence detected in Artibeus lituratus from Sabancuy, Campeche, Mexico, is marked in red. Labels before species names represent GenBank accession numbers of the sequences used for the phylogenetic analysis.
Discussion
This work reports for the first time the presence of OCEV in a bat in Mexico. Together with its previous identification in Peromyscus mexicanus rodents, this finding confirms the circulation of mammarenaviruses in southeastern Mexico. Although there are no reports that the Mammarenaviruses detected in bats of the genus Artibeus are pathogenic, epidemiological surveillance is essential to obtain more information about them, particularly because these bats can adapt to urban environments, which raises the possibility of a zoonotic spillover to humans and other animal species (Bentim Góes et al., 2022).
The existence of seemingly healthy bats carrying mammarenaviruses suggests two possibilities: either arenaviruses do infect bats persistently, or these viruses have low infectious potential in bat hosts, contrary to what has been found in rodents that carry them (Fischer et al., 2024), which suffer chronic infections (Buchmeier et al., 2007). This evidence may suggest that Artibeus bats are natural reservoirs of mammarenaviruses (Malmlov et al., 2017), which contribute to the circulation of Mammarenavirus in the Americas, or at least from southern Mexico to Brazil (Bentim Góes et al., 2022). The persistence of the mammarenavirus in Artibeus bats is relevant for public health because it indicates the potential of mammarenaviruses to spread through bats.
In the laboratory, it has previously been shown that OCEV could infect bat cells through its transferrin receptor 1 (TfR1) but not human cells (Cai et al., 2013), and this is the first time that its presence in the wild has been documented. While this represents the first documentation of OCEV in a bat, it remains unknown whether environmental stressors could facilitate potential transmission to humans, particularly given the close association between Artibeus bats and human populations. Further additional studies and larger sample sizes will be needed to isolate the virus and better understand how it is transmitted from rodents to bats or vice versa.
Conclusions
This work documents the first detection of OCEV in an Artibeus lituratus bat in Mexico. This finding is particularly significant, as it demonstrates that this bat species can host multiple mammarenaviruses species that are grouped in clade B.
Authors’ Contributions
G.T.-R.: Investigation, conceptualization, funding acquisition, methodology, writing—original draft, review and editing. M.S.-N.: Investigation, methodology, writing—review and editing. C.L.: Investigation, conceptualization, funding acquisition, project administration, methodology, writing—original draft, review and editing. S.Á.-R.: Investigation, methodology, writing—original draft, review and editing. D.T.-T.: Methodology, writing—original draft, review and editing. M.M.-T.: Methodology, writing—original draft, review and editing.
Footnotes
Acknowledgments
The present study was supported by the Sectoral Research Fund for Education SEP-CONACyT (251053) and the Rufford Foundation (34742-1). The authors wish to thank Jorge Bolaños Citalán for his assistance in the field. Silvia Hernández-Betancourt and Itandehui Hernández Aguilar and their teams provided tissue samples. Thanks to Rocío Carrasco and Anahí Canedo Texon for the coordination efforts, and Maricela García, and Aaron. Hernández Núñez for their support in the genetic analyses. The authors thank the local inhabitants and landowners in the different localities visited kindly allowed us access to their homes and land. María Elena Sánchez-Salazar translated the article into English.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This study was part of a major project sponsored by Fondo Sectorial de Investigación para la Educación SEP-CONACyT (251053), Rufford Foundation (34742-1) and Idea Wild (TAPIMEXI0920).