Bats are classified as the fourth largest group of mammals globally, with their habitat spanning every continent except Antarctica [1]. Ecuador is distinguished as the fourth richest country in bat biodiversity, hosting 171 different species [2]. Serving as reservoirs for various pathogens, bats notably harbor coronaviruses and paramyxoviruses. About 16% of species carry coronaviruses — evidenced by the discovery of 4,000 unique coronavirus sequences. These viruses are linked to significant zoonotic viruses, such as SARS-CoV, SARS-CoV2, and MERS-CoV [3]. Paramyxoviruses, also widespread among bat populations, with 657 unique sequences identified, underline their role as progenitors of numerous diseases affecting humans and animals [4].
The surfacing of zoonotic viral diseases could be driven by factors such as human population growth, urbanization, and agricultural practices. These factors intensify human-animal interactions, elevating the likelihood of zoonosis [5]. In 2002, the outbreak of SARS-CoV-1, originating from bats and transmitted through civet cats to humans, resulted in 8,098 infections and 774 fatalities, marking a mortality rate of 9.6% [6]. Considering the recent pandemic, a consensus among epidemiologists’ points to SARS-CoV-2 originating from a market trading wild animals for human consumption in Wuhan, China. The prevailing hypothesis is that bats, the initial hosts, transmitted the virus to pangolins, which in turn infected humans [7, 8].
Human-bat interactions can also enhance paramyxovirus transmission. A study in Cameroon found antibodies against paramyxoviruses, like Nipah and Hendra, in seven people [9]. In Malaysia's Tioman Island, the Tioman virus, a member of the Paramyxoviridae family affecting bats, pigs, and humans with flu-like symptoms, was studied. Antibodies against the Tioman virus were found in 5 of the 169 individuals tested, indicating its presence and potential health impact [10]. While zoonotic diseases pose a substantial threat, Ecuador lacks data on them. A review covering 25 studies with 9,371 subjects highlighted Brazil, Mexico, US, Jamaica, Peru, Bolivia, and Argentina for coronavirus prevalence in bats, but Ecuador remains unstudied [11]. A similar scene occurs for paramyxovirus, indicating a critical gap in understanding and managing potential zoonotic risks within the country [12–16]. This study's objective was to assess the presence of these viruses in bats from the Fundación para la Conservación de los Andes Tropicales (FCAT) in Northwestern Ecuador.
During November 2022, bats were systematically trapped through mist nets placed along known bat flight and feeding paths over ten nights at the FCAT Reserve in Esmeraldas province in three differently forested areas (Figure 1). Individuals were subsequently identified to species level with the Guide to the Mammals of Ecuador [17]. A buccal swab sample was collected from all captured bats using sterile polystyrene swabs. Swabs were preserved in 500 uL of DNA Shield (Ecogen) until analysis.
For viral RNA extraction the AccuPrep Dx Viral RNA Extraction kit (Bioneer) was used, following manufacturer's instructions. A total of 33 samples were analyzed with a two-step RT-PCR and 90 of them with a one-step RT-PCR. For the reverse transcription was performed at 50°C for 15 minutes and 85°C for 5 minutes with the following protocol: 10 μL of nuclease-free water, 4 μL of 5X RT Buffer, 2 μL of RNA, and 1 μL of reverse transcriptase. Primers for the helicase gene were used for coronavirus while primers the RNA-dependent RNA polymerase gene (RdRp) were used for paramyxovirus.
For the PCR reagents were added as follows: 10X Buffer, 100 pmol/μL of each primer, 1,5 μM MgCl2, 10μM dNTPs, 1 μL (1X) Fastgen Taq DNA polimerase and 2 μL of cDNA. For coronavirus the PCR began with a 95°C denaturation – 15 min, followed by 45 cycles of denaturation 95°C – 1 min, 48°C – 1 min and 72°C – 1 min, with a final extension of 10 minutes [18]. For paramyxovirus the PCR conditions were initial denaturation 60°C – 15 min, 98°C denaturation – 30 sec, followed by 40 cycles of denaturation 98°C – 10 sec, 50°C – 30 sec and 72°C for 30 sec, concluding with a 72°C extension of 7 minutes [19].
For the 90 samples processed through one step RT-PCR; 25 μL of 2X One-Step RT-PCR Buffer, 4 μL of RT-PCR Enzyme mix, 2.5 μL of each primer (100pmol/μL), 5 μL of RNA, and 10 μL of nuclease-free water, were used. The temperature conditions were the same as those described previously for PCR. The resultant amplicon fragments were analyzed via 2% agarose gel electrophoresis. To determine the presence of coronavirus the expected fragment was 591 bp, whilst for paramyxovirus, the expected fragment size was 580 bp.
In the study, 123 bats spanning 20 species were captured (see Table 1). Analysis revealed that 28 samples were positive for paramyxovirus, and 18 samples were positive for coronavirus. A total of 42 bats were infected with coronavirus and/or paramyxovirus, involving 10 species across 8 genera (see Table 2).
Coinfections were found in 4 individuals. These include: Carollia perspicilata, Vampyrodes major, Artibeus lituratus and Dermanura rosenbergi.
In examining the species captured, the forest zone boasted the greatest diversity and number of individuals, succeeded by the reforestation zone, and lastly, the agricultural zone (Table 1). This observation aligns with ecological principles. Research comparing bat biodiversity in untouched forest zones to that in fragmented areas revealed a direct correlation between the extent of forest coverage and the diversity and abundance of bat species [20]. Moreover, research in Colombia evaluated the species composition and richness of bats in three different sites within Nariño's tropical dry forest. From a total of 60 bats, the highest individual count was observed in the preserved dry spiny scrub. In contrast, areas subjected to disturbance showed reduced species richness. This aligns with the outcomes of the current study [21].
In a study carried out in Peru, the species identified as most prevalent were Carollia perspicillata, Carollia brevicauda, and Artibeus planirostris. Conversely, in our study, the most numerous species identified were C. perspicillata, C. brevicauda, and Dermanura rava. The overlap of two out of three predominant species in both studies can be attributed to their shared habitat preferences. C. brevicauda and C. perspicillata have a wide distribution, thriving in both the moist and arid forests located on the eastern and western slopes of the Andes encompassing a large altitudinal range [22]. Moreover, these forest environments abound with key food sources for C. perspicillata and C. brevicauda [23]. Specifically, in northwestern Ecuador, within the FCAT region, bats consume Piper spikes, which are prevalent in this area. The diet of bats in this part of Ecuador is diversified to include Cecropia concolor, Ficus eximia, Piper aduncum, Piper longistylosum, Piper tuberculatum, and Solanum sp. The widespread availability of these plant species ensures their presence in both the Peruvian study and this research. Furthermore, Carollia bats play a crucial role as seed dispersers for these plants, establishing a mutualistic relationship [24].
In the context of coronavirus and paramyxovirus detection using PCR, a study in Sao Paulo, Brazil, collected fecal samples from bats in 73 municipalities to screen for coronavirus. From the 305 samples collected, 9 samples were identified as coronavirus positive from five species: Cynomops abrasus, Cynomops planirostris, Desmodus rotundus, Glossophaga soricina, and Platyrrhinus lineatus. In contrast, the coronavirus positive species in our study were Artibeus lituratus, C. brevicauda, C. perspicillata, Dermanura rava, Dermanura rosenbergi, Vampyrodes major, and Sturnira luisi (Table 2.). It is notable that the species identified as positive in our investigation do not overlap with those found in the Brazilian study [25]. From the current study, two positive species are also distributed in the Sao Paulo area: A. lituratus and C. perspicillata [26]. However, these two species present in both regions, were not found to be infected in Brazil. This discrepancy can be attributed to environmental variations affecting each group, including differences in predator presence, trophic interactions, coinfections, and exposure to pathogens. As a result, the likelihood of identical bat species being infected with coronavirus across different regions is minimal [27].
A separate investigation into paramyxovirus in French Guiana's bat population found 103 individuals positive for the virus. The detection rates varied with the biological fluid examined; D. rotundus, for example, tested positive for paramyxovirus only in kidney and urine samples, not in saliva, lung, or blood [28]. Contrary to this, the current research identified positive paramyxovirus cases in saliva samples from A. lituratus, C. brevicauda, C. perspicillata, D. rava, D. rosenbergi, Lonchophylla concava, S. luisi, V. major, Platyrrhinus vittatus, and Vampyriscus nymphaea. Similarly, a study focused on identifying coronavirus in North American bats through RT-PCR, revealed that all 22 saliva samples tested were negative. Conversely, 6 out of 28 fecal samples were positive for the virus [29]. In comparison, the current study found coronavirus in 18 saliva samples from a total of 123 tested. This variation could be due to each virus's specific replication and excretion mechanisms [28]. Furthermore, in the second study the number of analyzed saliva samples was relatively low compared to the current research.
Bats' social behavior, particularly their use of communal feeding areas, facilitates the transmission of different viruses among individuals and across colonies of the same species [29]. These communal habits extend to resting sites, where bats also dispose of their waste. Since evidence suggests that bat feces may harbor paramyxovirus and coronavirus, it's plausible to deduce that the communal lifestyle contributes to the spread of infections [30]. Consequently, the species found in abundance in this study, C. brevicauda, C. perspicillata, D. rosenbergi, and D. rava, also exhibited the highest instances of coronavirus and paramyxovirus infections.
Prevalence of coronavirus was 14.63%, and 22.76% for paramyxovirus. These values are considered high in comparison with other studies, where significantly lower values were recorded with larger sample sizes. For example, a study conducted in Italy, analyzing bat saliva and feces, found 12% and 1% prevalence for coronavirus and paramyxovirus respectively, out of 302 captured bats [14]. In Costa Rica, from 421 fecal samples, only 4 of them were positive for coronavirus [31]. Similarly, in Peru, from 436 monitored individuals, a prevalence of 10.3% was recorded for paramyxovirus [15].
Due to these high values, meticulous epidemiological surveillance must be maintained in bats to detect coronavirus, paramyxovirus, and other viruses in general. This is crucial for several reasons, one of which being the variety of viruses harbored by bats. Some of the other viral families found include adenovirus, astrovirus, bornavirus, circovirus, herpesvirus, parvovirus, polyomavirus, and rhabdovirus [32]. Another crucial reason is that it could allow us to identify potential sites of zoonotic outbreaks. This enables the possibility for taking preemptive measures to prevent the spread of such diseases [33].
A total of 4 coinfections were recorded. A systematic review noted that out of 725 bats with coinfections, only 4 individuals had a coinfection with only two viruses, namely paramyxovirus and coronavirus. These findings imply that the prevalence of coinfections in the current study is also high, in relation to the number of samples [32]. This phenomenon could be attributed to peak viral proliferation phases, resulting in higher viral shedding [34]. Additionally, it might be explained by the possibility that the viral strains infecting these individuals possess molecular mechanisms that hinder the action of interferons, which are crucial for bats in regulating viral replication [29]. The likelihood of coinfections is also affected by the phylogenetic relationships among bat species. The ability of a virus to infect is contingent upon overcoming physiological and molecular defenses. Viruses are more likely to infect two bat species that are phylogenetically close, as they may share similar defenses. For example, the SARS CX1 coronavirus is evolutionarily akin to SARS-CoV-2, and both can infect identical bat species because the receptor-binding domain of their spike proteins only differs by five amino acids [35].
In this study, the bulk of positive cases were identified in C. brevicauda with 15 positive samples, followed by C. perspicillata with 10, and D. rosenbergi with 7 positive samples. Notably, among these, two cases of coinfections were detected: one in C. perspicillata and another in D. rosenbergi. This observation of coinfections may be attributed to the dense populations of the species, as well as to the phylogenetic distance between the mentioned species [35]. Findings of this study highlight a significant prevalence of coronavirus and paramyxovirus among the bat populations examined. This marks the first documentation in Ecuador of various bat species harboring infections of coronavirus and paramyxovirus. Given these findings, it underscores the imperative need for further research within Ecuador to track the viral diversity of coronavirus and paramyxovirus, particularly in regions proximate to human communities.