Wildlife has been known as a major reservoir of viruses causing old and new infectious human diseases. The emergence of new pathogens with zoonotic potential represents a constant threat to global public health as it has been seen with the current pandemic produced by SARS-CoV2. This has been the most notorious incident of a zoonotic event extending worldwide but events of this nature had happened before, as for example, with HCov-NL63, HCov-229E, SARS-CoV1 and MERS-Cov (1). Despite these early warnings, research of the viral population circulating in wildlife is very limited, especially in bats. Bats are the only mammal with the capacity to fly and is also one of the most diverse groups within this class (Mammalia) of animals. Bats are known reservoirs of viruses with zoonotic potential such as paramyxovirus, filoviruses, and lyssaviruses but new ones are being nowadays identified through NGS (Next Generation Sequencing) (2, 3). Coronaviruses infecting bats have been implicated as predecessors of those generating the human infections. Bats, as well, may become new reservoirs of viruses infecting other species through a process of adaptation which is a possible scenario with the current pandemic produced by SARS-Cov2 (4). Thus, surveillance of viral populations, in particular coronaviruses, in these mammals has become a priority to learn more about the events leading to interspecies and intraspecies transmissions. The family Coronaviridae is divided in four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Bat coronaviruses ancestors of human diseases, belong to the Alpha and Betacoronavirus genera.
The work described in this manuscript was based on bat and rodent sample collection carried out in two regions of Argentina. One was the north-western region of Argentina which is in an area where some of the most important phytogeographic/ecological units of South America converge, with desert/semi-arid regions alongside subtropical rainforests. This has given rise to one of the areas of Argentina with the greatest animal diversity. Unlike the north, the central part of Argentina is characterized by extensive plains in which one of the most important agricultural and livestock activities in the country is concentrated. Due to the high level of agricultural production and the large number of food storage facilities, mainly grains and cereals, the region provides an environment conducive to the presence and development of populations of wild animals that inevitably cohabit with domestic animals, production animals, and humans.
Several surveys along the years have contributed to the knowledge of the different bat species coexisting within these areas. In Argentina, field work has recorded 67 species of bats spanning 5 families (Emballonuridae, Noctilionidae, Phyllostomidae, Molossidae, and Vespertilionidae) and 29 genera (5). Despite the increasing interest worldwide to study the population of viruses circulating within any wildlife animal species, this kind of investigations have been scarcely done in bats in Argentina. In this work, we report, to our knowledge, the first alphacoronavirus sequences from bats in Argentina.
Bat and rodent samples were collected between 2020 and 2021 in northern and central Argentina, the Yunga region in the province of Jujuy and the northeast region of the province of La Pampa, respectively. The sampling sites were selected based on their favourable characteristics for the nesting of these animals and their contact with humans and other domestic or wild animals. Thus, ruined buildings, abandoned iron mines and cereal storage sheds in rural or peri-urban areas, in which the existence of nests of different bat species was already known, were chosen for sampling.
The samples were actively obtained by mist nets in the case of bats, and trap cages in the case of rodents, to guarantee the safety of both the animals and the operators. The nets were placed at the exit of the nests before sunset, to ensure the capture of the bats at the time of their exit. All animal procedures were performed according to a protocol approved by the Faculty of Veterinary Sciences, University of Buenos Aires (CICUAL 2020/9).
Each bat caught in the net was immediately handled by our team, taking the necessary precautions for such handling. Oropharyngeal swabs and, when possible, individual faecal samples were obtained from these animals. Due to their small size, swabbing of nasal or rectal areas were not pursued. The identification of species and, when possible, gender, were made based on morphological characteristics. Each animal, prior to its release, underwent local hair removal on the back to identify it as a "sampled animal" to avoid repeated captures. In addition, tissue samples were obtained from carcasses of some of these animals, provided by local individuals who hunt these animals regularly. In these cases, samples of spleen, liver, stomach, and intestine were taken. Several samples were pooled of faecal material collected from areas inhabited by several bat colonies. For this purpose, a plastic layer was prepared in strategic areas during the night and after two hours several individual depositions were collected.
All samples were collected in the field in sterile tubes filled with nucleic acid preservative solutions to prevent degradation of viral RNA until processing. Swab and stool samples were placed in DNA/RNA Shield 1X solution (Zymo Research, Irvine, CA, USA); tissue samples were transported in RNAlater™ stabilization solution (Invitrogen™, Waltham, MA USA) and stored at -80º C until processing.
Faecal material samples were homogenized in 250 µL of sterile 1X PBS for approximately 1 minute, followed by centrifugation at 3000 rpm for 5 minutes. Then, 20 µL of the supernatant were taken and brought to a final volume of 100 µL with 1X PBS before RNA extraction to reduce the interference of PCR inhibitors. The oropharyngeal swabs were vortexed for 5 minutes, after which 100 µL were used for RNA extraction using the Quick-RNA Viral Kit (Zymo Research, Irvine, CA, USA) following the manufacturer's protocol. RNA extraction from tissues was performed using TRIzol™ reagent (Invitrogen™, Waltham, MA USA ).
cDNA synthesis was performed by reverse transcription using random primers (EasyScript® First-Strand cDNA Synthesis SuperMix kit, Transgene Biotech®, Beijing,China) following manufacturer’ instructions. The presence of coronavirus RNA was evaluated by nested PCR (nPCR) with 5 µl of cDNA, 200 uM dNTPs, 1x buffer, 1 unit of GoTaq (Promega, Madison, WI, USA) and 25 pmoles of pan-coronavirus primers (6 In a first round the primers RdRp For1 : 5’-GGKTGG GAYTAYCCKAARTG-3’ and RdRp Rev2 : 5’-TGYTGTS WRCARAAYTCRTG-3’ were used. For the second round, 5 µl of the first round were utilized with primers RdRp For3: 5’-GGTTGGGACTATCCTAAGTGTGA-3’ and RdRp Rev4A: 5’-CCATCATCAGATAGAATCATCAT-3’. Both PCR reactions were run with the following program: 2 minutes at 94°C, 40 cycles of 94°C 1 minute, 48°C (first round) or 55°C (second round) ,1 minute 72°C and a final cycle at 72°C 5 minutes.
These primers target a 440 bp conserved region of the ORF1ab gene that encodes part of the viral replication complex, specifically the Nsp12 protein. Prior to processing samples from bats and rodents, degenerate primers were tested with different coronaviruses (canine, feline, bovine, avian and human) to assess their specificity.
The nested PCR products obtained by electrophoresis were purified with the EasyPure® rapid gel extraction kit (TransGene Biotech®, Beijing, China). DNA concentration was estimated using a NanoDrop™ Lite instrument (Thermo Scientific™, Waltham, MA USA). Sequencing was performed by the Sanger method and homologies to known coronaviruses were assessed by Blast analysis. Multiple sequence alignments were performed with MAFFT version 7.0. The alignments were constructed with the bat and rat sequences identified in this work, and selected sequences from GenBank with which they shared the highest nucleotide identity. Subsequently, they were manually edited with the BioEdit version 7.2 program to achieve the greatest possible gap reduction. The analysis of the evolutionary model and phylogenetic tree were performed with the efficient maximum likelihood phylogenomic software IQ-TREE version 1.6.1. The analysis showed TN + F + G as the best nucleotide substitution model (7). To evaluate the reliability of groups and branches, the support measure ultrafast bootstrap of 10,000 interactions was used(8). Sequences from coronaviruses from different genera were also added to the analysis as outgroups. The visualization and edition of the phylogenetic tree was carried out with Fig Tree version 1.4.3.
Three hundred sixty-one samples belonging to hematophagous, insectivorous, and frugivorous bats from the areas selected for sampling were analysed: 178 corresponded to oropharyngeal swabs, 117 to individual stool samples, and 34 to colony stool pools (Table 1). Likewise, 93 samples of wild rodents, 29 of individual faeces, 13 oropharyngeal swabs and 3 pools of faeces were collected and analysed. A total of 80 tissue samples (spleen, liver, stomach, and intestine) from 8 bats and 12 rats were also analysed (Table 1).
All samples were processed by RT-nPCR, obtaining 17 positives; fifteen belonged to bat samples (9 oropharyngeal swabs, 4 individual stool samples and stool pools from two bat colonies) whereas the remaining two positives were detected in two individual rodent faeces. All tissue samples and rodent oral swabs were negative. Most of the positive samples originated in the Yunga region in the province of Jujuy and only one from the northeast region of La Pampa province. (Table 1)
Sequence analysis of the partial RdRp sequences demonstrated a high nucleotide identity with alphacoronaviruses previously reported in bats from South America (particularly from Brazil) and the United States. Interestingly, we also found alphacoronavirus sequences in bat species different from those that had already been reported for those species, such as the case of an oropharyngeal swab from an insectivorous bat (Myotis sp.) in which we found sequences of alphacoronaviruses reported in hematophagous bats (Desmodus rotundus) (Table 1). In addition, two faecal samples from wild rodents were found to contain alphacoronavirus sequences related to insectivorous bats (Table 1). All sequences were submitted to GenBank with accession numbers ON22822-23, ON237741-43, ON246265-73, ON256703-05.
The phylogenetic analysis of the RdRp partial sequences confirmed that the all viral sequences identified belonged to the Alphacoronavirus genus, with a branch support value greater than 0.9 in most cases (Fig. 1).Previous published virome studies performed in Argentina on several bat species (Tadarida brasiliensis, Molossus molossus, Eumops bonariensis, Eumops patagonicus, and Eptesicus diminutus ( 9,10) reports the finding of alphacoronavirus sequences at least in Tadarida brasiliensis, but no GenBank accession numbers could be found associated with in order to be included in the phylogenetic analysis.
The results presented in this work represent one of the few reports on the coronavirus genera circulating in bats from Argentina. As expected, the sequences reported in this work show high similarities to previously reported alphacoronaviruses in the same bat species from Brazil (11,12 ) (Fig. 1). This observation may just be based on the greater number of alphacoronaviruses reported by this country versus others from South America. Thus, more representative sequences are needed to evaluate whether the amplified fragment really allows the identification of subtle differences among alphacoronaviruses within South American species. We did not detect bats infected by two different alphacoronaviruses, as described by others (13, 14) but in several cases, a particular bat species was found to harbour alphacoronavirus sequences from another bat species (alphacoronavirus from Molossus rufus detected in Myotis species and Myotis sp alphacoronavirus sequences detected in Histiotus laephotis and Tadarida brasiliensis bats). This result is not surprising since we sampled sites where several bat colonies were cohabitating, a finding also previously described in bats (15, 16). As seen for the two rodent samples from Jujuy, in which bat coronavirus sequences could be also detected, these observations may just indicate an environmental source and not necessarily active infection. However, it is also evidence that viral circulation among the different bat species is present leading to the speculation of eventual coinfections and the potential of viral recombination.
Still, some results require further investigation, as we found hematophagous alphacoronavirus sequences in insectivorous bats (Myotis sp) (Table 1, Fig. 1) when proximity between these two species could not be seen. Another interesting finding has been the presence of alphacoronaviruses in Myotis sp from central Argentina, which by the BLAST alignments showed great similarity with those described in several North American bat species (Rocky Mountain alphacoronavirus). These viruses, with a close phylogenetic association with the bat coronavirus HKU6, described in China (17), show sufficient sequence differences to represent a unique group of coronaviruses (18). However, the phylogenetic analysis also showed that this sequence was evolutionarily more distant than all the sequences included in the analysis, which emphasizes the importance of continuous work on the identification of new viral lineages in this region.
No betacoronaviruses could be identified in the samples collected for this study. Betacoronaviruses have been described in south American bat species, some of them represented in this study such as Artibeus sp and Desmodus rotundus (19, 12). The reduced number of samples and the few locations surveyed would not allow to exclude their presence but seem to be enough to conclude that alphacoronaviruses are the most common coronaviruses found in bats in Argentina, in agreement with reports from other south American countries (20, 21, 22).
Considering the crucial role bat coronaviruses play generating zoonotic events like the one produced by SARS-CoV2, any information about their circulation in this mammalian species is extremely valuable to continue monitoring and understanding their evolution. Thus, the first bat alphacoronavirus sequences reported in this manuscript contribute to the global surveillance efforts to monitor these viruses and their potential as human pathogens.