Investigation of astrovirus, coronavirus and paramyxovirus co-infections in bats in the western Indian Ocean

Co-infections have a key role in virus transmission in wild reservoir hosts. We investigated the simultaneous presence of astroviruses, coronaviruses, and paramyxoviruses in bats from Madagascar, Mayotte, Mozambique, and Reunion Island. A total of 871 samples from 28 bat species representing 8 families were tested by polymerase chain reactions (PCRs) targeting the RNA-dependent RNA-polymerase genes. Overall, 2.4% of bats tested positive for the presence of at least two viruses, only on Madagascar and in Mozambique. Significant variation in the proportion of co-infections was detected among bat species, and some combinations of co-infection were more common than others. Our findings support that co-infections of the three targeted viruses occur in bats in the western Indian Ocean region, although further studies are needed to assess their epidemiological consequences.

Co-infection (sometimes written as coinfection) can be defined as the simultaneous infection by at least two genetically different infectious agents in the same host [1][2][3][4][5]. It can affect both host fitness and disease transmission dynamics, therefore playing a critical role in the epidemiology of infectious agents [4][5][6][7]. In bats, although many studies have focused on the detection of emerging viruses such as astroviruses (AstVs), coronaviruses (CoVs), and paramyxoviruses (PMVs), limited data is available regarding co-infection patterns and its potential effect on host fitness and disease transmission dynamics [8][9][10][11][12][13].
Biological material was collected on Madagascar, in Mozambique, on Mayotte, and on Reunion Island as part of previous investigations on infectious agents circulation in bats (details relating to the collection of biological material are available in [23]). The list of samples included in this study (e.g. bat species, location, date, type of samples) is provided in the Additional file 1. All samples were previously tested for the presence of CoV [23]; some of them were also tested for the presence of AstV (516 samples; [20,21]) and PMV (167 samples; [19]). Additional assays were thus performed for Open Access *Correspondence: axel.hoarau@univ-reunion.fr 1 Processus Infectieux en Milieu Insulaire Tropical, INSERM 1187, CNRS 9192, IRD 249, Université de La Réunion, Sainte-Clotilde, La Réunion, France Full list of author information is available at the end of the article the detection of the AstV (355 samples) and PMV (704 samples) RNA-dependent RNA-polymerase (RdRp) genes. Molecular detection was performed using seminested polymerase chain reactions (PCRs), as previously described [16, 17, 19-21, 24, 25]. PCR products were visualized on 2% agarose gels stained with 2% Gelred (Biotium, Hayward, CA, USA). Pearson Chi square tests were conducted to examine the effect of the roost sites (i.e. cave, building, tree), host species, sex, and sampling location (i.e. country or island), on virus detection, and to investigate potential associations between AstV, CoV, and PMV. Analyses were conducted with R, version 4.0.5 [26].
PCR products of expected size were submitted for direct Sanger sequencing (Genoscreen, Lille, France). Nucleotide sequences were aligned to generate consensus sequences, and were edited manually using ChromasLite 2.6.5 (Technelysium Pty, South Brisbane, Australia). The 33 partial AstV sequences and 13 partial PMV sequences generated in this study were deposited in GenBank respectively under the accession numbers MZ614404 to MZ614436 and MZ614437 to MZ614449. Genetic diversity was explored with pairwise distance values obtained from phangorn package in R, version 2.6.3 [27]. Sequences were compared to reference sequences in NCBI GenBank using the Basic Local Alignment Search Tool (BLAST) with the standard nucleotide BLAST (BLASTn) algorithm (BLAST was performed on August 18th, 2021) [28,29]. Then, AstV and PMV sequences generated in this study were respectively aligned with 105 and 74 reference partial nucleotide sequences, using CLC Sequence Viewer version 7.6.1 (CLC Bio, Aarhus, Denmark). Phylogenetic trees were generated by maximum-likelihood using PhyML software 3.1 [30], with a GTR evolutionary model, and 1000 bootstrap replicates.
The overall proportion of positive bats detected for either AstVs or PMVs was consistent with previous studies performed in the WIO region, and in other tropical regions [31][32][33][34]. Interestingly, higher detection rates were found for both viruses in bats using caves as dayroost sites, suggesting that cave-roosting behavior maybe favorable for horizontal transmission between bats [35]. Differences between locations, sex, and bat species may be explained by a range of factors. For example, seasonality has been identified as a major driver of the infection dynamics of many pathogens, affecting both host susceptibility and transmission [17,[36][37][38][39]. Important seasonal variation in the prevalence of infected animals can depend on the period the samples were collected, and, in turn, can lead to misrepresentative conclusions regarding the level of bat exposure to viruses, in particular in crosssectional studies. Longitudinal studies in wild animals are thus important to precisely assess prevalence of infected animals and its temporal variation [17,37,40].
High genetic diversity was detected for AstV, with pairwise differences up to 46% between sequences, without support for host family or species association (Fig. 4). Based on BLASTn comparisons, we found that our sequences had a high level of identity (between 80 and 92%) with AstV previously described in bats of the WIO region [20,21], as well as with AstVs detected in bats from continental Africa (e.g. Gabon, Democratic Republic of Congo), and in other regions in the world (e.g. China, Thailand) (Additional file 4). However, one sequence obtained from a Triaenops menamena, a species endemic to Madagascar, showed 92% identity to an AstV sequence detected in a mouse from China (Additional file 4). Another example, even more unexpected, one AstV sequence obtained from Chaerephon leucogaster on Madagascar had 96% identity with an AstV sequence from a bird of the order Passeriformes (Additional file 4). These findings were consistent with phylogenetic results and were statistically supported (Fig. 4). A recent study also reported AstVs related to avastrovirus in environmental samples collected in a colony of Mormopterus francoismoutoui, a member of the family Molossidae endemic to Reunion Island [41]. These findings may suggest introduction of AstVs on the island by non-native rodents, and could also support environmental transmission of AstVs between species of different taxa, as previously suggested [42]. Nevertheless, studies investigating the circulation of AstVs in terrestrial small mammals in the WIO region are required to assess these potential host-shifts.
Genetic diversity was less important for PMV sequences, with pairwise differences up to 29%. All our sequences were genetically related to PMVs previously described on Madagascar [16,19] or in continental Africa (e.g. Ghana, Kenya), with sequence identity ranging from 78 to 99% (Additional file 5 and Fig. 5). Phylogenetic analyses highlighted some degree of host-specificity, as previously described for PMV in the western Indian Ocean (Fig. 5) [16]. For instance, most sequences clustered either with PMV sequences detected in bats on the same genus captured in the region or elsewhere (e.g. Hipposideros caffer from Mozambique), or with sequences obtained in bats from the same family (e.g. Mops condylurus sequence from Mozambique, and Chaerephon leucogaster sequence from Madagascar clustered with a sequence detected in Mops leucostigma on Madagascar). However, some sequences were included in more diversified groups including different bat families (e.g. two sequences obtained from Paratriaenops furculus on Madagascar that clustered with sequences detected in Miniopterus griveaudi and Chaerephon leucogaster on Madagascar).
We report co-infections in bats on Madagascar and in Mozambique, ranging from 3.2% to 15.7% of the positive samples, and depending on the tested bat species. Although our cross-sectional sampling precludes detailed interpretation of the biological drivers of such variation, our results nevertheless highlight that interactions between infectious agents in bats may exist with potential consequences on their epidemiology. AstVs, CoVs, and PMVs are emerging viruses that represent a major challenge for human and animal health. Further knowledge on virus interaction in wildlife, based on long-term longitudinal sampling is needed to fully assess the epidemiological consequences of coinfections [5].