Bats (Order: Chiroptera) are one of the most speciose mammalian clades (1, 2), and this diversity is matched by an equally high richness of viruses, many of which are zoonotic (3, 4). Bat species are the confirmed reservoir hosts for Hendra and Nipah viruses, lyssaviruses, Marburg virus, and SARS-like coronaviruses (CoVs) (5–8). Yet with few exceptions (e.g., lyssaviruses), these pathogens seem to not cause disease in their bat hosts (9–11). This tolerance of otherwise virulent infections is likely driven by distinct aspects of bat immunity that evolved alongside their unique ability of powered flight and their very long lifespans (12–14). These include but are not limited to constitutive expression of interferons (IFNs) and IFN-stimulated genes (ISGs), robust complement proteins, and a dampened inflammatory response (15–20).

Relationships between bats and CoVs specifically have been of particular interest for zoonotic risk assessment (21, 22). CoVs are RNA viruses found across mammals and birds, with at least seven known human viruses: two and five in the genera Alphacoronavirus and Betacoronavirus (23, 24). A novel α-CoV with canine origins was recently detected in humans, but its role in zoonotic disease remains unclear (25). CoVs are highly diverse in bats, which are the likely ancestral hosts of α- and β-CoVs (26–28). The evolutionary origins of highly pathogenic CoVs (i.e., SARS-CoV, MERS-CoV, SARS-CoV-2) have also been ascribed to bats, but spillover has typically involved intermediate hosts rather than direct bat-to-human transmission (5, 29, 30).

Our understanding of bat–CoV interactions and the bat immune response to infection remains limited and has stemmed mostly from experimental studies of a few select species, including Rousettus leschenaulti, R. aegyptiacus, Artibeus jamaicensis, and Eptesicus fuscus (10, 11, 28, 31, 32). In vivo studies have typically found short-term CoV replication and shedding without substantial weight loss or pathology, supporting viral tolerance, although some species seem to entirely resist infection. Tolerance appears driven by innate immune processes, such as increased expression of ISGs, with little adaptive immune response. In vitro studies have further supported bat receptor affinity for CoVs (i.e., susceptibility) but little host inflammatory response in bats (10, 11, 16, 33).

Despite the clear insights afforded by experiments, model bat systems are heavily limited by logistical constraints (e.g., necessity for specialized facilities, colony maintenance) and have been mostly focused on frugivorous bats, which are relatively easy to keep in captivity compared to other dietary guilds (34, 35). Transcriptomics of key tissues (e.g., spleen) has helped advance the field by identifying immune responses to infection in a wider array of species (36–38). However, such approaches are restrictive when lethal bat sampling is limited, such as when working with threatened species, in protected habitats, or in longitudinal studies to assess how these viruses persist in bat populations. Blood transcriptomes overcome such challenges to a degree and are increasingly feasible (39, 40), yet such assays are only informative about the immune response in the blood itself. Proteomics, on the other hand, provides a unique and more nuanced perspective into the immune system, because the blood proteome includes proteins secreted from not only blood cells but also proximal organs such as the liver (41).

Proteomics holds special promise for illuminating the innate and adaptive immune factors involved in bat responses to infection, because it can accommodate the small bat blood volumes typical of field studies (e.g., <10 μL). Recently, we surveyed the serum proteome of wild vampire bats (Desmodus rotundus) (42). Owing to its diet of mainly mammal blood, this species is the primary reservoir host of rabies lyssavirus in Latin America, and alpha- and betacoronaviruses have also been identified in this species (43–46). Using only 2 μL sera from these small (25–40 gram) bats, we identified 361 proteins across five orders of magnitude, including antiviral and antibacterial components, 20S proteasome complex, and redox activity proteins. Mass spectrometry–based proteomics can thus facilitate the relative quantification of classical immunological proteins while also providing insight into proteins yet to be fully recognized for their importance in resolving viral infection (47–49).

Here, we expand our recent work on serum proteomics in vampire bats in the context of CoV infection. First, by profiling the serum proteome of the same host species in an additional year of study, we provide a more general and comprehensive characterization of the wild bat immune phenotype. Second, owing to changes in regulations for importing vampire bat samples into the United States, we also assess the impact of heat inactivation, a common method of inactivating bat sera (50), on the proteome. Lastly, and most importantly, we assess differences in the serum proteomes of wild bats with and without acute CoV infection. We aimed to identify up- and downregulated immune responses of wild bats to CoV infection, with particular interest in comparisons with results from experimental infections. We also aimed to guide the discovery of candidate serum biomarkers of viral infection. By taking an agnostic approach via discovery proteomics (51), such biomarkers could provide new and mechanistic insight into CoV pathogenesis in wild bats (52).

Despite an increasing interest in bat–virus interactions, especially for CoVs given their human health relevance (22, 23), we still have limited insights into the immune mechanisms involved in infection of bats (28). Here, we used serum proteomics to broadly profile the immune phenotype of wild vampire bats in the presence of relatively common CoV infections. Novel α-CoVs detected in these bats had little association with serum protein composition nor abundance, although ROC curve analyses identified 7–32 candidate biomarkers of CoV infection, including AHSG, C4A, F12, GPI, DSG2, GSTO1, and RNH1. Enrichment analyses using these protein classifiers identified strong downregulation of complement, regulation of proteolysis, immune effector processes, and humoral immunity in CoV-infected bats alongside an upregulation of neutrophil immunity, overall granulocyte activation, myeloid cell responses, and glutathione processes. Such results denote a mostly cellular immune response of vampire bats to CoVs and further identify putative biomarkers that could provide new insights into CoV pathogenesis in both wild and experimental populations.

Much bat immunology work to date has understandably focused on model bat systems under captive conditions (14, 18). However, identifying the immune correlates of infection is especially important for wild populations, where susceptibility and tolerance to infection, alongside other immunological processes, can vary based on habitat quality and host life history (e.g., reproduction) (78). Such efforts could provide a mechanistic basis for establishing when and where pathogen pressure from bats is greatest and thus help predict viral spillover (79, 80). Unlike some other global profiling techniques, proteomics has the benefit of leveraging the small blood volumes that can be obtained non-lethally from most small bats (e.g., members of the Yangochiroptera, including over 1000 species) and thus is especially amenable to the long-term, mark–recapture studies required to study bat virus dynamics (52, 81). To facilitate this work, we here built on our prior proteomic characterization of Desmodus rotundus (42). We identified a core serum protein phenotype for this species from multiple years of sampling that could serve as a reference for long-term proteomic studies, although technical advances (e.g., DIA-NN) likely contributed to an expanded protein repertoire in the current study. We also assessed the impacts of heat treatment, a common inactivation method for sera, given recent shifts in United States importation regulations. Artifacts from heat inactivation were not sufficiently conserved to be statistically significant, and most serum proteins had small changes in abundance before and after this treatment. Yet given the extent of such changes, we suggest original samples should typically not be analyzed with heated samples for comparative purposes. In contexts where sera inactivation is required, however, heat treatment across samples should not bias characterization of bat serum proteomes. Such optimizations could next be applied across longitudinal timepoints to more broadly study bat–virus interactions in the wild.

We here focused this initial study on CoVs, which have been previously characterized as genetically diverse (α- and β-CoVs) in Neotropical bats, including but not limited to Desmodus rotundus (43, 45, 82, 83). Our detection of CoVs from a northern Belize colony of vampire bats in swab samples with paired sera (4/19) was higher than other CoV surveys in this species (46, 76, 82, 84), although future studies with larger sample sizes are needed to test if this represents a true geographic difference in virus prevalence. Yet despite many available CoV sequences in GenBank from diverse bat surveys, all CoVs detected here fell within the genus Alphacoronavirus but outside of known bat α-CoV clades. Instead, these viruses were either entirely novel or more closely related to human α-CoVs, specifically HCoV-NL63 and HCoV-229E, suggesting greater genetic diversity of CoVs within vampire bats than previously recognized. Such results could be further interrogated through whole-genome sequencing of these viruses (45). Similarly, such results further suggest the possibility of high zoonotic potential (or spillback from humans to bats) for vampire bat CoVs, which likely varies based on geography given the high degree of genetic differentiation across the broad distribution of Desmodus rotundus (85–87). Such findings should be confirmed with larger sample sizes and characterization, including in vitro assessments and attempts at virus isolation.

Despite identifying CoV infection in a small number of bat samples, we were unable to detect CoV proteins in the serum proteomes. Previously, we detected two CoV peptides in sera from this population, but these were likely at the edge of detection limits (42). As such detection limits are susceptible to technical artifacts, including but not limited to sample handling, protein processing, or instrument performance, heat inactivation could have affected our ability to identify similar peptides in these bat samples, especially for bats positive for CoVs by PCR. Additionally, our ability to detect viral proteins may have been further restricted by ongoing limitations in applying proteomics to wild species. In humans, over 3000 serum proteins can be detected by mass spectrometry after depletion of the most abundant proteins (41). However, using antibody-based depletion techniques is not an effective strategy in non-human mammals (47), such that undepleted serum proteomics in bats will be limited to the top 300–600 proteins, with false negatives for low abundance proteins such as those of viruses (88). Alternatively, lack of detection of CoV proteins in sera despite detection of CoV RNA in oral and rectal swabs could indicate tropism, as CoVs have been more readily detected in bat feces and saliva than in blood (89).

Using our novel α-CoVs, we then tested for differential composition and abundance of serum proteins between uninfected and infected vampire bats. In both cases, we found negligible overall differences in serum proteomes with CoV infection. This lack of differences is not necessarily surprising, because bats and their α- and β-CoVs share a long coevolutionary history (27, 90). However, such null results should also be qualified by the challenges posed to differential abundance tests by sample imbalance, given the small number of infected relative to uninfected bats (91). To partly address this imbalance, we used ROC curve analyses to identify proteins with strict (AuROC ≥ 0.9; n = 7) and less conservative (AuROC ≥ 0.8; n = 25) classifier ability for infection (47, 92). The unbiased query of proteins in relation to infection through proteomics can in turn result in detection of unexpected candidate biomarkers and new insights into CoV pathogenesis in bats. For example, we identified increased ribonuclease inhibitor (RNH1) as a putative biomarker. RNH1 inhibits RNase 1 and blocks extracellular RNA degradation, possibly resulting in increased tumor necrosis factor (TNF)–α activation (93). Prior cell line studies of Eptesicus fuscus have shown limited production of TNF-α upon stimulation (94), ​​whereas those of Pteropus alecto have suggested an induced TNF-α response (16). Whether greater abundance of pro-inflammatory cytokines such as TNF-α occur with CoV infection in bats would thus be a fruitful area for future work based on RNH1 differences here. We also identified lower complement C4A (one of two C4 isotypes) as another putative biomarker. In humans, lower complement C4A and C3 can signal elevated autoimmunity (95, 96), and decreased complement C4 and C3 in COVID-19 patients also corresponds to disease severity (97). The processes that shape serum complement, namely complement synthesis, activation, and clearance, remain poorly characterized in bats (17), but the identification of C4A as a classifier could suggest specific explorations into how complement affects CoV infection.

Other candidate biomarkers also had more direct implications for the antiviral response in bats. AHSG (alpha-2-HS-glycoprotein) is a negative acute phase reactant (98) and here was a positive predictor of CoV infection. In humans, elevated AHSG is accordingly protective against progression of disease caused by SARS-CoV (99), and decreased inflammation could also contribute to viral tolerance in bats. We also identified poly(rC)-binding protein 1 (PCBP1) as a positive, albeit weaker, predictor of CoV infection (AuROC = 0.87). This RNA-binding protein is upregulated in activated T cells to control effector T cells converting into regulatory T cells and thus stabilizes the innate immune response (100, 101); PCBP1 may also prevent virus-related inflammation (102). Whereas human patients with prolonged SARS-CoV-2 infections showed lower PCB1 compared to patients with short-term infections (103), bats with CoV infection here had elevated PCBP1 and more generally harbor more PCBP1 than humans (42). Despite focusing on two different viral genera, such results suggest both similarities and differences in how bats and humans may respond to CoV infection. More generally, the list of such candidate biomarkers identified here can be used to create accurate, sensitive, quantitative, and bat-specific parallel reaction monitoring mass spectrometry-based protein assays (104, 105). Such assays could facilitate more thorough investigations into bat immune response to CoV infection. Further, such putative biomarkers are only observational correlates of naturally occurring CoV infection, but bats can be infected by a high diversity of viruses that could elicit similar immune responses (106, 107). Experimental infection with CoVs would be an important future step to identify the roles of such proteins in pathogenesis.

In addition to identifying candidate biomarkers, we also leveraged these proteins to more generally assess broad up- and downregulated biological processes with CoV infection through enrichment analyses. Using all candidate biomarkers, we found that CoV-infected bats displayed downregulation of the complement system, regulation of proteolysis, immune effector processes, and humoral immunity while also showing upregulated neutrophil-mediated immunity, overall granulocyte activation, myeloid cell responses, and glutathione processes. These results in part support findings from limited experimental infections of select bat species, which have shown little humoral response to CoVs (11, 31). Yet while Rousettus aegyptiacus challenged with SARS-like CoVs did not show hematological changes following infection (31), CoV-infected vampire bats here had largely upregulated cellular immune responses. Similarly, experimental studies have suggested bat tolerance of CoVs to be driven by upregulation of cytokine responses and a downregulated inflammatory response (11, 16, 33), but we did not find GO terms related to cytokines or inflammation in our analyses. Such discrepancies could again result from our ability to only detect the top 300–600 proteins without antibody-based depletion, which could cause low-abundance proteins (including but not limited to IFNs) being especially difficult to characterize here (88). Alternatively, these differences could reflect distinct immune responses of bats for α-CoV infection, given that experimental studies to date have focused on β-CoVs. Additionally, these findings could also signal immunological variation within and among bat clades, given that Desmodus rotundus and the closely related Artibeus jamaicensis may respond differently to CoVs (11).

Future proteomic analyses across bat species in the wild could provide a tractable means to broadly characterize host responses to viruses, including but not limited to hypothesized immune mechanisms of tolerance in this order of mammals and to infection with diverse CoVs. By leveraging the benefits of proteomics to quantify hundreds of proteins from the small sera volumes that can be obtained from most bat species (41, 42), such analyses could evaluate whether particular immune responses to viruses such as CoVs are conserved across bats (e.g., downregulation of humoral immunity) and which may be a feature of particular bat clades. In particular, further comparative proteomic analyses across Neotropical bats, including both additional members of the Phyllostomidae as well as sister families such as the Mormoopidae (108), would illuminate whether vampire bats have particular immunological relationships with CoVs that may facilitate viral tolerance. As suggested in our work here on Desmodus rotundus, such studies could also identify putative biomarkers that may suggest novel mechanisms of pathogenesis and facilitate development of protein-specific assays to improve the resource base for studying the immunology of wild bats and bat–virus dynamics.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the American Museum of Natural History (AMNHIACUC-20190129).

DB, MF, and NS conducted fieldwork. G-SL and RR ran CoV diagnostics. MJ, AB, and BN conducted proteomic analyses and bioinformatics. DB and BN analyzed data. DB and BN wrote the manuscript with contributions from all co-authors. All authors contributed to the article and approved the submitted version.

This work was supported by National Geographic (NGS-55503R-19 to DB, MF, and NS) and funds provided by Indiana University (G-SL, RR) and College of Charleston (MJ, AB).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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