Introduction: This article presents comprehensive safety guidelines and risk management strategies for conducting research involving bats in laboratory environments, building upon previously established field safety recommendations. With the increasing use of bats in biomedical and ecological research, proper handling, housing, biosafety, and biosecurity measures are critical for protecting researchers, the surrounding community, and animal welfare.Methods: This document discusses essential topics such as institutional review processes, medical surveillance, regulatory compliance, and the transportation of live bats and their biological materials. It also includes a discussion of pathogens of concern that may be found in bats or bat samples, from rabies and other lyssaviruses to filoviruses, henipaviruses, and others. It emphasizes the need for robust risk assessments tailored to various laboratory settings, highlighting procedures for handling potentially infectious tissues and fluids and managing bat housing in vivarium facilities. In addition, it addresses considerations for quarantine, environmental controls, and biosafety and biosecurity protocols that are critical for protecting researchers and the community, specifically the use of personal protective equipment and training requirements for laboratory personnel.Discussion: By detailing these safety practices using a holistic approach from a broad background of expertise, this article aims to equip researchers and biosafety professionals with the tools necessary to establish a safe and ethical framework for bat-related studies, facilitating responsible and effective research while minimizing zoonotic risks and supporting conservation efforts.

Bats are major reservoirs of viruses that can be transmitted to humans in zoonotic outbreaks. Antibody-mediated immunity plays an important role in shaping viral evolution and immune evasion but remains understudied in bats. All known mammals have a single immunoglobulin heavy chain (IgH) gene locus and up to two light chain loci. We have identified dual IgH loci on separate chromosomes in 26 bat species, highlighting extreme variation of immunogenetic architecture in order Chiroptera. In a model species, Eptesicus fuscus, we leveraged single-cell transcriptomes to confirm functional rearrangement and expression of both loci, but with different mechanisms for generating antibody diversity and function. These results provide a foundation for analysis of humoral immunity and pathogen response in bats.

Tacaribe virus (TCRV) was the first arenavirus discovered in the New World and was isolated from Artibeus bats in Trinidad and Tobago in the 1950s. One isolate, TRVL-11573, remains but it was passaged by intracranial inoculation of newborn mice 22 times that likely changed its biology. This isolate has been extensively used for arenavirus research, including our previous work that showed it can cause fatal neurological disease in Jamaican fruit bats (Artibeus jamaicensis). Another divergent TCRV, DOM2014, was recently identified in transcriptome data from a Jamaican fruit bat captured in the Dominican Republic that contained TCRV genome. A kidney fragment homogenate from this bat was inoculated into Jamaican fruit bats and all became infected with signs of mild liver disease. Experimental challenge of Jamaican fruit bats with DOM2014 led to nonfatal infection that persisted through the end of the study on day 21 and with contact transmission to naive bats. Histopathology, immunohistochemistry and serum chemistry confirmed infection and mild liver disease, but none of the bats produced neutralizing antibodies. B cell receptor transcripts suggested limited somatic hypermutation that could explain the lack of detectable neutralizing antibodies. Transcriptome profiling of livers and spleens showed signatures of a typical innate antiviral response; however, evidence of adaptive immune suppression was also present. Similarly, liver transcriptome analysis showed signatures of an expected innate antiviral response and metabolic dysfunction. The isolation of TCRV DOM2014 provides a relevant model for the study of a bat reservoir host, and which may challenge the extensive work previously conducted with TRVL-11573.

Mouse lemurs (Microcebus spp.) are an emerging primate model organism, but their genetics, cellular and molecular biology remain largely unexplored. In an accompanying paper1, we performed large-scale single-cell RNA sequencing of 27 organs from mouse lemurs. We identified more than 750 molecular cell types, characterized their transcriptomic profiles and provided insight into primate evolution of cell types. Here we use the generated atlas to characterize mouse lemur genes, physiology, disease and mutations. We uncover thousands of previously unidentified lemur genes and hundreds of thousands of new splice junctions including over 85,000 primate splice junctions missing in mice. We systematically explore the lemur immune system by comparing global expression profiles of key immune genes in health and disease, and by mapping immune cell development, trafficking and activation. We characterize primate-specific and lemur-specific physiology and disease, including molecular features of the immune program, lemur adipocytes and metastatic endometrial cancer that resembles the human malignancy. We present expression patterns of more than 400 primate genes missing in mice, many with similar expression patterns to humans and some implicated in human disease. Finally, we provide an experimental framework for reverse genetic analysis by identifying naturally occurring nonsense mutations in three primate immune genes missing in mice and by analysing their transcriptional phenotypes. This work establishes a foundation for molecular and genetic analyses of mouse lemurs and prioritizes primate genes, isoforms, physiology and disease for future study.

The ability of multiple bat species to host zoonotic pathogens without often showing disease has fostered a growing interest in bat immunology to discover the ways immune systems may differ between bats and other vertebrates. However, interspecific variation in immunological diversity among bats has only begun to be recognized. The order Chiroptera accounts for over 20% of all mammalian species and shows extreme diversity in a suite of correlated ecological traits, such that bats should not be expected to be immunologically homogenous. We review the ecological and evolutionary diversity of chiropteran hosts and highlight case studies emphasizing the range of immune strategies thus far observed across bat species, including responses to SARS-CoV-2. Next, we synthesize and propose hypotheses to explain this immunological diversity, focusing on pathogen exposure, biogeography, host energetics, and environmental stability. We then analyze immunology-related citations across bat species to motivate discussions of key research priorities. Broad sampling is needed to remedy current biases, as only a fraction of bat species has been immunologically studied. Such work should integrate methodological advancements, in vitro and in vivo studies, and phylogenetic comparative methods to robustly test evolutionary hypotheses and understand the drivers and consequences of immunological diversity among bats.

We generated a highly contiguous, annotated genome of the Jamaican fruit bat, Artibeus jamaicensis, including annotated germline immunoglobulin heavy-chain (IGH) and light-chain lambda (IGL) loci to understand bat B cell receptor repertoires. The bat germline shares many structures and features described in human immunoglobulin loci. However, some features are unique to A. jamaicensis, including an expansion of cysteine-rich IGHV genes. To investigate the relationship between the germline IGH locus and expressed B cell receptors (BCRs), we sequenced the BCRs of wild-caught and captive A. jamaicensis, finding an enrichment of IGHV3 and IGHV4 genes. Compared to humans, A. jamaicensis had shorter CDRH3s and lower levels of somatic hypermutation. Our results demonstrate that while immunoglobulin loci are largely conserved between bats and humans, distinct differences exist in the bat germline, highlighting the need for more detailed genetic characterization of these mammals.

Bats (order Chiroptera) are known carriers of lethal human viruses yet rarely exhibit disease symptoms. Research suggests bats have evolved unique mechanisms to tolerate viruses, reducing disease severity through long-term co-evolution. Studying these adaptations could provide critical insights for developing antiviral therapies, which remain challenging due to viral diversity, rapid evolution, and host toxicity. Bat immunology is a relatively new field. Early studies show that bats possess immune cell functions similar to those in humans and other mammals, alongside evidence of a dampened innate inflammatory response. However, how bats maintain immune tolerance while regulating virus replication remains poorly understood. Our research addresses this gap using in vitro models with the recombinantly recovered H18N11 influenza A virus in immortalized bat epithelial lung cells. We assess infection kinetics—including adhesion, entry, replication, and release—and compare them to human and mouse epithelial cells infected with species-specific influenza A viruses. Transcriptomics and RNAseq are employed to identify innate immune transcripts and differentially expressed proteins in response to infection. By isolating antiviral proteins and their effector mechanisms, we aim to uncover therapeutic insights inspired by bats’ evolved immune responses. Leveraging bat’s unique immune phenotype could lead to innovative antiviral stratgies, strengthening pandemic preparedness. Viral Immunology (VIR)

Over the past two decades, research on bat-associated microbes such as viruses, bacteria and fungi has dramatically increased. Here, we synthesize themes from a conference symposium focused on advances in the research of bats and their microbes, including physiological, immunological, ecological and epidemiological research that has improved our understanding of bat infection dynamics at multiple biological scales. We first present metrics for measuring individual bat responses to infection and challenges associated with using these metrics. We next discuss infection dynamics within bat populations of the same species, before introducing complexities that arise in multi-species communities of bats, humans and/or livestock. Finally, we outline critical gaps and opportunities for future interdisciplinary work on topics involving bats and their microbes.

Emerging infectious diseases are increasingly understood as a hallmark of the Anthropocene. Most experts agree that anthropogenic ecosystem change and high-risk contact among people, livestock, and wildlife have contributed to the recent emergence of new zoonotic, vector-borne, and environmentally-transmitted pathogens. However, the extent to which these factors also structure landscapes of human infection and outbreak risk is not well understood, beyond certain well-studied disease systems. Here, we consolidate 58,319 unique records of outbreak events for 32 emerging infectious diseases worldwide, and systematically test the influence of 16 hypothesized social and environmental drivers on the geography of outbreak risk, while adjusting for multiple detection, reporting, and research biases. Across diseases, outbreak risks are widely associated with mosaic landscapes where people live alongside forests and fragmented ecosystems, and are commonly exacerbated by long-term decreases in precipitation. The combined effects of these drivers are particularly strong for vector-borne diseases (e.g., Lyme disease and dengue fever), underscoring that policy strategies to manage these emerging risks will need to address land use and climate change. In contrast, we find little evidence that spillovers of directly-transmitted zoonotic diseases (e.g., Ebola virus disease and mpox) are consistently associated with these factors, or with other anthropogenic drivers such as deforestation and agricultural intensification. Most importantly, we find that observed spatial outbreak intensity is primarily an artefact of the geography of healthcare access, indicating that existing disease surveillance systems remain insufficient for comprehensive monitoring and response: across diseases, outbreak reporting declined by a median of 32% (range 1.2%-96.7%) for each additional hour's travel time from the nearest health facility. Our findings underscore that disease emergence is a multicausal feature of social-ecological systems, and that no one-size-fits-all global strategy can prevent epidemics and pandemics. Instead, ecosystem-based interventions should follow regional priorities and system-specific evidence, and be paired with investment in One Health surveillance and health system strengthening. ### Competing Interest Statement Related research funding: BVS, CJC, DWR, KEJ, and RG have received research grants from the Coalition for Epidemic Preparedness Innovations. Consulting: BH has been a consultant to the Wellcome Trust on emerging infectious diseases. CJC has been a consultant for the US Department of State on Global Health issues. Government advisory roles: RK is a senior advisor at the U.S. Department of State Bureau of Global Health Security and Diplomacy. Non-governmental advisory roles: DJB is a current member of the Lancet-PPATS Commission on Prevention of Viral Spillover. CJC, CHT, and SJR have been contributing authors on related reports by the Intergovernmental Panel on Climate Change. CJC has been a contributing author on related reports by the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services. HC-E has been a contributor to related reports by the International Union for the Conservation of Nature. RK is a current member of the Pandemic Fund Technical Advisory Panel. ### Funding Statement This work was supported by an NSF Biology Integration Institute grant (NSF DBI 2021909 and 2213854), which supported RG, SJR, RM, GFA, DJB, EAE, HKF, BAH, SNS, and CJC, as well as the Verena Institute collaborative platform under which this work was organized (viralemergence.org). Further support came from the Trinity Challenge (RG, KEJ, DWR), the Wolfson Foundation (via a UCL Excellence Fellowship; RG), the Bill and Melinda Gates foundation (grant OPP#1181128; DMP), Bryce Carmine and Anne Carmine (nee Percival) through the Massey University Foundation (RLM), the Wellcome Trust (award no. 101103/Z/13/Z; DL), and Schmidt Sciences (CHT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All code, data (where not subject to sharing constraints) and disease-specific results objects (e.g. CSVs of parameter estimates; rasters of fitted geospatial effects) are available on GitHub at github.com/viralemergence/fingerprint-preprint.

The genetic locus encoding immunoglobulin heavy chains (IgH) is critical for vertebrate humoral immune responses and diverse antibody repertoires. Immunoglobulin and T cell receptor loci of most bat species have not been annotated, despite the recurrent role of bats as viral reservoirs and sources of zoonotic pathogens. We investigated the genetic structure and function of IgH loci across the largest bat family, Vespertilionidae, focusing on big brown bats (Eptesicus fuscus ). We discovered that E. fuscus and ten other species within Vespertilionidae have two complete, functional, and distinct immunoglobulin heavy chain loci on separate chromosomes. This locus organization is previously unknown in mammals, but is reminiscent of more limited duplicated loci in teleost fish. Single cell transcriptomic data validate functional rearrangement and expression of immunoglobulin heavy chains of both loci in the expressed repertoire of Eptesicus fuscus , with maintenance of allelic exclusion, bias of usage toward the smaller and more compact IgH locus, and evidence of differential selection of antigen-experienced B cells and plasma cells varying by IgH locus use. This represents a unique mechanism for mammalian humoral immunity and may contribute to bat resistance to viral pathogenesis.

Accumulating data suggest that some bat species host emerging viruses that are highly pathogenic in humans and agricultural animals. Laboratory-based studies have highlighted important adaptations in bat immune systems that allow them to better tolerate viral infections compared to humans. Simultaneously, ecological studies have discovered critical extrinsic factors, such as nutritional stress, that correlate with virus shedding in wild-caught bats. Despite some progress in independently understanding the role of bats as reservoirs of emerging viruses, there remains a significant gap in the molecular understanding of factors that drive virus spillover from bats. Driven by a collective goal of bridging the gap between the fields of bat virology, immunology, and disease ecology, we hosted a satellite symposium at the 2024 American Society for Virology meeting. Bringing together virologists, immunologists, and disease ecologists, we discussed the intrinsic and extrinsic factors such as virus receptor engagement, adaptive immunity, and virus ecology that influence spillover from bat hosts. This article summarizes the topics discussed during the symposium and emphasizes the need for interdisciplinary collaborations and resource sharing.

Leukocyte profiles are broadly used to assess the health status of many species. Reference intervals, and an understanding of the factors that may influence these intervals, are necessary for adequate interpretation of leukograms. Using a data set that spans over three decades, we investigated variation in leukocyte profile in several populations of the evolutionarily unique reptile, the tuatara (Sphenodon punctatus). To do this, we first established reference intervals for each leukocyte type according to best practices. Next, we determined that source population and sampling date were the two most important predictors of leukocyte makeup. We found significant differences in the ratio of heterophils: lymphocytes (H:L) between populations, with tuatara on the more resource-stressed sampling island having a significantly higher ratio of H:L. Finally, we found that sampling location, sex, and life stage did not explain variation in the responses of tuatara to stimulation with Concanavalin A and lipopolysaccharide in both 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide and Griess assay experiments. Our results offer important insight into the function of leukocytes in reptiles.

Animals and their viruses are connected by a sprawling, tangled network of species interactions. Data on the host-virus network are available from several sources, which use different naming conventions and often report metadata in different levels of detail.

ABSTRACT Mouse lemurs ( Microcebus spp.) are an emerging primate model organism. However, little is known about their genetics or cellular and molecular biology. In the accompanying paper, we used large-scale single cell RNA-sequencing of 27 organs and tissues to identify over 750 molecular cell types, characterize their full transcriptomic profiles, and study evolution of primate cell types. Here we use the atlas to characterize mouse lemur genes, mutations, physiology, and disease. We uncover thousands of previously unidentified lemur genes and hundreds of thousands of new splice junctions that globally define lemur gene structures and reveal over 85,000 primate splice junctions missing in mice. We systematically explore the lemur immune system, comparing the global expression profiles of key immune genes in health and disease, and molecular mapping of immune cell development, trafficking, and their local and global activation to infection. We characterize primate/lemur-specific physiology and disease including molecular features of the immune program, of lemur adipocytes that exhibit dramatic seasonal rhythms, and of metastatic endometrial cancer that resembles the human malignancy. We identify and describe the expression patterns of over 400 primate genes missing in mice, many with similar expression patterns in human and lemur and some implicated in human disease. Finally, we provide an experimental framework for reverse genetic analysis by identifying naturally-occurring nonsense (null) mutations in three primate genes missing in mice and analyzing their transcriptional phenotypes. This work establishes mouse lemur as a tractable primate model organism for genetic and molecular analysis, and it prioritizes primate genes, splice junctions, physiology, and disease for future study.

Pandemics originating from non-human animals highlight the need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection and/or potential reservoirs to mitigate public health and conservation concerns. Multiple zoonotic coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2 and Middle Eastern respiratory syndrome-associated coronavirus (MERS-CoV), are hypothesized to have evolved in bats. We investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2, and dipeptidyl-peptidase 4 (DPP4 or CD26), the host protein bound by MERS-CoV, in the largest bat datasets to date. Both the ACE2 and DPP4 genes are under strong selection pressure in bats, more so than in other mammals, and in residues that contact viruses. Additionally, mammalian groups vary in their similarity to humans in residues that contact SARS-CoV, SARS-CoV-2 and MERS-CoV, and increased similarity to humans in binding residues is broadly predictive of susceptibility to SARS-CoV-2. This work augments our understanding of the relationship between coronaviruses and mammals, particularly bats, provides taxonomically diverse data for studies of how host proteins are bound by coronaviruses and can inform surveillance, conservation and public health efforts.

Introduction: Field work with bats is an important contribution to many areas of research in environmental biology and ecology, as well as microbiology. Work with bats poses hazards such as bites and scratches, and the potential for exposure to infectious pathogens such as rabies virus. It also exposes researchers to many other potential hazards inherent to field work, such as environmental conditions, delayed emergency responses, or challenging work conditions. Methods: This article discusses the considerations for a thorough risk assessment process around field work with bats, pre- and post-occupational health considerations, and delves into specific considerations for areas related to biosafety concerns—training, personal protective equipment, safety consideration in field methods, decontamination, and waste. It also touches on related legal and ethical issues that sit outside the realm of biosafety, but which must be addressed during the planning process. Discussion: Although the focal point of this article is bat field work located in northern and central America, the principles and practices discussed here are applicable to bat work elsewhere, as well as to field work with other animal species, and should promote careful considerations of how to safely conduct field work to protect both researchers and animals.

Pandemics originating from non-human animals highlight the need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection and/or potential reservoirs to mitigate public health and conservation concerns. Multiple zoonotic coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2 and Middle Eastern respiratory syndrome-associated coronavirus (MERS-CoV), are hypothesized to have evolved in bats. We investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2, and dipeptidyl-peptidase 4 (DPP4 or CD26), the host protein bound by MERS-CoV, in the largest bat datasets to date. Both the ACE2 and DPP4 genes are under strong selection pressure in bats, more so than in other mammals, and in residues that contact viruses. Additionally, mammalian groups vary in their similarity to humans in residues that contact SARS-CoV, SARS-CoV-2 and MERS-CoV, and increased similarity to humans in binding residues is broadly predictive of susceptibility to SARS-CoV-2. This work augments our understanding of the relationship between coronaviruses and mammals, particularly bats, provides taxonomically diverse data for studies of how host proteins are bound by coronaviruses and can inform surveillance, conservation and public health efforts.

SignificanceIsolated and infrequently colonized, islands harbor many of nature’s most renowned evolutionary radiations. Despite this evolutionary exuberance, island occupation has long been considered irreversible: The much tougher competitive and predatory milieu on mainlands prevents colonization, much less evolutionary diversification, from islands to continents. To test these postulates, we examined neotropicalAnolislizards, asking what happens when mainland and island evolutionary radiations collide. Far from being a dead end, we show that island-to-mainland colonization seeded an extensive radiation that achieved its ecomorphological disparity in ways distinct from their island ancestors. Moreover, when the incumbent and island-derived radiations collided, the ensuing interactions favored the latter, together highlighting a persistent role of both historical contingency and determinism in adaptive radiation.

The SARS-CoV-2 pandemic has led to increased concern over transmission of pathogens from humans to animals (“spillback”) and its potential to threaten conservation and public health. To assess this threat, we reviewed published evidence of spillback events, including instances where spillback could threaten conservation and human health. We identified 97 verified examples of spillback, involving a wide range of pathogens; however, infected hosts were mostly non-human primates or large, long-lived captive animals. Relatively few spillback events resulted in morbidity and mortality, and very few led to maintenance of a human pathogen in a new reservoir or subsequent “secondary spillover” back into humans. Together, these results imply that spillback represents an apparently minor threat to conservation and public health, particularly relative to other anthropogenic stressors like land use and climate change. Lastly, we outline how researchers can collect experimental and observational evidence that will expand our capacity for spillback risk assessment.