The translational potential of studying bat immunity.

Molecular studies in bats have led to the discovery of antiviral adaptations that may explain how some bat species have evolved enhanced immune tolerance towards viruses. Accumulating data suggest that some bat species have also evolved remarkable features of longevity and low rates of cancer. Furthermore, recent research strongly suggests that discovering immune adaptations in bat models can be translated to develop immune modulators and recognize alternate therapeutic strategies for diseases affecting humans. We posit that research in bat immunology will lead to discoveries that can potentially be translated to improve health outcomes in humans.

Inflammasome activity is controlled by ZBTB16-dependent SUMOylation of ASC.

Inflammasome activity is important for the immune response and is instrumental in numerous clinical conditions. Here we identify a mechanism that modulates the central Caspase-1 and NLR (Nod-like receptor) adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). We show that the function of ASC in assembling the inflammasome is controlled by its modification with SUMO (small ubiquitin-like modifier) and identify that the nuclear ZBTB16 (zinc-finger and BTB domain-containing protein 16) promotes this SUMOylation. The physiological significance of this activity is demonstrated through the reduction of acute inflammatory pathogenesis caused by a constitutive hyperactive inflammasome by ablating ZBTB16 in a mouse model of Muckle-Wells syndrome. Together our findings identify an further mechanism by which ZBTB16-dependent control of ASC SUMOylation assembles the inflammasome to promote this pro-inflammatory response.

Massively-multiplexed epitope mapping techniques for viral antigen discovery.

Following viral infection, viral antigens bind specifically to receptors on the surface of lymphocytes thereby activating adaptive immunity in the host. An epitope, the smallest structural and functional unit of an antigen, binds specifically to an antibody or antigen receptor, to serve as key sites for the activation of adaptive immunity. The complexity and diverse range of epitopes are essential to study and map for the diagnosis of disease, the design of vaccines and for immunotherapy. Mapping the location of these specific epitopes has become a hot topic in immunology and immune therapy. Recently, epitope mapping techniques have evolved to become multiplexed, with the advent of high-throughput sequencing and techniques such as bacteriophage-display libraries and deep mutational scanning. Here, we briefly introduce the principles, advantages, and disadvantages of the latest epitope mapping techniques with examples for viral antigen discovery.

Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.

Introduction: Bats are important providers of ecosystem services such as pollination, seed dispersal, and insect control but also act as natural reservoirs for virulent zoonotic viruses. Bats host multiple viruses that cause life-threatening pathology in other animals and humans but, themselves, experience limited pathological disease from infection. Despite bats' importance as reservoirs for several zoonotic viruses, we know little about the broader viral diversity that they host. Bat virus surveillance efforts are challenged by difficulties of field capture and the limited scope of targeted PCR- or ELISA-based molecular and serological detection. Additionally, virus shedding is often transient, thus also limiting insights gained from nucleic acid testing of field specimens. Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a broad serological tool used previously to comprehensively profile viral exposure history in humans, offers an exciting prospect for viral surveillance efforts in wildlife, including bats. Methods: Here, for the first time, we apply PhIP-Seq technology to bat serum, using a viral peptide library originally designed to simultaneously assay exposures to the entire human virome. Results: Using VirScan, we identified past exposures to 57 viral genera-including betacoronaviruses, henipaviruses, lyssaviruses, and filoviruses-in semi-captive Pteropus alecto and to nine viral genera in captive Eonycteris spelaea. Consistent with results from humans, we find that both total peptide hits (the number of enriched viral peptides in our library) and the corresponding number of inferred past virus exposures in bat hosts were correlated with poor bat body condition scores and increased with age. High and low body condition scores were associated with either seropositive or seronegative status for different viruses, though in general, virus-specific age-seroprevalence curves defied assumptions of lifelong immunizing infection, suggesting that many bat viruses may circulate via complex transmission dynamics. Discussion: Overall, our work emphasizes the utility of applying biomedical tools, like PhIP-Seq, first developed for humans to viral surveillance efforts in wildlife, while highlighting opportunities for taxon-specific improvements.

Antiviral effects of interferon-stimulated genes in bats.

The interferon pathway is the first line of defense in viral infection in all mammals, and its induction stimulates broad expression of interferon-stimulated genes (ISGs). In mice and also humans, the antiviral function of ISGs has been extensively studied. As an important viral reservoir in nature, bats can coexist with a variety of pathogenic viruses without overt signs of disease, yet only limited data are available for the role of ISGs in bats. There are multiple species of bats and work has begun deciphering the differences and similarities between ISG function of human/mouse and different bat species. This review summarizes the current knowledge of conserved and bat-specific-ISGs and their known antiviral effector functions.

Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Of bats and men: Immunomodulatory treatment options for COVID-19 guided by the immunopathology of SARS-CoV-2 infection.

In humans, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is highly infective, often causes severe acute and/or long-term illness, and elicits a high rate of mortality, even in countries with sophisticated medical systems. Detailed knowledge on the immune responses underpinning COVID-19 (coronavirus disease 2019), and on strategies SARS-CoV-2 uses to evade them, can provide pivotal guidance to researchers and clinicians developing and administering potentially life-saving immunomodulatory therapies. The need for such therapies in COVID-19 is unlikely to abate soon given the emergence of variants of concern that may pose new challenges for some vaccines and neutralizing antibodies. Here, we summarize current knowledge on COVID-19 immunopathogenesis in relation to three clinical disease stages and focus on immune evasion strategies used by pathogenic coronaviruses such as skewing type I, II, and III interferon responses and inhibiting detection via pattern recognition and antigen presentation. Insights gained from bats, which exhibit minimal disease in response to SARS-CoV-2 infection, offer an informative perspective and may guide future development of new therapies. We also discuss how knowledge of immunopathology may inform therapeutic decisions, for example, on selecting the most appropriate immunotherapeutic agents and timing their administration, to reduce morbidity and mortality of COVID-19.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses.

Type I interferons (IFNs) are our first line of defense against virus infection. Recent studies have suggested the ability of SARS-CoV-2 proteins to inhibit IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wild-type SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are scarce. Here we demonstrate that SARS-CoV-2 infection induces a type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Furthermore, we show that physiological levels of IFNα detected in patients with moderate COVID-19 is sufficient to suppress SARS-CoV-2 replication in human airway cells.

Lessons from the host defences of bats, a unique viral reservoir.

There have been several major outbreaks of emerging viral diseases, including Hendra, Nipah, Marburg and Ebola virus diseases, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS)-as well as the current pandemic of coronavirus disease 2019 (COVID-19). Notably, all of these outbreaks have been linked to suspected zoonotic transmission of bat-borne viruses. Bats-the only flying mammal-display several additional features that are unique among mammals, such as a long lifespan relative to body size, a low rate of tumorigenesis and an exceptional ability to host viruses without presenting clinical disease. Here we discuss the mechanisms that underpin the host defence system and immune tolerance of bats, and their ramifications for human health and disease. Recent studies suggest that 64 million years of adaptive evolution have shaped the host defence system of bats to balance defence and tolerance, which has resulted in a unique ability to act as an ideal reservoir host for viruses. Lessons from the effective host defence of bats would help us to better understand viral evolution and to better predict, prevent and control future viral spillovers. Studying the mechanisms of immune tolerance in bats could lead to new approaches to improving human health. We strongly believe that it is time to focus on bats in research for the benefit of both bats and humankind.

Unraveling the Zoonotic Origin and Transmission of SARS-CoV-2.

The origin and zoonotic transmission route of SARS-CoV-2 remain speculative. We discuss scenarios for the zoonotic emergence of SARS-CoV-2, and also explore the missing evidence and ecological considerations that are necessary to confidently identify the origin and transmission route of SARS-CoV-2 and to prevent future pandemics of zoonotic viruses.

Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection.

Bat cells and tissue have elevated basal expression levels of antiviral genes commonly associated with interferon alpha (IFNα) signaling. Here, we show Interferon Regulatory Factor 1 (IRF1), 3, and 7 levels are elevated in most bat tissues and that, basally, IRFs contribute to the expression of type I IFN ligands and high expression of interferon regulated genes (IRGs). CRISPR knockout (KO) of IRF 1/3/7 in cells reveals distinct subsets of genes affected by each IRF in an IFN-ligand signaling-dependent and largely independent manner. As the master regulators of innate immunity, the IRFs control the kinetics and maintenance of the IRG response and play essential roles in response to influenza A virus (IAV), herpes simplex virus 1 (HSV-1), Melaka virus/Pteropine orthoreovirus 3 Melaka (PRV3M), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) infection. With its differential expression in bats compared to that in humans, this highlights a critical role for basal IRF expression in viral responses and potentially immune cell development in bats with relevance for IRF function in human biology.

Complementary regulation of caspase-1 and IL-1ß reveals additional mechanisms of dampened inflammation in bats.

Bats have emerged as unique mammalian vectors harboring a diverse range of highly lethal zoonotic viruses with minimal clinical disease. Despite having sustained complete genomic loss of AIM2, regulation of the downstream inflammasome response in bats is unknown. AIM2 sensing of cytoplasmic DNA triggers ASC aggregation and recruits caspase-1, the central inflammasome effector enzyme, triggering cleavage of cytokines such as IL-1ß and inducing GSDMD-mediated pyroptotic cell death. Restoration of AIM2 in bat cells led to intact ASC speck formation, but intriguingly resulted in a lack of caspase-1 or consequent IL-1ß activation. We further identified two residues undergoing positive selection pressures in Pteropus alecto caspase-1 that abrogate its enzymatic function and are crucial in human caspase-1 activity. Functional analysis of another bat lineage revealed a targeted mechanism for loss of Myotis davidii IL-1ß cleavage and elucidated an inverse complementary relationship between caspase-1 and IL-1ß, resulting in overall diminished signaling across bats of both suborders. Thus we report strategies that additionally undermine downstream inflammasome signaling in bats, limiting an overactive immune response against pathogens while potentially producing an antiinflammatory state resistant to diseases such as atherosclerosis, aging, and neurodegeneration.

Positive Selection of a Serine Residue in Bat IRF3 Confers Enhanced Antiviral Protection.

Compared with other mammals, bats harbor more zoonotic viruses per species and do not demonstrate signs of disease on infection with these viruses. To counteract infections with viruses, bats have evolved enhanced mechanisms to limit virus replication and immunopathology. However, molecular and cellular drivers of antiviral responses in bats largely remain an enigma. In this study, we demonstrate that a serine residue in IRF3 is positively selected for in multiple bat species. IRF3 is a central regulator of innate antiviral responses in mammals. Replacing the serine residue in bat IRF3 with the human leucine residue decreased antiviral protection in bat cells, whereas the addition of this serine residue in human IRF3 significantly enhanced antiviral protection in human cells. Our study provides genetic and functional evidence for enhanced IRF3-mediated antiviral responses in bats and adds support to speculations that bats have positively selected for multiple adaptations in their antiviral immune responses.

Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia.

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

High basal heat-shock protein expression in bats confers resistance to cellular heat/oxidative stress.

Bats, unique among mammals with powered flight, have many species with the longest size-proportionate lifespan of all mammals. Evolutionary adaptations would have been required to survive the elevated body temperatures during flight. Heat shock protein (HSP), highly conserved master regulators of cell stress, expression was examined across tissues and various cell lines in bats. Basal expression level of major HSPs (HSP70 and HSP90) is significantly higher in two different bat species compared to other mammals. This HSP expression could be a bat-unique, key factor to modulate cellular stress and death. Consequently, bat cells survive prolonged heat treatment, along with other stress stimuli, in a HSP-dependent manner, whereas other mammalian cells succumbed. This suggests HSP expression in bats could be an important adaption to intrinsic metabolic stresses like flight and therefore an important model to study stress resilience and longevity in general.

Studies on B Cells in the Fruit-Eating Black Flying Fox (Pteropus alecto).

The ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Here, we report a panel of cross-reactive antibodies against MHC-II, NK1.1, CD3, CD21, CD27, and immunoglobulin (Ig), that allows flow cytometry analysis of B, T and NK cell populations in two different fruit-eating bat species namely, Pteropus alecto and E. spelaea. Results confirmed predominance of T cells in the spleen and blood of bats, as previously reported by us. However, the percentages of B cells in bone marrow and NK cells in spleen varied greatly between wild caught P. alecto bats and E. spelaea colony bats, which may reflect inherent differences of their immune system or different immune status. Other features of bat B cells were investigated. A significant increase in sIg+ B cell population was observed in the spleen and blood from LPS-injected bats but not from poly I:C-injected bats, supporting T-independent polyclonal B cell activation by LPS. Furthermore, using an in vitro calcium release assay, P. alecto B cells exhibited significant calcium release upon cross-linking of their B cell receptor. Together, this work contributes to improve our knowledge of bat adaptive immunity in particular B cells.

Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host.

Bats are special in their ability to host emerging viruses. As the only flying mammal, bats endure high metabolic rates yet exhibit elongated lifespans. It is currently unclear whether these unique features are interlinked. The important inflammasome sensor, NLR family pyrin domain containing 3 (NLRP3), has been linked to both viral-induced and age-related inflammation. Here, we report significantly dampened activation of the NLRP3 inflammasome in bat primary immune cells compared to human or mouse counterparts. Lower induction of apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and secretion of interleukin-1ß in response to both 'sterile' stimuli and infection with multiple zoonotic viruses including influenza A virus (-single-stranded (ss) RNA), Melaka virus (PRV3M, double-stranded RNA) and Middle East respiratory syndrome coronavirus (+ssRNA) was observed. Importantly, this reduction of inflammation had no impact on the overall viral loads. We identified dampened transcriptional priming, a novel splice variant and an altered leucine-rich repeat domain of bat NLRP3 as the cause. Our results elucidate an important mechanism through which bats dampen inflammation with implications for longevity and unique viral reservoir status.

Exploring the genome and transcriptome of the cave nectar bat Eonycteris spelaea with PacBio long-read sequencing.

Background: In the past two decades, bats have emerged as an important model system to study host-pathogen interactions. More recently, it has been shown that bats may also serve as a new and excellent model to study aging, inflammation, and cancer, among other important biological processes. The cave nectar bat or lesser dawn bat (Eonycteris spelaea) is known to be a reservoir for several viruses and intracellular bacteria. It is widely distributed throughout the tropics and subtropics from India to Southeast Asia and pollinates several plant species, including the culturally and economically important durian in the region. Here, we report the whole-genome and transcriptome sequencing, followed by subsequent de novo assembly, of the E. spelaea genome solely using the Pacific Biosciences (PacBio) long-read sequencing platform. Findings: The newly assembled E. spelaea genome is 1.97 Gb in length and consists of 4,470 sequences with a contig N50 of 8.0 Mb. Identified repeat elements covered 34.65% of the genome, and 20,640 unique protein-coding genes with 39,526 transcripts were annotated. Conclusions: We demonstrated that the PacBio long-read sequencing platform alone is sufficient to generate a comprehensive de novo assembled genome and transcriptome of an important bat species. These results will provide useful insights and act as a resource to expand our understanding of bat evolution, ecology, physiology, immunology, viral infection, and transmission dynamics.