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.

Bat Employs a Conserved MDA5 Gene to Trigger Antiviral Innate Immune Responses.

Bats are important hosts for various zoonotic viral diseases. However, they rarely show signs of disease infection with such viruses. As the first line for virus control, the innate immune system of bats attracted our full attention. In this study, the Tadarida brasiliensis MDA5 gene (batMDA5), a major sensor for anti-RNA viral infection, was first cloned, and its biological functions in antiviral innate immunity were identified. Bioinformatics analysis shows that the amino acid sequence of batMDA5 is poorly conserved among species, and it is evolutionarily closer to humans. The mRNA of batMDA5 was significantly upregulated in Newcastle disease virus (NDV), avian influenza virus (AIV), and vesicular stomatitis virus (VSV)-infected bat TB 1 Lu cells. Overexpression of batMDA5 could activate IFNß and inhibit vesicular stomatitis virus (VSV-GFP) replication in TB 1 Lu cells, while knockdown of batMDA5 yielded the opposite result. In addition, we found that the CARD domain was essential for MDA5 to activate IFNß by constructing MDA5 domain mutant plasmids. These results indicated that bat employs a conserved MDA5 gene to trigger anti-RNA virus innate immune response. This study helps understand the biological role of MDA5 in innate immunity during evolution.

Exploring the Role of Innate Lymphocytes in the Immune System of Bats and Virus-Host Interactions.

Bats are reservoirs of a large number of viruses of global public health significance, including the ancestral virus for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the causative agent of coronavirus disease 2019 (COVID-19). Although bats are natural carriers of multiple pathogenic viruses, they rarely display signs of disease. Recent insights suggest that bats have a more balanced host defense and tolerance system to viral infections that may be linked to the evolutionary adaptation to powered flight. Therefore, a deeper understanding of bat immune system may provide intervention strategies to prevent zoonotic disease transmission and to identify new therapeutic targets. Similar to other eutherian mammals, bats have both innate and adaptive immune systems that have evolved to detect and respond to invading pathogens. Bridging these two systems are innate lymphocytes, which are highly abundant within circulation and barrier tissues. These cells share the characteristics of both innate and adaptive immune cells and are poised to mount rapid effector responses. They are ideally suited as the first line of defense against early stages of viral infections. Here, we will focus on the current knowledge of innate lymphocytes in bats, their function, and their potential role in host-pathogen interactions. Moreover, given that studies into bat immune systems are often hindered by a lack of bat-specific research tools, we will discuss strategies that may aid future research in bat immunity, including the potential use of organoid models to delineate the interplay between innate lymphocytes, bat viruses, and host tolerance.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

Constitutive IFNα Protein Production in Bats.

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this symbiotic relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this, we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but were not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

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.

Comparative Computational Modeling of the Bat and Human Immune Response to Viral Infection with the Comparative Biology Immune Agent Based Model.

Given the impact of pandemics due to viruses of bat origin, there is increasing interest in comparative investigation into the differences between bat and human immune responses. The practice of comparative biology can be enhanced by computational methods used for dynamic knowledge representation to visualize and interrogate the putative differences between the two systems. We present an agent based model that encompasses and bridges differences between bat and human responses to viral infection: the comparative biology immune agent based model, or CBIABM. The CBIABM examines differences in innate immune mechanisms between bats and humans, specifically regarding inflammasome activity and type 1 interferon dynamics, in terms of tolerance to viral infection. Simulation experiments with the CBIABM demonstrate the efficacy of bat-related features in conferring viral tolerance and also suggest a crucial role for endothelial inflammasome activity as a mechanism for bat systemic viral tolerance and affecting the severity of disease in human viral infections. We hope that this initial study will inspire additional comparative modeling projects to link, compare, and contrast immunological functions shared across different species, and in so doing, provide insight and aid in preparation for future viral pandemics of zoonotic origin.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals.

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen's kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading worldwide, causing a global pandemic. Bat-origin RaTG13 is currently the most phylogenetically related virus. Here we obtained the complex structure of the RaTG13 receptor binding domain (RBD) with human ACE2 (hACE2) and evaluated binding of RaTG13 RBD to 24 additional ACE2 orthologs. By substituting residues in the RaTG13 RBD with their counterparts in the SARS-CoV-2 RBD, we found that residue 501, the major position found in variants of concern (VOCs) 501Y.V1/V2/V3, plays a key role in determining the potential host range of RaTG13. We also found that SARS-CoV-2 could induce strong cross-reactive antibodies to RaTG13 and identified a SARS-CoV-2 monoclonal antibody (mAb), CB6, that could cross-neutralize RaTG13 pseudovirus. These results elucidate the receptor binding and host adaption mechanisms of RaTG13 and emphasize the importance of continuous surveillance of coronaviruses (CoVs) carried by animal reservoirs to prevent another spillover of CoVs.

Gut microbiota in antiviral strategy from bats to humans: a missing link in COVID-19.

Bats are a potential natural reservoir for SARS-CoV-2 virus and other viruses detrimental to humans. Accumulated evidence has shown that, in their adaptation to a flight-based lifestyle, remodeling of the gut microbiota in bats may have contributed to immune tolerance to viruses. This evidence from bats provides profound insights into the potential influence of gut microbiota in COVID-19 disease in humans. Here, we highlight recent advances in our understanding of the mechanisms by which the gut microbiota helps bats tolerate deadly viruses, and summarize the current clinical evidence on the influence of gut microbiota on the susceptibility to SARS-CoV-2 infection and risk of COVID-19 leading to a fatal outcome. In addition, we discuss the implications of gut microbiota-targeted approaches for preventing infection and reducing disease severity in COVID-19 patients.

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.

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.

Novel Insights Into Immune Systems of Bats.

In recent years, viruses similar to those that cause serious disease in humans and other mammals have been detected in apparently healthy bats. These include filoviruses, paramyxoviruses, and coronaviruses that cause severe diseases such as Ebola virus disease, Marburg haemorrhagic fever and severe acute respiratory syndrome (SARS) in humans. The evolution of flight in bats seem to have selected for a unique set of antiviral immune responses that control virus propagation, while limiting self-damaging inflammatory responses. Here, we summarize our current understanding of antiviral immune responses in bats and discuss their ability to co-exist with emerging viruses that cause serious disease in other mammals. We highlight how this knowledge may help us to predict viral spillovers into new hosts and discuss future directions for the field.

Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence.

Bats host virulent zoonotic viruses without experiencing disease. A mechanistic understanding of the impact of bats' virus hosting capacities, including uniquely constitutive immune pathways, on cellular-scale viral dynamics is needed to elucidate zoonotic emergence. We carried out virus infectivity assays on bat cell lines expressing induced and constitutive immune phenotypes, then developed a theoretical model of our in vitro system, which we fit to empirical data. Best fit models recapitulated expected immune phenotypes for representative cell lines, supporting robust antiviral defenses in bat cells that correlated with higher estimates for within-host viral propagation rates. In general, heightened immune responses limit pathogen-induced cellular morbidity, which can facilitate the establishment of rapidly-propagating persistent infections within-host. Rapidly-transmitting viruses that have evolved with bat immune systems will likely cause enhanced virulence following emergence into secondary hosts with immune systems that diverge from those unique to bats.

Bats can carry viruses that are deadly to other mammals without themselves showing serious symptoms. In fact, bats are natural reservoirs for viruses that have some of the highest fatality rates of any viruses that people acquire from wild animals ­ including rabies, Ebola and the SARS coronavirus. Bats have a suite of antiviral defenses that keep the amount of virus in check. For example, some bats have an antiviral immune response called the interferon pathway perpetually switched on. In most other mammals, having such a hyper-vigilant immune response would cause harmful inflammation. Bats, however, have adapted anti-inflammatory traits that protect them from such harm, include the loss of certain genes that normally promote inflammation. However, no one has previously explored how these unique antiviral defenses of bats impact the viruses themselves. Now, Brook et al. have studied this exact question using bat cells grown in the laboratory. The experiments made use of cells from one bat species ­ the black flying fox ­ in which the interferon pathway is always on, and another ­ the Egyptian fruit bat ­ in which this pathway is only activated during an infection. The bat cells were infected with three different viruses, and then Brook et al. observed how the interferon pathway helped keep the infections in check, before creating a computer model of this response. The experiments and model helped reveal that the bats' defenses may have a potential downside for other animals, including humans. In both bat species, the strongest antiviral responses were countered by the virus spreading more quickly from cell to cell. This suggests that bat immune defenses may drive the evolution of faster transmitting viruses, and while bats are well protected from the harmful effects of their own prolific viruses, other creatures like humans are not. The findings may help to explain why bats are often the source for viruses that are deadly in humans. Learning more about bats' antiviral defenses and how they drive virus evolution may help scientists develop better ways to predict, prevent or limit the spread of viruses from bats to humans. More studies are needed in bats to help these efforts. In the meantime, the experiments highlight the importance of warning people to avoid direct contact with wild bats.

A Potent Postentry Restriction to Primate Lentiviruses in a Yinpterochiropteran Bat.

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.

The World Goes Bats: Living Longer and Tolerating Viruses.

For centuries, people believed that bats possessed sinister powers. Bats are thought to be ancestral hosts to many deadly viruses affecting humans including Ebola, rabies, and most recently SARS-CoV-2 coronavirus. However, bats themselves tolerate these viruses without ill effects. The second power that bats have is their longevity. Bats live much longer than similar-sized land mammals. Here we review how bats' ability to control inflammation may be contributing to their longevity. The underlying mechanisms may hold clues to developing new treatments for age-related diseases. Now may be the time to use science to exploit the secret powers of bats for human benefit.

Suppressed anti-inflammatory heat shock response in high-risk COVID-19 patients: lessons from basic research (inclusive bats), light on conceivable therapies.

The major risk factors to fatal outcome in COVID-19 patients, i.e., elderliness and pre-existing metabolic and cardiovascular diseases (CVD), share in common the characteristic of being chronic degenerative diseases of inflammatory nature associated with defective heat shock response (HSR). The molecular components of the HSR, the principal metabolic pathway leading to the physiological resolution of inflammation, is an anti-inflammatory biochemical pathway that involves molecular chaperones of the heat shock protein (HSP) family during homeostasis-threatening stressful situations (e.g., thermal, oxidative and metabolic stresses). The entry of SARS coronaviruses in target cells, on the other hand, aggravates the already-jeopardized HSR of this specific group of patients. In addition, cellular counterattack against virus involves interferon (IFN)-mediated inflammatory responses. Therefore, individuals with impaired HSR cannot resolve virus-induced inflammatory burst physiologically, being susceptible to exacerbated forms of inflammation, which leads to a fatal "cytokine storm". Interestingly, some species of bats that are natural reservoirs of zoonotic viruses, including SARS-CoV-2, possess an IFN-based antiviral inflammatory response perpetually activated but do not show any sign of disease or cytokine storm. This is possible because bats present a constitutive HSR that is by far (hundreds of times) more intense and rapid than that of human, being associated with a high core temperature. Similarly in humans, fever is a physiological inducer of HSR while antipyretics, which block the initial phase of inflammation, impair the resolution phase of inflammation through the HSR. These findings offer a rationale for the reevaluation of patient care and fever reduction in SARS, including COVID-19.

In silico studies on the comparative characterization of the interactions of SARS-CoV-2 spike glycoprotein with ACE-2 receptor homologs and human TLRs.

Coronavirus disease-2019 (COVID-19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has come out as a major threat for mankind in recent times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS-CoV-2), its mode of interaction with the angiotensin-converting enzyme-2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll-like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS-CoV-2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS-CoV-2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS-CoV-2 from a bat and also revealed that TLR4 may have a crucial role in the virus-induced inflammatory consequences associated with COVID-19. Therefore, selective targeting of TLR4-spike protein interaction by designing competitive TLR4-antagonists could pave a new way to treat COVID-19. Finally, this study is expected to improve our understanding on the immunobiology of SARS-CoV-2 and could be useful in adopting spike protein, ACE2, or TLR-guided intervention strategy against COVID-19 shortly.