Among their many unique biological features, bats are increasingly recognized as a key reservoir of many emerging viruses that cause massive morbidity and mortality in humans. Bats are capable of harboring many of these deadly viruses without any apparent signs of pathology, in a mechanism known as viral disease tolerance. However, the immunological mechanisms behind viral tolerance remain poorly understood. As a non-model organism species, there are very limited research resources and tools available to study bat immunology. In the cave nectar bat Eonycteris spelaea, we have a panel of monoclonal antibodies (mAbs) against major immune markers. An immunophenotyping survey of major immune compartments and barrier sites using these mAbs reveals differences in the immunological landscape of bats.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in patients who are severely ill, and the pathophysiology of disease is thought to be immune mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens and often promote inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and nonhuman primates. Using a mouse model of MC deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed through detection of MC-specific proteases, including chymase, the levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.

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 in-flammasome 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.

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats (Eonycteris spelaea). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

As the only mammalian group capable of powered flight, bats have many unique biological traits. Previous comparative genomic studies in bats have focused on long-term evolution. However, the micro-evolutionary processes driving recent evolution are largely under-explored. Using resequencing data from 50 black flying foxes (Pteropus alecto), one of the model species for bats, we find that black flying fox has much higher genetic diversity and lower levels of linkage disequilibrium than most of the mammalian species. Demographic inference reveals strong population fluctuations (>100 fold) coinciding with multiple historical events including the last glacial change and Toba super eruption, suggesting that the black flying fox is a very resilient species with strong recovery abilities. While long-term adaptation in the black flying fox is enriched in metabolic genes, recent adaptation in the black flying fox has a unique landscape where recently selected genes are not strongly enriched in any functional category. The demographic history and mode of adaptation suggest that black flying fox might be a well-adapted species with strong evolutionary resilience. Taken together, this study unravels a vibrant landscape of recent evolution for the black flying fox and sheds light on several unique evolutionary processes for bats comparing to other mammalian groups.

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.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcrip-tome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a dou-ble-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector func-tion early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the pre-dominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

Bats are known natural reservoirs of several highly pathogenic zoonotic viruses, including Hendra virus, Nipah virus, rabies virus, SARS-like coronaviruses, and suspected ancestral reservoirs of SARS-CoV-2 responsible for the ongoing COVID-19 pandemic. The capacity to survive infections of highly pathogenic agents without severe disease, together with many other unique features, makes bats an ideal animal model for studying the regulation of infection, cancer, and longevity, which is likely to translate into human health outcomes. A key factor that limits bat research is lack of breeding bat colonies. To address this need, a captive bat colony was established in Singapore from 19 wild-caught local cave nectar bats. The bats were screened for specific pathogens before the start of captive breeding. Custom-made cages and an optimized diet inclusive of Wombaroo dietary formula, liquid diet, and supplement of fruits enabled the bats to breed prolifically in our facility. Cages are washed daily and disinfected once every fortnight. Bats are observed daily to detect any sick bat or abnormal behavior. In addition, bats undergo a thorough health check once every 3 to 4 mo to check on their overall wellbeing, perform sampling, and document any potential pregnancy. The current colony houses over 80 bats that are successfully breeding, providing a valuable resource for research in Singapore and overseas.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Introduction: Globally, SARS-CoV-2 variants with varying virulence and transmissibility are emerging. Higher risks of severe COVID-19 including death in chronic obstructive pulmonary disease (COPD) are proposed and therefore assessing airway tissue tropism in relation to COPD is critical to better understand COVID-19 outcomes in this high-risk group. Methods: We established human airway organoid models from adult stem cells representative of the upper and lower airway in healthy individuals and COPD and evaluated infectivity, replication competence, and the innate immune response to natural SARS-CoV-2 infection with the ancestral Wuhan strain and its variant containing the spike D614G mutation. Results: SARS-CoV-2 replicates to higher viral titres in the upper rather than lower airway in healthy organoids. The D614G variant induced higher CXCL10, IL-6 and TNF-α expression and exhibits prolonged replication indicative of enhanced transmissibility. In contrast, SARS-CoV-2 infection of COPD organoids demonstrates increased replication in the lower airway, the key site of severe COVID-19 infection with additional impairment of the type-I interferon response following D614G infection. Conclusions: Our findings provide important insight into SARS-CoV-2 pathogenesis in COPD and, establish a novel human model system amenable for studying emerging viral variants. Funding: This research is supported by the National Research Foundation Singapore under its COVID-19 Research Fund (MOH-000409) and administered by the Singapore Ministry of Health's National Medical Research Council.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.

Background/Purpose: The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in significant mortality, social disorder and economic hardships. We set out to understand why certain individuals could resolve the disease successfully without serious complications by immune profiling of PBMCs from convalescent COVID-19 patients (n = 14) with mild disease trajectory. Methods: We designed 7 peptides targeting the receptor binding region (RBD) of the spike protein of SARS-CoV-2, that encompass broad HLA class I / II alleles, with the aid of the Immune Epitope Database and Analysis Resource (IEDB) website. The RBD site was chosen as it was shown to be protective when neutralising antibodies against this region could negate binding of viral spike protein to host cell ACE-2 receptor. Convalescent COVID-19 patients enrolled in this study were screened and selected for positive antibody titre against the RBD region. PBMCs from convalescent COVID-19 patients were stimulated with/without the peptides for 72hrs and immune profiled (n = 70 markers across two panels) with the high dimensional single cell mass cytometry platform (CyToF). Results: Convalescent COVID-19 patients elicited a robust recall memory T follicular helper response (CD3 + CD4 + CD45RO + CXCR5 + Tigit + ;∗∗∗∗ p < 0.0001) demonstrating peptide efficacy. Unsupervised clustering (FlowSOM) of the CD4 T cell immune landscape reaffirmed increase in memory T follicular subsets and additionally CD4 + CD45RO + CXCR5 -subsets. Further gating of antigen specific memory cells (CD45RO + CD69 + ) revealed an increase in Tbet + CXCR3 + T effectors in both CD4 and CD8 compartments. Strikingly we detected a parallel increase in CD4 + Treg (CD25 + FoxP3 + ) CXCR3 + Tbet + CD45RO + CD152 + Tigit + expression. Conclusions: COVID-19 patients that successfully resolve the viral infection not only mount a robust T effector and follicular response but also in tandem a similar T regulatory profile.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype. MC activation in humans was confirmed, through detection of the MC-specific protease, chymase, levels of which were significantly correlated with disease severity. These results support the association of MC activation with severe COVID-19, suggesting potential strategies for intervention.

Bats harbor viruses of global public health significance. Understanding bat immune systems may provide intervention strategies to prevent zoonotic disease transmission and identify therapeutic targets. This protocol describes how to culture and expand pteropid bat unconventional T cells, restricted by the MHC-I-related protein 1 (MR1), an MHC-I-like protein. Using multicolor flow-cytometry-based techniques, we examine pteropid MR1T cell functionality, including proliferative capacity, cytotoxicity, and cytokine production. This protocol can be adapted to aid immunological research in other bat species.For complete details on the use and execution of this protocol, please refer to Leeansyah et al. (2020b)

An accurate depiction of the convalescent COVID-19 immunome will help delineate the immunological milieu crucial for disease resolution and protection. Using mass cytometry, we characterized the immune architecture in patients recovering from mild COVID-19. We identified a virus-specific immune rheostat composed of an effector T (T eff ) cell recall response that is balanced by the enrichment of a highly specialized regulatory T (T reg ) cell subset. Both components were reactive against a peptide pool covering the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We also observed expansion of IFNγ + memory CD4 + T cells and virus-specific follicular helper T (T FH ) cells. Overall, these findings pinpoint critical immune effector and regulatory mechanisms essential for a potent, yet harmless resolution of COVID-19 infection.

Bats are rich reservoirs of viruses, including several high consequence zoonoses. In this study, high throughput sequencing was used to characterize the virome through a longitudinal study of a captive colony of lesser dawn bats, species Eonycteris spelaea, in Singapore. This study utilized viral RNA extracted from swabs of four body sites per bat per timepoint. Swabs of the exterior of the bat (head and body) were used to evaluate virus populations and demonstrate utility as a sample site for future surveillance to extrapolate population-level infection. Through unbiased shotgun and target-enrichment sequencing, we identified both the known and previously unknown viruses of zoonotic relevance and defined the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. We observed the population persistence of three zoonotic-related viral families that are known to be associated with spillover from bats to humans: Paramyxoviridae, Reoviridae, and Coronaviridae. To our knowledge, this is the first study that combines probe-based viral enrichment with high-throughput sequencing or that creates a viral profile from multiple swab sites on individual bats and their cohorts. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Noninvasive surveillance methods that target the body of bats not only detect viruses shed within the colony but can also represent viral populations dispersed throughout the entire colony. New knowledge of persistent viral families should inform future directions for the biosurveillance of viruses that have the potential to cross the species barrier from bats to humans or other amplifying hosts.

Bats are asymptomatic reservoir hosts for several highly pathogenic viruses. Understanding this enigmatic relationship between bats and emerging zoonotic viruses requires tools and approaches which enable the comparative study of bat immune cell populations and their functions. We show that bat genomes have a conservation of immune marker genes which delineate phagocyte populations in humans, while lacking key mouse surface markers such as Ly6C and Ly6G. Cross-reactive antibodies against CD44, CD11b, CD14, MHC II, and CD206 were multiplexed to characterize circulating monocytes, granulocytes, bone-marrow derived macrophages (BMDMs) and lung alveolar macrophages (AMs) in the cave nectar bat Eonycteris spelaea. Transcriptional profiling of bat monocytes and BMDMs identified additional markers – including MARCO, CD68, CD163, CD172α, and CD88 – which can be used to further characterize bat myeloid populations. Bat cells often resembled their human counterparts when comparing immune parameters that are divergent between humans and mice, such as the expression patterns of certain immune cell markers. A genome-wide comparison of immune-related genes also revealed a much closer phylogenetic relationship between bats and humans compared to rodents. Taken together, this study provides a set of tools and a comparative framework which will be important for unravelling viral disease tolerance mechanisms in bats.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.