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.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Detection of filovirus-reactive antibodies in Australian bat species.

Bats have been implicated as the reservoir hosts of filoviruses in Africa, with serological evidence of filoviruses in various bat species identified in other countries. Here, serum samples from 190 bats, comprising 12 different species, collected in Australia were evaluated for filovirus antibodies. An in-house indirect microsphere assay to detect antibodies that cross-react with Ebola virus (Zaire ebolavirus; EBOV) nucleoprotein (NP) followed by an immunofluorescence assay (IFA) were used to confirm immunoreactivity to EBOV and Reston virus (Reston ebolavirus; RESTV). We found 27 of 102 Yinpterochiroptera and 19 of 88 Yangochiroptera samples were positive to EBOV NP in the microsphere assay. Further testing of these NP positive samples by IFA revealed nine bat sera that showed binding to ebolavirus-infected cells. This is the first report of filovirus-reactive antibodies detected in Australian bat species and suggests that novel filoviruses may be circulating in Australian bats.

Acute experimental infection of bats and ferrets with Hendra virus: Insights into the early host response of the reservoir host and susceptible model species.

Bats are the natural reservoir host for a number of zoonotic viruses, including Hendra virus (HeV) which causes severe clinical disease in humans and other susceptible hosts. Our understanding of the ability of bats to avoid clinical disease following infection with viruses such as HeV has come predominantly from in vitro studies focusing on innate immunity. Information on the early host response to infection in vivo is lacking and there is no comparative data on responses in bats compared with animals that succumb to disease. In this study, we examined the sites of HeV replication and the immune response of infected Australian black flying foxes and ferrets at 12, 36 and 60 hours post exposure (hpe). Viral antigen was detected at 60 hpe in bats and was confined to the lungs whereas in ferrets there was evidence of widespread viral RNA and antigen by 60 hpe. The mRNA expression of IFNs revealed antagonism of type I and III IFNs and a significant increase in the chemokine, CXCL10, in bat lung and spleen following infection. In ferrets, there was an increase in the transcription of IFN in the spleen following infection. Liquid chromatography tandem mass spectrometry (LC-MS/MS) on lung tissue from bats and ferrets was performed at 0 and 60 hpe to obtain a global overview of viral and host protein expression. Gene Ontology (GO) enrichment analysis of immune pathways revealed that six pathways, including a number involved in cell mediated immunity were more likely to be upregulated in bat lung compared to ferrets. GO analysis also revealed enrichment of the type I IFN signaling pathway in bats and ferrets. This study contributes important comparative data on differences in the dissemination of HeV and the first to provide comparative data on the activation of immune pathways in bats and ferrets in vivo following infection.

An argument for pandemic risk management using a multidisciplinary One Health approach to governance: an Australian case study.

The emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic has resulted in significant global impact. However, COVID-19 is just one of several high-impact infectious diseases that emerged from wildlife and are linked to the human relationship with nature. The rate of emergence of new zoonoses (diseases of animal origin) is increasing, driven by human-induced environmental changes that threaten biodiversity on a global scale. This increase is directly linked to environmental drivers including biodiversity loss, climate change and unsustainable resource extraction. Australia is a biodiversity hotspot and is subject to sustained and significant environmental change, increasing the risk of it being a location for pandemic origin. Moreover, the global integration of markets means that consumption trends in Australia contributes to the risk of disease spill-over in our regional neighbours in Asia-Pacific, and beyond. Despite the clear causal link between anthropogenic pressures on the environment and increasing pandemic risks, Australia's response to the COVID-19 pandemic, like most of the world, has centred largely on public health strategies, with a clear focus on reactive management. Yet, the span of expertise and evidence relevant to the governance of pandemic risk management is much wider than public health and epidemiology. It involves animal/wildlife health, biosecurity, conservation sciences, social sciences, behavioural psychology, law, policy and economic analyses to name just a few.The authors are a team of multidisciplinary practitioners and researchers who have worked together to analyse, synthesise, and harmonise the links between pandemic risk management approaches and issues in different disciplines to provide a holistic overview of current practice, and conclude the need for reform in Australia. We discuss the adoption of a comprehensive and interdisciplinary 'One Health' approach to pandemic risk management in Australia. A key goal of the One Health approach is to be proactive in countering threats of emerging infectious diseases and zoonoses through a recognition of the interdependence between human, animal, and environmental health. Developing ways to implement a One Health approach to pandemic prevention would not only reduce the risk of future pandemics emerging in or entering Australia, but also provide a model for prevention strategies around the world.

Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity.

Bats have attracted attention in recent years as important reservoirs of viruses deadly to humans and other mammals. These infections are typically nonpathogenic in bats raising questions about innate immune differences that might exist between bats and other mammals. The APOBEC3 gene family encodes antiviral DNA cytosine deaminases with important roles in the suppression of diverse viruses and genomic parasites. Here, we characterize pteropid APOBEC3 genes and show that species within the genus Pteropus possess the largest and most diverse array of APOBEC3 genes identified in any mammal reported to date. Several bat APOBEC3 proteins are antiviral as demonstrated by restriction of retroviral infectivity using HIV-1 as a model, and recombinant A3Z1 subtypes possess strong DNA deaminase activity. These genes represent the first group of antiviral restriction factors identified in bats with extensive diversification relative to homologues in other mammals.

Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats.

Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses.

Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus.

Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis.IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

Characterization of the Antigen Processing Machinery and Endogenous Peptide Presentation of a Bat MHC Class I Molecule.

Bats are a major reservoir of emerging and re-emerging infectious diseases, including severe acute respiratory syndrome-like coronaviruses, henipaviruses, and Ebola virus. Although highly pathogenic to their spillover hosts, bats harbor these viruses, and a large number of other viruses, with little or no clinical signs of disease. How bats asymptomatically coexist with these viruses is unknown. In particular, little is known about bat adaptive immunity, and the presence of functional MHC molecules is mostly inferred from recently described genomes. In this study, we used an affinity purification/mass spectrometry approach to demonstrate that a bat MHC class I molecule, Ptal-N*01:01, binds antigenic peptides and associates with peptide-loading complex components. We identified several bat MHC class I-binding partners, including calnexin, calreticulin, protein disulfide isomerase A3, tapasin, TAP1, and TAP2. Additionally, endogenous peptide ligands isolated from Ptal-N*01:01 displayed a relatively broad length distribution and an unusual preference for a C-terminal proline residue. Finally, we demonstrate that this preference for C-terminal proline residues was observed in Hendra virus-derived peptides presented by Ptal-N*01:01 on the surface of infected cells. To our knowledge, this is the first study to identify endogenous and viral MHC class I ligands for any bat species and, as such, provides an important avenue for monitoring and development of vaccines against major bat-borne viruses both in the reservoir and spillover hosts. Additionally, it will provide a foundation to understand the role of adaptive immunity in bat antiviral responses.

Insights into the ancestral organisation of the mammalian MHC class II region from the genome of the pteropid bat, Pteropus alecto.

BACKGROUND: Bats are an extremely successful group of mammals and possess a variety of unique characteristics, including their ability to co-exist with a diverse range of pathogens. The major histocompatibility complex (MHC) is the most gene dense and polymorphic region of the genome and MHC class II (MHC-II) molecules play a vital role in the presentation of antigens derived from extracellular pathogens and activation of the adaptive immune response. Characterisation of the MHC-II region of bats is crucial for understanding the evolution of the MHC and of the role of pathogens in shaping the immune system. RESULTS: Here we describe the relatively contracted MHC-II region of the Australian black flying-fox (Pteropus alecto), providing the first detailed insight into the MHC-II region of any species of bat. Twelve MHC-II genes, including one locus (DRB2) located outside the class II region, were identified on a single scaffold in the bat genome. The presence of a class II locus outside the MHC-II region is atypical and provides evidence for an ancient class II duplication block. Two non-classical loci, DO and DM and two classical, DQ and DR loci, were identified in P. alecto. A putative classical, DPB pseudogene was also identified. The bat's antigen processing cluster, though contracted, remains highly conserved, thus supporting its importance in antigen presentation and disease resistance. CONCLUSIONS: This detailed characterisation of the bat MHC-II region helps to fill a phylogenetic gap in the evolution of the mammalian class II region and is a stepping stone towards better understanding of the immune responses in bats to viral, bacterial, fungal and parasitic infections.

Marsupials and monotremes possess a novel family of MHC class I genes that is lost from the eutherian lineage.

BACKGROUND: Major histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes. RESULTS: Application of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT, which has not previously been characterized. These class I genes are found in both American and Australian marsupials, and in monotremes, at an evolutionary chromosomal breakpoint, but are not present in non-mammalian genomes and have been lost from the eutherian lineage. We show that UT family members are expressed in the thymus of the gray short-tailed opossum and in other immune tissues of several Australian marsupials. Structural homology modelling shows that the proteins encoded by this family are predicted to have an open, though short, antigen-binding groove. CONCLUSIONS: We have identified a novel sub-family of putatively non-classical MHC class I genes that are specific to marsupials and monotremes. This family was present in the ancestral mammal and is found in extant marsupials and monotremes, but has been lost from the eutherian lineage. The function of this family is as yet unknown, however, their predicted structure may be consistent with presentation of antigens to T-cells.

Characterisation of novel microRNAs in the Black flying fox (Pteropus alecto) by deep sequencing.

BACKGROUND: Bats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats. RESULTS: This study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction. CONCLUSIONS: MicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

Deep RNA sequencing reveals complex transcriptional landscape of a bat adenovirus.

Bat adenoviruses are a group of recently identified adenoviruses (AdVs) which are highly prevalent in bats yet share low similarity to known AdVs from other species. In this study, deep RNA sequencing was used to analyze the transcriptome at five time points following the infection of a bat AdV in a kidney cell line derived from a myotis bat species. Evidence of AdV replication was observed with the proportion of viral RNAs ranging from 0.01% at 6 h to 1.3% at 18 h. Further analysis of viral temporal gene expression revealed three replication stages, the early-stage genes encoding mainly host interaction proteins, the intermediate-stage genes for the DNA replication and assembly proteins, and the late-stage genes for most structural proteins. Several bat AdV genes were expressed at stages that differed from those of their counterpart genes previously reported for human AdV type 2. In addition, single-base resolution splice sites of several genes and promoter regions of all 30 viral genes were fully determined. Simultaneously, the temporal cellular gene expression profiles were identified. The most overrepresented functional categories of the differentially expressed genes were related to cellular immune response, transcription, translation, and DNA replication and repair. Taken together, the deep RNA sequencing provided a global, transcriptional profile of the novel bat AdV and the virus-host interactions which will be useful for the understanding and investigation of AdV replication, pathogenesis, and specific virus-bat interactions in future research.

Profile of ocular trauma in the Solomon Islands.

BACKGROUND: The objective of this study was to characterize the causes of ocular trauma and determine the risk factors for infection and vision loss following ocular trauma in the Solomon Islands. DESIGN: A prospective clinic-based study. PARTICIPANTS: A total of 507 patients with ocular trauma who were reviewed at the National Referral Hospital in Honiara or one of five provincial eye clinics were included. METHODS: An interview-based questionnaire to determine the circumstances of ocular trauma, and an ocular examination to elicit the trauma sustained,infectious sequelae and the visual outcome. MAIN OUTCOME MEASURE: Visual acuity. RESULTS: Males were significantly more likely to have ocular trauma than females (P = 0.01). The major cause of ocular trauma in young boys and girls was being poked by a stick, followed by lime burns in young boys. For both genders, physical violence resulted in most injuries across all adult age groups. Microbial keratitis complicated 4.4% of ocular trauma. Monocular vision impairment (<6/18) occurred in 5.5% of participants and was more likely to occur if female (P = 0.02). CONCLUSIONS: Ocular trauma is a significant cause of visual morbidity in the Solomon Islands. The results from this prospective study provide a basis for planning blindness prevention programmes in the Western Pacific.

Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity.

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.

Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences.

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

The swan genome and transcriptome, it is not all black and white.

BACKGROUND: The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related northern hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious diseases, notably infectious diseases from which Australia has been largely shielded. Unlike mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to highly pathogenic avian influenza. Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. RESULTS: Here, we generate the first chromosome-length black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We use these genomes and transcriptomes to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to highly pathogenic avian influenza. We also implicate genetic differences in SLC45A2 gene in the iconic plumage of the black swan. CONCLUSION: Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat, the black swan would be in a significant peril.

The immune gene repertoire of an important viral reservoir, the Australian black flying fox.

BACKGROUND: Bats are the natural reservoir host for a range of emerging and re-emerging viruses, including SARS-like coronaviruses, Ebola viruses, henipaviruses and Rabies viruses. However, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. Massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. Although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. In this study, we describe the immune transcriptome of the Australian flying fox, Pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity. RESULTS: Towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from P. alecto. We identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. A small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts. CONCLUSIONS: This study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. In addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection.

ACE2 Expression in Organotypic Human Airway Epithelial Cultures and Airway Biopsies.

Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.