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

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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

Culture, expansion, and flow-cytometry-based functional analysis of pteropid bat MR1-restricted unconventional T cells

Summary: 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

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.</p

Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host–pathogen interactions in the bat airway epithelium

ABSTRACTBats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host–pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air–liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents

Image_2_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.PDF

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p

Image_1_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.JPEG

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p

Image_4_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.JPEG

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p

Image_3_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.JPEG

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p

Table_1_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.DOCX

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p

Image_5_Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses.JPEG

IntroductionBats 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.MethodsHere, 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.ResultsUsing 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.DiscussionOverall, 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.</p